WorldWideScience

Sample records for membrane-bound random dna

  1. Structure Biology of Membrane Bound Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Dax [Johns Hopkins Univ., Baltimore, MD (United States). School of Medicine. Dept. of Physiology

    2016-11-30

    The overall goal of the proposed research is to understand the membrane-associated active processes catalyzed by an alkane $\\square$-hydroxylase (AlkB) from eubacterium Pseudomonase oleovorans. AlkB performs oxygenation of unactivated hydrocarbons found in crude oils. The enzymatic reaction involves energy-demanding steps in the membrane with the uses of structurally unknown metal active sites featuring a diiron [FeFe] center. At present, a critical barrier to understanding the membrane-associated reaction mechanism is the lack of structural information. The structural biology efforts have been challenged by technical difficulties commonly encountered in crystallization and structural determination of membrane proteins. The specific aims of the current budget cycle are to crystalize AlkB and initiate X-ray analysis to set the stage for structural determination. The long-term goals of our structural biology efforts are to provide an atomic description of AlkB structure, and to uncover the mechanisms of selective modification of hydrocarbons. The structural information will help elucidating how the unactivated C-H bonds of saturated hydrocarbons are oxidized to initiate biodegradation and biotransformation processes. The knowledge gained will be fundamental to biotechnological applications to biofuel transformation of non-edible oil feedstock. Renewable biodiesel is a promising energy carry that can be used to reduce fossil fuel dependency. The proposed research capitalizes on prior BES-supported efforts on over-expression and purification of AlkB to explore the inner workings of a bioenergy-relevant membrane-bound enzyme.

  2. Heterologous expression and purification of membrane-bound pyrophosphatases

    DEFF Research Database (Denmark)

    Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg

    2011-01-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low l...

  3. Tunable Tensor Voting Improves Grouping of Membrane-Bound Macromolecules

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A.; Bebis, George; Parvin, Bahram

    2009-04-15

    Membrane-bound macromolecules are responsible for structural support and mediation of cell-cell adhesion in tissues. Quantitative analysis of these macromolecules provides morphological indices for damage or loss of tissue, for example as a result of exogenous stimuli. From an optical point of view, a membrane signal may have nonuniform intensity around the cell boundary, be punctate or diffused, and may even be perceptual at certain locations along the boundary. In this paper, a method for the detection and grouping of punctate, diffuse curvilinear signals is proposed. Our work builds upon the tensor voting and the iterative voting frameworks to propose an efficient method to detect and refine perceptually interesting curvilinear structures in images. The novelty of our method lies on the idea of iteratively tuning the tensor voting fields, which allows the concentration of the votes only over areas of interest. We validate the utility of our system with synthetic and annotated real data. The effectiveness of the tunable tensor voting is demonstrated on complex phenotypic signals that are representative of membrane-bound macromolecular structures.

  4. NMR spectroscopic studies of membrane-bound biological systems

    International Nuclear Information System (INIS)

    Hohlweg, W.

    2013-01-01

    In the course of this thesis, biological NMR spectroscopy was employed in studying membrane-bound peptides and proteins, for which structural information is still comparatively hard to obtain. Initial work focused on various model peptides bound to membrane-mimicking micelles, studying the protonation state of arginine in a membrane environment. Strong evidence for a cation-π complex was found in TM7, a peptide which forms the seventh transmembrane helix of subunit a of the vacuolar-type H+-ATPase (V-ATPase). V-ATPase is a physiologically highly relevant proton pump, which is present in intracellular membranes of all eukaryotic organisms, as well as the plasma membrane of several specialized cells. Loss of functional V-ATPase is associated with human diseases such as osteopetrosis, distal renal tubular acidosis or the spreading of cancer. V-ATPase is considered a potential drug target in the treatment of osteoporosis and cancer, or in the development of novel contraceptives. Results from NMR solution structure determination, NMR titration experiments, paramagnetic relaxation enhancement experiments and tryptophan fluorescence spectroscopy confirm the existence of a buried cation-? complex formed between arginine residue R735, which is essential for proton transport, and neighbouring tryptophan and tyrosine residues. In vivo experiments in the yeast Saccharomyces cerevisiae using selective growth tests and fluorescence microscopy showed that formation of the cation-π complex is essential for V-ATPase function. Deletion of both aromatic residues, as well as only the one tryptophan residue leads to growth defects and inability to maintain vacuolar pH homeostasis. These findings shine new light on the still elusive mechanism of proton transport in V-ATPase, and show that arginine R735 may be directly involved in proton transfer across the membrane. (author) [de

  5. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-01-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  6. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

    NARCIS (Netherlands)

    Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

  7. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

  8. Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

    Science.gov (United States)

    Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

    2017-10-01

    Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

    Science.gov (United States)

    Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

    2016-04-19

    Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

  10. Application of randomly amplified polymorphic DNA (RAPD ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... Polymerase chain reaction (PCR) based molecular markers have become increasingly popular for fingerprinting and cultivars identification since the development of PCR technology. (Saiki et al., 1988). RAPD-PCR (randomly amplified polymorphic DNA) was first conducted by Williams et al. (1990).

  11. Random amplified polymorphic DNA based genetic characterization ...

    African Journals Online (AJOL)

    Random amplified polymorphic DNA based genetic characterization of four important species of Bamboo, found in Raigad district, Maharashtra State, India. ... Bambusoideae are differentiated from other members of the family by the presence of petiolate blades with parallel venation and stamens are three, four, six or more, ...

  12. The impact of physiological crowding on the diffusivity of membrane bound proteins.

    Science.gov (United States)

    Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C

    2016-02-21

    Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

  13. Synthetic activity enhancement of membrane-bound lipase from Rhizopus chinensis by pretreatment with isooctane.

    Science.gov (United States)

    Wang, Dong; Xu, Yan; Teng, Yun

    2007-05-01

    The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4 degrees C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.

  14. Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Oksana G. Luneva

    2016-06-01

    Full Text Available Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

  15. Compartmentalized system with membrane-bound glycerol kinase. Activity and product distribution versus asymmetrical substrate supply.

    Science.gov (United States)

    Girard, A; Merchie, B; Maïsterrena, B

    1991-03-15

    An artificial-membrane-bound glycerokinase chosen as a membrane-bound two-substrate-enzyme model has been used to separate two unequal compartments of a specially designed diffusion cell. An interesting feature is the asymmetry of compartments and the existence of a diffusion layer adjacent to only one face of the enzymic membrane. In such a situation the apparent enzyme activity and the product distribution in the system have been studied versus all the possibilities of combination of ATP and glycerol supply. Our approach has lead us to differentiate two different roles played by a diffusion layer adjacent to a permeable enzymic membrane. Depending on the spatial origin of the enzymic substrates (i.e. from which compartment they derive), the diffusion layer can play either the role of a passive additional resistance to that of the membrane or the role of a third compartment in which the reaction product can partially accumulate before splitting on both parts of the membrane. Our results mainly demonstrate that a membrane-bound enzyme activity and the resulting product distribution occurring in a compartmentalized system may be regulated by the cumulative effect due to the asymmetry in volumes of the compartments, the presence of a diffusion layer and the different possibilities of substrate supply. With the topography studied, which is close to that reported for many 'in vivo' situations, the product may be diffused lead to vectorial metabolism processes.

  16. RANDNA: a random DNA sequence generator.

    Science.gov (United States)

    Piva, Francesco; Principato, Giovanni

    2006-01-01

    Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

  17. Molecular cloning, mRNA expression and characterization of membrane-bound hemoglobin in oriental river prawn Macrobrachium nipponense.

    Science.gov (United States)

    Sun, Shengming; Xuan, Fujun; Fu, Hongtuo; Zhu, Jian; Ge, Xianping; Wu, Xugan

    2017-05-01

    Most hemoglobins are respiratory proteins and are ubiquitous in animals, bacteria, fungi, protists, and plants. In this study, we describe a membrane-bound hemoglobin in the oriental river prawn Macrobrachium nipponense (MnHb), which also expresses hemocyanin. MnHb cDNA was cloned using the rapid amplification of cDNA ends (RACE) approach, which afforded a 1201bp gene encoding a 193 amino acid polypeptide. Bioinformatic evaluation suggested MnHb is membrane anchored by N-myristoylation, and immunofluorescence confirmed its location in the membrane of chief cells in the gill. The effect of hypoxia on MnHb expression was investigated, and reverse transcription PCR (RT-PCR) and Western blotting showed that MnHb was expressed almost exclusively in the gill. Quantitative RT-PCR revealed a significant increase in expression after 6h of hypoxia, and levels peaked at 24h due to oxidative stress. Exposure of cultured prawns to the stress inducer H 2 O 2 significantly up-regulated the expression of MnHb in a dose-dependent manner. MnHb may have a role in protecting cell membrane lipids from damage by reactive oxygen species. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Application of random amplified polymorphic DNA (RAPD) markers ...

    African Journals Online (AJOL)

    SAM

    2014-06-11

    1998). DNA fingerprinting of strawberry (Fragaria X ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers. Euphytica. 102:247-253. Doyle JJ, Doyle LJ (1990). Isolation of plant DNA from fresh tissue.

  19. Identification and characterization of a naturally occurring, efficiently cleaved, membrane-bound, clade A HIV-1 Env, suitable for immunogen design, with properties comparable to membrane-bound BG505.

    Science.gov (United States)

    Das, Supratik; Boliar, Saikat; Samal, Sweety; Ahmed, Shubbir; Shrivastava, Tripti; Shukla, Brihaspati N; Goswami, Sandeep; Bansal, Manish; Chakrabarti, Bimal K

    2017-10-01

    Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine. We have recently reported that Envs 4-2.J41 (clade C) and JRCSF (clade B) are also efficiently cleaved and show similar properties. However, an efficiently cleaved, membrane-bound clade A Env suitable for genetic vaccination has not been directly demonstrated. Here we report that BG505 and a new clade A Env, QB726.70M.ENV.C4 (or A5) are efficiently cleaved on cell membrane. A5 shows desirable antigenic properties comparable with BG505 on cell surface. A5SOSIP in supernatant displays majority of bNAb binding epitopes. Thus, both BG505 and A5 Envs can be used in DNA prime-protein boost vaccination studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    Energy Technology Data Exchange (ETDEWEB)

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  1. Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner

    2011-01-01

    In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets...... or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could...

  2. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

    Science.gov (United States)

    Jurat-Fuentes, Juan Luis; Karumbaiah, Lohitash; Jakka, Siva Rama Krishna; Ning, Changming; Liu, Chenxi; Wu, Kongming; Jackson, Jerreme; Gould, Fred; Blanco, Carlos; Portilla, Maribel; Perera, Omaththage; Adang, Michael

    2011-03-01

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  3. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

    Directory of Open Access Journals (Sweden)

    Juan Luis Jurat-Fuentes

    Full Text Available Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP were detected by two dimensional differential in-gel electrophoresis (2D-DIGE analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  4. Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

    Energy Technology Data Exchange (ETDEWEB)

    Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu (Univ. of Tokyo (Japan))

    1990-01-09

    Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

  5. Membrane-bound globin X protects the cell from reactive oxygen species.

    Science.gov (United States)

    Koch, Jonas; Burmester, Thorsten

    2016-01-08

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Identification and characterization of GmPDIL7, a soybean ER membrane-bound protein disulfide isomerase family protein.

    Science.gov (United States)

    Okuda, Aya; Matsusaki, Motonori; Masuda, Taro; Urade, Reiko

    2017-02-01

    Most proteins synthesized in the endoplasmic reticulum (ER) possess intramolecular and intermolecular disulfide bonds, which play an important role in the conformational stability and function of proteins. Hence, eukaryotic cells contain protein disulfide bond formation pathways such as the protein disulfide isomerase (PDI)-ER oxidoreductin 1 (Ero1) system in the ER lumen. In this study, we identified soybean PDIL7 (GmPDIL7), a novel soybean ER membrane-bound PDI family protein, and determined its enzymatic properties. GmPDIL7 has a putative N-terminal signal sequence, a thioredoxin domain with an active center motif (CGHC), and a putative C-terminal transmembrane region. Likewise, we demonstrated that GmPDIL7 is ubiquitously expressed in soybean tissues and is localized in the ER membrane. Furthermore, GmPDIL7 associated with other soybean PDI family proteins in vivo and GmPDIL7 mRNA was slightly upregulated under ER stress. The redox potential of recombinant GmPDIL7 expressed in Escherichia coli was -187 mV, indicating that GmPDIL7 could oxidize unfolded proteins. GmPDIL7 exhibited a dithiol oxidase activity level that was similar to other soybean PDI family proteins. However, the oxidative refolding activity of GmPDIL7 was lower than other soybean PDI family proteins. GmPDIL7 was well oxidized by GmERO1. Taken together, our results indicated that GmPDIL7 primarily plays a role as a supplier of disulfide bonds in nascent proteins for oxidative folding on the ER membrane. The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Protein disulfide isomerase: EC 5.3.4.1. © 2016 Federation of European Biochemical Societies.

  7. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  8. Effect of narcotics on membrane-bound mitochondrial processes in fish

    DEFF Research Database (Denmark)

    Vergauwen, Lucia; Nørgaard Schmidt, Stine; Michiels, Ellen

    in vivo (zebrafish embryo) and in vitro tests. We applied a passive dosing method to expose zebrafish embryos up to 5 days post fertilization to linear dilution series of a set of non-polar narcotics (phenanthrene and three chlorobenzene structure analogues). In addition to increasing mortality, we...... membrane-bound process and is therefore a potential target. We found that in zebrafish embryos ETC activity was increased at low exposure concentrations, suggesting a compensatory response, while it decreased when exposure concentrations reached levels causing reduced motility, heart rate and eventually...... mortality. The effect of narcotic compounds on ETC activity was confirmed in vitro: we observed inhibition of the ETC after adding the compounds directly to a homogenate of control embryos. To further investigate effects on the energy production system, and to characterize the observed compensatory response...

  9. Membrane-bound alcohol dehydrogenase is essential for glyceric acid production in Acetobacter tropicalis.

    Science.gov (United States)

    Habe, Hiroshi; Sato, Shun; Fukuoka, Tokuma; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2011-01-01

    Acetobacter tropicalis NBRC16470 can produce highly enantiomerically pure D-glyceric acid (D-GA; >99 % enantiomeric excess) from glycerol. To investigate whether membrane-bound alcohol dehydrogenase (mADH) is involved in GA production in A. tropicalis, we amplified part of the gene encoding mADH subunit I (adhA) using polymerase chain reaction and constructed an adhA-disrupted mutant of A. tropicalis (ΔadhA). Because ΔadhA did not produce GA, we confirmed that mADH is essential for the conversion of glycerol to GA. We also cloned and sequenced the entire region corresponding to adhA and adhB, which encodes mADH subunit II. The sequences showed high identities (84-86 %) with the equivalent mADH subunits from other Acetobacter spp.

  10. Free and membrane-bound ribosomes and polysomes in hippocampal neurons during a learning experiment.

    Science.gov (United States)

    Wenzel, J; David, H; Pohle, W; Marx, I; Matthies, H

    1975-01-24

    The ribosomes of the CA1 and CA3 pyramidal cells of hipocampus were investigated by morphometric methods after the acquisition of a shock-motivated brightness discrimination in rats. A significant increase in the total number of ribosomes was observed in CA1 cells of trained animals and in CA3 cells of both active controls and trained rats. A significant increase in membrane-bound ribosomes was obtained in CA1 and CA3 cells after training only. The results confirm the suggestion of an increased protein synthesis in hippocampal neurons during and after the acquisition of a brightness discrimination, as we have concluded from out previous investigations on the incorporation of labeled amino acids under identical experimental conditions. The results lead to the assumption that the protein synthesis in some neuronal cells may probably differ not only quantitatively, but also qualitatively in trained and untrained animals.

  11. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    Correia, Cristina; Besson, Stéphane; Brondino, Carlos D; González, Pablo J; Fauque, Guy; Lampreia, Jorge; Moura, Isabel; Moura, José J G

    2008-11-01

    Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

  12. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

    DEFF Research Database (Denmark)

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas

    2017-01-01

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosyla......Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes...

  13. Membrane-bound mucins and mucin terminal glycans expression in idiopathic or Helicobacter pylori, NSAID associated peptic ulcers.

    Science.gov (United States)

    Niv, Yaron; Boltin, Doron; Halpern, Marisa; Cohen, Miriam; Levi, Zohar; Vilkin, Alex; Morgenstern, Sara; Manugian, Vahig; St Lawrence, Erica; Gagneux, Pascal; Kaur, Sukhwinder; Sharma, Poonam; Batra, Surinder K; Ho, Samuel B

    2014-10-28

    To determine the expression of membrane-bound mucins and glycan side chain sialic acids in Helicobacter pylori (H. pylori)-associated, non-steroidal inflammatory drug (NSAID)-associated and idiopathic-gastric ulcers. We studied a cohort of randomly selected patients with H. pylori (group 1, n = 30), NSAID (group 2, n = 18), combined H. pylori and NSAID associated gastric ulcers (group 3, n = 24), and patients with idiopathic gastric ulcers (group 4, n = 20). Immunohistochemistry for MUC1, MUC4, MUC17, and staining for Erythrina cristagalli agglutinin and Sambucus nigra agglutinin (SNA) lectins was performed on sections from the ulcer margins. Staining intensity of MUC17 was higher in H. pylori ulcers (group 1) than in idiopathic ulcers (group 4), 11.05 ± 3.67 vs 6.93 ± 4.00 for foveola cells, and 10.29 ± 4.67 vs 8.00 ± 3.48 for gland cells, respectively (P < 0.0001). In contrast, MUC1 expression was higher in group 4 compared group 1, 9.89 ± 4.17 vs 2.93 ± 5.13 in foveola cells and 7.63 ± 4.60 vs 2.57± 4.50 for glands, respectively (P < 0.0001). SNA lectin staining was increased in group 4, in parallel to elevated MUC1 expression, indicating more abundant α2-6 sialylation in that group. Cytoplasmic MUC17 staining was significantly decreased in the cases with idiopathic ulcer. The opposite was observed for both MUC1 and SNA lectin. This observation may reflect important pathogenic mechanisms, since different mucins with altered sialylation patterns may differ in their protection efficiency against acid and pepsin.

  14. Membrane-bound organelles versus membrane-less compartments and their control of anabolic pathways in Drosophila

    NARCIS (Netherlands)

    Aguilera-Gomez, Angelica; Rabouille, Catherine

    2017-01-01

    Classically, we think of cell compartmentalization as being achieved by membrane-bound organelles. It has nevertheless emerged that membrane-less assemblies also largely contribute to this compartmentalization. Here, we compare the characteristics of both types of compartmentalization in term of

  15. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    NARCIS (Netherlands)

    Lundin, A.; Dijkman, R.; Bergstrom, T.; Kann, N.; Adamiak, B.; Hannoun, C.; Kindler, E.; Jonsdottir, H.R.; Muth, D.; Kint, J.; Forlenza, M.

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound

  16. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    Directory of Open Access Journals (Sweden)

    Karolína Šubrtová

    2015-02-01

    Full Text Available In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt membrane potential (Δψm is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC. Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

  17. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

    DEFF Research Database (Denmark)

    Kruse, Torben; Bork-Jensen, J.; Gerdes, Kenn

    2005-01-01

    . subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became......D. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based......MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B...

  18. Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

    Science.gov (United States)

    Meisrimler, Claudia-Nicole; Planchon, Sebastien; Renaut, Jenny; Sergeant, Kjell; Lüthje, Sabine

    2011-08-12

    Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor

    Science.gov (United States)

    Lister, Francis G. A.; Le Bailly, Bryden A. F.; Webb, Simon J.; Clayden, Jonathan

    2017-05-01

    Signal transduction through G-protein-coupled receptors (GPCRs) involves binding to signalling molecules at the cell surface, which leads to global changes in molecular conformation that are communicated through the membrane. Artificial mechanisms for communication involving ligand binding and global conformational switching have been demonstrated so far only in the solution phase. Here, we report a membrane-bound synthetic receptor that responds to binding of a ligand by undergoing a conformational change that is propagated over several nanometres, deep into the phospholipid bilayer. Our design uses a helical foldamer core, with structural features borrowed from a class of membrane-active fungal antibiotics, ligated to a water-compatible, metal-centred binding site and a conformationally responsive fluorophore. Using the fluorophore as a remote reporter of conformational change, we find that binding of specific carboxylate ligands to a Cu(II) cofactor at the binding site perturbs the foldamer's global conformation, mimicking the conformational response of a GPCR to ligand binding.

  20. Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

    Science.gov (United States)

    Ahrens, M L

    1981-04-06

    In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

  1. Steric pressure between membrane-bound proteins opposes lipid phase separation.

    Science.gov (United States)

    Scheve, Christine S; Gonzales, Paul A; Momin, Noor; Stachowiak, Jeanne C

    2013-01-30

    Cellular membranes are densely crowded with a diverse population of integral and membrane-associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to concentrate proteins and lipids locally, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase separations, resulting in a homogeneous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains that increases in significance as the molecular weight of the proteins increases. Comparison with a simple analytical model reveals that domains are destabilized when the steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.

  2. Molecular characterization of soluble and membrane-bound trehalases of the whitefly, Bemisia tabaci.

    Science.gov (United States)

    Wang, Jia; He, Wen-Bo; Su, Yun-Lin; Bing, Xiao-Li; Liu, Shu-Sheng

    2014-04-01

    Trehalases (Tres) have been demonstrated to be the key enzymes that are involved in various trehalose-associated physiological processes in insects. However, little attention has been devoted to the Tres in the whitefly, Bemisia tabaci. In this study, a soluble Tre (BtTre-1) and a membrane-bound Tre (BtTre-2) were cloned in the invasive cryptic species Middle East-Asia Minor 1 (MEAM1) of the whitefly B. tabaci complex. Alignment of deduced amino acids sequences of both BtTres revealed that they share common consensus regions and residues with Tres of other insect species. Levels of BtTres expression in various stages and tissues of the whitefly suggested that BtTre-2 may play a key role in trehalose catabolism during development of the whitefly, especially for oocyte development, while BtTre-1 may prevent trehalose in salivary gland from leaking and entering into plants along with saliva. Potential roles of trehalose catabolism in response to direct and/or plant-mediated indirect effects of Tomato Yellow Leaf Curl China Virus (TYLCCNV) were also detected. Whiteflies feeding on virus-infected tobacco plants showed higher BtTres expressions and accordingly higher BtTres activity but lower trehalose content than those feeding on uninfected plants. The enhanced trehalose catabolism may be beneficial to oocyte development in ovary and attenuate plant defensive responses induced by trehalose in saliva. Viruliferous and nonviruliferous whiteflies feeding on cotton, a nonhost plant for TYLCCNV, differed significantly only in trehalose content. The higher trehalose content in viruliferous whiteflies may be conducive to resisting the stress inflicted by TYLCCNV. © 2014 Wiley Periodicals, Inc.

  3. Conversion of the random amplified polymorphic DNA (RAPD ...

    African Journals Online (AJOL)

    Conversion of the random amplified polymorphic DNA (RAPD) marker UBC#116 linked to Fusarium crown and root rot resistance gene (Frl) into a co-dominant sequence characterized amplified region (SCAR) marker for marker-assisted selection of tomato.

  4. Mapping of randomly amplified polymorphic DNA primer (RAPD) on ...

    African Journals Online (AJOL)

    Mapping of randomly amplified polymorphic DNA primer (RAPD) on chromosome 2A of common wheat. Khamsa Parveen, Inamullah Inamullah, Habib Ahmad, Muhammad Sajjad Iqbal, Fida Muhammad Abbassi, Aziz ud-Din, Abdullah Khan, Imtiaz Ahmad Khan ...

  5. Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

    Science.gov (United States)

    Zakharov, S D; Lindeberg, M; Griko, Y; Salamon, Z; Tollin, G; Prendergast, F G; Cramer, W A

    1998-04-14

    Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

  6. Random amplified polymorphic DNA (RAPD) markers reveal genetic ...

    African Journals Online (AJOL)

    The present study evaluated genetic variability of superior bael genotypes collected from different parts of Andaman Islands, India using fruit characters and random amplified polymorphic DNA (RAPD) markers. Genomic DNA extracted from leaf material using cetyl trimethyl ammonium bromide (CTAB) method was ...

  7. Mapping of randomly amplified polymorphic DNA primer (RAPD) on ...

    African Journals Online (AJOL)

    Genet & Botany only

    2012-08-14

    Aug 14, 2012 ... 500. Figure 1. PCR amplification profile of the two genetic stocks of common wheat,. NT2A2B and NT1D1B using RAPD primer GLC-07. M = Molecular size marker (100 bp DNA ladder, Gene Link, USA). important crops species including wheat (T. aestivum L.). Among the DNA markers, randomly amplified.

  8. Random Coding Bounds for DNA Codes Based on Fibonacci Ensembles of DNA Sequences

    Science.gov (United States)

    2008-07-01

    Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188) Washington, DC...COVERED (From - To) 6 Jul 08 – 11 Jul 08 4. TITLE AND SUBTITLE RANDOM CODING BOUNDS FOR DNA CODES BASED ON FIBONACCI ENSEMBLES OF DNA SEQUENCES...sequences which are generalizations of the Fibonacci sequences. 15. SUBJECT TERMS DNA Codes, Fibonacci Ensembles, DNA Computing, Code Optimization 16

  9. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    Science.gov (United States)

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Al-Numair, Khalid S; Veeramani, Chinnadurai; Alsaif, Mohammed A; Chandramohan, Govindasamy

    2015-01-01

    Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) was administered orally once daily for 45 d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. In our study, diabetic rats had significantly (p kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) for a period of 45 d resulted in significant (p kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

  11. The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Carlo Barnaba

    2017-05-01

    Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

  12. Randomly amplified polymorphic DNA-polymerase chain reaction ...

    Indian Academy of Sciences (India)

    Genetic similarity and diversity of cultured catfish Silurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic ...

  13. Evaluation of membrane-bound and soluble forms of HLA-G in Systemic Sclerosis.

    Science.gov (United States)

    Contini, Paola; Negrini, Simone; Murdaca, Giuseppe; Borro, Matteo; Puppo, Francesco

    2018-04-16

    Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and wide-spread fibrosis. Human leukocyte antigen-G (HLA-G) is a non-classic class I major histocompatibility complex (MHC) molecule characterized by complex immuno-modulating properties. HLA-G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA-G is also detectable in soluble form (sHLA-G) deriving from the shedding of surface isoforms (sHLA-G1) or the secretion of soluble isoforms (HLA-G5). Several immunosuppressive functions have been attributed to both membrane-bound and soluble HLA-G molecules. The plasma levels of sHLA-G were higher in SSc patients (444.27 ± 304.84 U/ml) as compared to controls (16.74 ± 20.58 U/ml) (p G (r: 0.65; p G1 (r: 0.60; p = 0.003) and HLA-G5 (r: 0.47; p = 0.02). The percentage of HLA-G-positive monocytes (0.98 ± 1.72), CD4+ (0.37 ± 0.68), CD8+ (2.05 ± 3.74) and CD4+CD8+ double positive cells (14.53 ± 16.88) was higher in SSc patients than in controls (0.11 ± 0.08, 0.01 ± 0.01, 0.01 ± 0.01 and 0.39 ± 0.40, respectively) (p G molecules and the membrane expression of HLA-G by PBMC is clearly elevated suggesting an involvement of HLA-G molecules in the immune dysregulation of SSc. This article is protected by copyright. All rights reserved. © 2018 British Society for Immunology.

  14. Structural and dynamical insights into the membrane-bound α-synuclein.

    Directory of Open Access Journals (Sweden)

    Neha Jain

    Full Text Available Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson's disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES, which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could

  15. (ISSR) and randomly amplified polymorphic DNA

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... The phylogenic relationship within four species of Polygonum (including Polygonum hyrcanicum Rech. f. (three samples), Polygonum persicaria Boiss & Bushe Boiss, Polygonum avicular L., and Polygonum hydropiper L. subsp. Hydropiper) was investigated by randomly amplified polymorphic DNA ...

  16. DNA based random key generation and management for OTP encryption.

    Science.gov (United States)

    Zhang, Yunpeng; Liu, Xin; Sun, Manhui

    2017-09-01

    One-time pad (OTP) is a principle of key generation applied to the stream ciphering method which offers total privacy. The OTP encryption scheme has proved to be unbreakable in theory, but difficult to realize in practical applications. Because OTP encryption specially requires the absolute randomness of the key, its development has suffered from dense constraints. DNA cryptography is a new and promising technology in the field of information security. DNA chromosomes storing capabilities can be used as one-time pad structures with pseudo-random number generation and indexing in order to encrypt the plaintext messages. In this paper, we present a feasible solution to the OTP symmetric key generation and transmission problem with DNA at the molecular level. Through recombinant DNA technology, by using only sender-receiver known restriction enzymes to combine the secure key represented by DNA sequence and the T vector, we generate the DNA bio-hiding secure key and then place the recombinant plasmid in implanted bacteria for secure key transmission. The designed bio experiments and simulation results show that the security of the transmission of the key is further improved and the environmental requirements of key transmission are reduced. Analysis has demonstrated that the proposed DNA-based random key generation and management solutions are marked by high security and usability. Published by Elsevier B.V.

  17. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Science.gov (United States)

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. PMID:25258549

  18. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

    Science.gov (United States)

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

  19. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

    OpenAIRE

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas; Madsen, Bjørn; Kongstad, Kenneth T.; Staerk, Dan; Bennedsen, Mads; Okkels, Finn T.; Rasmussen, Silas A.; Larsen, Thomas O.; Frandsen, Rasmus J. N.; Møller, Birger Lindberg

    2017-01-01

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, contain...

  20. DNA-based random number generation in security circuitry.

    Science.gov (United States)

    Gearheart, Christy M; Arazi, Benjamin; Rouchka, Eric C

    2010-06-01

    DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. This research focuses on further developing DNA-based methodologies to mimic digital data manipulation. While exhibiting fundamental principles, this work was done in conjunction with the vision that DNA-based circuitry, when the technology matures, will form the basis for a tamper-proof security module, revolutionizing the meaning and concept of tamper-proofing and possibly preventing it altogether based on accurate scientific observations. A paramount part of such a solution would be self-generation of random numbers. A novel prototype schema employs solid phase synthesis of oligonucleotides for random construction of DNA sequences; temporary storage and retrieval is achieved through plasmid vectors. A discussion of how to evaluate sequence randomness is included, as well as how these techniques are applied to a simulation of the random number generation circuitry. Simulation results show generated sequences successfully pass three selected NIST random number generation tests specified for security applications.

  1. Random access in large-scale DNA data storage.

    Science.gov (United States)

    Organick, Lee; Ang, Siena Dumas; Chen, Yuan-Jyue; Lopez, Randolph; Yekhanin, Sergey; Makarychev, Konstantin; Racz, Miklos Z; Kamath, Govinda; Gopalan, Parikshit; Nguyen, Bichlien; Takahashi, Christopher N; Newman, Sharon; Parker, Hsing-Yeh; Rashtchian, Cyrus; Stewart, Kendall; Gupta, Gagan; Carlson, Robert; Mulligan, John; Carmean, Douglas; Seelig, Georg; Ceze, Luis; Strauss, Karin

    2018-03-01

    Synthetic DNA is durable and can encode digital data with high density, making it an attractive medium for data storage. However, recovering stored data on a large-scale currently requires all the DNA in a pool to be sequenced, even if only a subset of the information needs to be extracted. Here, we encode and store 35 distinct files (over 200 MB of data), in more than 13 million DNA oligonucleotides, and show that we can recover each file individually and with no errors, using a random access approach. We design and validate a large library of primers that enable individual recovery of all files stored within the DNA. We also develop an algorithm that greatly reduces the sequencing read coverage required for error-free decoding by maximizing information from all sequence reads. These advances demonstrate a viable, large-scale system for DNA data storage and retrieval.

  2. A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

    Directory of Open Access Journals (Sweden)

    Davide Danovi

    2010-07-01

    Full Text Available Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF, a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.

  3. ( Quercus spp. ) using random amplified polymorphic DNA (RAPD)

    African Journals Online (AJOL)

    Quercus is one of the most important woody genera of the Northern hemisphere and considered as one of the main forest tree species in Iran. In this study, genetic relationships in the genus Quercus, using random amplified polymorphic DNA (RAPD) was examined. Five species, including: Quercus robur, Quercus ...

  4. Application of random amplified polymorphic DNA (RAPD) markers ...

    African Journals Online (AJOL)

    The random amplified polymorphic DNA (RAPD) technique has been widely applied to identify different varieties of plants for molecular breeding. However, application of RAPD markers to identify parthenogenesis in plants has not been reported. In this investigation, we used pedigree and RAPD markers to differentiate ...

  5. Virulence properties and random amplification of polymorphic DNA ...

    African Journals Online (AJOL)

    Genotypic and phenotypic characterization as well as studies on the virulence factors of Candida albicans isolates obtained from oral cavity of patients was carried out using random amplified polymorphic DNA (RAPD) fingerprinting and epithelial cells adherence assay, respectively. RAPD patterns revealed the presence of ...

  6. Simulations Using Random-Generated DNA and RNA Sequences

    Science.gov (United States)

    Bryce, C. F. A.

    1977-01-01

    Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

  7. Efficiency of random amplified polymorphic DNA (RAPD) and inter ...

    African Journals Online (AJOL)

    Efficiency of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers for genotype fingerprinting and genetic diversity studies in canola ( ) ... The number of amplified fragments with RAPD primers ranged from 8 to 21, with the size of amplicons ranging from 162 to 3154 bp.

  8. Random amplified polymorphic DNA (RAPD) and simple sequence ...

    African Journals Online (AJOL)

    Knowledge as to genetic diversity and relationships among maize hybrids is important for breeding strategies. The main aims of this study were to (1) estimate molecular genetic diversity among 30 maize hybrids by random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers; and (2) compare ...

  9. Randomly Amplified Polymorphic DNA tests (RAPDs) cannot be ...

    African Journals Online (AJOL)

    The three South African crane species — the Blue Crane (Anthropoides paradisea), the Wattled Crane (Bugeranus carunculatus) and the Grey Crowned Crane (Balearica regulorum regulorum) — are listed as threatened by the IUCN. This study investigated the suitability of Randomly Amplified Polymorphic DNA markers in ...

  10. A Rewritable, Random-Access DNA-Based Storage System

    Science.gov (United States)

    Tabatabaei Yazdi, S. M. Hossein; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-09-01

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

  11. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    Directory of Open Access Journals (Sweden)

    Leśniewicz Krzysztof

    2012-10-01

    Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

  12. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins.

    Science.gov (United States)

    Velasco, Gloria; Cal, Santiago; Quesada, Victor; Sánchez, Luis M; López-Otín, Carlos

    2002-10-04

    We have identified and cloned a fetal liver cDNA encoding a new serine proteinase that has been called matriptase-2. This protein exhibits a domain organization similar to other members of an emerging family of membrane-bound serine proteinases known as type II transmembrane serine proteinases. Matriptase-2 contains a short cytoplasmic domain, a type II transmembrane sequence, a central region with several modular structural domains including two CUB (complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domains and three low density lipoprotein receptor tandem repeats, and finally, a C-terminal catalytic domain with all typical features of serine proteinases. The human matriptase-2 gene maps to 22q12-q13, a location that differs from all type II transmembrane serine proteinase genes mapped to date. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA confirmed that matriptase-2 is anchored to the cell surface. Matriptase-2 was expressed in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying serine proteinases and endogenous proteins such as type I collagen, fibronectin, and fibrinogen. Matriptase-2 could also activate single-chain urokinase plasminogen activator, albeit with low efficiency. These activities were abolished by inhibitors of serine proteinases but not by inhibitors of other classes of proteolytic enzymes. Northern blot analysis demonstrated that matriptase-2 transcripts are only detected at significant levels in both fetal and adult liver, suggesting that this novel serine proteinase may play a specialized role in matrix remodeling processes taking place in this tissue during development or in adult tissues.

  13. Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis.

    OpenAIRE

    Bansal, N S; McDonell, F

    1997-01-01

    The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same ...

  14. Protective effect of Lagenaria siceraria (Mol) against membrane-bound enzyme alterations in isoproterenol-induced cardiac damage in rats.

    Science.gov (United States)

    Vijayakumar, M; Selvi, V; Krishnakumari, S

    2012-01-01

    This study was aimed at evaluating the preventive role of the ethanolic extract of Lagenaria siceraria (Mol) fruit on membrane-bound enzymes, such as sodium potassium-dependent adenosine triphosphatase (Na(+)/K(+) ATPase), calcium-dependent adenosine triphosphatase (Ca(2+) ATPase) and magnesium-dependent adenosine triphosphatase (Mg(2+) ATPase) on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Male albino Wistar rats were pretreated with the ethanolic extract of L. siceraria (Mol) fruit (125, 250 and 500 mg kg(-1) body weight) for a period of 30 days. After the treatment period, ISO (85mg kg(-1) body weight) was subcutaneously injected into rats at 24-h intervals for 2 days. ISO-induced rats showed a significant (p Mol) fruit for a period of 30 days exhibited a significant (p Mol) fruit has membrane-stabilising role in ISO-induced MI in rats.

  15. Release of membrane-bound vesicles and inhibition of tumor cell adhesion by the peptide Neopetrosiamide A.

    Directory of Open Access Journals (Sweden)

    Pamela Austin

    2010-05-01

    Full Text Available Neopetrosiamide A (NeoA is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown.We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of beta1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: beta1 integrin and numerous alpha integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that beta1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane.NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface.

  16. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  17. Molecular Dynamics Simulations to Provide Insights into Epitopes Coupled to the Soluble and Membrane-Bound MHC-II Complexes

    Science.gov (United States)

    Bello, Martiniano; Correa-Basurto, Jose

    2013-01-01

    Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P7w, and MHC-II-P22w) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P7m, and MHC-II-P22m). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are

  18. Differential expression of growth factor receptors and membrane-bound tumor markers for imaging in male and female breast cancer.

    Directory of Open Access Journals (Sweden)

    Jeroen F Vermeulen

    Full Text Available INTRODUCTION: Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. METHODS: Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. RESULTS: Growth factor receptors were variably expressed in 4.5% (MET up to 38.5% (IGF1-R of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. CONCLUSIONS: Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of

  19. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  20. Random amplified polymorphic DNA analysis of genetically modified organisms.

    Science.gov (United States)

    Yoke-Kqueen, Cheah; Radu, Son

    2006-12-15

    Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.

  1. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

    International Nuclear Information System (INIS)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A.

    1988-01-01

    A partially purified, membrane-bound Na + -K + -ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H 2 O 2 for either 15 or 30 min at 37 degree C. The activity of ouabain-sensitive Na + -K + -ATPase was reduced proportionally to the concentration of H 2 O 2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na + -K + -ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na + K + -ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degree C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na + -K + -ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney

  2. Structure and dynamics of the membrane-bound form of the filamentous bacteriophage coat proteins by NMR spectroscopy

    International Nuclear Information System (INIS)

    Bogusky, M.J.

    1987-01-01

    The structure and dynamics of the Pf1 and fd bacteriophage coat proteins in detergent micelles are characterized in solution by nuclear magnetic resonance spectroscopy. The coat proteins are found to exist within the bacterial inner cell membrane during viral infection and assembly. The coat proteins serve as a model system to investigate integral membrane proteins as well as the viral infection and assembly processes. The coat protein is insoluble in aqueous or organic solvents and can only be effectively solubilized in the presence of detergents that form micelles or phospholipids that form vesicles. The effective molecular weight of the detergent-micelle complex is ca. 30K daltons. Sequential assignment strategies were ineffective due to short T/sub 2s/ and severe resonance degeneracy. The backbone resonance assignments were completed by the combination of several homo- and heteronuclear correlation techniques with biosynthetic 15 N labelling. 2D NOE experiments were used to locate and characterize the secondary structure of the membrane bound form of the proteins showing them to be largely helical with the hydrophobic core existing in a very stable helix

  3. Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

    Science.gov (United States)

    Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

    2017-10-01

    Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  4. Membrane-bound catechol-O-methyl transferase in cortical neurons and glial cells is intracellularly oriented

    Directory of Open Access Journals (Sweden)

    Björn H Schott

    2010-10-01

    Full Text Available Catechol-O-methyl transferase (COMT is involved in the inactivation of dopamine in brain regions in which the dopamine transporter (DAT1 is sparsely expressed. The membrane-bound isoform of COMT (MB-COMT is the predominantly expressed form in the mammalian central nervous system (CNS. It has been a matter of debate whether in neural cells of the CNS the enzymatic domain of MB-COMT is oriented towards the cytoplasmic or the extracellular compartment. Here we used live immunocytochemistry on cultured neocortical neurons and glial cells to investigate the expression and membrane orientation of native COMT and of transfected MB-COMT fused to green fluorescent protein (GFP. After live staining, COMT immunoreactivity was reliably detected in both neurons and glial cells after permeabilization, but not on unpermeabilized cells. Similarly, autofluorescence of COMT-GFP fusion protein and antibody fluorescence showed overlap only in permeabilized neurons. Our data provide converging evidence for an intracellular membrane orientation of MB-COMT in neurons and glial cells, suggesting the presence of a DAT1-independent postsynaptic uptake mechanism for dopamine, prior to its degradation via COMT.

  5. Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa.

    Science.gov (United States)

    Kannangara, Rubini; Siukstaite, Lina; Borch-Jensen, Jonas; Madsen, Bjørn; Kongstad, Kenneth T; Staerk, Dan; Bennedsen, Mads; Okkels, Finn T; Rasmussen, Silas A; Larsen, Thomas O; Frandsen, Rasmus J N; Møller, Birger Lindberg

    2017-12-07

    Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function.

  6. Aluminium and Acrylamide Disrupt Cerebellum Redox States, Cholinergic Function and Membrane-Bound ATPase in Adult Rats and Their Offspring.

    Science.gov (United States)

    Ghorbel, Imen; Amara, Ibtissem Ben; Ktari, Naourez; Elwej, Awatef; Boudawara, Ons; Boudawara, Tahia; Zeghal, Najiba

    2016-12-01

    Accumulation of aluminium and acrylamide in food is a major source of human exposure. Their adverse effects are well documented, but there is no information about the health problems arising from their combined exposure. The aim of the present study was to examine the possible neurotoxic effects after co-exposure of pregnant and lactating rats to aluminium and acrylamide in order to evaluate redox state, cholinergic function and membrane-bound ATPases in the cerebellum of adult rats and their progeny. Pregnant female rats have received aluminium (50 mg/kg body weight) via drinking water and acrylamide (20 mg/kg body weight) by gavage, either individually or in combination from the 14th day of pregnancy until day 14 after delivery. Exposure to these toxicants provoked an increase in malondialdehyde (MDA) and advanced oxidation protein product (AOPP) levels and a decrease in SOD, CAT, GPx, Na + K + -ATPase, Mg 2+ -ATPase and AChE activities in the cerebellum of mothers and their suckling pups. A reduction in GSH, NPSH and vitamin C levels was also observed. These changes were confirmed by histological results. Interestingly, co-exposure to these toxicants exhibited synergism based on physical and biochemical variables in the cerebellum of mothers and their progeny.

  7. Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

    Science.gov (United States)

    Ferroni, Felix M; Rivas, María G; Rizzi, Alberto C; Lucca, María E; Perotti, Nora I; Brondino, Carlos D

    2011-10-01

    The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

  8. Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

    Science.gov (United States)

    Li, Mei-Hui

    2012-04-01

    Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period. Copyright © 2012 SETAC.

  9. The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

    International Nuclear Information System (INIS)

    Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N.

    2011-01-01

    The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners

  10. Aldopentoses as new substrates for the membrane-bound, pyrroloquinoline quinone-dependent glycerol (polyol) dehydrogenase of Gluconobacter sp.

    Science.gov (United States)

    Yakushi, Toshiharu; Terada, Yuka; Ozaki, Seishiro; Kataoka, Naoya; Akakabe, Yoshihiko; Adachi, Osao; Matsutani, Minenosuke; Matsushita, Kazunobu

    2018-04-01

    Membrane-bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH, or polyol dehydrogenase) of Gluconobacter sp. oxidizes various secondary alcohols to produce the corresponding ketones, such as oxidation of D-sorbitol to L-sorbose in vitamin C production. Substrate specificity of GLDH is considered limited to secondary alcohols in the D-erythro configuration at the next to the last carbon. Here, we suggest that L-ribose, D- and L-lyxoses, and L-tagatose are also substrates of GLDH, but these sugars do not meet the substrate specificity rule of GLDH. The oxygen consumption activity of wild-type Gluconobacter frateurii cell membranes depends on several kinds of sugars as compared with that of the membranes of a GLDH-negative variant. Biotransformation of those sugars with the membranes was examined to determine the reaction products. A time course measuring the pH in the reaction mixture and the increase or decrease in substrates and products on TLC suggested that oxidation products of L-lyxose and L-tagatose were ketones with unknown structures, but those of L-ribose and D-lyxose were acids. The oxidation product of L-ribose was purified and revealed to be L-ribonate by HRMS and NMR analysis. Biotransformation of L-ribose with the membranes and also with the whole cells produced L-ribonate in nearly stoichiometric amounts, indicating that the specific oxidation site in L-ribose is recognized by GLDH. Since purified GLDH produced L-ribonate without any intermediate-like compounds, we propose here a reaction model where the first carbon in the pyranose form of L-ribose is oxidized by GLDH to L-ribonolactone, which is further hydrolyzed spontaneously to produce L-ribonate.

  11. Depression of membrane-bound Na sup + -K sup + -ATPase activity induced by free radicals and by ischemia of kidney

    Energy Technology Data Exchange (ETDEWEB)

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. (Univ. of Ottawa, Ontario (Canada))

    1988-02-01

    A partially purified, membrane-bound Na{sup +}-K{sup +}-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H{sub 2}O{sub 2} for either 15 or 30 min at 37{degree}C. The activity of ouabain-sensitive Na{sup +}-K{sup +}-ATPase was reduced proportionally to the concentration of H{sub 2}O{sub 2} and the duration of incubation. There were decreases in SH contents and turnover rates of the Na{sup +}-K{sup +}-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na{sup +}K{sup +}-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4{degree}C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na{sup +}-K{sup +}-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney.

  12. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, J.; Norby, J.G.

    1988-12-05

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.

  13. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    International Nuclear Information System (INIS)

    Jensen, J.; Norby, J.G.

    1988-01-01

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper

  14. The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2011-12-01

    The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.

  15. Tumor Cell Clone Expressing the Membrane-bound Form of IL-12p35 Subunit Stimulates Antitumor Immune Responses Dominated by CD8(+) T Cells.

    Science.gov (United States)

    Lim, Hoyong; Do, Seon Ah; Park, Sang Min; Kim, Young Sang

    2013-04-01

    IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membrane-bound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-α. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the CD8(+) T cell-depleted mice than in CD4(+) T cell-depleted or normal mice. These results suggest that CD8(+) T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and CD4(+) helper T cells.

  16. An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures.

    Science.gov (United States)

    Pedro, Augusto Q; Martins, Luís M; Dias, João M L; Bonifácio, Maria J; Queiroz, João A; Passarinha, Luís A

    2015-08-07

    Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson's disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN). The optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks. The ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure

  17. Composting oily sludges: Characterizing microflora using randomly amplified polymorphic DNA

    International Nuclear Information System (INIS)

    Persson, A.; Quednau, M.; Ahrne, S.

    1995-01-01

    Laboratory-scale composts in which oily sludge was composted under mesophilic conditions with amendments such as peat, bark, and fresh or decomposed horse manure, were studied with respect to basic parameters such as oil degradation, respirometry, and bacterial numbers. Further, an attempt was made to characterize a part of the bacterial flora using randomly amplified polymorphic DNA (RAPD). The compost based on decomposed horse manure showed the greatest reduction of oil (85%). Comparison with a killed control indicated that microbial degradation actually had occurred. However, a substantial part of the oil was stabilized rather than totally broken down. Volatiles, on the contrary, accounted for a rather small percentage (5%) of the observed reduction. RAPD indicated that a selection had taken place and that the dominating microbial flora during the active degradation of oil were not the same as the ones dominating the different basic materials. The stabilized compost, on the other hand, had bacterial flora with similarities to the ones found in peat and bark

  18. Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach

    Directory of Open Access Journals (Sweden)

    Iñiguez Alena M

    2003-01-01

    Full Text Available The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.

  19. Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.

    Science.gov (United States)

    Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2015-05-29

    The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Membrane-bound steel factor maintains a high local concentration for mouse primordial germ cell motility, and defines the region of their migration.

    Directory of Open Access Journals (Sweden)

    Ying Gu

    Full Text Available Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.

  1. Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

    Directory of Open Access Journals (Sweden)

    Nimgampalle Mallikarjuna

    2016-12-01

    Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20% in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration.

  2. Genetic variability of cultivated cowpea in Benin assessed by random amplified polymorphic DNA

    NARCIS (Netherlands)

    Zannou, A.; Kossou, D.K.; Ahanchédé, A.; Zoundjihékpon, J.; Agbicodo, E.; Struik, P.C.; Sanni, A.

    2008-01-01

    Characterization of genetic diversity among cultivated cowpea [Vigna unguiculata (L.) Walp.] varieties is important to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the

  3. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, C.J.; Weinstein, J.D. (Clemson Univ, SC (United States))

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  4. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that l5-nucleotide-long random...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...

  5. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  6. Randomly amplified polymorphic DNA-polymerase chain reaction ...

    Indian Academy of Sciences (India)

    Unknown

    The polymorphic DNA markers that were shown to genetically link to a trait of interest could be used for .... 2.3 Primers, markers and amplification conditions. Out of 20 decamer primers (Operon Technologies, ..... and stock structure of school mackerel and spotted mackerel in northern Australian waters; J. Fish Biol. 53 543– ...

  7. Mapping of randomly amplified polymorphic DNA primer (RAPD) on ...

    African Journals Online (AJOL)

    Genet & Botany only

    2012-08-14

    Aug 14, 2012 ... (Islam and Shepherd, 1992). But these markers were not considered suitable for large scale mapping. With the recent introduction of molecular biology, DNA based markers including polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), single nucleotide polymorphisms ...

  8. Randomly amplified polymorphic DNA-polymerase chain reaction ...

    Indian Academy of Sciences (India)

    Unknown

    Department of Animal Science, College of Industry Science, Kongju National University,. Yesan-kun ..... Appl. Genet. 97 1314–1320. Mohd-Azmi M, Ali A S and Kheng W K 2000 DNA finger- printing of red jungle fowl, village chicken and broilers;.

  9. induced by cadmium using random amplified polymorphic DNA

    African Journals Online (AJOL)

    darya

    2013-04-17

    Apr 17, 2013 ... under different concentration of cadmium showed some changes (increase or decrease). Changes observed in the DNA profiles such as modifications in band intensity and loss of bands may be due to the changes in oligonucleotide priming sites leading to genomic rearrangements, and less likely to point ...

  10. Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    International Nuclear Information System (INIS)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-01-01

    In this work, we examined occlusion of 22 Na+ and 86 Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22 Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22 Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32 P incorporation from [gamma- 32 P]CrATP. Maximum capacity for phosphorylation from [gamma- 32 P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [ 3 H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport

  11. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    Directory of Open Access Journals (Sweden)

    Roberts Paul C

    2009-04-01

    Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

  12. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  13. Occlusion of /sup 22/Na+ and /sup 86/Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Vilsen, B.; Andersen, J.P.; Petersen, J.; Jorgensen, P.L.

    1987-08-05

    In this work, we examined occlusion of /sup 22/Na+ and /sup 86/Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of /sup 22/Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of /sup 22/Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by /sup 32/P incorporation from (gamma-/sup 32/P)CrATP. Maximum capacity for phosphorylation from (gamma-/sup 32/P)CrATP was 6 nmol/mg of protein and equal to capacities for binding of (48V)vanadate and (/sup 3/H)ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.

  14. Random amplified polymorphic DNA (RAPD) based assessment of ...

    African Journals Online (AJOL)

    Knowledge of genetic distances between genotypes is important for efficient organization and conservation of plant genetic resources for crop improvement programs. In this study genetic distances between genotype pairs (complements of Jaccard's similarity coefficient) were estimated from Random Amplified Polymorphic ...

  15. Random amplified polymorphic DNA (RAPD) based assessment of ...

    African Journals Online (AJOL)

    SAM

    2014-05-07

    May 7, 2014 ... Knowledge of genetic distances between genotypes is important for efficient organization and conservation of plant genetic resources for crop improvement programs. In this study genetic distances between genotype pairs (complements of Jaccard's similarity coefficient) were estimated from Random ...

  16. A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.

    Science.gov (United States)

    Larrasa, J; Garcia, A; Ambrose, N C; Alonso, J M; Parra, A; de Mendoza, M Hermoso; Salazar, J; Rey, J; de Mendoza, J Hermoso

    2002-04-01

    Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

  17. Physically transient photonics: random versus distributed feedback lasing based on nanoimprinted DNA.

    Science.gov (United States)

    Camposeo, Andrea; Del Carro, Pompilio; Persano, Luana; Cyprych, Konrad; Szukalski, Adam; Sznitko, Lech; Mysliwiec, Jaroslaw; Pisignano, Dario

    2014-10-28

    Room-temperature nanoimprinted, DNA-based distributed feedback (DFB) laser operation at 605 nm is reported. The laser is made of a pure DNA host matrix doped with gain dyes. At high excitation densities, the emission of the untextured dye-doped DNA films is characterized by a broad emission peak with an overall line width of 12 nm and superimposed narrow peaks, characteristic of random lasing. Moreover, direct patterning of the DNA films is demonstrated with a resolution down to 100 nm, enabling the realization of both surface-emitting and edge-emitting DFB lasers with a typical line width of <0.3 nm. The resulting emission is polarized, with a ratio between the TE- and TM-polarized intensities exceeding 30. In addition, the nanopatterned devices dissolve in water within less than 2 min. These results demonstrate the possibility of realizing various physically transient nanophotonics and laser architectures, including random lasing and nanoimprinted devices, based on natural biopolymers.

  18. How DNA is damaged by external electric fields: selective mutation vs. random degradation.

    Science.gov (United States)

    Cerón-Carrasco, José Pedro; Cerezo, Javier; Jacquemin, Denis

    2014-05-14

    DNA is constantly exposed to exogenous agents that randomly damage the genetic code. However, external perturbations might also be used to induce malignant cell death if the mutation processes are controlled. The present communication reports a set of parameters allowing DNA mutation through the use of intense external electric fields. This is a step towards the design of pulsed electric field therapy for genetic diseases.

  19. Aggressive re-warming at 38.5 degrees C following deep hypothermia at 21 degrees C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release

    NARCIS (Netherlands)

    Tang, Min; Zhao, Xiao-gang; He, Yi; Gu, Yan; Mei, Ju

    2016-01-01

    Background: Cardiopulmonary bypass (CPB) is often performed under hypothermic condition. The effects of hypothermia and re-warming on neutrophil activity are unclear. This study aimed to compare the effects of different hypothermia and re-warming regimens on neutrophil membrane bound elastase (MBE)

  20. Pathogen-expanded CD11b+ invariant NKT cells feedback inhibit T cell proliferation via membrane-bound TGF-β1.

    Science.gov (United States)

    Han, Yanmei; Jiang, Zhengping; Chen, Zhubo; Gu, Yan; Liu, Yanfang; Zhang, Xiang; Cao, Xuetao

    2015-04-01

    Natural killer T cells (NKT cells) are effector cells, but also regulator of immune response, which either promote or suppress immune response through production of different cytokines. However, the subsets of NKT cells with definite phenotype and regulatory function need to be further identified. Furthermore, the mechanisms for NKT cells to regulate immune response remain to be fully elucidated. Here we identified CD11b(+) invariant NKT (CD11b(+) iNKT) cells as a new subset of regulatory NKT cells in mouse models with infection. αGalCer:CD1d complex(+)TCRβ(+)NK1.1(+) NKT cells could be categorized to CD11b(+) and CD11b(-) subsets. NKT cells are enriched in liver. During Listeria monocytogenes infection, hepatic CD11b(+) iNKT cells were significantly induced and expanded, with peak expansion on day 8. CD11b(+) iNKT cells were also expanded significantly in spleen and mesenteric lymph nodes. As compared to CD11b(-) iNKT cells, CD11b(+) iNKT cells expressed higher levels of CD27, FasL, B7H1, CD69, and particularly higher level of membrane-bound TGF-β1 (mTGF-β1), but produced less IFN-γ, IL-4, IL-10 and TGF-β1. Hepatic CD11b(+) iNKT cells suppressed antigen-nonspecific and OVA-specific CD4 and CD8 T cell proliferation through mTGF-β1 both in vitro and in vivo, meanwhile, they did not interfere with activation of CD4 T cells and cytotoxicity of the activated CD8 T cells. Thus, we have identified a new subset of pathogen-expanded CD11b(+) invariant NKT cells which can feedback inhibit T cell response through cell-to-cell contact via cell surface (membrane-bound) TGF-β1, especially at the late stage of immune response against infection. CD11b(+) regulatory iNKT cells may contribute to protect host from pathological injure by preventing immune overactivation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

    NARCIS (Netherlands)

    Wang, G.; van Dam, A. P.; Spanjaard, L.; Dankert, J.

    1998-01-01

    To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA

  2. Transformations of membrane-bound organelles in sec 14 mutants of the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

    Science.gov (United States)

    Rambourg, A; Clermont, Y; Nicaud, J M; Gaillardin, C; Kepes, F

    1996-07-01

    were dispersed throughout the cytoplasm. Later on, the faintly stained ER elements and related tubular networks decreased in number, whereas the intensely stained nodular tubular networks increased in frequency. The incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Widemeshed, intensely stained fenestrated spheres were often encountered and increased in number, in parallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as they seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cerevisiae sec14 mutant, the main ultrastructural modification observed in Y. lipolytica transferred to the YPD medium was the formation of deep plasma membrane invaginations. It appears that two functionally homologous PI/PC transfer proteins (Sec14psc and Sec14pyl) control distinct physiological processes in the two sec14 mutants examined. Such differences are perhaps related to the regulatory role of these proteins in different target organelles, i.e., the Golgi apparatus in S. cerevisiae or the plasma membrane in Y. lipolytica.

  3. Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

    1985-08-01

    The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

  4. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  5. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  6. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

    Science.gov (United States)

    Kondo, K; Beppu, T; Horinouchi, S

    1995-09-01

    The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

  7. The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

    Science.gov (United States)

    Vanshylla, Kanika; Opazo, Felipe; Gronke, Konrad; Wienands, Jürgen; Engels, Niklas

    2018-03-01

    Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

    Directory of Open Access Journals (Sweden)

    Julia Gunzenhäuser

    Full Text Available The assembly process of the human immunodeficiency virus 1 (HIV-1 is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

  9. In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

    Science.gov (United States)

    Lei, Li; Egli, Martin

    2016-04-01

    Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

  10. Identification of Species Related to Anopheles (Nyssorhynchus) albitarsis by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (Diptera: Culicidae)

    Science.gov (United States)

    1995-11-01

    plified polymorphic DNA in the population genet- ics and systematics of grasshoppers . Genome 35: 569-574. Galvgo ALA, Damesceno RG 1942. Sobre urn...iynchus) albitarsis by Random Amplified Polymorphic DNA -Polymerase Chain Reaction (Diptera: Culicidae) Richard C Wilkerson/+, Thomas V Caffigan, Jo...Instituto de Biologia do ExCrcito, Rua Francisco Manuel 102, 2091 l-270 Rio de Janeiro, RJ, Brasil Species-specific Random Amplified Polymorphic DNA

  11. rMotifGen: random motif generator for DNA and protein sequences

    Directory of Open Access Journals (Sweden)

    Hardin C Timothy

    2007-08-01

    Full Text Available Abstract Background Detection of short, subtle conserved motif regions within a set of related DNA or amino acid sequences can lead to discoveries about important regulatory domains such as transcription factor and DNA binding sites as well as conserved protein domains. In order to help assess motif detection algorithms on motifs with varying properties and levels of conservation, we have developed a computational tool, rMotifGen, with the sole purpose of generating a number of random DNA or protein sequences containing short sequence motifs. Each motif consensus can be user-defined, randomly generated, or created from a position-specific scoring matrix (PSSM. Insertions and mutations within these motifs are created according to user-defined parameters and substitution matrices. The resulting sequences can be helpful in mutational simulations and in testing the limits of motif detection algorithms. Results Two implementations of rMotifGen have been created, one providing a graphical user interface (GUI for random motif construction, and the other serving as a command line interface. The second implementation has the added advantages of platform independence and being able to be called in a batch mode. rMotifGen was used to construct sample sets of sequences containing DNA motifs and amino acid motifs that were then tested against the Gibbs sampler and MEME packages. Conclusion rMotifGen provides an efficient and convenient method for creating random DNA or amino acid sequences with a variable number of motifs, where the instance of each motif can be incorporated using a position-specific scoring matrix (PSSM or by creating an instance mutated from its corresponding consensus using an evolutionary model based on substitution matrices. rMotifGen is freely available at: http://bioinformatics.louisville.edu/brg/rMotifGen/.

  12. Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: Two identification techniques for food-borne yeasts

    NARCIS (Netherlands)

    Baleiras Couto, M.M.; Vogels, J.T.W.E.; Hofstra, H.; Veld, J.H.J. Huis in't; Vossen, J.M.B.M. van der

    1995-01-01

    The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be

  13. Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano.

    Science.gov (United States)

    Verdoodt, Freija; Willems, Maxime; Mouton, Stijn; De Mulder, Katrien; Bert, Wim; Houthoofd, Wouter; Smith, Julian; Ladurner, Peter

    2012-01-01

    Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.

  14. Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano.

    Directory of Open Access Journals (Sweden)

    Freija Verdoodt

    Full Text Available Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs. Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.

  15. Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

    Science.gov (United States)

    Qian, Li; Ji, Ming-Chun; Pan, Xin-Yuan; Gong, Wei-Juan; Tian, Fang; Duan, Qiu-Fang

    2011-05-01

    Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1ε, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1ε gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1ε gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1ε was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1ε in recombinant BaF3/mb15/RAE-1ε cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1ε cells had the ability of promoting NK cells proliferation and IFN-γ secretion. In conclusion, BaF3/mb15/RAE-1ε cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions.

    Science.gov (United States)

    Bhubhanil, Sakkarin; Chamsing, Jareeya; Sittipo, Panida; Chaoprasid, Paweena; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2014-05-01

    Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)-vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

  17. Unexpected Potency Differences between B-Cell-Activating Factor (BAFF) Antagonist Antibodies against Various Forms of BAFF: Trimer, 60-Mer, and Membrane-Bound.

    Science.gov (United States)

    Nicoletti, Amy M; Kenny, Cynthia Hess; Khalil, Ashraf M; Pan, Qi; Ralph, Kerry L M; Ritchie, Julie; Venkataramani, Sathyadevi; Presky, David H; DeWire, Scott M; Brodeur, Scott R

    2016-10-01

    Therapeutic agents antagonizing B-cell-activating factor/B-lymphocyte stimulator (BAFF/BLyS) are currently in clinical development for autoimmune diseases; belimumab is the first Food and Drug Administration-approved drug in more than 50 years for the treatment of lupus. As a member of the tumor necrosis factor superfamily, BAFF promotes B-cell survival and homeostasis and is overexpressed in patients with systemic lupus erythematosus and other autoimmune diseases. BAFF exists in three recognized forms: membrane-bound and two secreted, soluble forms of either trimeric or 60-mer oligomeric states. To date, most in vitro pharmacology studies of BAFF neglect one or more of these forms. Here, we report a comprehensive in vitro cell-based analysis of BAFF in assay systems that measure all forms of BAFF-mediated activation. We demonstrate the effects of these BAFF forms in both a primary human B-cell proliferation assay and in nuclear factor κB reporter assay systems in Chinese hamster ovary cells expressing BAFF receptors and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). In contrast to the mouse system, we find that BAFF trimer activates the human TACI receptor. Further, we profiled the activities of two clinically advanced BAFF antagonist antibodies, belimumab and tabalumab. Unexpectedly, we revealed differences in inhibitory potencies against the various BAFF forms, in particular that belimumab does not potently inhibit BAFF 60-mer. Through this increased understanding of the activity of BAFF antagonists against different forms of BAFF, we hope to influence the discovery of BAFF antagonist antibodies with distinct therapeutic mechanisms for improvement in the treatment of lupus or other related autoimmune pathologies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  18. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: Resolution of the activity into soluble and membrane-bound fractions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, C.J.; Weinstein, J.D. (Clemson Univ., SC (United States))

    1991-07-01

    The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelatase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzymen was sensitive to the sulfhydryl reagent N-ethylmaleimide (I{sub 50}, 20 {mu}M). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.

  19. Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108.

    Science.gov (United States)

    Yakushi, Toshiharu; Fukunari, Seiya; Kodama, Tomohiro; Matsutani, Minenosuke; Nina, Shun; Kataoka, Naoya; Theeragool, Gunjana; Matsushita, Kazunobu

    2018-04-03

    Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent K M values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent K M value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

  20. Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

    Science.gov (United States)

    Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

    1999-03-01

    Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

  1. Application of random amplified polymorphic DNA (RAPD) to detect the genotoxic effect of heavy metals.

    Science.gov (United States)

    Enan, Mohamed R

    2006-03-01

    This paper presents the results of a study on the influence of lead, copper, manganese and cadmium on DNA integrity in plant cells. Plants, as biological indicators, can measure the potential effects of pollutants when they are used to measure effects of heavy metals. The genotoxicity of heavy metals in kidney-bean (Phaseolus vulgaris) seedlings was subjected to RAPD (random amplified polymorphic DNA) analysis. An RAPD 'fingerprinting' technique was used to detect DNA damage in the kidney-bean seedlings treated with two selected heavy metals at concentrations of 150 and 350 mg x l(-1). Polymorphisms became evident as the presence and/or absence of DNA fragments in treated samples compared with the untreated one. At 350 mg x l(-1), a high number of both missing bands and new amplified fragment were observed. Results suggested that a qualitative measure reflecting changes in RAPD profiles were significantly affected at higher concentrations (350 mg x l(-1)) of the tested heavy metals. A total of 467 RAPD fragments in RAPD profiles were detected by using six random primers (decamers) and 224 of these fragments showed polymorphism. There was a distinct distance between the band patterns of treated plants and the control samples when the cluster method was applied. In addition, the result derived from numerical analysis revealed a considerable distance between the band pattern of the plant samples treated with 350 mg x l(-1) heavy metals and the control sample. Finally, a comparison between untreated and treated genomes shows that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.

  2. Non-random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region.

    Science.gov (United States)

    Ruiz-Estévez, M; Ruiz-Ruano, F J; Cabrero, J; Bakkali, M; Perfectti, F; López-León, M D; Camacho, J P M

    2015-06-01

    We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA-derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8-28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation-free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated. © 2015 The Royal Entomological Society.

  3. Radiation breakage of DNA: a model based on random-walk chromatin structure

    Science.gov (United States)

    Ponomarev, A. L.; Sachs, R. K.

    2001-01-01

    Monte Carlo computer software, called DNAbreak, has recently been developed to analyze observed non-random clustering of DNA double strand breaks in chromatin after exposure to densely ionizing radiation. The software models coarse-grained configurations of chromatin and radiation tracks, small-scale details being suppressed in order to obtain statistical results for larger scales, up to the size of a whole chromosome. We here give an analytic counterpart of the numerical model, useful for benchmarks, for elucidating the numerical results, for analyzing the assumptions of a more general but less mechanistic "randomly-located-clusters" formalism, and, potentially, for speeding up the calculations. The equations characterize multi-track DNA fragment-size distributions in terms of one-track action; an important step in extrapolating high-dose laboratory results to the much lower doses of main interest in environmental or occupational risk estimation. The approach can utilize the experimental information on DNA fragment-size distributions to draw inferences about large-scale chromatin geometry during cell-cycle interphase.

  4. Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

    Directory of Open Access Journals (Sweden)

    Massimo Giuliani

    Full Text Available BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP, of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg. The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+ PB-HP. Finally, a small subset of NKp46(+ HLA-G(+ IL-10(+ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+ CD16(+ NKp30(+ NKp44(+ NKp46(+ CD94(+ CD69(+ CCR7(+ generated from specific pSTAT6(+ GATA3(+ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant

  5. Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae.

    Science.gov (United States)

    Ashayeri-Panah, M; Eftekhar, F; Feizabadi, M M

    2012-04-01

    To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae. Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 μmol 1(-1) primer, 4 mmol 1(-1) MgCl(2), 0·4 mmol 1(-1) dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 μl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson's diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson's similarity coefficient ≈ 100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates. Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae. This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  6. [The random amplified polymorphic DNA identification of 9 Taenia saginata isolates from four provinces].

    Science.gov (United States)

    Zhang, Ke; Yang, Ming; Bao, Huai-En

    2006-12-01

    To make molecular identification for 9 isolates of Taenia saginata from 4 provinces. Genomic DNA was extracted from the segments of adult tapeworms collected from Taoyuan of Taiwan (TW1), Duyun of Guizhou (DY1, DY2), Congjiang of Guizhou (CJ1, CJ2, CJ3, CJ4), Dali of Yunnan (DL1) and Wushi of Xinjiang (XJ1) respectively. PCRs were carried out with 13 random primers. A phylogenetic tree of different geographical strains was constructed. 331 DNA fragments were amplified. The number of DNA fragments amplified by single primer was between 3 and 28. The average number of amplified DNA fragments by the 13 primers was 14.15. The average number of fragments from the 9 isolates of T. saginata was 14.08. Phylogenetic tree revealed that there were two branches in the tree, DY1, DY2, DL1 and TW1 occupied one branch, while CJ1, CJ2, CJ3, CJ4 and XJ1 occupied the other one. By the RAPD analysis, the isolates DY1, DY2, DL1 and TW1 belong to Taenia saginata asiatica, and the isolates CJ1, CJ2, CJ3, CJ4 and XJ1 belong to T. saginata saginata.

  7. Development of Randomly Amplified Polymorphic DNA Based SCAR Marker for Identification of Ipomoea mauritiana Jacq (Convolvulaceae

    Directory of Open Access Journals (Sweden)

    Kambiranda Devaiah

    2011-01-01

    Full Text Available Vidari is an Ayurvedic herbal drug used as aphrodisiac, galactagogue and is also used in the preparation of Chyavanaprash. Tubers of Ipomoea mauritiana Jacq. (Convolvulaceae, Pueraria tuberosa (Roxb. ex Willd. DC (Fabaceae, Adenia hondala (Gaertn. de Wilde (Passifloraceae and pith of Cycas circinalis L. (Cycadaceae are all traded in the name of Vidari, creating issues of botanical authenticity of the Ayurvedic raw drug. DNA-based markers have been developed to distinguish I. mauritiana from the other Vidari candidates. A putative 600-bp polymorphic sequence, specific to I. mauritiana was identified using randomly amplified polymorphic DNA (RAPD technique. Furthermore, sequence characterized amplified region (SCAR primers (IM1F and IM1R were designed from the unique RAPD amplicon. The SCAR primers produced a specific 323-bp amplicon in authentic I. mauritiana and not in the allied species.

  8. Development of Randomly Amplified Polymorphic DNA Based SCAR Marker for Identification of Ipomoea mauritiana Jacq (Convolvulaceae)

    Science.gov (United States)

    Devaiah, Kambiranda; Balasubramani, Subramani Paranthaman; Venkatasubramanian, Padma

    2011-01-01

    Vidari is an Ayurvedic herbal drug used as aphrodisiac, galactagogue and is also used in the preparation of Chyavanaprash. Tubers of Ipomoea mauritiana Jacq. (Convolvulaceae), Pueraria tuberosa (Roxb. ex Willd.) DC (Fabaceae), Adenia hondala (Gaertn.) de Wilde (Passifloraceae) and pith of Cycas circinalis L. (Cycadaceae) are all traded in the name of Vidari, creating issues of botanical authenticity of the Ayurvedic raw drug. DNA-based markers have been developed to distinguish I. mauritiana from the other Vidari candidates. A putative 600-bp polymorphic sequence, specific to I. mauritiana was identified using randomly amplified polymorphic DNA (RAPD) technique. Furthermore, sequence characterized amplified region (SCAR) primers (IM1F and IM1R) were designed from the unique RAPD amplicon. The SCAR primers produced a specific 323-bp amplicon in authentic I. mauritiana and not in the allied species. PMID:21738554

  9. Development of Random Amplified Polymorphic DNA markers for authentification of Cissus repanda vahl.

    Science.gov (United States)

    Harisha, C R; Acharya, Rabinarayan; Chauhan, Maltiben G

    2012-04-01

    Cissus repanda Vahl. belongs to the family Vitaceae, commonly known in Hindi as "Panivel," is a large climber distributed all over India. The crushed or powder of root is prescribed by tribal people and traditional medical practitioners of Orissa for its healing properties in cases of bone fracture, cuts and wounds, swellings, and so on. In spite of its reputation, its leaves have not been investigated scientifically. The present study deals with pharmacognostical and molecular characterization by Random Amplified Polymorphic DNA (RAPD) markers and their role in laying down standardization and pharmacopoeial parameters. Genomic isolation of DNA from fresh leaves was amplified by RAPD markers. The diagnostic characters are mucilage, calcium oxalate rosette crystals, spiral vessels, and fibers. The unique bands obtained in Polymerase Chain Reaction (PCR) amplification clearly discriminated having, many bright and light bands indicating the genuinity of the plant. RAPD may serve as a complementary tool in quality control of many herbal sources.

  10. Intraspecific genetic diversity of Drechslera tritici-repentis as detected by random amplified polymorphic DNA analysis

    Directory of Open Access Journals (Sweden)

    Ana Maria Pujol Vieira dos Santos

    2002-01-01

    Full Text Available The phytopathogenic fungus Drechslera tritici-repentis causes tan spot, an important disease of wheat in the southern Brazilian state of Rio Grande do Sul. Twelve D. tritici-repentis isolates were obtained from wheat seeds from different locations in the state. Their colony morphology on potato dextrose agar and polymorphisms in genomic DNA by the random amplified polymorphic DNA (RAPD method were investigated. For the RAPD method, 23 primers were tested of which nine were selected for use in the study of D. tritici-repentis polymorphisms. The degree of similarity between isolates was calculated using a simple matching coefficient and dendrograms constructed by the unweighted pair-group method with arithmetical averages (UPGMA. The morphological and RAPD analyses showed intraspecific polymorphisms within the isolates, but it was not possible to establish a relationship between these polymorphisms and the geographical regions from where the host seeds were collected.

  11. Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae from Western and Northeastern Colombia

    Directory of Open Access Journals (Sweden)

    Carmen Elisa Posso

    2003-06-01

    Full Text Available Random amplified polymorphic DNA (RAPD markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8 but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8. According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001. The variation among individuals within populations (phiST 0.8869, P < 0.001was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

  12. Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers.

    Science.gov (United States)

    Howland, Shanshan W; Poh, Chek-Meng; Rénia, Laurent

    2011-09-01

    Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5'→3' exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Comparison of the value of pulsed-field gel electrophoresis, random amplified polymorphic DNA and amplified rDNA restriction analysis for subtyping Taylorella equigenitalis.

    Science.gov (United States)

    Kagawa, S; Moore, J E; Murayama, O; Matsuda, M

    2001-05-01

    Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.

  14. Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

    Science.gov (United States)

    Oefner, P J; Hunicke-Smith, S P; Chiang, L; Dietrich, F; Mulligan, J; Davis, R W

    1996-10-15

    Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

  15. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1 and membrane progestin receptor (mPR beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    Directory of Open Access Journals (Sweden)

    Fostier Alexis

    2006-02-01

    Full Text Available Abstract Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2 has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma, was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

  16. Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: A 19F nuclear magnetic resonance study

    International Nuclear Information System (INIS)

    Peersen, O.B.; Pratt, E.A.; Truong, H.T. N.; Ho, C.; Rule, G.S.

    1990-01-01

    The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The 19 F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wide-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes

  17. The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner

    Science.gov (United States)

    Kondrashov, F. A.; Toshchakov, S. V.; Dominova, I.; Shvyreva, U. S.; Vrublevskaya, V. V.; Morenkov, O. S.; Panyukov, V. V.

    2017-01-01

    Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase

  18. Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

    Science.gov (United States)

    Bidochka, M J; Walsh, S R; Ramos, M E; Leger, R J; Silver, J C; Roberts, D W

    1995-01-01

    The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers. PMID:7574596

  19. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    Science.gov (United States)

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

  20. Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

    Science.gov (United States)

    He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

    2003-06-01

    Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

  1. Molecular Characterization of Some Turkish Olive Cultivars Using Random Amplified Polymorphic DNA (RAPD Markers

    Directory of Open Access Journals (Sweden)

    Ergün KAYA

    2015-03-01

    Full Text Available Olive (Olea europea L. is one of the oldest cultivated plants characteristic in the Mediterranean area, where it is the most important oilproducing crop. The cultivated olive (O. europaea L. var. europaea is propagated by cutting or grafting, whereas wild olive (O. europaea L. var. sylvestris is reproduced from seeds. These two olive types are interfertile and have led to a large number of varieties. Morphological descriptions are not entirely reliable, due to numerous synonyms and homonyms in designations, labelling mistakes, the presence of varietal clones, and the uncertain identification methods thus far applied. Molecular markers, as random amplified polymorphic DNA (RAPD markers, are environment-independent and efficient to identify olive varieties and to detect synonymous and homonymous. In this study, fifteen selected RAPD markers are used for determination of relationships among twenty individuals belonging to four important Turkish olive cultivars. Our results showed that RAPD markers can be used to differentiate olive cultivars

  2. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    Science.gov (United States)

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. © 2016 Wiley Periodicals, Inc.

  3. Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

    Science.gov (United States)

    Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

    2015-12-28

    The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

  4. Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars.

    Science.gov (United States)

    Stiles, J I; Lemme, C; Sondur, S; Morshidi, M B; Manshardt, R

    1993-02-01

    We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.

  5. Molecular epidemiologic investigations of Mycoplasma gallisepticum conjunctivitis in songbirds by random amplified polymorphic DNA analyses.

    Science.gov (United States)

    Ley, D. H.; Berkhoff, J. E.; Levisohn, S.

    1997-01-01

    An ongoing outbreak of conjunctivitis in free-ranging house finches (Carpodacus mexicanus) began in 1994 in the eastern United States. Bacterial organisms identified as Mycoplasma gallisepticum (MG) were isolated from lesions of infected birds. MG was also isolated from a blue jay (Cyanocitta cristata) that contracted conjunctivitis after being housed in a cage previously occupied by house finches with conjunctivitis, and from free-ranging American goldfinches (Carduelis tristis) in North Carolina in 1996. To investigate the molecular epidemiology of this outbreak, we produced DNA fingerprints of MG isolates by random amplification of polymorphic DNA (RAPD). We compared MG isolates from songbirds examined from 1994 through 1996 in 11 states, representing three host species, with vaccine and reference strains and with contemporary MG isolates from commercial poultry. All MG isolates from songbirds had RAPD banding patterns identical to each other but different from other strains and isolates tested. These results indicate that the outbreak of MG in songbirds is caused by the same strain, which suggests a single source; the outbreak is not caused by the vaccine or reference strains analyzed; and MG infection has not been shared between songbirds and commercial poultry. PMID:9284386

  6. Genetic variability in yam cultivars from Guinea-Sudan of Benin assessed by random amplified polymorphic DNA

    NARCIS (Netherlands)

    Zannou, A.; Agbicodo, E.; Zoundjihékpon, J.; Struik, P.C.; Ahanchédé, A.; Kossou, D.K.; Sanni, A.

    2009-01-01

    Yam (Dioscorea spp.) is an important food and cash crop in the Guinea-Sudan zone of Benin. The genetic diversity of about 70 cultivars of Dioscorea cayenensis/Dioscorea rotundata (Guinea yam) and about 20 cultivars of Dioscorea alata (water yam) was analysed using random amplified polymorphic DNA

  7. Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

    OpenAIRE

    Bidochka, M J; Walsh, S R; Ramos, M E; Leger, R J; Silver, J C; Roberts, D W

    1995-01-01

    The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplifica- tion of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, or...

  8. Non-radioactive chemical sequencing of biotin labelled DNA.

    OpenAIRE

    Richterich, P

    1989-01-01

    Methods for the nonradioactive chemical sequencing of DNA are described. A biotin marker molecule, attached chemically to an oligonucleotide primer or enzymatically in an endfilling reaction of restriction enzyme sites, is stable during the base-specific chemical modification and strand scission reactions. Following fragment separation by direct blotting electrophoresis, the membrane bound sequence pattern can be visualized by a streptavidin-bridged enzymatic color reaction. The biotin labeli...

  9. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  10. Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

    Science.gov (United States)

    Shami, Gerald; Cheng, Delfine; Henriquez, Jeffrey; Braet, Filip

    2014-12-01

    Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. A microassay for the determination of soluble and membrane-bound glutamate decarboxylase activity--influences of cations, lipid composition, and pyridoxal 5'-phosphate on the glutamate decarboxylase binding to liposomes

    International Nuclear Information System (INIS)

    Hagel, C.; Fleissner, A.; Seifert, R.

    1989-01-01

    A radiochemical microassay for soluble and membrane-bound glutamate decarboxylase (GAD) is described. Up to 180 samples can be determined per day with a variation coefficient of 2%. The method detects newly synthesized gamma-amino-n-butyric acid in the picomole range and can easily be applied to other enzymes whose substrate and product differ by charge. In an aqueous homogenate of brain (1 + 10; w/v) about 15% of the total GAD activity are spun down by centrifugation (1 h, 100,000g) increasing to 35% of the total GAD activity in solutions with 8 mM calcium chloride or 100 mM potassium acetate. There is similar dependence on the cation concentration when GAD binds to phospholipid vesicles (liposomes) as well as dependence on lipid concentration and lipid composition. The coenzyme pyridoxal 5'-phosphate has no influence on GAD binding to liposomes

  12. Molecular characterization of Pasteurella multocida isolates from swine lungs by Randomly Amplified Polymorphic DNA

    Directory of Open Access Journals (Sweden)

    Cristiane Silva Chitarra

    2016-01-01

    Full Text Available ABSTRACT: Swine respiratory diseases such as atrophic rhinitis and bronchopneumonia caused by Pasteurella (P. multocida cause important economic losses to the modern swine industry. The purpose of this study was to characterize P. multocida strains isolated from swine lungs by RAPD (Randomly Amplified Polymorphic DNA to demonstrate their genetic diversity. Ninety-four samples of fragments from lungs with pneumonia and sixty one samples without pneumonia were collected in slaughterhouses in Mato Grosso during the period from December 2009 to March 2010. Clinical cases in 2012 and 2013 were also included in this study. Among the lung fragments with macroscopic lesions, without macroscopic lesions and clinical samples, 40.42%, 4.49% and 100% were positive for P. multocida, respectively. Bacterial identification culturing was confirmed by PCR (polymerase chain reaction by means of the amplification of the gene kmt1. RAPD technique was performed for 46 isolates, and in every isolate, a total of 7 to 11 amplification bands were detected, composed of 8 clusters based on genetic similarity. Thus, treatment, control and preventive measures should consider the genetic diversity of P. multocida populations in swine herds in order to improve the development of new protocols to produce antimicrobials and vaccines.

  13. Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae

    Directory of Open Access Journals (Sweden)

    Brenière Simone F

    2003-01-01

    Full Text Available In Mexico, Triatoma longipennis (Usinger, Triatoma picturata (Usinger, and Triatoma pallidipennis (Stal, primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD. Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug and Triatoma barberi (Usinger. Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

  14. Consumption of a dark roast coffee decreases the level of spontaneous DNA strand breaks: a randomized controlled trial.

    Science.gov (United States)

    Bakuradze, T; Lang, R; Hofmann, T; Eisenbrand, G; Schipp, D; Galan, J; Richling, E

    2015-02-01

    Coffee consumption has been reported to decrease oxidative damage in peripheral white blood cells (WBC). However, effects on the level of spontaneous DNA strand breaks, a well established marker of health risk, have not been specifically reported yet. We analyzed the impact of consuming a dark roast coffee blend on the level of spontaneous DNA strand breaks. Healthy men (n = 84) were randomized to consume daily for 4 weeks either 750 ml of fresh coffee brew or 750 ml of water, subsequent to a run in washout phase of 4 weeks. The study coffee was a blend providing high amounts of both caffeoylquinic acids (10.18 ± 0.33 mg/g) and the roast product N-methylpyridinium (1.10 ± 0.05 mg/g). Before and after the coffee/water consumption phase, spontaneous strand breaks were determined by comet assay. At baseline, both groups exhibited a similar level of spontaneous DNA strand breaks. In the intervention phase, spontaneous DNA strand breaks slightly increased in the control (water only) group whereas they significantly decreased in the coffee group, leading to a 27% difference within both arms (p = 0.0002). Food frequency questionnaires indicated no differences in the overall diet between groups, and mean body weight during the intervention phases remained stable. The consumption of the study coffee substantially lowered the level of spontaneous DNA strand breaks in WBC. We conclude that regular coffee consumption contributes to DNA integrity.

  15. Molecular Typing of Borrelia burgdorferi Sensu Lato by Randomly Amplified Polymorphic DNA Fingerprinting Analysis

    Science.gov (United States)

    Wang, Guiqing; van Dam, Alje P.; Spanjaard, Lodewijk; Dankert, Jacob

    1998-01-01

    To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species. PMID:9508310

  16. Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus.

    Science.gov (United States)

    Pinochet, P; Cenis, J L; Fernández, C; Doucet, M; Maruli, J

    1994-09-01

    The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus.

  17. Perceptions of genetic testing for personalized nutrition: a randomized trial of DNA-based dietary advice.

    Science.gov (United States)

    Nielsen, Daiva E; Shih, Sarah; El-Sohemy, Ahmed

    2014-01-01

    Direct-to-consumer (DTC) genetic tests have facilitated easy access to personal genetic information related to health and nutrition; however, consumer perceptions of the nutritional information provided by these tests have not been evaluated. The objectives of this study were to assess individual perceptions of personalized nutrition and genetic testing and to determine whether a personalized nutrition intervention modifies perceptions. A double-blind, parallel-group, randomized controlled trial was conducted among healthy men and women aged 20-35 years (n = 138). Participants in the intervention group (n = 92) were given a report of DNA-based dietary advice and those in the control group (n = 46) were given a general dietary advice report. A survey was completed at baseline and 3 and 12 months after distributing the reports to assess perceptions between the two groups. No significant differences in perceptions of personalized nutrition and genetic testing were observed between the intervention and control group, so responses of both groups were combined. As compared to baseline, participant responses increased significantly toward the positive end of a Likert scale at 3 months for the statement 'I am interested in the relationship between diet and genetics' (mean change ± SD: 0.28 ± 0.99, p = 0.0002). The majority of participants indicated that a university research lab (47%) or health care professional (41%) were the best sources for obtaining accurate personal genetic information, while a DTC genetic testing company received the fewest selections (12%). Most participants (56%) considered dietitians to be the best source of personalized nutrition followed by medical doctors (27%), naturopaths (8%) and nurses (6%). These results suggest that perceptions of personalized nutrition changed over the course of the intervention. Individuals view a research lab or health care professional as better providers of genetic information than a DTC genetic testing company

  18. Continuous-time random walk models of DNA electrophoresis in a post array: part I. Evaluation of existing models.

    Science.gov (United States)

    Olson, Daniel W; Ou, Jia; Tian, Mingwei; Dorfman, Kevin D

    2011-02-01

    Several continuous-time random walk (CTRW) models exist to predict the dynamics of DNA in micropost arrays, but none of them quantitatively describes the separation seen in experiments or simulations. In Part I of this series, we examine the assumptions underlying these models by observing single molecules of λ DNA during electrophoresis in a regular, hexagonal array of oxidized silicon posts. Our analysis takes advantage of a combination of single-molecule videomicroscopy and previous Brownian dynamics simulations. Using a custom-tracking program, we automatically identify DNA-post collisions and thus study a large ensemble of events. Our results show that the hold-up time and the distance between collisions for consecutive collisions are uncorrelated. The distance between collisions is a random variable, but it can be smaller than the minimum value predicted by existing models of DNA transport in post arrays. The current CTRW models correctly predict the exponential decay in the probability density of the collision hold-up times, but they fail to account for the influence of finite-sized posts on short hold-up times. The shortcomings of the existing models identified here motivate the development of a new CTRW approach, which is presented in Part II of this series. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Complexes of poly(ethylene glycol)-based cationic random copolymer and calf thymus DNA: a complete biophysical characterization.

    Science.gov (United States)

    Nisha, C K; Manorama, Sunkara V; Ganguli, Munia; Maiti, Souvik; Kizhakkedathu, Jayachandran N

    2004-03-16

    Complete biophysical characterization of complexes (polyplexes) of cationic polymers and DNA is needed to understand the mechanism underlying nonviral therapeutic gene transfer. In this article, we propose a new series of synthesized random cationic polymers (RCPs) from methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride with different mole ratios (32:68, 11:89, and 6:94) which could be used as a model system to address and answer the basic questions relating to the mechanism of the interaction of calf thymus DNA (CT-DNA) and cationic polymers. The solubility of the complexes of CT-DNA and RCP was followed by turbidity measurements. It has been observed that complexes of RCP with 68 mol % MePEGMA precipitate near the charge neutralization point, whereas complexes of the other two polymers are water-soluble and stable at all compositions. Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with RCP. Ethidium bromide exclusion and gel electrophoretic mobility show that both polymers are capable of binding with CT-DNA. Atomic force microscopy images in conjunction with light scattering experiments showed that the complexes are spherical in nature and 75-100 nm in diameter. Circular dichroism spectroscopy studies indicated that the secondary structure of DNA in the complexes is not perturbed due to the presence of poly(ethylene glycol) segments in the polymer. Furthermore, we used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the helix-coil transition of CT-DNA in the complexes. UV and differential scanning calorimetry melting experiments revealed that DNA in the complexes is more stable than in the free state and the extent of stability depends on the polymer composition. Isothermal titration calorimetry experiments showed that the binding of these RCPs to CT-DNA is associated with small exothermic

  20. A polymer, random walk model for the size-distribution of large DNA fragments after high linear energy transfer radiation

    Science.gov (United States)

    Ponomarev, A. L.; Brenner, D.; Hlatky, L. R.; Sachs, R. K.

    2000-01-01

    DNA double-strand breaks (DSBs) produced by densely ionizing radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. Stochastic DSB clustering at large scales, from > 100 Mbp down to random-walk, coarse-grained polymer model for chromatin is combined with a simple track structure model in Monte Carlo software called DNAbreak and is applied to data on alpha-particle irradiation of V-79 cells. The chromatin model neglects molecular details but systematically incorporates an increase in average spatial separation between two DNA loci as the number of base-pairs between the loci increases. Fragment-size distributions obtained using DNAbreak match data on large fragments about as well as distributions previously obtained with a less mechanistic approach. Dose-response relations, linear at small doses of high linear energy transfer (LET) radiation, are obtained. They are found to be non-linear when the dose becomes so large that there is a significant probability of overlapping or close juxtaposition, along one chromosome, for different DSB clusters from different tracks. The non-linearity is more evident for large fragments than for small. The DNAbreak results furnish an example of the RLC (randomly located clusters) analytic formalism, which generalizes the broken-stick fragment-size distribution of the random-breakage model that is often applied to low-LET data.

  1. Random Amplified Polymorphic DNA (RAPD) Markers Readily Distinguish Cryptic Mosquito Species (Diptera: Culicidae: Anopheles)

    Science.gov (United States)

    1993-01-01

    DNA isolation. Individual larvae or adults were ground with a strong diagnostic bands and simple patterns. Primers pro- plastic pestle in...V. (1988) Com- peninsular Malaysia and Thailand (Diptera: Culicidae). Mosq parison of DNA probe and cytogenic methods for identifying field Syst 20

  2. Cryptic Species in the Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) Complex: Incongruence Between Random Amplified Polymorphic DNA-Polymerase Chain Reaction Identification and Analysis of Mitochondrial DNA COI Gene Sequences

    OpenAIRE

    LEHR, M. A.; KILPATRICK, C. W.; WILKERSON, R. C.; CONN, J. E.

    2005-01-01

    Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga, A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galvão & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses s...

  3. Uso do Random Amplified Polymorphic DNA (RAPD no estudo populacional do Triatoma brasiliensis Neiva, 1911 Use of Random Amplified Polymorphic DNA (RAPD in the populational study of Triatoma brasiliensis Neiva, 1911

    Directory of Open Access Journals (Sweden)

    Érika C. Borges

    2000-01-01

    Full Text Available Para o estudo de variabilidade genética em Triatoma brasiliensis, o principal vetor da doença de Chagas no Nordeste brasileiro, espécimes de três diferentes populações intradomiciliares foram analisados. Regiões do DNA genômico foram amplificadas utilizando dois iniciadores randômicos através da técnica de RAPD (Random Amplified Polymorphic DNA, visualizados em géis de poliacrilamida corados pela prata. Os perfis originados se mostraram bastante homogêneos quando comparados intrapopulacionalmente. Populações capturadas em duas regiões diferentes do Estado do Ceará também apresentaram homogeneidade entre si, mas, quando comparadas com a população proveniente do Piauí, foi possível diferenciá-las. Esses resultados, preliminares, indicam que o RAPD pode ser usado com sucesso nos estudos de variabilidade em triatomíneos, bem como sugerem a existência de variabilidade entre diferentes populações de T. brasiliensis pertencentes a uma mesma subespécie.We evaluated the genetic variability of Triatoma brasiliensis, the main vector of Chagas disease in Northeast Brazil, using specimens from three populations. Regions of genomic DNA were amplified by RAPD (Random Amplified Polimorphic DNA, using two primers. The products were visualized after polyacrylamide gel electrophoresis followed by silver staining. A dendrogram constructed through the Dice similarity coefficient allowed for separation of the tested specimens into three distinct groups. The populations captured in areas from Ceará State showed similar profiles, but different from that captured in Piauí State. Our results indicate that RAPD can be used successfully in triatomine studies and suggest the presence of genetic variability between different populations of T. brasiliensis.

  4. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

    2011-01-01

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O 2 ) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  5. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kukat, Alexandra [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Edgar, Daniel [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Bratic, Ivana [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Maiti, Priyanka [Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Trifunovic, Aleksandra, E-mail: aleksandra.trifunovic@ki.se [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany)

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  6. First-trimester risk assessment based on ultrasound and cell-free DNA vs combined screening: a randomized controlled trial.

    Science.gov (United States)

    Kagan, K O; Sroka, F; Sonek, J; Abele, H; Lüthgens, K; Schmid, M; Wagner, P; Brucker, S; Wallwiener, D; Hoopmann, M

    2017-09-19

    This was a randomized controlled trial to compare risk assessment by first-trimester combined screening (FTCS) with an approach that combines a detailed ultrasound examination at 11-13 weeks' gestation and cell-free DNA (cfDNA) analysis. Pregnant women with a normal first-trimester ultrasound examination at 11-13 weeks' gestation (fetal nuchal translucency (NT) ≤ 3.5 mm and no fetal defects) were randomized into one of two groups. In the first group, risk of aneuploidy was assessed using FTCS based on the most recent UK Fetal Medicine Foundation algorithm. In the second group, risk assessment was based on ultrasound findings and cfDNA analysis. An additional tube of blood was collected for FTCS in case the cfDNA analysis was uninformative. Primary outcome was false-positive rate in screening for trisomy 21. A case was considered false positive if the karyotype was not trisomy 21 and if the risk for trisomy 21 was >1:100, irrespective of the method of risk calculation. Results were compared using 95% CIs using the Clopper-Pearson method. Between October 2015 and December 2016, 1518 women with singleton pregnancy underwent first-trimester screening. Thirty-one (2.0%) pregnancies were not eligible for randomization due to increased NT (> 3.5 mm) and/or fetal defect. After exclusion of women who declined randomization (n = 87) and cases of fetal death and loss to follow-up (n = 24), 688 pregnancies were randomized into the FTCS arm and 688 into the ultrasound + cfDNA analysis arm. There were no differences in maternal and gestational age, maternal weight and BMI, ethnicity, use of assisted reproduction and cigarette smoking between the two arms. In the ultrasound + cfDNA analysis arm, median risk for trisomy 21 was 1 in 10 000. None of the cases had a risk above 1: 100 (95% CI, 0.0-0.5%). In the FTCS arm, the median risk for trisomy 21 was 1 in 3787 and in 17 cases, the risk was higher than 1:100, which corresponds to 2.5% (95% CI, 1.5-3.9%) of

  7. Colloidal Dancers: Designing networks of DNA-functionalized colloids for non-random walks

    Science.gov (United States)

    Gehrels, Emily W.; Rogers, W. Benjamin; Zeravcic, Zorana; Manoharan, Vinothan N.

    2014-03-01

    We present experimental developments of a system of DNA-functionalized colloidal particles with the goal of creating directed motion (`dancing') along patterned substrates in response to temperature cycling. We take advantage of toehold exchange in the design of the DNA sequences that mediate the colloidal interactions to produce broadened, flat, or even re-entrant binding and unbinding transitions between the particles and substrate. Using this new freedom of design, we devise systems where, by thermal ratcheting, we can externally control the direction of motion and sequence of steps of the colloidal dancer. In comparison to DNA-based walkers, which move autonomously and whose motion is controlled by the substrate, our colloidal dancers respond to external driving, and their motion can be controlled in situ. Our use of DNA-functionalized colloidal particles instead of pure DNA systems also enables walking on the mesoscale in contrast to the molecular length scales previously demonstrated, allowing for the future prospect of directed transport over larger distances.

  8. Random amplified polymorphic DNA markers of the Brassica alboglabra chromosome of a B. campestris-alboglabra addition line

    DEFF Research Database (Denmark)

    Bagger Jørgensen, Rikke; Chen, B.Y.; Cheng, B.F.

    1996-01-01

    The alien C-genome chromosome in a Brassica campestris-alboglabra monosomic addition line was characterized by random amplified polymorphic DNA (RAPD) analysis. The alien chromosome carried three loci, E(c), W-c and Lap-1C, controlling synthesis of erucic acid, white flower colour and a fast......-migrating band of leucine aminopeptidase (Lap-1C(c)) respectively. The RAPD analysis revealed 17 markers specific to the alien chromosome. Among 45 offspring plants from the selfed addition line the alien C-chromosome was transmitted to 15 plants, four plants had only parts of this chromosome and the remaining...

  9. Characterization of plasma membrane bound inorganic ...

    African Journals Online (AJOL)

    ... N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F1Fo-ATPase inhibitor quercetin. Conclusion: We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase ...

  10. A membrane-bound vertebrate globin.

    Directory of Open Access Journals (Sweden)

    Miriam Blank

    Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

  11. Genetic analysis of Apuleia leiocarpa as revealed by random amplified polymorphic DNA markers: prospects for population genetic studies.

    Science.gov (United States)

    Lencina, K H; Konzen, E R; Tsai, S M; Bisognin, D A

    2016-12-19

    Apuleia leiocarpa (Vogel) J.F. MacBride is a hardwood species native to South America, which is at serious risk of extinction. Therefore, it is of prime importance to examine the genetic diversity of this species, information required for developing conservation, sustainable management, and breeding strategies. Although scarcely used in recent years, random amplified polymorphic DNA markers are useful resources for the analysis of genetic diversity and structure of tree species. This study represents the first genetic analysis based on DNA markers in A. leiocarpa that aimed to investigate the levels of polymorphism and to select markers for the precise characterization of its genetic structure. We adapted the original DNA extraction protocol based on cetyltrimethyl ammonium bromide, and describe a simple procedure that can be used to obtain high-quality samples from leaf tissues of this tree. Eighteen primers were selected, revealing 92 bands, from which 75 were polymorphic and 61 were sufficient to represent the overall genetic structure of the population without compromising the precision of the analysis. Some fragments were conserved among individuals, which can be sequenced and used to analyze nucleotide diversity parameters through a wider set of A. leiocarpa individuals and populations. The individuals were separated into 11 distinct groups with variable levels of genetic diversity, which is important for selecting desirable genotypes and for the development of a conservation and sustainable management program. Our results are of prime importance for further investigations concerning the genetic characterization of this important, but vulnerable species.

  12. Development of Random Amplified Polymorphism DNA Markers Linked to CMV-B2 Resistance Gene in Melon

    Directory of Open Access Journals (Sweden)

    BUDI SETIADI DARYONO

    2009-12-01

    Full Text Available Two random amplified polymorphic DNA (RAPD markers linked to CMV-B2 resistance gene (Creb-2 in melon cultivar Yamatouri were cloned and sequenced to design sequence characterized amplified region (SCAR markers for detection of CMV-B2 resistance gene (Creb-2 in melon. SCOPE14 derived from OPE-14 yielded a single DNA band at 541 bp, while SCAPB05 derived from APB-05, yielded a single DNA band at 1,046 bp, respectively. Segregation of SCOPE14 and SCAPB05 markers in bulk of F2 plants demonstrated that they were co-segregated with RAPD markers from which the SCAR markers were originated. Furthermore, results of SCAR analysis in diverse melons showed SCAPB05 primers obtained a single 1,046 bp linked to Creb-2 in resistant cultivars Sanuki-shirouri and Kohimeuri. However, SCOPE14 failed to detect Creb-2 in diverse melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to CMV-B2 by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers.

  13. Antiviral Therapy Reduces Hepatocellular Carcinoma Recurrence in Patients With Low HBV-DNA Levels: A Randomized Controlled Trial.

    Science.gov (United States)

    Huang, Gang; Li, Peng-Peng; Lau, Wan Yee; Pan, Ze-Ya; Zhao, Ling-Hao; Wang, Zhen-Guang; Wang, Meng-Chao; Zhou, Wei-Ping

    2018-03-08

    Despite antiviral treatment has been shown to reduce hepatocellular carcinoma (HCC) recurrence after curative treatment for hepatitis B virus (HBV)-related HCC in patients with high preoperative HBV-DNA levels, it is still unclear whether antiviral therapy is useful in reducing recurrence in patients with low preoperative HBV-DNA levels. In this randomized controlled trial, 200 patients who underwent curative resection for HCC with low baseline HBV-DNA levels were randomly assigned to receive preemptive antiviral therapy or not. The primary endpoints were recurrence-free survival. This study was censored on March 31, 2015 when all surviving patients had a minimum follow-up of 60 months. The analysis was done on an intention-to-treat basis. The baseline clinical, laboratory, and tumor characteristics of the 2 groups were comparable. The 1-, 3-, and 5-year recurrence-free survival rates for the antiviral group and the control group were 85.9%, 55.2%, and 52.0% and 80.6%, 40.9%, and 32.3%, respectively. The corresponding overall survival rates for the 2 groups were 94.0%, 75.7%, and 64.1% and 90.0%, 62.4%, and 43.7%, respectively. The recurrence-free survival and overall survival for the antiviral group were significantly better than the control group (P = 0.016, P = 0.004, respectively). After adjusting for confounding prognostic factors in a Cox model, the relative risks of recurrence and death for antiviral treatment were 0.601 [95% confidence interval (CI), 0.409-0.884; P = 0.010] and 0.509 (95% CI, 0.333-0.778; P = 0.002), respectively. Antiviral therapy was an independent protective factor of late tumor recurrence (hazard ratio [HR] = 0.316, 95% CI 0.157-0.637; P = 0.001) but not of early tumor recurrence (HR = 0.782, 95% CI, 0.493-1.240; P = 0.296). In patients with low preoperative HBV-DNA levels, antiviral therapy significantly reduced HCC recurrence after R0 hepatic resection.

  14. GENETIC VARIABILITY OF GIANT GOURAMY STRAINS REVEALED BY RANDOM AMPLIFIED POLYMORPHISM DNA

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2013-12-01

    Full Text Available Genetic variability of giant gouramy strains has been observed using RAPD Marker. Genomic DNA was extracted from giant gouramy fin clip, and amplified using primers OPA 1-20. There was no significant difference among three strains of giant gouramy analyzed. Based on two primers of RAPD (OPA 4 and 7, the highest variability was observed in Bluesafir with heterogeneity value of 0.3050 and followed by Paris (0.2832 and Soang (0.2360 respectively. The average of Nei’s genetic distance is 0.118, and the smallest on is between Paris and Bluesafir.

  15. Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba

    Directory of Open Access Journals (Sweden)

    Alves J.M.P.

    2000-01-01

    Full Text Available The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed.

  16. Genetic variation in wild and hatchery populations of giant freshwater prawn (Macrobrachium rosenbergii revealed by randomly amplified polymorphic DNA markers

    Directory of Open Access Journals (Sweden)

    Babul Bala

    2017-06-01

    Full Text Available Sustainable improvement and conservation of any genetic resource depend on the assessment of its intra- and inter-population genetic variation. In order to estimate genetic variation in both wild and hatchery populations of Macrobrachium rosenbergii, randomly amplified polymorphic DNA (RAPD analysis was performed. Analyses of 51 polymorphic loci amplified from genomic DNA by three decamer random primers revealed different degrees of genetic variation in two wild (Bhairab and Rupsha rivers and hatchery-derived gher (Gher-1 and Gher-2 populations. The proportion of polymorphic loci was found to be higher in wild populations (0.90 and 0.65 for the Bhairab and Rupsha populations, respectively than the hatchery-derived gher populations (0.29 and 0.16 for Gher-1 and Gher-2, respectively. Likewise, the river populations contained higher levels of gene diversity (0.221 and 0.179 for Bhairab and Rupsha populations, respectively than the gher populations (0.114 and 0.045 for Gher-1 and Gher-2, respectively. These results suggest reduction of genetic variation and heterozygosity in the hatchery-derived gher populations. Inter-population similarity indices and pairwise genetic distance values showed that variation between the wild or between the gher populations were lower than those between the wild and hatchery populations. On average, 14 loci exhibited significant deviation from homogeneity in wild vs hatchery population pairs, whereas 2 and 3 loci showed heterogeneity in Gher-1 vs Gher-2 and Bhairab vs Rupsha population pairs, respectively. A genetic distance-based UPGMA dendrogram segregated river populations from the gher populations. Our study, therefore, revealed substantial levels of genetic variation between wild and hatchery populations of M. rosenbergii.

  17. Identification of velvet antler by random amplified polymorphism DNA combined with non-gel sieving capillary electrophoresis.

    Science.gov (United States)

    Yuan, Guangxin; Sun, Jiyan; Li, Hongyu; Fu, Guilian; Xu, Guangyu; Li, Mingcheng; Zhang, Lihua; Fan, Xintian

    2016-01-01

    Mitochondrial DNA of velvet antler was amplified with random amplified polymorphic DNA (RAPD) technique and the PCR products were detected with non-gel sieving capillary electrophoresis to establish a RAPD-HPCE method used for identifying the authenticity of velvet antler or it counterfeits. Factors that could affect the PCR amplification and capillary electrophoresis were optimized. Under the optimized conditions, namely, 20 mmol L(-1) NaH2PO4-Na2HPO4-2 mmol L(-1) EDTA buffer solution [0.8% (W/V) HPMC, 15 mmol L(-1) TBAP and pH 7.3], -10 kV injection voltage and -8 kV separation voltage, Cervus nippon Temminck antler, Cervus elaphus Linnaeus antler, Rangifer tarandus antler, Cervus canadensis antler and Elaphurus davidianus antler were analyzed. The analysis on the similarity of obtained elctrophoretograms showed that there were significant differences in similarities of different velvet antlers, which could be used for the quick identification of the authenticity of velvet antler samples. It can be found that the technique of RAPD combined with HPCE is advantageous in rich polymorphism, high detection rate, simple and convenient performance, high efficiency, rapidness and sensitivity, indicating that it should be suitable for the quick identification of the authenticity of velvet antler samples.

  18. Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis.

    Science.gov (United States)

    Larrasa, José; García-Sánchez, Alfredo; Ambrose, Nicholas C; Parra, Alberto; Alonso, Juan M; Rey, Joaquín M; Hermoso-de-Mendoza, Miguel; Hermoso-de-Mendoza, Javier

    2004-11-01

    This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

  19. Identification of Leishmania Species Causing Cutaneous Leishmaniasis Using Random Amplified Polymorphic DNA (RAPD-PCR in Kharve, Iran

    Directory of Open Access Journals (Sweden)

    Fatemeh Saadabadi

    2013-05-01

    Full Text Available Background: Leishmaniasis, especially cutaneous leishmaniasis, is considered an important health problem in many parts of Iran including Kharve, Khorasan Razavi province. Cutaneous leishmaniasis is caused by various species of Leishmania, each having a different secondary host. Thus, identifying the parasites’ specie is of paramount importance for containment strategy planning. The morphological differentiation of Leishmania species is not possible, rendering the molecular methods as the sole means to this purpose. Therefore, to identify the causative agent of cutaneous leishmaniasis in Kharve, Random Amplified Polymorphic DNA-PCR (RAPD-PCR was used. Methods: The disease was first confirmed by direct smears. Samples were gathered from 22 patients with established cutaneous leishmaniasis. The samples were immediately cultured in NNN medium, followed by sub-culture in RPMI-1640. Afterwards, DNA was extracted and amplified using RAPD-PCR. Electrophoresis patterns from each isolate were compared with reference strains of Leishmania major (L. major and Leishmania tropica (L. tropica. Results: The results of this study indicated that the parasite causing cutaneous leishmaniasis in Kharve is L. tropica. Conclusion: It seems that L. tropica is the only causative agent of cutaneous leishmaniasis in Kharve, and RAPD-PCR is a suitable tool for Leishmania characterization in epidemiological studies.

  20. Use of Random Amplified Polymorphic DNA (RAPD) Technique to Study the Genetic Diversity of Eight Aloe Species.

    Science.gov (United States)

    Ezzat, Shahira M; El Sayed, Abeer M; Salama, Maha M

    2016-10-01

    The genus Aloe comprises over 400 species of flowering succulent plants. Aloe leaves are used in the treatment of asthma, gastrointestinal ulcers, cardiovascular disease, tumors, burns, and diabetes. They are rich in anthraquinones, such as aloin, aloe-emodin, chrysophanol, aloinoside A, and aloinoside B. The various species of Aloe show chemical and morphological similarity and diversity, which depend on the genotype and environmental conditions. In a continuity to our interest in the genus Aloe , this study targets the authentication of eight different Aloe species, Aloe vera (A 1 ) , Aloe arborescens (A 2 ) , Aloe eru (A 3 ) , Aloe grandidentata (A 4 ) , Aloe perfoliata (A 5 ), Aloe brevifolia (A 6 ) , Aloe saponaria (A 7 ), and Aloe ferox (A 8 ), grown in Egypt by using the technique of random amplified polymorphic DNA. Twelve decamer primers were screened in amplification with genomic DNA extracted from all species, of which five primers yielded species-specific reproducible bands. Out of 156 loci detected, the polymorphic, monomorphic, and unique loci were 107, 26, and 23, respectively. Based on a dendrogram and similarity matrix, the eight Aloe species were differentiated from each other and showed more divergence. Aloe species prevailed similarity coefficients of 54-70 % by which they could be classified into three major groups. Thus, this technique may contribute to the identification of these Aloe species that have great morphological similarity in the Egyptian local markets. Georg Thieme Verlag KG Stuttgart · New York.

  1. Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis.

    Science.gov (United States)

    Radu, S; Elhadi, N; Hassan, Z; Rusul, G; Lihan, S; Fifadara, N; Yuherman; Purwati, E

    1998-08-01

    Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.

  2. Random amplified polymorphic DNA typing of clinical and environmental Aeromonas hydrophila strains from Limpopo province, South Africa.

    Science.gov (United States)

    Ramalivhana, J N; Obi, C L; Samie, A; Labuschagne, C; Weldhagen, G F

    2010-02-01

    The aim of the present study was to determine the genetic relatedness of strains isolated from diarrhoeal stool and water specimens collected from water-storage containers from different geographical areas in the Limpopo province. In total, 32 Aeromonas strains isolated from stool specimens collected from HIV/AIDS patients suffering from gastroenteritis and their household drinking-water stored in 20-L and 25-L containers were analyzed by random amplified polymorphic DNA PCR (RAPD). The RAPD fingerprints obtained proved reproducible when repeated on three different occasions using whole-cell DNA isolated from the Aeromonas strains. In total, 12 unique RAPD fingerprints were found. The results revealed a tendency of the isolates to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). However, a certain degree of similarity was also observed between isolates of water sources and clinical sources which indicated genetic relatedness. There were also genetic similarities between the clinical and the environmental strains of Aeromonas spp. isolated from different geographical areas. This study has demonstrated the genetic relatedness of Aeromonas hydrophila isolates from household drinking-water and clinical sources in South Africa, which may be due to cross-contamination from water to patients or vice-versa. This observation is of public-health significance, particularly in the era of HIV/AIDS. This study points to the importance of monitoring and evaluating infection-control measures for improved hygiene and to prevent cross-contaminations.

  3. Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

    Directory of Open Access Journals (Sweden)

    El mashad Noha

    2009-01-01

    Full Text Available Abstract Background Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB. The aim of this study was to identify the presence of Isoniazid (INH and Rifampicin(RIF drug resistance in new and previously treated tuberculosis (TB cases using DNA sequencing. Methods This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli. Results Of the 153 patients, 105 (68.6% were new cases and 48 (31.4% were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases and INH resistant specimens (26 cases to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9% of RIF resistance were having rpoB gene mutation versus 3 cases (13.1% having no mutation with a high statistical significant difference between them (P Conclusion We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

  4. Fuzzy ensemble clustering based on random projections for DNA microarray data analysis.

    Science.gov (United States)

    Avogadri, Roberto; Valentini, Giorgio

    2009-01-01

    Two major problems related the unsupervised analysis of gene expression data are represented by the accuracy and reliability of the discovered clusters, and by the biological fact that the boundaries between classes of patients or classes of functionally related genes are sometimes not clearly defined. The main goal of this work consists in the exploration of new strategies and in the development of new clustering methods to improve the accuracy and robustness of clustering results, taking into account the uncertainty underlying the assignment of examples to clusters in the context of gene expression data analysis. We propose a fuzzy ensemble clustering approach both to improve the accuracy of clustering results and to take into account the inherent fuzziness of biological and bio-medical gene expression data. We applied random projections that obey the Johnson-Lindenstrauss lemma to obtain several instances of lower dimensional gene expression data from the original high-dimensional ones, approximately preserving the information and the metric structure of the original data. Then we adopt a double fuzzy approach to obtain a consensus ensemble clustering, by first applying a fuzzy k-means algorithm to the different instances of the projected low-dimensional data and then by using a fuzzy t-norm to combine the multiple clusterings. Several variants of the fuzzy ensemble clustering algorithms are proposed, according to different techniques to combine the base clusterings and to obtain the final consensus clustering. We applied our proposed fuzzy ensemble methods to the gene expression analysis of leukemia, lymphoma, adenocarcinoma and melanoma patients, and we compared the results with other state of the art ensemble methods. Results show that in some cases, taking into account the natural fuzziness of the data, we can improve the discovery of classes of patients defined at bio-molecular level. The reduction of the dimension of the data, achieved through random

  5. Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

    Science.gov (United States)

    David E. Schreiber; Karen J. Garner; James M. Slavicek

    1997-01-01

    Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

  6. Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

    Science.gov (United States)

    Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

    2016-03-01

    Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. © 2015 Wiley Periodicals, Inc.

  7. Genetic diversity of pinus roxburghii sarg. Collected from different himalayan regions of India assessed by random amplified polymorphic DNA analysis.

    Science.gov (United States)

    Sinha, Dwaipayan; Singh, Jyotsna; Tandon, P K; Kakkar, Poonam

    2013-09-01

    Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species.

  8. The influence of ginger (Zingiber officinale on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial

    Directory of Open Access Journals (Sweden)

    Jalil Hosseini

    2016-08-01

    Full Text Available Background: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. Objective: This study was designed to investigate the effects of ginger (Zingiber officinale on sperm DNA fragmentation (SDF in infertile men. Materials and Methods: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group. Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. Results: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02 in cases and (56.75, 95%CI: 40.01-73.5 in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39 was lower compared with placebo (40.54, 95%CI: 23.94-57.13 after three month of treatment (p=0.02. In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009. There were no significant differences between two groups regarding to semen parameters. Conclusion: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

  9. [Isolation of DNA from museum exhibits of butterflies (Lepidoptera, Papilionidae) and PCR analysis with random and universal genes-specific primers].

    Science.gov (United States)

    Zakharov, E V; Chelomina, G N; Zhuravlev, Iu N

    2000-09-01

    The effect of the duration of storage of entomological material on DNA preservation was estimated. The results of the optimization of conditions for the analysis of random amplified polymorphic DNA in a polymerase chain reaction (RAPD-PCR) are presented as applied to the DNA of lepidopterans of the family Papilionidae. RAPD patterns are shown for the first time in Atrophaneura alcinous and four species of the genus Parnassius (sensu lata). The applicability of museum specimens of butterflies for RAPD analysis was demonstrated. The results of PCR analysis using DNA obtained from different collection specimens stored for up to five years were compared. The authenticity of DNA obtained from collection specimens was proved using PCR with universal primers, which are specific to the COI and COII cytochrome genes of mitochondrial DNA (mt DNA). The lengths of individuals gene fragments obtained by the amplification of both museum and live specimens were 800 and 1600 bp. The conservative regions of mitochondrial genome were shown to be slightly different in two A. alcinous subspecies.

  10. Genetic variations of Lansium domesticum Corr. accessions from Java, Sumatra and Ceram based on Random Amplified Polymorphic DNA fingerprints

    Directory of Open Access Journals (Sweden)

    KUSUMADEWI SRI YULITA

    2011-07-01

    Full Text Available Yulita KS (2011 Genetic variations of Lansium domesticum Corr. accessions from Java, Bengkulu and Ceram based on Random Amplified Polymorphic DNA fingerprints. Biodiversitas 12: 125-130. Duku (Lansium domesticum Corr. is one of popular tropical fruits in SE Asia. The spesies has three varieties, known as duku, langsat and kokosan; and duku is the most popular one for being the sweetiest fruit. Indonesia has several local varieties of duku, such as duku Condet, duku Sumber and duku Palembang. This present study aimed to assess genetic diversity of 47 accessions of duku from Java, Sumatra, and Ceram based on RAPD fingerprints. Ten RAPD’s primers were initially screened and five were selected for the analysis. These five primers (OPA 7, 13, 18, OPB 7, and OPN 12 generated 53 scorable bands with an average of 10.6 polymorphic fragment per primer. Percentage of polymorphism ranged from 16.89% (OPA 7 and OPN 12 to 24.54% (OPB 7 with an average of 20.16% polymorphism. OPB 7 at 450 bp was exclusively possessed by accession 20 (Java, OPA 18 at 500 bp was by accession 6 (Java, 550 bp by 3 clones from Bengkulu. While OPN 12 at 300 bp and OPA 13 at 450 bp were shared among the accessions. Clustering analysis was performed based on RAPD profiles using the UPGMA method. The range of genetic similarity value among accessions was 0.02-0.65 suggesting high variation of gene pool existed among accessions.

  11. Identification of species related to Anopheles (Nyssorhynchus albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Richard C. Wilkerson

    1995-12-01

    Full Text Available Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR markers were used to identify four species related to Anopheles (Nyssorhynchus albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995, which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys. albitarsis, species B is undescribed, species C is An. (Nys marajoara Galvão and Damasceno and species D is An. (Nys. deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys. deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of São Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.

  12. Comparison of statistical methods for identification of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium from randomly amplified polymorphic DNA patterns.

    Science.gov (United States)

    Moschetti, G; Blaiotta, G; Villani, F; Coppola, S; Parente, E

    2001-05-01

    Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus, Enterococcus faecium, and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25-36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus, E. faecium, and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.

  13. Estimating Genetic Conformism of Korean Mulberry Cultivars Using Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeat Profiling

    Directory of Open Access Journals (Sweden)

    Sunirmal Sheet

    2018-03-01

    Full Text Available Apart from being fed to silkworms in sericulture, the ecologically important Mulberry plant has been used for traditional medicine in Asian countries as well as in manufacturing wine, food, and beverages. Germplasm analysis among Mulberry cultivars originating from South Korea is crucial in the plant breeding program for cultivar development. Hence, the genetic deviations and relations among 8 Morus alba plants, and one Morus lhou plant, of different cultivars collected from South Korea were investigated using 10 random amplified polymorphic DNA (RAPD and 10 inter-simple sequence repeat (ISSR markers in the present study. The ISSR markers exhibited a higher polymorphism (63.42% among mulberry genotypes in comparison to RAPD markers. Furthermore, the similarity coefficient was estimated for both markers and found to be varying between 0.183 and 0.814 for combined pooled data of ISSR and RAPD. The phenogram drawn using the UPGMA cluster method based on combined pooled data of RAPD and ISSR markers divided the nine mulberry genotypes into two divergent major groups and the two individual independent accessions. The distant relationship between Dae-Saug (SM1 and SangchonJo Sang Saeng (SM5 offers a possibility of utilizing them in mulberry cultivar improvement of Morus species of South Korea.

  14. Estimating Genetic Conformism of Korean Mulberry Cultivars Using Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeat Profiling.

    Science.gov (United States)

    Sheet, Sunirmal; Ghosh, Kuntal; Acharya, Satabdi; Kim, Kwang-Pyo; Lee, Yang Soo

    2018-03-15

    Apart from being fed to silkworms in sericulture, the ecologically important Mulberry plant has been used for traditional medicine in Asian countries as well as in manufacturing wine, food, and beverages. Germplasm analysis among Mulberry cultivars originating from South Korea is crucial in the plant breeding program for cultivar development. Hence, the genetic deviations and relations among 8 Morus alba plants, and one Morus lhou plant, of different cultivars collected from South Korea were investigated using 10 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) markers in the present study. The ISSR markers exhibited a higher polymorphism (63.42%) among mulberry genotypes in comparison to RAPD markers. Furthermore, the similarity coefficient was estimated for both markers and found to be varying between 0.183 and 0.814 for combined pooled data of ISSR and RAPD. The phenogram drawn using the UPGMA cluster method based on combined pooled data of RAPD and ISSR markers divided the nine mulberry genotypes into two divergent major groups and the two individual independent accessions. The distant relationship between Dae-Saug (SM1) and SangchonJo Sang Saeng (SM5) offers a possibility of utilizing them in mulberry cultivar improvement of Morus species of South Korea.

  15. Penentuan Marka Genetik Escherichia coli O157:H7 Asal Hewan dan Manusia dengan Metode Random Amplified Polymorphic DNA (GENETIC MARKERS IDENTIFICATION OF ESCHERICHIA COLI O157:H7 ORIGINATED FROM ANIMALS AND HUMAN BY USING RANDOM AMPLIFIED POLYMORPHIC DNA

    Directory of Open Access Journals (Sweden)

    I Wayan Suardana

    2014-10-01

    Full Text Available The use of random amplified polymorphic DNA (RAPD as a method to identify a genetic markerof bacteria is widely used by researcher. This method is known as a simple, faster, and reliabletechnicque. This study is to find out the aplication of RAPD method in order to identify specific markersof E. coli O157:H7 as a zoonotic agent. The study began by cultivating of 20 isolates of E. coli O157:H7colected by previous study that consist of 2 isolates originated from cattle feces, 2 isolates originatedfrom beef, 2 isolates originated from chicken feces, 2 isolates originated from healthy human and 11isolates originated from unhealthy human (human with kidney failure. All isolates were confirmed byculturing on selective medium sorbitol MacConkey agar (SMAC. Confirmation were followed by testingon O157 latex aglutination, and finally by testing on H7 antiserum. RAPD method as molecularanalysis was performed using decamer primers mixture OPA-01, OPA-02, OPA-03, and OPA-04.Results of study showed both bands 1721 and 700 bp are specifically to differentiate of isolatesoriginated from cases of healthy and unhealthy human. On the other hand, bands with position 1721 bp,300 bp, and 250 bp indicate the isolates originated from unhealthy human, healthy human and chicken,respectively. Isolates from beef are characterized by both bands 1400 and 429 bp, and the isolates fromcattle feces are identified by band with position 342 bp. The specific bands are considered as markers inorder to know the source of E. coli O157:H7 fastly.

  16. Structured lifestyle intervention based on a trans-cultural diabetes-specific nutrition algorithm (tDNA) in individuals with type 2 diabetes: a randomized controlled trial

    Science.gov (United States)

    Chee, Winnie S S; Gilcharan Singh, Harvinder Kaur; Hamdy, Osama; Mechanick, Jeffrey I; Lee, Verna K M; Barua, Ankur; Mohd Ali, Siti Zubaidah; Hussein, Zanariah

    2017-01-01

    Objective Trans-cultural diabetes nutrition algorithm (tDNA) was created by international task force and culturally customized for Malaysian population. This study was designed to evaluate its effectiveness versus usual diabetes care in primary care settings. Research design and methods We randomized 230 patients with overweight/obesity, type 2 diabetes, and glycated hemoglobin (A1c) 7%–11% to receive usual care (UC) or UC with tDNA for 6 months. The tDNA intervention consisted of structured low-calorie meal plan, diabetes-specific meal replacements, and increased physical activity. Participants were counseled either through motivational interviewing (tDNA-MI) or conventional counseling (tDNA-CC). The UC group received standard dietary and exercise advice through conventional counseling. All patients were followed for another 6 months after intervention. Results At 6 months, A1c decreased significantly in tDNA-MI (−1.1±0.1%, pdiabetes control and body weight in primary care setting. PMID:29435347

  17. Randomly amplified polymorphic DNA analysis provides rapid differentiation of methicillin-resistant coagulase-negative staphylococcus bacteremia isolates in pediatric hospital.

    Science.gov (United States)

    Bingen, E; Barc, M C; Brahimi, N; Vilmer, E; Beaufils, F

    1995-01-01

    Coagulase-negative staphylococci (CoNS) are now recognized as the most common cause of nosocomial bacteremia in pediatric patients. Randomly amplified polymorphic DNA analysis was used to study the relationships among 12 isolates of CoNS obtained from eight patients with catheter-related bacteremia in two distinct wards of our hospital and 6 epidemiologically unrelated strains. With this method, we were able to discriminate isolates that otherwise were indistinguishable by conventional criteria such as biochemical typing and antibiotic susceptibility patterns. Our results indicate that there were episodes of cross-infections among four patients in one ward but independent infectious episodes among four patients in the other ward. Randomly amplified polymorphic DNA analysis is a rapid method which seems particularly well suited to the epidemiological study of CoNS isolates. PMID:7650208

  18. Comparative assessment of fungal cellobiohydrolase I richness and composition in cDNA generated using oligo(dT) primers or random hexamers.

    Science.gov (United States)

    Weber, Carolyn F; Kuske, Cheryl R

    2012-02-01

    Understanding soil fungal distribution and activities, particularly at the level of gene expression, is important in unveiling mechanisms regulating their activities in situ. Recent identification of fungal genes involved in carbon cycling has provided the foundation for developing reverse-transcriptase PCR assays to monitor spatiotemporal gene expression patterns in soils and other complex microbial systems. The polyadenylated 3' ends of eukaryotic mRNA transcripts enables the use of oligo(dT) primers for cDNA synthesis, but this can result in the overrepresentation of the 3' end of transcripts in cDNA pools. In an effort to increase the uniformity of transcripts represented in cDNA pools, random hexamers have been used. The use of both priming methods is abundant in the literature, but we do not know how these methods perform relative to each other. We performed comparative richness and compositional analyses of the fungal glycosyl hydrolase family 7 cellobiohydrolase I gene cbhI amplified from soil cDNAs that had been generated using either oligo(dT) primers or random hexamers. Our results demonstrate that similar cbhI richness and composition were recovered using both approaches. Richness estimates and compositional profiles of cbhI sequence libraries generated from random hexamer-primed cDNA were more variable than from libraries generated from oligo(dT) primed cDNA. However, our overall results indicate that, on average, comparable richness and composition were recovered from soil cDNAs when either priming method was used. Published by Elsevier B.V.

  19. Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China.

    Science.gov (United States)

    Wang, Dong; Zhang, Limei; Zhou, Xuezhang; He, Yulong; Yong, Changfu; Shen, Mingliang; Szenci, Otto; Han, Bo

    2016-12-01

    Staphylococcus aureusis the leading pathogen involved inbovine mastitis, but knowledgeabout antimicrobial resistance, virulence factors, and genotypes of Staphylococcus aureus resulting in bovine mastitis in Ningxia, China, is limited. Therefore, antimicrobial susceptibility, virulence gene, and randomly amplified polymorphic DNA (RAPD) analyses of Staph. aureus were carried out. A total of 327 milk samples from cows with clinical and subclinical mastitis in 4 regions of Ningxia were used for the isolation and identification of pathogens according to phenotypic and molecular characteristics. Antimicrobial susceptibility against 22 antimicrobial agents was determined by disk diffusion. The presence of 8 virulence genes in Staph. aureus isolates was tested by PCR. Genotypes of isolates were investigated based on RAPD. Results showed that 35 isolates obtained from mastitis milk samples were identified as Staph. aureus. The isolates were resistant to sulfamethoxazole (100%), penicillin G (94.3%), ampicillin (94.3%), erythromycin (68.6%), azithromycin (68.6%), clindamycin (25.7%), amoxicillin (11.4%), and tetracycline (5.7%). All of the isolates contained one or more virulence genes with average (standard deviation) of 6.6±1.6. The most prevalent virulence genes were hlb (97.1%), followed by fnbpA, hla, coa (94.3% each), nuc (85.7%), fnbpB (80%), clfA (77.1%), and tsst-1 (40%). Nine different gene patterns were found and 3 of them were the dominant gene combinations (77.1%). Staphylococcus aureus isolates (n=35) were divided into 6 genotypes by RAPD tying, the genotypes III and VI were the most prevalent genotypes. There was greatvariation in genotypes of Staph. aureus isolates, not only among different farms, but also within the same herd in Ningxia province. The study showed a high incidence of Staph. aureus with genomic variation of resistance genes, which is matter of great concern in public and animal health in Ningxia province of China. Copyright © 2016 American

  20. Discovery of cell-type specific DNA motif grammar in cis-regulatory elements using random Forest.

    Science.gov (United States)

    Wang, Xin; Lin, Peijie; Ho, Joshua W K

    2018-01-19

    It has been observed that many transcription factors (TFs) can bind to different genomic loci depending on the cell type in which a TF is expressed in, even though the individual TF usually binds to the same core motif in different cell types. How a TF can bind to the genome in such a highly cell-type specific manner, is a critical research question. One hypothesis is that a TF requires co-binding of different TFs in different cell types. If this is the case, it may be possible to observe different combinations of TF motifs - a motif grammar - located at the TF binding sites in different cell types. In this study, we develop a bioinformatics method to systematically identify DNA motifs in TF binding sites across multiple cell types based on published ChIP-seq data, and address two questions: (1) can we build a machine learning classifier to predict cell-type specificity based on motif combinations alone, and (2) can we extract meaningful cell-type specific motif grammars from this classifier model. We present a Random Forest (RF) based approach to build a multi-class classifier to predict the cell-type specificity of a TF binding site given its motif content. We applied this RF classifier to two published ChIP-seq datasets of TF (TCF7L2 and MAX) across multiple cell types. Using cross-validation, we show that motif combinations alone are indeed predictive of cell types. Furthermore, we present a rule mining approach to extract the most discriminatory rules in the RF classifier, thus allowing us to discover the underlying cell-type specific motif grammar. Our bioinformatics analysis supports the hypothesis that combinatorial TF motif patterns are cell-type specific.

  1. Absence of zero-temperature transmission rate of a double-chain tight-binding model for DNA with random sequence of nucleotides in thermodynamic limit

    International Nuclear Information System (INIS)

    Xiong Gang; Wang, X.R.

    2005-01-01

    The zero-temperature transmission rate spectrum of a double-chain tight-binding model for real DNA is calculated. It is shown that a band of extended-like states exists only for finite chain length with strong inter-chain coupling. While the whole spectrum tends to zero in thermodynamic limit, regardless of the strength of inter-chain coupling. It is also shown that a more faithful model for real DNA with periodic sugar-phosphate chains in backbone structures can be mapped into the above simple double-chain tight-binding model. Combined with above results, the transmission rate of real DNA with long random sequence of nucleotides is expected to be poor

  2. Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

    OpenAIRE

    Antolin, M. F.; Bosio, C. F.; Cotton, J.; Sweeney, W.; Strand, M. R.; Black-IV, W. C.

    1996-01-01

    The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a d...

  3. Assessment of the genotoxicity of heavy metals in Phaseolus vulgaris L. as a model plant system by Random Amplified Polymorphic DNA (RAPD) analysis.

    Science.gov (United States)

    Gjorgieva, Darinka; Kadifkova-Panovska, Tatjana; Mitrev, Saša; Kovacevik, Biljana; Kostadinovska, Emilija; Bačeva, Katerina; Stafilov, Trajče

    2012-01-01

    Impact assessments of environmental pollutants are important in eco-genotoxicology. A random amplified polymorphic DNA (RAPD) technique was used to detect genotoxicity-induced DNA damage in Phaseolus vulgaris L. from heavy metals at two different concentrations. The results from six 10-base pair (bp) random RAPD primers with 60-70% GC content used, showed a total of 295 RAPD fragments of 700-4000 bp in molecular size in the seedlings of untreated and treated samples, of which only 163 fragments were polymorphic. Polymorphisms became evident as the disappearance and/or appearance of DNA fragments in treated samples compared to the control. A dendrogram constructed using the Numerical Taxonomy and Multivariate Analysis System (NTSYSps), showed that the control group merged with groups treated with CuSO(4)·5H(2)O (150 mg L(-1)) and MnSO(4)·H(2)O (150 mg L(-1)) in a separate cluster. These groups were linked with all of the other samples treated with metals at concentrations of 150 mg L(-1) and CuSO(4)·5H(2)O and Cd(NO(3))(2) at concentrations of 350 mg L(-1). Finally, the samples treated with metals at concentrations of 350 mg L(-1) together with NiSO(4) at the concentration of 150 mg L(-1), clustered separately. The DNA polymorphism detected by RAPD analysis offered a useful biomarker assay for the detection of toxic chemicals genotoxicity in plant model systems.

  4. Placing and shaping liposomes with reconfigurable DNA nanocages

    Science.gov (United States)

    Zhang, Zhao; Yang, Yang; Pincet, Frederic; C. Llaguno, Marc; Lin, Chenxiang

    2017-07-01

    The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

  5. Electron microscopic observations and DNA chain fragmentation studies on apoptosis in bone tumor cells induced by 153Sm-EDTMP

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Xiao Dong; Han Xiaofeng

    1997-01-01

    The morphological changes observed by electron microscopy indicate that after internal irradiation with 153 Sm-EDTMP bone tumor cells displayed feature of apoptosis, such as margination of condensed chromatin, chromatin fragmentation, as well as the membrane bounded apoptotic bodies formation. The quantification analysis of fragmentation DNA for bone tumor cells induced by 153 Sm-EDTMP shows that the DNA fragmentation is enhanced with the prolongation of internally irradiated time. These characteristics suggest that 153 Sm-EDTMP internal irradiation could induce bone tumor cells to go to apoptosis

  6. Bevacizumab Plus Irinotecan Versus Temozolomide in Newly Diagnosed O6-Methylguanine-DNA Methyltransferase Nonmethylated Glioblastoma: The Randomized GLARIUS Trial.

    Science.gov (United States)

    Herrlinger, Ulrich; Schäfer, Niklas; Steinbach, Joachim P; Weyerbrock, Astrid; Hau, Peter; Goldbrunner, Roland; Friedrich, Franziska; Rohde, Veit; Ringel, Florian; Schlegel, Uwe; Sabel, Michael; Ronellenfitsch, Michael W; Uhl, Martin; Maciaczyk, Jaroslaw; Grau, Stefan; Schnell, Oliver; Hänel, Mathias; Krex, Dietmar; Vajkoczy, Peter; Gerlach, Rüdiger; Kortmann, Rolf-Dieter; Mehdorn, Maximilian; Tüttenberg, Jochen; Mayer-Steinacker, Regine; Fietkau, Rainer; Brehmer, Stefanie; Mack, Frederic; Stuplich, Moritz; Kebir, Sied; Kohnen, Ralf; Dunkl, Elmar; Leutgeb, Barbara; Proescholdt, Martin; Pietsch, Torsten; Urbach, Horst; Belka, Claus; Stummer, Walter; Glas, Martin

    2016-05-10

    In patients with newly diagnosed glioblastoma that harbors a nonmethylated O(6)-methylguanine-DNA methyltransferase promotor, standard temozolomide (TMZ) has, at best, limited efficacy. The GLARIUS trial thus explored bevacizumab plus irinotecan (BEV+IRI) as an alternative to TMZ. In this phase II, unblinded trial 182 patients in 22 centers were randomly assigned 2:1 to BEV (10 mg/kg every 2 weeks) during radiotherapy (RT) followed by maintenance BEV (10 mg/kg every 2 weeks) plus IRI(125 mg/m(2) every 2 weeks) or to daily TMZ (75 mg/m(2)) during RT followed by six courses of TMZ (150-200 mg/m(2)/d for 5 days every 4 weeks). The primary end point was the progression-free survival rate after 6 months (PFS-6). In the modified intention-to-treat (ITT) population, PFS-6 was increased from 42.6% with TMZ (95% CI, 29.4% to 55.8%) to 79.3% with BEV+IRI (95% CI, 71.9% to 86.7%; P <.001). PFS was prolonged from a median of 5.99 months (95% CI, 2.7 to 7.3 months) to 9.7 months (95% CI, 8.7 to 10.8 months; P < .001). At progression, crossover BEV therapy was given to 81.8% of all patients who received any sort of second-line therapy in the TMZ arm. Overall survival (OS) was not different in the two arms: the median OS was 16.6 months (95% CI, 15.4 to 18.4 months) with BEV+IRI and was 17.5 months (95% CI, 15.1 to 20.5 months) with TMZ. The time course of quality of life (QOL) in six selected domains of the European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire (QLQ) -C30 and QLQ-BN20 (which included cognitive functioning), of the Karnofsky performance score, and of the Mini Mental State Examination score was not different between the treatment arms. BEV+IRI resulted in a superior PFS-6 rate and median PFS compared with TMZ. However, BEV+IRI did not improve OS, potentially because of the high crossover rate. BEV+IRI did not alter QOL compared with TMZ. © 2016 by American Society of Clinical Oncology.

  7. Prevention of alcohol-induced DNA damage by a proprietary glycyrrhizin/D-mannitol product: A randomized, placebo-controlled, cross-over human study.

    Science.gov (United States)

    Chigurupati, Harsha; Auddy, Biswajit; Biyani, Manish; Chakrabarti, Shrabana; Stohs, Sidney J

    2017-11-06

    The purpose of the present study was to evaluate the ability of a proprietary combination of glycyrrhizin and D-mannitol to protect against oxidative damage to DNA associated with acute alcohol consumption by human subjects in a randomized, placebo-controlled cross-over designed study. Excessive alcohol consumption is associated with numerous diseases. Alcohol has been shown to generate reactive oxygen species that can result in DNA damage, leading to genetic and epigenetic changes. A total of 25 subjects (13 male and 12 female) were enrolled. Alcohol intake in the form of vodka (40% ethanol) was adjusted based on 1.275 g of 100% ethanol/kg body weight for men and 1.020 g/kg body weight for women, which was consumed with and without the study product. Blood samples were drawn at 2 h after alcohol consumption, lymphocytes were isolated, and were subjected to DNA comet electrophoresis on a blinded basis. Acute alcohol consumption increased lymphocyte DNA damage by approximately 8.36%. Co-consumption of the glycyrrhizin/D-mannitol study product with alcohol reduced DNA damage to baseline levels. No adverse effects were associated with use of the study product, and no differences were observed in blood alcohol concentrations in the presence or absence of the study product in males and females. Acute alcohol ingestion resulted in measurable increases in DNA damage, which were prevented by the addition of the proprietary glycyrrhizin/D-mannitol (NTX ® ) study product to the alcohol, suggesting that the tissue-damaging effects of alcohol consumption can be ameliorated. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Prognostic value of DNA cytometry in 281 premenopausal patients with lymph node negative breast carcinoma randomized in a control trial: multivariate analysis with Ki-67 index, mitotic count, and microvessel density

    NARCIS (Netherlands)

    Mandard, A. M.; Denoux, Y.; Herlin, P.; Duigou, F.; van de Vijver, M. J.; Clahsen, P. C.; van den Broek, L.; Sahmoud, T. M.; Henry-Amar, M.; van de Velde, C. J.

    2000-01-01

    BACKGROUND: The clinical relevance of DNA image cytometry (ICM) and flow cytometry (FCM) remains under investigation in breast carcinoma. The objective of the current work was to study the prognostic value of DNA ICM and FCM in a series of patients randomized in a control trial. A multivariate

  9. Ancient DNA from hunter-gatherer and farmer groups from Northern Spain supports a random dispersion model for the Neolithic expansion into Europe.

    Directory of Open Access Journals (Sweden)

    Montserrat Hervella

    Full Text Available BACKGROUND/PRINCIPAL FINDINGS: The phenomenon of Neolithisation refers to the transition of prehistoric populations from a hunter-gatherer to an agro-pastoralist lifestyle. Traditionally, the spread of an agro-pastoralist economy into Europe has been framed within a dichotomy based either on an acculturation phenomenon or on a demic diffusion. However, the nature and speed of this transition is a matter of continuing scientific debate in archaeology, anthropology, and human population genetics. In the present study, we have analyzed the mitochondrial DNA diversity in hunter-gatherers and first farmers from Northern Spain, in relation to the debate surrounding the phenomenon of Neolithisation in Europe. METHODOLOGY/SIGNIFICANCE: Analysis of mitochondrial DNA was carried out on 54 individuals from Upper Paleolithic and Early Neolithic, which were recovered from nine archaeological sites from Northern Spain (Basque Country, Navarre and Cantabria. In addition, to take all necessary precautions to avoid contamination, different authentication criteria were applied in this study, including: DNA quantification, cloning, duplication (51% of the samples and replication of the results (43% of the samples by two independent laboratories. Statistical and multivariate analyses of the mitochondrial variability suggest that the genetic influence of Neolithisation did not spread uniformly throughout Europe, producing heterogeneous genetic consequences in different geographical regions, rejecting the traditional models that explain the Neolithisation in Europe. CONCLUSION: The differences detected in the mitochondrial DNA lineages of Neolithic groups studied so far (including these ones of this study suggest different genetic impact of Neolithic in Central Europe, Mediterranean Europe and the Cantabrian fringe. The genetic data obtained in this study provide support for a random dispersion model for Neolithic farmers. This random dispersion had a different

  10. High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar...... as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates...

  11. Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardiometabolic Disease: A Mendelian Randomization Approach.

    Directory of Open Access Journals (Sweden)

    Michael M Mendelson

    2017-01-01

    Full Text Available The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain.We conducted an association study of body mass index (BMI and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1], demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83 of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination.We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases.

  12. Random subcloning.

    Science.gov (United States)

    Roach, J C

    1995-12-01

    Random subcloning strategies are commonly employed for analyzing pieces of DNA that are too large for direct analysis. Such strategies are applicable to gene finding, physical mapping, and DNA sequencing. Random subcloning refers to the generation of many small, directly analyzable fragments of DNA that represent random fragments of a larger whole, such as a genome. Following analysis of these fragments, a map or sequence of the original target may be reconstructed. Mathematical modeling is useful in planning such strategies and in providing a reference for their evaluation, both during execution and following completion. The statistical theory necessary for constructing these models has been developed independently over the last century. This paper brings this theory together into a statistical model for random subcloning strategies. This mathematical model retains its utility even at high subclone redundancies, which are necessary for project completion. The discussion here centers on shotgun sequencing, a random subcloning strategy envisioned as the method of choice for sequencing the human genome.

  13. Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmata.

    Science.gov (United States)

    Atienzar; Cordi; Donkin; Evenden; Jha; Depledge

    2000-08-01

    The random amplified polymorphic DNA (RAPD) technique was used to detect DNA damage in the sublittoral macroalgae Palmaria palmata (Rhodophyta) exposed to both ambient and elevated irradiances of UV-B (280-315 nm). To investigate the potential of this method in ecotoxicological assessments, the qualitative and quantitative modifications in RAPD profiles were compared with changes in a number of physiological and fitness parameters. RAPD detectable modifications in DNA profiles were observed in all UV exposed individuals compared with controls. Changes in chlorophyll fluorescence (F(v)/F(m) ratio), in vivo pigment absorptance, thallus growth and RAPD profiles, examined simultaneously, provided a sensitive measure of UV-induced toxicity. In conclusion, the application of the RAPD method in conjunction with other suitable physiological and fitness measurements, may prove to be a valuable tool for investigating the specific effects of genotoxic agents upon marine algal populations. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organisation.

  14. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used...... to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were...... of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potential....

  15. Membrane-bound versus soluble major histocompatibility complex Class I-related chain A and major histocompatibility complex Class I-related chain B differential expression: Mechanisms of tumor eradication versus evasion and current drug development strategies

    Directory of Open Access Journals (Sweden)

    P K Suresh

    2016-01-01

    Full Text Available Major histocompatibility complex Class I-related chain A/chain B (MICA/MICB is stress-inducible, highly polymorphic ligands whose expression at the transcript level has been detected in all tissues except the central nervous system. However, their restricted protein expression is due to their regulation at the posttranslational level. Its levels are elevated in virally infected and neoplastically transformed cells. Membrane expression of this NKG2DL marks the aberrant cells for elimination by those immune effector cells that express the cognate NKG2D receptor. Among the evasion strategies developed by tumors, the metalloprotease-dependent shedding of MICA/MICB from tumors (either the free or the exosome form can contribute to the inhibition of cytolysis by the immune effector cells (all NK cells, most NKT cells; γδ CD8+ T cells and αβ CD8+ T cells, as well as some αβ CD4+ T cells. There are micro-RNA clusters that regulate surface expression and shedding. Polymorphic variants can be used as susceptibility/associative markers and can also possibly be used to correlate with tumor survival as well as staging/grading of tumors. Variations in the expression level require quantification of this marker for diagnostic/prognostic and therapeutic purposes. Mechanism-based studies would provide a better tumor-specific understanding of their relative roles in the processes of tumor cell elimination versus growth and progression. Last but not least, conventional, interlaboratory validated assays (for, e.g., antibody-based methods should be replaced by robust, reproducible, feasible biophysics-based methods using tumor biopsies. Further, correlative DNA polymorphism-based studies can be done using biological fluids (for, e.g., human saliva that can be sampled by minimally invasive means.

  16. Membrane-bound versus soluble major histocompatibility complex Class I-related chain A and major histocompatibility complex Class I-related chain B differential expression: Mechanisms of tumor eradication versus evasion and current drug development strategies.

    Science.gov (United States)

    Suresh, P K

    2016-01-01

    Major histocompatibility complex Class I-related chain A/chain B (MICA/MICB) is stress-inducible, highly polymorphic ligands whose expression at the transcript level has been detected in all tissues except the central nervous system. However, their restricted protein expression is due to their regulation at the posttranslational level. Its levels are elevated in virally infected and neoplastically transformed cells. Membrane expression of this NKG2DL marks the aberrant cells for elimination by those immune effector cells that express the cognate NKG2D receptor. Among the evasion strategies developed by tumors, the metalloprotease-dependent shedding of MICA/MICB from tumors (either the free or the exosome form) can contribute to the inhibition of cytolysis by the immune effector cells (all NK cells, most NKT cells; γδ CD8+ T cells and αβ CD8+ T cells, as well as some αβ CD4+ T cells). There are micro-RNA clusters that regulate surface expression and shedding. Polymorphic variants can be used as susceptibility/associative markers and can also possibly be used to correlate with tumor survival as well as staging/grading of tumors. Variations in the expression level require quantification of this marker for diagnostic/prognostic and therapeutic purposes. Mechanism-based studies would provide a better tumor-specific understanding of their relative roles in the processes of tumor cell elimination versus growth and progression. Last but not least, conventional, interlaboratory validated assays (for, e.g., antibody-based methods) should be replaced by robust, reproducible, feasible biophysics-based methods using tumor biopsies. Further, correlative DNA polymorphism-based studies can be done using biological fluids (for, e.g., human saliva) that can be sampled by minimally invasive means.

  17. The use of random amplified polymorphic DNA to evaluate the genetic variability of Ponkan mandarin (Citrus reticulata Blanco accessions

    Directory of Open Access Journals (Sweden)

    Coletta Filho Helvécio Della

    2000-01-01

    Full Text Available RAPD analysis of 19 Ponkan mandarin accessions was performed using 25 random primers. Of 112 amplification products selected, only 32 were polymorphic across five accessions. The absence of genetic variability among the other 14 accessions suggested that they were either clonal propagations with different local names, or that they had undetectable genetic variability, such as point mutations which cannot be detected by RAPD.

  18. First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

    Directory of Open Access Journals (Sweden)

    Bello Martha Darce

    2002-01-01

    Full Text Available A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis. The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%, C. tropicalis (23%, C. glabrata (14% and C. krusei (4%. This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

  19. Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

    Science.gov (United States)

    Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

    2015-09-01

    In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  20. Clonality Analysis of Helicobacter pylori in Patients Isolated from Several Biopsy Specimens and Gastric Juice in a Japanese Urban Population by Random Amplified Polymorphic DNA Fingerprinting

    Directory of Open Access Journals (Sweden)

    Nariaki Toita

    2013-01-01

    Full Text Available Background. The number of Helicobacter pylori clones infecting a single host has been discussed in numerous reports. The number has been suggested to vary depending on the regions in the world. Aim. The purpose of this study was to examine the number of clones infecting a single host in a Japanese urban population. Materials and Methods. Thirty-one Japanese patients undergoing upper gastrointestinal endoscopy were enrolled in this study. H. pylori isolates (total 104 strains were obtained from biopsy specimens (antrum, corpus, and duodenum and gastric juice. Clonal diversity was examined by the random amplified polymorphic DNA (RAPD fingerprinting method. Results. The RAPD fingerprinting patterns of isolates from each patient were identical or very similar. And the isolates obtained from several patients with 5- to 9-year intervals showed identical or very similar RAPD patterns. Conclusion. Each Japanese individual of an urban population is predominantly infected with a single H. pylori clone.

  1. EVALUASI KERAGAMAN GENTIK IKAN KALUI (Osphronemus goramy DARI KABUPATEN LIMA PULUH KOTA, SUMATERA BARAT BERDASARKAN MARKA RANDOM AMPLIFIED POLYMORPHISM DNA (RAPD

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2017-01-01

    Full Text Available Ikan kalui merupakan nama lokal dari ikan gurami di Kabupaten Lima Puluh Kota, Sumatera Barat termasuk jenis ikan ekonomis tinggi. Terdapat lima strain ikan kalui yang tersebar di pembudidaya, yaitu Tambago, Palapah, Krista, Jepun, dan Merah. Penelitian ini dilakukan untuk mengamati keragaman genetik strain ikan kalui atau gurami dengan menggunakan penanda RAPD. Sebanyak 50 sampel DNA ikan kalui diekstraksi dari sirip dan diamplifikasi secara random dengan menggunakan empat primer terbaik dari 20 primer OPA yaitu OPA-02, OPA-04, OPA-06, dan OPA-07. Hasil menunjukkan bahwa perbedaan yang nyata secara genetik hanya terdapat antara strain ikan kalui Merah dengan Tambago dan Krista. Variasi genetik tertinggi diamati pada ikan kalui strain Krista dengan nilai heterogenitas 0,1756 kemudian diikuti berturut-turut oleh strain Merah (0,1735; Palapah (0,1480; Jepun (0,0594; dan Tambago (0,0203. Jarak rata-rata Nei genetik adalah 0,407, dengan nilai terendah yang teramati antara strain Tambago dan Palapah. Kalui is a local name of giant gouramy fish in West Sumatera Province that categorized as an high economically fish. Five strains of kalui are distributed to farmers i.e. Tambago, Palapah, Krista, Jepun and Merah. This research was conducted to observe the genetic variation of Kalui strains using RAPD marker. A total of 50 samples of whole DNA was extracted from kalui-giant gouramy finclip and randomly amplified using four of the best 20 primers (OPA i.e. OPA-02, OPA-04, OPA-06 and OPA-07. The results showed that Significant genetic differences were only observed between strain Merah-Tambago and Merah-Krista. The highest variability was observed in Krista with heterogeneity value of 0.1735 followed by Merah (0,1735, Palapah (0,1480, Jepun (0,0594 and Tambago (0,0203. The average Nei genetic distance was 0.407, with the lowest was observed between Tambago and Palapah.

  2. Daily Nutritional Dose Supplementation with Antioxidant Nutrients and Phytochemicals Improves DNA and LDL Stability: A Double-Blind, Randomized, and Placebo-Controlled Trial

    Directory of Open Access Journals (Sweden)

    You Jin Kim

    2013-12-01

    Full Text Available Reactive oxygen species are important risk factors for age-related diseases, but they also act as signaling factors for endogenous antioxidative defense. The hypothesis that a multi-micronutrient supplement with nutritional doses of antioxidant nutrients and phytochemicals (MP may provide protection against oxidative damage and maintain the endogenous antioxidant defense capacity was assessed in subjects with a habitually low intake of fruits and vegetables. In a randomized, placebo-controlled, and parallel designed trial, 89 eligible subjects were assigned to either placebo or MP for eight weeks. Eighty subjects have completed the protocol and included for the analysis. MP treatment was superior at increasing serum folate (p < 0.0001 and resistance to DNA damage (p = 0.006, tail intensity; p = 0.030, tail moment by comet assay, and LDL oxidation (p = 0.009 compared with the placebo. Moreover, the endogenous oxidative defense capacity was not weakened after MP supplementation, as determined by the levels of glutathione peroxidase (p = 0.442, catalase (p = 0.686, and superoxide dismutase (p = 0.804. The serum folate level was negatively correlated with DNA damage (r = −0.376, p = 0.001 for tail density; r = −0.329, p = 0.003 for tail moment, but no correlation was found with LDL oxidation (r = −0.123, p = 0.275. These results suggest that MP use in healthy subjects with habitually low dietary fruit and vegetable intake may be beneficial in providing resistance to oxidative damage to DNA and LDL without suppressing the endogenous defense mechanisms.

  3. Molecular Diversity of Antagonistic Streptomyces spp. against Botrytis allii, the agent of onion gray mold using Random Amplified Polymorphic DNA (RAPD Markers

    Directory of Open Access Journals (Sweden)

    M. Jorjandi

    2014-08-01

    Full Text Available As an aim in sustainable agriculture, biological control of plant diseases has received intensive attention mainly as a response to public concern about the use of chemical fungicides in the environment. Soil Actinomycetes particularly Streptomyces spp. enhance soil fertility and have antagonistic activity against wide range of plant pathogens. To investigate for biocontrol means against the pathogen, 30 isolates of Actinomycetes have been isolated from agricultural soils of Kerman province of Iran and assayed for antagonistic activity against Botrytis allii, the agent of onion gray mold. RAPD DNA analysis has been used to determine the relatedness of active and non-active isolates based on their RAPD-PCR fingerprints. PCR amplifiable DNA samples have been isolated using the CTAB method and amplified fragments have been obtained from 5 random 10-mer primers. Different DNA fingerprinting patterns have been obtained for all of the isolates. Electrophoretic and cluster analysis of the amplification products has revealed incidence of polymorphism among the isolates. A total of 138 bands, ranging in size from 150-2800 bp, have been amplified from primers which 63.7% of the observed bands have been polymorphic. Genetic distances among different varieties have been analyzed with a UPGMA (Unweighted pair-group method, arithmetic average-derived dendrogram. Resulting dendrogram has showed from 0.65 to 0.91 similarities among varieties and divided the isolates into five major groups. Isolates which haven’t had any antagonistic activity against B. allii have been separated into a group and other isolates classified into four groups. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.

  4. Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but Not L. rhamnosus GG in the Human Vagina as Demonstrated by Randomly Amplified Polymorphic DNA

    Science.gov (United States)

    Gardiner, Gillian E.; Heinemann, Christine; Bruce, Andrew W.; Beuerman, Dee; Reid, Gregor

    2002-01-01

    Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 are well-characterized probiotic strains with efficacy in the prevention and treatment of urogenital infections in women. The aim of the present study was to apply a molecular biology-based methodology for the detection of these strains and L. rhamnosus GG (a commercially available intestinal probiotic) in the human vagina in order to assess probiotic persistence at this site. Ten healthy women inserted vaginally a capsule containing either a combination of strains GR-1 and RC-14 or the GG strain for 3 consecutive nights. Vaginal swabs taken before and at various time points after probiotic insertion were analyzed, and the Lactobacillus flora was assessed by randomly amplified polymorphic DNA (RAPD) analysis. This method generated discrete DNA fingerprints for GR-1, RC-14, and GG and enabled successful detection of these strains in the vagina. Strain GR-1 and/or strain RC-14 was found to persist in the vaginal tract for up to 19 days after vaginal instillation, while L. rhamnosus GG was detectable for up to 5 days postadministration. In conclusion, the fates of probiotic L. rhamnosus and L. fermentum strains were successfully monitored in the human vagina by RAPD analysis. This technique provides molecular biology-based evidence that RC-14 and GR-1, strains selected as urogenital probiotics, persist in the human vagina and may be more suited to vaginal colonization than L. rhamnosus GG. This highlights the importance of proper selection of strains for urogenital probiotic applications. PMID:11777835

  5. Population structure of a lodgepole pine (Pinus contorta) and jack pine (P. banksiana) complex as revealed by random amplified polymorphic DNA.

    Science.gov (United States)

    Ye, Terrance Z; Yang, Rong-Cai; Yeh, Francis C

    2002-06-01

    We studied the population structure of a lodgepole (Pinus contorta Dougl.) and jack pine (Pinus banksiana Lamb.) complex in west central Alberta and neighboring areas by assessing random amplified polymorphic DNA (RAPD) variability in 23 lodgepole pine, 9 jack pine, and 8 putative hybrid populations. Of 200 random primers screened, 10 that amplified 39 sharp and reproducible RAPDs were chosen for the study. None of the 39 RAPDs were unique to the parental species. RAPD diversity ranged from 0.085 to 0.190 among populations and averaged 0.143 for lodgepole pine, 0.156 for jack pine, 0.152 for hybrids, and 0.148 for all 40 populations. The estimated population differentiation based on G(ST) was 0.168 for hybrids, 0.162 for lodgepole pine, 0.155 for jack pine, and 0.247 across all 40 populations. Cluster analysis of genetic distances generally separated jack pine from lodgepole pine and hybrids, but no division could be identified that further separated lodgepole pine from hybrids. The observed weak to mild trend of "introgression by distance" in the complex and neighbouring areas was consistent with the view that introgressive hybridization between lodgepole and jack pines within and outside the hybrid zone may have been through secondary contact and primary intergradation, respectively.

  6. Genetic diversity of pigeon pea (Cajanus cajan (l.) Millsp.) based on molecular characterization using randomly amplified polymorphic DNA (RAPD) markers

    Science.gov (United States)

    Khoiriyah, N.; Yuniastuti, E.; Purnomo, D.

    2018-03-01

    Pigeon pea (Cajanus cajan (L.) Millsp.) is an annual leguminous crop (perennial) which has advantages over other local leguminous crops as drought resistant, hold collapsed and strong pods. The research on drought resistance plant is very important to adapt to climate change adverse impact to support food security. The potential of pigeon pie has not been supported by accurate data. To explore the potential of pigeon pea, it is necessary to record the important properties by characterization, one of which is molecular. Increasing genetic diversity can be done through mutation which widely used gamma ray for the induction. The purpose of this study was to identify the genetic diversity of pigeon pea of black, white and brown seeds type resulted by gamma-ray irradiation with a wavelength of 100, 200 and 300 grays by using RAPD method. The experiment resulted 14 bands, 12 of them are polymorphic bands and 2 of them are monomorphic with size varied from 300 bp to 1.3 kbp. The dendrogram showed from 30 accessions are divided into two main clusters, B shows clear genetical divergence from other clusters and some others split randomly. The range of similarity coefficient is from 0.43 to 1.00

  7. Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas.

    Science.gov (United States)

    Damasco, O P; Graham, G C; Henry, R J; Adkins, S W; Smiths, M K; Godwin, I D

    1996-11-01

    A RAPD marker specific to the dwarf off-type (hereafter known as dwarf) from micropropagation of Cavendish banana (Musa spp. AAA) cultivars New Guinea Cavendish and Williams was identified following an analysis of 57 normal (true-to-type) and 59 dwarf plants generated from several different micropropagation events. Sixty-six random decamer primers were used in the initial screen, of which 19 (28.8%) revealed polymorphisms between normal and dwarf plants. Primer OPJ-04 (5'-CCGAACACGG-3') was found to amplify an approx. 1.5 kb band which was consistently present in all normal but absent in all dwarf plants of both cultivars. Reliable detection of dwarf plants was achieved using this marker, providing the only available means ofin vitro detection of dwarfs. The use of this marker could facilitate early detection and elimination of dwarfs from batches of micropropagated bananas, and may be a useful tool in determining what factors in the tissue culture process lead to this off type production.Other micropropagation-induced RAPD polymorphisms were observed but were not associated with the dwarf trait.

  8. Genetic diversity and population structure of the narrow endemic and endangered species Heteroplexis microcephala Y. L. Chen. in China revealed by random amplified polymorphic DNA markers

    Directory of Open Access Journals (Sweden)

    Shi Yancai

    2016-01-01

    Full Text Available Heteroplexis microcephala Y. L. Chen. is an endemic and endangered species found only in karst limestone regions in the Yangshuo County of the Guangxi Zhuang Autonomous Region in China: it is a habitat representative of species in the Heteroplexis genus. To provide basic genetic information for its conservation, in this study we evaluated the genetic variation and differentiation among six wild populations of H. microcephala by random amplified polymorphic DNA markers (RAPD. The leaves of 141 individuals were sampled. Based on 12 primers, 113 DNA fragments were generated. Genetic diversity was low at the population level (Nei’s gene diversity (h=0.0579; Shannon information index (I=0.0924; percentage of polymorphic bands (PPB=23.30%, but relatively high at the species level (h=0.1701; I=0.2551; PPB=46.34%. The coefficient of genetic differentiation based on Nei’s genetic diversity analysis (0.6661 was high, indicating that there was significant genetic differentiation among populations, which was confirmed by AMOVA analysis exhibiting population differentiation among populations of 68.77%. Low gene flow among populations (0.2507 may result from several factors, such as a harsh pollination environment, population isolation and low seed dispersal distance. Limited gene flow and self-compatibility are the primary reasons for the high genetic differentiation observed for this species. We propose the collection of seeds from more populations with fewer individuals and core populations for ex situ conservation and suggest methods to increase seed germination rates.

  9. Synthesis and studies of polypeptide materials: Self-assembled block copolypeptide amphiphiles, DNA-condensing block copolypeptides and membrane-interactive random copolypeptides

    Science.gov (United States)

    Wyrsta, Michael Dmytro

    A new class of transition metal initiators for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs), has been developed by Deming et al. This discovery has allowed for the synthesis of well-defined "protein-like" polymers. Using this chemistry we have made distinct block/random copolypeptides for biomedical applications. Drug delivery, gene delivery, and antimicrobial polymers were the focus of our research efforts. The motivation for the synthesis and study of synthetic polypeptide based materials comes from proteins. Natural proteins are able to adopt a staggeringly large amount of uniquely well-defined folded structures. These structures account for the diversity in properties of proteins. As catalysts (enzymes) natural proteins perform some of the most difficult chemistry with ease and precision at ambient pressures and temperatures. They also exhibit incredible structural properties that directly result from formation of complex hierarchical assemblies. Self-assembling block copolymers were synthesized with various compositions and architectures. In general, di- and tri-block amphiphiles were studied for their self-assembling properties. Both spherical and tubular vesicles were found to assemble from di- and tri-block amphiphiles, respectively. In addition to self-assembly, pH responsiveness was engineered into these amphiphiles by the incorporation of basic residues (lysine) into the hydrophobic block. Another form of self-assembly studied was the condensation of DNA using cationic block copolymers. It was found that cationic block copolymers could condense DNA into compact, ordered, water-soluble aggregates on the nanoscale. These aggregates sufficiently protected DNA from nucleases and yet were susceptible to proteases. These studies form the basis of a gene delivery platform. The ease with which NCAs are polymerized renders them completely amenable to parallel synthetic methods. We have employed this technique to discover new

  10. Isolation and purification of membrane-bound cytochrome c from ...

    African Journals Online (AJOL)

    Administrator

    2007-05-02

    ferrochrome and redox spectra showed the presence of heme-c. Key words: Cytochrome c, respiratory chain and Proteus mirabilis. INTRODUCTION. Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium.

  11. Investigating membrane-bound Argonaute functions in Arabidopsis

    DEFF Research Database (Denmark)

    Barghetti, Andrea

    by direct cleavage or by inhibition of their translation. Arabidopsis genome encode for 10 different AGO proteins, of which AGO1 is the main effector of post-transcriptional gene silencing mediated by sRNA. Importantly, a fraction of AGO1 population is associated with endomembranes, in particular...

  12. Biogenesis of membrane bound respiratory complexes in Escherichia coli

    OpenAIRE

    Price, Claire E.; Driessen, Arnold J.M.

    2010-01-01

    Escherichia colt is one of the preferred bacteria for studies on the energetics and regulation of respiration Respiratory chains consist of primary dehydrogenases and terminal reductases or oxidases linked by quinones. In order to assemble this complex arrangement of protein complexes, synthesis of the subunits occurs in the cytoplasm followed by assembly in the cytoplasm and/or membrane, the incorporation of metal or organic cofactors and the anchoring of the complex to the membrane In the c...

  13. Biogenesis of membrane bound respiratory complexes in Escherichia coli

    NARCIS (Netherlands)

    Price, Claire E.; Driessen, Arnold J. M.

    Escherichia colt is one of the preferred bacteria for studies on the energetics and regulation of respiration Respiratory chains consist of primary dehydrogenases and terminal reductases or oxidases linked by quinones. In order to assemble this complex arrangement of protein complexes, synthesis of

  14. Isolation and purification of membrane-bound cytochrome c from ...

    African Journals Online (AJOL)

    In the present studies, respiratory chain pathogenic bacterium, Proteus mirabilis, was investigated. In the first phase, growth profile study was performed to optimize the P. mirabilis growth. Maximum bacterial growth could be obtained between 10 – 12 h of culturing time. Down-stream processing was performed by using ...

  15. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    Science.gov (United States)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  16. Multiple correspondence analysis and random amplified polymorphic DNA molecular typing to assess the sources of Staphylococcus aureus contamination in alheira production lines.

    Science.gov (United States)

    Esteves, A; Patarata, L; Aymerich, T; Garriga, M; Martins, C

    2007-03-01

    Sources and tracing of Staphylococcus aureus in alheira (garlic sausage) production were evaluated by multifactorial correspondence analysis (MCA) of occurrence data and a random amplified polymorphic DNA (RAPD) on S. aureus isolates. Samples from four production lines, four different production batches, and 14 different sampling sites (including raw material, different contact surfaces, and several stages of alheira manufacturing) were analyzed at four sampling times. From the 896 microbial analyses completed, a collection of 170 S. aureus isolates was obtained. Although analysis of the occurrence data alone was not elucidative enough, MCA and RAPD-PCR were able to assess the sources of contamination and to trace the spread of this microorganism along the production lines. MCA results indicated that the presence of S. aureus in alheira was related to its presence in the intermediate manufacturing stages after heat treatment but before stuffing in the casings. It was also possible to associate a cross-contamination path related to handler procedures. RAPD-PCR typing in accordance to MCA results confirmed the cross-contamination path between the raw material and casings and the role of handlers as an important cross-contamination vehicle.

  17. Characterization of Grain Amaranth (Amaranthus spp. Germplasm in South West Nigeria Using Morphological, Nutritional, and Random Amplified Polymorphic DNA (RAPD Analysis

    Directory of Open Access Journals (Sweden)

    Pamela E. Akin-Idowu

    2016-01-01

    Full Text Available Efficient utilization of plant genetic resources for nutrition and crop improvement requires systematic understanding of the important traits. Amaranthus species are distributed worldwide with an interesting diversity of landraces and cultivars whose leaves and seeds are consumed. Despite their potential to enhance food security and economic livelihoods, grain amaranth breeding to improve nutritional quality and adoption by farmers in sub-Saharan Africa is scanty. This study assessed the variation among 29 grain amaranth accessions using 27 phenotypic (10 morphological and 17 nutritional characters and 16 random amplified polymorphic DNA (RAPD primers. Multivariate analysis of phenotypic characters showed the first four principal components contributing 57.53% of observed variability, while cluster analysis yielded five groups at 87.5% similarity coefficient. RAPD primers generated a total of 193 amplicons with an average of 12.06 amplicons per primer, 81% of which were polymorphic. Genetic similarities based on Jaccard’s coefficient ranged from 0.61 to 0.88. The RAPD-based unweighted pair group method with arithmetic mean dendrogram grouped the accessions into nine clusters, with the same species clustering together. RAPD primers distinguished the accessions more effectively than phenotypic markers. Accessions in the different clusters as obtained can be exploited for heterotic gain in desired nutritional traits.

  18. Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting.

    Science.gov (United States)

    Yokota, Shin-ichi; Konno, Mutsuko; Fujiwara, Shin-ichi; Toita, Nariaki; Takahashi, Michiko; Yamamoto, Soh; Ogasawara, Noriko; Shiraishi, Tsukasa

    2015-10-01

    The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families. © 2015 John Wiley & Sons Ltd.

  19. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    International Nuclear Information System (INIS)

    Matsushima, H.; Bogenmann, E.

    1990-01-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment

  20. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  1. Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA.

    Science.gov (United States)

    Doria, Francesca; Napoli, Chiara; Costantini, Antonella; Berta, Graziella; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

    2013-08-01

    Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.

  2. Detection of genetic variability in Basmati and non-Basmati rice varieties and their radiation induced mutants through random amplified polymorphic DNA (RAPD)

    International Nuclear Information System (INIS)

    Farooq, S.; Iqbal, N.; Arif, M.

    1998-01-01

    Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ''fingerprinting'' of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F 2 plants and four F 3 families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author)

  3. Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates

    Science.gov (United States)

    Biller, Steven J; McDaniel, Lauren D; Breitbart, Mya; Rogers, Everett; Paul, John H; Chisholm, Sallie W

    2017-01-01

    Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time. PMID:27824343

  4. Gamma-ray induced double-strand breaks in DNA resulting from randomly-inflicted single-strand breaks: temporal local denaturation, a new radiation phenomenon?

    NARCIS (Netherlands)

    Schans, G.P. van der

    1978-01-01

    The induction of single- and double-strand breaks in DNA by γ-rays has been measured. The maximum number of nucleotide paris (a) between two independently induced single-strand breaks in opposite strands of the DNA which cannot prevent the occurrence of a double-strand break was found to amount to

  5. Clarithromycin-susceptible and -resistant Helicobacter pylori isolates with identical randomly amplified polymorphic DNA-PCR genotypes cultured from single gastric biopsy specimens prior to antibiotic therapy

    NARCIS (Netherlands)

    van der Ende, A.; van Doorn, L. J.; Rooijakkers, S.; Feller, M.; Tytgat, G. N.; Dankert, J.

    2001-01-01

    Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance,

  6. Prognostic Impact of Deficient DNA Mismatch Repair in Patients With Stage III Colon Cancer From a Randomized Trial of FOLFOX-Based Adjuvant Chemotherapy

    Science.gov (United States)

    Sinicrope, Frank A.; Mahoney, Michelle R.; Smyrk, Thomas C.; Thibodeau, Stephen N.; Warren, Robert S.; Bertagnolli, Monica M.; Nelson, Garth D.; Goldberg, Richard M.; Sargent, Daniel J.; Alberts, Steven R.

    2013-01-01

    Purpose The association of deficient DNA mismatch repair (dMMR) with prognosis in patients with colon cancer treated with adjuvant fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy remains unknown. Patients and Methods Resected, stage III colon carcinomas from patients (N = 2,686) randomly assigned to FOLFOX ± cetuximab (North Central Cancer Treatment Group N0147 trial) were analyzed for mismatch repair (MMR) protein expression and mutations in BRAFV600E (exon 15) and KRAS (codons 12 and 13). Association of biomarkers with disease-free survival (DFS) was determined using Cox models. A validation cohort (Cancer and Leukemia Group B 88903 trial) was used. Results dMMR was detected in 314 (12%) of 2,580 tumors, of which 49.3% and 10.6% had BRAFV600E or KRAS mutations, respectively. MMR status was not prognostic overall (adjusted hazard ratio [HR], 0.82; 95% CI, 0.64 to 1.07; P = .14), yet significant interactions were found between MMR and primary tumor site (Pinteraction = .009) and lymph node category (N1 v N2; Pinteraction = .014). Favorable DFS was observed for dMMR versus proficient MMR proximal tumors (HR, 0.71; 95% CI, 0.53 to 0.94; P = .018) but not dMMR distal tumors (HR, 1.71; 95% CI, 0.99 to 2.95; P = .056), adjusting for mutations and covariates. Any survival benefit of dMMR was lost in N2 tumors. Mutations in BRAFV600E (HR, 1.37; 95% CI, 1.08 to 1.70; P = .009) or KRAS (HR, 1.44; 95% CI, 1.21 to 1.70; P < .001) were independently associated with worse DFS. The observed MMR by tumor site interaction was validated in an independent cohort of stage III colon cancers (Pinteraction = .037). Conclusion The prognostic impact of MMR depended on tumor site, and this interaction was validated in an independent cohort. Among dMMR cancers, proximal tumors had favorable outcome, whereas distal or N2 tumors had poor outcome. BRAF or KRAS mutations were independently associated with adverse outcome. PMID:24019539

  7. Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

    Directory of Open Access Journals (Sweden)

    Anna Crescenti

    Full Text Available DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs, methylenetetrahydrofolate reductase (MTHFR, and methionine synthase reductase (MTRR genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, p<0.001. Additionally, we found an association between the cocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process.Clinicaltrials.govNCT00511420 and NCT00502047.

  8. Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

    Directory of Open Access Journals (Sweden)

    Patricia J Munseri

    Full Text Available Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools.In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46.129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%. There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS or in response rates and median titers for binding antibodies to Env gp160 between study groups.A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA.World Health Organization International Clinical Trials Registry

  9. High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.

    2004-01-01

    The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non- L. monocytogenes strains representing six other Listeria species...... of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included....... Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable...

  10. Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner =Purificação de uma enzima “tipo tripsina” não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner

    Directory of Open Access Journals (Sweden)

    Marcelo Matos Santoro

    2012-07-01

    Full Text Available Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima “tipo-tripsina” ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS e submetida

  11. High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.

    2004-01-01

    of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included...... for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains...

  12. DNA vaccine delivered by a needle-free injection device improves potency of priming for antibody and CD8+ T-cell responses after rAd5 boost in a randomized clinical trial.

    Directory of Open Access Journals (Sweden)

    Barney S Graham

    Full Text Available DNA vaccine immunogenicity has been limited by inefficient delivery. Needle-free delivery of DNA using a CO2-powered Biojector® device was compared to delivery by needle and syringe and evaluated for safety and immunogenicity.Forty adults, 18-50 years, were randomly assigned to intramuscular (IM vaccinations with DNA vaccine, VRC-HIVDNA016-00-VP, (weeks 0, 4, 8 by Biojector® 2000™ or needle and syringe (N/S and boosted IM at week 24 with VRC-HIVADV014-00-VP (rAd5 with N/S at 10(10 or 10(11 particle units (PU. Equal numbers per assigned schedule had low (≤500 or high (>500 reciprocal titers of preexisting Ad5 neutralizing antibody.120 DNA and 39 rAd5 injections were given; 36 subjects completed follow-up research sample collections. IFN-γ ELISpot response rates were 17/19 (89% for Biojector® and 13/17 (76% for N/S delivery at Week 28 (4 weeks post rAd5 boost. The magnitude of ELISpot response was about 3-fold higher in Biojector® compared to N/S groups. Similar effects on response rates and magnitude were observed for CD8+, but not CD4+ T-cell responses by ICS. Env-specific antibody responses were about 10-fold higher in Biojector-primed subjects.DNA vaccination by Biojector® was well-tolerated and compared to needle injection, primed for greater IFN-γ ELISpot, CD8+ T-cell, and antibody responses after rAd5 boosting.ClinicalTrials.gov NCT00109629.

  13. Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus Otimização do protocolo de amplificação randômica de DNA polimórfico para identificação molecular de Lophius gastrophysus

    Directory of Open Access Journals (Sweden)

    Micheline S. Ramella

    2005-12-01

    Full Text Available Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.Lophius gastrophysus apresenta importante valor comercial no Brasil, principalmente para a exportação. Neste estudo, descrevemos uma otimização do protocolo de amplificação aleatória de DNA polimórfico (RAPD para identificação de L. gastrophysus. Diferentes condições (temperatura de recozimento, quantidade de DNA e concentração de MgCl2 foram testadas para obter perfis reprodutíveis. Os iniciadores A01, A02 e A03 geraram os melhores resultados de amplificação quando utilizados temperatura de recozimento de 36ºC, 25 ng de DNA e 2,5 mM de MgCl2. A identificação exata de espécies e da origem dos produtos marinhos faz-se necessária e a RAPD é uma ferramenta rápida e precisa para expor fraudes comerciais.

  14. Randomly detected genetically modified (GM maize (Zea mays L. near a transport route revealed a fragile 45S rDNA phenotype.

    Directory of Open Access Journals (Sweden)

    Nomar Espinosa Waminal

    Full Text Available Monitoring of genetically modified (GM crops has been emphasized to prevent their potential effects on the environment and human health. Monitoring of the inadvertent dispersal of transgenic maize in several fields and transport routes in Korea was carried out by qualitative multiplex PCR, and molecular analyses were conducted to identify the events of the collected GM maize. Cytogenetic investigations through fluorescence in situ hybridization (FISH of the GM maize were performed to check for possible changes in the 45S rDNA cluster because this cluster was reported to be sensitive to replication and transcription stress. Three GM maize kernels were collected from a transport route near Incheon port, Korea, and each was found to contain NK603, stacked MON863 x NK603, and stacked NK603 x MON810 inserts, respectively. Cytogenetic analysis of the GM maize containing the stacked NK603 x MON810 insert revealed two normal compact 5S rDNA signals, but the 45S rDNA showed a fragile phenotype, demonstrating a "beads-on-a-string" fragmentation pattern, which seems to be a consequence of genetic modification. Implications of the 45S rDNA cluster fragility in GM maize are also discussed.

  15. Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: Duplication and non-random loss

    Energy Technology Data Exchange (ETDEWEB)

    Lavrov, Dennis V.; Boore, Jeffrey L.; Brown, Wesley M.

    2001-11-08

    We determined the complete mtDNA sequences of the millipedes Narceus annularus and Thyropygus sp. (Arthropoda: Diplopoda) and identified in both genomes all 37 genes typical for metazoan mtDNA. The arrangement of these genes is identical in the two millipedes, but differs from that inferred to be ancestral for arthropods by the location of four genes/gene clusters. This novel gene arrangement is unusual for animal mtDNA, in that genes with opposite transcriptional polarities are clustered in the genome and the two clusters are separated by two non-coding regions. The only exception to this pattern is the gene for cysteine tRNA, which is located in the part of the genome that otherwise contains all genes with the opposite transcriptional polarity. We suggest that a mechanism involving complete mtDNA duplication followed by the loss of genes, predetermined by their transcriptional polarity and location in the genome, could generate this gene arrangement from the one ancestral for arthropods. The proposed mechanism has important implications for phylogenetic inferences that are drawn on the basis of gene arrangement comparisons.

  16. Rho GTPases: Novel Players in the Regulation of the DNA Damage Response?

    Directory of Open Access Journals (Sweden)

    Gerhard Fritz

    2015-09-01

    Full Text Available The Ras-related C3 botulinum toxin substrate 1 (Rac1 belongs to the family of Ras-homologous small GTPases. It is well characterized as a membrane-bound signal transducing molecule that is involved in the regulation of cell motility and adhesion as well as cell cycle progression, mitosis, cell death and gene expression. Rac1 also adjusts cellular responses to genotoxic stress by regulating the activity of stress kinases, including c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK and p38 kinases as well as related transcription factors. Apart from being found on the inner side of the outer cell membrane and in the cytosol, Rac1 has also been detected inside the nucleus. Different lines of evidence indicate that genotoxin-induced DNA damage is able to activate nuclear Rac1. The exact mechanisms involved and the biological consequences, however, are unclear. The data available so far indicate that Rac1 might integrate DNA damage independent and DNA damage dependent cellular stress responses following genotoxin treatment, thereby coordinating mechanisms of the DNA damage response (DDR that are related to DNA repair, survival and cell death.

  17. KARAKTER GENETIK INDUK (F-0) DAN TURUNANNYA (F-1) PADA IKAN HIAS LAUT CLOWN (Amphiprion percula) MENGGUNAKAN MARKER RAPD (Random Amplified Polymorfism DNA)

    OpenAIRE

    Sari Budi Moria Sembiring; Ketut Maha Setiawati; Haryanti Haryanti; Ida Komang Wardana

    2016-01-01

    Studi analisis karakter genetik ikan hias laut clown menggunakan metode penanda DNA RAPD dilakukan dalam upaya membantu pengembangan perbenihan dan budidaya ikan hias laut clown di Indonesia. Tujuan penelitian ini adalah untuk mendeterminasi karakter genetik dengan menggunakan analisis individu dari populasi induk (F-0) dan turunannya (F-1) sehingga diperoleh tingkat penurunan keragaman genetik dan keterkaitannya dengan karakter morfologi. Sampel yang dianalisis terdiri atas 5 pasang induk ik...

  18. Microchip capillary electrophoresis-based genetic comparison of closely related cyathostomin nematode parasites of horses using randomly amplified polymorphic DNA polymerase chain reaction.

    Science.gov (United States)

    Posedi, Janez; Drögemüller, Michaela; Schnieder, Thomas; Höglund, Johan; Lichtenfels, J Ralph; von Samson-Himmelstjerna, Georg

    2004-03-01

    The microchip-based capillary electrophoresis technology represents a valuable recent development for the analysis of complex DNA banding patterns. We have used this technology for the differentiation of the closely related cyathostomin species Cylicocyclus elongatus and C. insigne from the horse. We found that the Agilent 2100 bioanalyser in combination with the DNA 7500 Lab Chip were suited to perform a phylogenetic DNA fingerprinting analysis of the parasite species studied. The analysis of the electrophoretic data was optimised and it was possible to resolve a phylogenetic tree where all 12 individual worms of the two Cylicocyclus species studied were assigned to their species as determined by microscopic identification based on morphological traits. Thus, our data indicated that the procedure described here provides an additional powerful tool that can be employed for species delineation of closely related strains or species, such as the two taxa of Cylicocyclus investigated in the present study. Furthermore, by determining the second internal transcribed spacer region of three and nine individual worms for C. elongatus and C. insigne, respectively, low intraspecific variations of only up to 0.3% were demonstrated.

  19. Phase I randomized clinical trial of VRC DNA and rAd5 HIV-1 vaccine delivery by intramuscular (i.m., subcutaneous (s.c. and intradermal (i.d. administration (VRC 011.

    Directory of Open Access Journals (Sweden)

    Mary E Enama

    Full Text Available Phase 1 evaluation of the VRC HIV DNA and rAd5 vaccines delivered intramuscularly (i.m. supported proceeding to a Phase 2 b efficacy study. Here we report comparison of the i.m., subcutaneous (s.c. and intradermal (i.d. routes of administration.Sixty subjects were randomized to 6 schedules to evaluate the i.m., s.c. or i.d. route for prime injections. Three schedules included DNA primes (Wks 0,4,8 and 3 schedules included rAd5 prime (Wk0; all included rAd5 i.m. boost (Wk24. DNA vaccine dosage was 4 mg i.m. or s.c., but 0.4 mg i.d., while all rAd5 vaccinations were 1010 PU. All injections were administered by needle and syringe.Overall, 27/30 subjects completed 3 DNA primes; 30/30 subjects completed rAd5 primes. Mild local pruritus (itchiness, superficial skin lesions and injection site nodules were associated with i.d. and s.c., but not i.m. injections. All routes induced T-cell and antibody immune responses after rAd5 boosting. Overall, >95% had Env antibody and >80% had Env T-cell responses.The pattern of local reactogenicity following i.d. and s.c. injections differed from i.m. injections but all routes were well-tolerated. There was no evidence of an immunogenicity advantage following s.c. or i.d. delivery, supporting i.m. delivery as the preferred route of administration.Clinicaltrials.gov NCT00321061.

  20. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

    Science.gov (United States)

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlevic, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

    2017-06-02

    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Influence of HLA-A, HLA-B, and HLA-DR matching on rejection of random corneal grafts using corneal tissue for retrospective DNA HLA typing

    NARCIS (Netherlands)

    M.C. Bartels (Marjolijn); H.G. Otten; B.E. van Gelderen; A. van der Lelij (Allegonda)

    2001-01-01

    textabstractAIM: To establish if coincidental HLA-A, HLA-B, and HLA-DR tissue matching is associated with a reduced likelihood of corneal graft rejection. METHODS: Organ culture preserved random donor corneas were used for penetrating keratoplasty (PKP). Corneal tissue from all

  2. KARAKTER GENETIK INDUK (F-0 DAN TURUNANNYA (F-1 PADA IKAN HIAS LAUT CLOWN (Amphiprion percula MENGGUNAKAN MARKER RAPD (Random Amplified Polymorfism DNA

    Directory of Open Access Journals (Sweden)

    Sari Budi Moria Sembiring

    2016-11-01

    Full Text Available Studi analisis karakter genetik ikan hias laut clown menggunakan metode penanda DNA RAPD dilakukan dalam upaya membantu pengembangan perbenihan dan budidaya ikan hias laut clown di Indonesia. Tujuan penelitian ini adalah untuk mendeterminasi karakter genetik dengan menggunakan analisis individu dari populasi induk (F-0 dan turunannya (F-1 sehingga diperoleh tingkat penurunan keragaman genetik dan keterkaitannya dengan karakter morfologi. Sampel yang dianalisis terdiri atas 5 pasang induk ikan clown (10 sampel dan masing-masing turunannya sebanyak 10 ekor (50 sampel sehingga total 60 sampel. Nilai rata-rata keragaman genetik induk ikan clown dari semua lokus primer sebesar 0,253, sedangkan pada turunannya (F-1 adalah 0,157. Hal ini menggambarkan adanya pengaruh genetik terhadap perbedaan pola pemunculan band putih. Study genetic characteristic of clownfish, Amphiprion percula using RAPD DNA marker was conducted in order to support development of breeding and culture program of marine ornamental clownfish in Indonesia. The objective of this research was to determine of genetic characteristic of clown fish using individual analysis from F-0 population and its generations (F-1 to find specific marker which is related to its morphology. Total samples analyzed were 60, consist of 5 pairs of clownfish broodstock (10 samples and 10 ind each generations (50 samples. Mean value of genetic diversity of clown fish broodstock from all primer loci was 0.253, while on its generation F-1 was 0.157. This result showed there was effect of genetic on the differences of white band pattern appearance.

  3. The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis.

    Science.gov (United States)

    Reich, Charles F; Pisetsky, David S

    2009-03-10

    Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for beta-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

  4. The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

    International Nuclear Information System (INIS)

    Reich, Charles F.; Pisetsky, David S.

    2009-01-01

    Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death

  5. EVALUASI VARIASI FENOTIPE DAN GENOTIPE POPULASI IKAN TAMBAKAN DARI JAWA BARAT, KALIMANTAN TENGAH, DAN JAMBI DENGAN TRUSS MORFOMETRIK DAN RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD

    Directory of Open Access Journals (Sweden)

    Anang Hari Kristanto

    2018-01-01

    Full Text Available Ikan tambakan (Helostoma temminckii digemari sebagai ikan konsumsi, di daerah Sumatera dan Kalimantan. Dalam rangka pengembangan budidayanya melalui program domestikasi, informasi terkait variasi fenotipe dan genotipe induk asal perlu dilakukan. Penelitian ini bertujuan untuk mendapatkan karakteristik fenotipe dan genotipe ikan tambakan dari Jawa Barat, Kalimantan Tengah, dan Jambi. Penelitian dilakukan di Balai Riset Perikanan Budidaya Air Tawar dan Penyuluhan Perikanan (BRPBATPP, Bogor. Data diperoleh melalui pengukuran jarak bagian tubuh berdasarkan metode truss morphometric dan analisis DNA menggunakan metode RAPD. Hasil yang diperoleh menunjukkan bahwa nilai polimorfisme (81,25% dan heterozigositas (0,3544 tertinggi terdapat pada populasi ikan tambakan asal Kalimantan Tengah. Jarak genetik tertinggi antara populasi Jambi dengan Kalimantan Tengah sebesar 0,1452; sedangkan jarak genetik terendah adalah 0,1044 yaitu antara populasi Jambi dengan Jawa Barat. Berdasarkan uji karakter morfometrik diketahui terdapat 13 karakter yang berbeda nyata yaitu A1, A2, A4, A5, B3, C1, C3, C4, C5, D3, D4, D5, dan D6. Populasi Jambi dengan Jawa Barat memiliki hubungan kekerabatan lebih dekat dibanding dengan populasi Kalimantan Tengah. Kissing gouramy (Helostoma temminckii is a favored fish for consumption in Sumatra and Kalimantan area. Currently, information related to phenotypic variation and genotypes of the original broodstocks of kissing gouramy is limited to develop the fish’s culture technology through a domestication program. Therefore, the study was conducted to determine the characteristics of phenotype, and genotype of original kissing gouramy broodstocks. The research was conducted at the Institute for Freshwater Aquaculture Research and Development, Bogor. Data collection was carried out by measuring the body length using truss morphometric method and DNA analysis using the RAPD method. The results showed that the highest polymorphism (81

  6. Analysis of genetic variability in soursop Annona muricata L populations from Central Java and East Java based on random amplified polymorphic DNA RAPD marker

    Directory of Open Access Journals (Sweden)

    Suratman Suratman

    2014-08-01

    Full Text Available The objective of this research was to determine genetic variability of the soursop (Annona muricata L. populations from Central Java and East Java based on RAPD markers. Leaves of 40 individuals were collected from 4 soursop populations in Central Java and East Java, include : Sukoharjo, Karanganyar (Central Java, and Ngawi, Pacitan (East Java. Genomic DNA was extracted from the leaves by the CTAB extraction procedure with some modifications. A total of 15 RAPD primers were purchased from commercial source and tested to find specific diagnostic markers for each individuals by RAPD-PCR. The measurement of soursop population genetic distance was based on similarity coefficient using method of Group Average Clustering and Unweight Pair Group Method Arithmetic (UPGMA of NTSYS program version 2.02i. Results showed that each soursop population collected from different localities seemed have variability in RAPD profiles by using different primers. Four RAPD polymorphic primer was selected from 15 RAPD primers, namely A18, A20, P10 and P11. A total of 58 bands produced, varying from 9 to 20 bands per primer. The selected four RAPD primers produced 57 polymorphic bands, whereas polymorphism for each primer ranged from 95 % to 100 %. Dendrogram indicated that four soursop populations tend to segregate form two separated clade. The sample collected from Sukoharjo formed a separate cluster while the sample collected from Ngawi, Pacitan and Karanganyar grouped together in other cluster and diverged from population Sukoharjo.

  7. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

  8. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells

    International Nuclear Information System (INIS)

    Ahamed, Maqusood; Karns, Michael; Goodson, Michael; Rowe, John; Hussain, Saber M.; Schlager, John J.; Hong Yiling

    2008-01-01

    Silver nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and life sciences. Ag NPs are of interest to defense and engineering programs for new material applications as well as for commercial purposes as an antimicrobial. However, little is known about the genotoxicity of Ag NPs following exposure to mammalian cells. This study was undertaken to examine the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag NPs in two types of mammalian cells; mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Both types of Ag NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated-H2AX expression. Furthermore both of them induced cell death as measured by the annexin V protein expression and MTT assay. Our observations also suggested that the different surface chemistry of Ag NPs induce different DNA damage response: coated Ag NPs exhibited more severe damage than uncoated Ag NPs. The results suggest that polysaccharide coated particles are more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles

  9. Mitochondrial DNA.

    Science.gov (United States)

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  10. Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage.

    Science.gov (United States)

    Martínez-García, Belén; Fernández, Xavier; Díaz-Ingelmo, Ofelia; Rodríguez-Campos, Antonio; Manichanh, Chaysavanh; Roca, Joaquim

    2014-02-01

    By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.

  11. Modeling DNA

    Science.gov (United States)

    Robertson, Carol

    2016-01-01

    Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

  12. Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

    Directory of Open Access Journals (Sweden)

    Roger Le Grand

    2013-07-01

    Full Text Available HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb. We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

  13. Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

    Directory of Open Access Journals (Sweden)

    Emma-Jo Hayton

    Full Text Available HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern.These data demonstrate safety and good tolerability of the pSG2

  14. Radiation-induced electron migration along DNA

    International Nuclear Information System (INIS)

    Fuciarelli, A.F.; Sisk, E.C.; Miller, J.H.; Zimbrick, J.D.

    1994-04-01

    Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to nonrandom types of damage along DNA manifested distal to the sites of the initial energy deposition. Electron migration along DNA is significantly influenced by the DNA base sequence and DNA conformation. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution which compares to average migration distances of 6 to 10 bases for Escherichia coli DNA irradiated in solution and 5.5 base pairs for Escherichia coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along DNA. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation

  15. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  16. Genotype characterization of the Haematobia Irritans (diptera: muscidae from Brazil, Dominican Republic and Colombia based on randomly amplified polymorphic dna (rapd analysis Caracterização genotípica de Haematobia irritans (Diptera: Muscidae procedentes do Brasil, República Dominicana e Colômbia baseada na análise do DNA polimórfico amplificado ao acaso (RAPD

    Directory of Open Access Journals (Sweden)

    Luciana Gatto Brito

    2008-12-01

    Full Text Available Blood-sucking flies are important parasites in animal production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. H. irritans is one of the parasites of cattle that cause significant economic losses in many parts of the world, including South America. In the present work, Brazilian, Colombian and Dominican Republic populations of this species were studied by Random Amplified Polymorphic DNA (RAPD to assess basically genetic variability between populations. Fifteen different decamer random primers were employed in the genomic DNA amplification, yielding 196 fragments in the three H. irritans populations. Among H. irritans samples, that from Colombia produced the smallest numbers of polymorphic bands. This high genetic homogeneity may be ascribed to its geographic origin, which causes high isolation, low gene flow, unlike the other American populations, from Brazil and Dominican Republic. Molecular marker fragments, which its produced exclusive bands, detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian, Colombian and Dominican Republic populations of horn fly from South America. Similarity indices produced by chemo metric analysis showed the closest relationships between flies from Brazil and Dominican Republic, while flies from Colombia showed the greatest genotypic differentiation relative to the others populations.Moscas hematófagas são importantes parasitas em sistemas de produção animal, especialmente em condições confinamento. Haematobia irritans, a mosca-dos-chifres, é uma das espécies que mais causam problemas em sistemas de produção de bovinos, devido ao intenso estresse que impõe aos animais. H. irritans é um dos parasitas de bovinos que

  17. Randomization tests

    CERN Document Server

    Edgington, Eugene

    2007-01-01

    Statistical Tests That Do Not Require Random Sampling Randomization Tests Numerical Examples Randomization Tests and Nonrandom Samples The Prevalence of Nonrandom Samples in Experiments The Irrelevance of Random Samples for the Typical Experiment Generalizing from Nonrandom Samples Intelligibility Respect for the Validity of Randomization Tests Versatility Practicality Precursors of Randomization Tests Other Applications of Permutation Tests Questions and Exercises Notes References Randomized Experiments Unique Benefits of Experiments Experimentation without Mani

  18. Evaluation of bilge water-induced plant genotoxicity using randomly ...

    African Journals Online (AJOL)

    Evaluation of bilge water-induced plant genotoxicity using randomly amplified polymorphic dna (rapd) ... Ife Journal of Science ... The Random Amplified Polymorphic DNA (RAPD) technique was employed to evaluate the level of DNA alteration in Allium cepa root tips exposed to different concentrations of bilge water (1, 5, ...

  19. A phase IIA randomized clinical trial of a multiclade HIV-1 DNA prime followed by a multiclade rAd5 HIV-1 vaccine boost in healthy adults (HVTN204.

    Directory of Open Access Journals (Sweden)

    Gavin J Churchyard

    Full Text Available The safety and immunogenicity of a vaccine regimen consisting of a 6-plasmid HIV-1 DNA prime (envA, envB, envC, gagB, polB, nefB boosted by a recombinant adenovirus serotype-5 (rAd5 HIV-1 with matching inserts was evaluated in HIV-seronegative participants from South Africa, United States, Latin America and the Caribbean.480 participants were evenly randomized to receive either: DNA (4 mg i.m. by Biojector at 0, 1 and 2 months, followed by rAd5 (10(10 PU i.m. by needle/syringe at 6 months; or placebo. Participants were monitored for reactogenicity and adverse events throughout the 12-month study. Peak and duration of HIV-specific humoral and cellular immune responses were evaluated after the prime and boost.The vaccine was well tolerated and safe. T-cell responses, detected by interferon-γ (IFN-γ ELISpot to global potential T-cell epitopes (PTEs were observed in 70.8% (136/192 of vaccine recipients overall, most frequently to Gag (54.7% and to Env (54.2%. In U.S. vaccine recipients T-cell responses were less frequent in Ad5 sero-positive versus sero-negative vaccine recipients (62.5% versus 85.7% respectively, p = 0.035. The frequency of HIV-specific CD4+ and CD8+ T-cell responses detected by intracellular cytokine staining were similar (41.8% and 47.2% respectively and most secreted ≥2 cytokines. The vaccine induced a high frequency (83.7%-94.6% of binding antibody responses to consensus Group M, and Clades A, B and C gp140 Env oligomers. Antibody responses to Gag were elicited in 46% of vaccine recipients.The vaccine regimen was well-tolerated and induced polyfunctional CD4+ and CD8+ T-cells and multi-clade anti-Env binding antibodies.ClinicalTrials.gov NCT00125970.

  20. Low Genetic Diversity Among Garlic (Allium sativum L. Accessions Detected Using Random Amplified Polymorphic DNA (RAPD Escasa Diversidad Genética entre Accesiones de Ajo (Allium sativum L. Detectada Mediante ADN Polimórfico Amplificado al Azar (RAPD

    Directory of Open Access Journals (Sweden)

    Mario Paredes C

    2008-03-01

    Full Text Available Garlic (Allium sativum L. is a species of vegetative propagation, showing high morphological diversity. Besides, its clones have specific adaptations to different agroclimatic regions. The objective of this study was to determine the genetic diversity of 65 garlic clones collected in Chile and introduced from different countries, by using RAPD (Random Amplified Polymorphic DNA. Fourty random primers of 10 mers generated a total of 398 bands with an 87% of polymorphism. Each primer amplified between two and 20 bands. The size of the fragments obtained fluctuated between 3200 and 369 bp. The results showed that the clones analyzed had a genetic similarity rate of 94%. In addition, 70% of them were clustered in one major group. However, in spite of that situation several clones have different agronomic characteristicsEl ajo (Allium sativum L. es una especie de propagación vegetativa, que presenta una amplia variabilidad morfológica. Los clones de esta especie tienen una adaptación específica a diferentes regiones agroclimáticas. El objetivo de este estudio fue determinar la diversidad genética existente en 65 clones de ajos colectados en Chile e introducidos desde diferentes países, utilizando RAPD (ADN Polimórfico Amplificado al Azar. Para esta evaluación se utilizaron 40 partidores de 10-mers. Los partidores generaron entre dos y 20 bandas, observándose un alto número de patrones con bandas múltiples. Los fragmentos generados difieren en su tamaño entre 3.200 y 369 pb. Los partidores generaron 398 bandas, de las cuales un 87% fueron polimórficas. El análisis estadístico realizado detectó una similitud genética alta, de un 94% entre las accesiones evaluadas, donde aproximadamente un 70% de los clones formaron un grupo homogéneo. Sin embargo, este grupo incluye clones que presentan diferentes características agronómicas

  1. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  2. DNA Vaccines

    Indian Academy of Sciences (India)

    DNA vaccine, immune response, antibodies, infectious diseases. GENERAL I ARTICLE. DNA Vaccines. P N Rangarajan. History of Vaccine Development. The year 1996 marked the 200th anniversary of the first vaccine developed against smallpox by Edward Jenner. In the now- famous 1796 experiment, Jenner scratched ...

  3. Hyperstretching DNA

    NARCIS (Netherlands)

    Schakenraad, Koen; Biebricher, Andreas S.; Sebregts, Maarten; Ten Bensel, Brian; Peterman, Erwin J.G.; Wuite, Gijs J L; Heller, Iddo; Storm, Cornelis; Van Der Schoot, Paul

    2017-01-01

    The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely

  4. Random amplified polymorphic DNA (RAPD) marker associated ...

    African Journals Online (AJOL)

    Jane

    2011-06-29

    Jun 29, 2011 ... by combined RAPD and ISSR markers. Afr. J. Biotechnol. 7: 2333-. 2340. Cony MA, Trione SO (1998). Inter- and interspecific variability in. Prosopis flexuosa and P. chilensis: seed germination under salt and moisture stress. J. Arid Environ. 40: 307-317. Crawford RMM (1978). Mineral Nutrition (Chapter 6).

  5. Phytohormones content and random amplified polymorphic DNA ...

    African Journals Online (AJOL)

    In Egypt, Citrus represents one of the main fruit tree crops for both local and export potentials. In this study, leaf and vegetative bud samples were studied for cultivars of sour orange (seedless, sweet seeded, Brazilian and Spanish), common sweet orange (Florida, Fsido, Shamoty, and Valencia) and navel orange B29 from ...

  6. Random amplified polymorphic DNA (RAPD) marker associated ...

    African Journals Online (AJOL)

    Jane

    2011-06-29

    Jun 29, 2011 ... stage (Maas, 1993). Furthermore, plants are more tolerant to salt stress during seed germination than the seedling stage (Maas, 1993; Cony and Trione, 1998;. Meloni et al., 2008). However, tolerance to salinity during seeds germination is the most viable criteria used for selection of salt tolerant plants ...

  7. Random amplified polymorphic DNA based genetic characterization ...

    African Journals Online (AJOL)

    DIRECTOR

    2013-07-10

    Jul 10, 2013 ... Due to the unusually long sexual cycle and unavaila- bility of any other diagnostic tool, identification of bamboo .... To our knowledge, nothing along these lines has been done. Some approaches are use- .... (Dipterocarpaceae), in Malaysia: Implications for conservation of genetic resources and tree.

  8. Comparison between random amplified polymorphic DNA (RAPD ...

    African Journals Online (AJOL)

    Understanding the genetic diversity of germplasm is essential in plant breeding programmes and germplasm management. Molecular markers are efficient and effective tools widely used for assessing genetic diversity among crop genotypes. Recently, high resolution melt analysis (HRM) has been reported for detecting ...

  9. using random amplified polymorphic DNA (RAPD)

    African Journals Online (AJOL)

    To study the pattern of genetic diversity in 45 genotypes of common bean, 19 RAPD primers were used. Of 253 bands produced, 236 bands (94.22%) were polymorphic in which maximum number (20 polymorphic bands) were observed in the profiles of the primer OPB-07. Highest PIC value (0.79) was observed for the ...

  10. Random amplified polymorphic DNA (RAPD) marker associated ...

    African Journals Online (AJOL)

    Seed germination and seedling growth of 20 provenances of Acacia senegal were evaluated under salinity conditions. For germination study, seeds irrigated with NaCl in five concentrations had electrical conductivities (EC) of 0 (only distilled water), 2, 4, 6 and 10 dSm-1. Final germination percentage (FG) and germination ...

  11. DNA Translocation and Cell Electroporation in Micro and Nanofluidic Devices

    Science.gov (United States)

    Gupta, Cherry

    The cell membrane is made of a thin (˜ 5nm) lipid bilayer which serves as an effective insulator and diffusion barrier for entities external to the cell from entering the cell. However, for research, diagnostic and therapeutic purposes, there is a need to deliver molecules of interest to the interior of live cells. This is usually accomplished by two methods: (a) carrier mediated delivery which consists of encapsulating the gene/molecule of interest either in an empty viral capsid or in chemically formulated lipoplex or polyplex nanoparticles, or (b) physical methods of delivery, which include the use of different kinds of forces to create reversible pores on the cell membrane (sonoporation, electroporation) or directly inject molecules to the cell cytosol (Gene Gun, microinjection). Of the aforementioned techniques, electroporation is the most commonly used due to it simplicity and ease of use. Our group recently demonstrated a nanochannel based electroporation (NEP) technique, in which two microchannels (˜40 m diameter) are connected by a nanochannel (˜ 200-400 mum diameter) in the center. A cell is positioned in one microchannel such that it makes contact with the nanochannel and transfection agents are placed in the other microchannel. An external electric field applied across the device locally porates the cell where it touches the nancohannel and drives the transfection agents into the cell. Besides maintaining high cell viability and achieving dose control, an important feature of NEP is the delivery of large molecules such as plasmids and quantum dots directly into the cell cytosol. In contrast, delivery of large plasmids during bulk electroporation, wherein cells and genes/plasmids are mixed in a buffered medium and an external electric field is applied across the mixture which electroporates the cells, is via formation of cell membrane bound aggregates which get endocytosed post pulsation. Various mechanisms of DNA transport across the membrane have

  12. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  13. Hydrogen-Rich Saline Ameliorates Allergic Rhinitis by Reversing the Imbalance of Th1/Th2 and Up-Regulation of CD4+CD25+Foxp3+Regulatory T Cells, Interleukin-10, and Membrane-Bound Transforming Growth Factor-β in Guinea Pigs.

    Science.gov (United States)

    Xu, Feifei; Yu, Shaoqing; Qin, Mali; Mao, Yong; Jin, Ling; Che, Na; Liu, Shuangxi; Ge, Rongming

    2018-02-01

    It is well known that CD4+CD25+Foxp3+Treg cells play an important role in the development of allergic rhinitis (AR); the defect of cell numbers and functions contribute to AR. Hydrogen has been proven effective in alleviating symptoms of AR. We herein aim to verify the protective effects of hydrogen on CD4+CD25+Foxp3+Treg cells in guinea pigs with AR and to explore the effect of hydrogen-rich saline (HRS) on CD4+CD25+Foxp3+Treg cells in animals with AR and investigate the underlying anti-inflammatory mechanism. Eighteen guinea pigs were randomly divided into three groups (control group/AR group/AR-HRS group). The guinea pigs were injected with hydrogen-rich saline (AR-HRS group) for 10 days after sensitization. The control group was injected with an equal volume of normal saline. The number of sneezes, degree of runny nose, and nasal-rubbing movements were scored. Peripheral blood eosinophil count was recorded. The proportions of Th1/Th2 of the peripheral blood and the CD4+CD25+Foxp3+T cells in the CD4+T cells of the spleen and peripheral blood were determined by flow cytometry. The content of interleukin (IL)-10 and transforming growth factor (TGF)-β in the serum was detected by enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expression of Foxp3, IL-10, and TGF-β were determined by Western blot, immunofluorescence, and real-time PCR analysis, respectively. Scores of symptoms, number of eosinophils,and nasal mucosa damage were dramatically reduced after HRS treatment. HRS increased the expression of Foxp3, IL-10, TGF-β, and number of CD4+CD25+Foxp3+Treg cells, which were reduced in AR. HRS also revised the dysregulation of Th1/Th2 balance. Both the number and biological activity of CD4+CD25+Foxp3+Treg cells increased with up-regulation of Th1/Th2 after HRS administration. HRS could play a protective role in attenuating AR through improving the proportion and functions of CD4+CD25+Foxp3+Treg cells.

  14. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  15. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  16. DNA nanotechnology

    Science.gov (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.

    2018-01-01

    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  17. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  18. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Braddock, M.

    1985-07-01

    The hydroxyl radical (OH radical) is the most damaging radical produced by the effect of ionizing radiation in water. The rate of reaction of the OH radical with purified, native and isodisperse DNA has been determined as compared with calf thymus DNA. This has been achieved by direct observation of the rate of formation of the DNA-OH radical adduct, and by competition with SCN - . Results obtained from direct observation are consistent with calculations which have been performed using the encounter frequency model of Braams and Ebert. However, results obtained for OH radical with DNA derived from competition plots suggest a rate constant somewhat lower than that obtained from direct observation. The relative merits of both techniques are discussed. In order to study the effect of energy deposited directly in the DNA, dry films of purified plasmid DNA have been irradiated in a system where the indirect effects of radical interaction have been minimized. The present results indicate that with different molecular lengths of plasmid DNA, non-random breakage may occur, and that additional damage may be brought about at sites of previously existing damage. Differences in the sensitivity of plasmid DNA molecules of varying lengths to radiation induced double strand breaks have been demonstrated. (author)

  19. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  20. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA

    Science.gov (United States)

    1989-01-01

    A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized and stored within membrane-bound vesicles in the cytoplasm of transfected fibroblasts. BBTY-1 detected a 2.4-kb mRNA transcript in nine of nine pigmented, tyrosinase-positive melanoma cell lines. Tyrosinase transcripts of the same size and abundance were detected in a subset (three of eight) of nonpigmented, tyrosinase-negative melanoma cell lines, suggesting that post-transcriptional events are important in regulating tyrosinase activity. Two melanocyte antigens, recognized by mAbs TA99 and CF21, that are specifically located within melanosomes and are coexpressed with tyrosinase activity, did not react with transfected mouse fibroblasts expressing human tyrosinase, supporting the conclusion that these antigenic determinants are distinct from the tyrosinase molecule coded for by BBTY-1. PMID:2499655

  1. Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics

    Directory of Open Access Journals (Sweden)

    Domenica Musumeci

    2017-12-01

    Full Text Available Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement (“signal-on” or a quenching (“signal-off” effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment.

  2. Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics.

    Science.gov (United States)

    Musumeci, Domenica; Platella, Chiara; Riccardi, Claudia; Moccia, Federica; Montesarchio, Daniela

    2017-12-20

    Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement ("signal-on") or a quenching ("signal-off") effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment.

  3. Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays.

    Science.gov (United States)

    Cho, J C; Tiedje, J M

    2001-08-01

    Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.

  4. DNA nanotechnology

    OpenAIRE

    Nadrian C Seeman

    2003-01-01

    Since Watson and Crick’s determination of its structure nearly 50 years ago, DNA has come to fill our lives in many areas, from genetic counseling to forensics, from genomics to gene therapy. These, and other ways in which DNA affects human activities, are related to its function as genetic material, not just our genetic material, but the genetic material of all living organisms. Here, we will ignore DNA’s biological role; rather, we will discuss how the properties that make it so successful ...

  5. CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge.

    Science.gov (United States)

    Kwa, Suefen; Lai, Lilin; Gangadhara, Sailaja; Siddiqui, Mariam; Pillai, Vinod B; Labranche, Celia; Yu, Tianwei; Moss, Bernard; Montefiori, David C; Robinson, Harriet L; Kozlowski, Pamela A; Amara, Rama Rao

    2014-09-01

    CD40L in a membrane-bound form, along with SIV antigens, in a nucleic acid (DNA) vector. We tested the immunogenicity and efficacy of the CD40L-adjuvanted vaccine in macaques using a heterologous mucosal SIV infection. The CD40L-adjuvanted vaccine enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV T cell responses and improved protection. These results demonstrate that VLP-membrane-bound CD40L serves as a novel adjuvant for an HIV vaccine. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Genetic differentiation and inheritance of random amplified ...

    African Journals Online (AJOL)

    ufuoma

    2013-09-11

    populations; the pectoral spine phenotypic groups ... Key words: Random amplified polymorphic DNA (RAPD) marker, morphologic and genetic variability, Clarias gariepinus. ..... However, genetic variability of the stock will have to be.

  7. DNA Sequences of RAPD Fragments in the Egyptian cotton ...

    African Journals Online (AJOL)

    Random Amplified Polymorphic DNAs (RAPDs) is a DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. Despite the fact that the RAPD technique has become a very powerful tool and has found use in numerous applications, yet, the nature of ...

  8. Random magnetism

    International Nuclear Information System (INIS)

    Tahir-Kheli, R.A.

    1975-01-01

    A few simple problems relating to random magnetic systems are presented. Translational symmetry, only on the macroscopic scale, is assumed for these systems. A random set of parameters, on the microscopic scale, for the various regions of these systems is also assumed. A probability distribution for randomness is obeyed. Knowledge of the form of these probability distributions, is assumed in all cases [pt

  9. Molecular mechanisms of DNA photodamage

    Energy Technology Data Exchange (ETDEWEB)

    Starrs, S.M

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA){sub n} and (GA){sub n}, and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a

  10. Molecular mechanisms of DNA photodamage

    International Nuclear Information System (INIS)

    Starrs, S.M.

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA) n and (GA) n , and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a dimeric

  11. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  12. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length ...

  13. Use of DNA markers in forest tree improvement research

    Science.gov (United States)

    D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

    1992-01-01

    DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

  14. DNA Investigations.

    Science.gov (United States)

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  15. Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed

    International Nuclear Information System (INIS)

    Raap, J.; Hollander, J.; Ovchinnikova, T. V.; Swischeva, N. V.; Skladnev, D.; Kiihne, S.

    2006-01-01

    Interactions between 15 N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortunately, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in 13 C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C α signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the 31 P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The 13 C-peptide peaks were selectively detected in a 13 C-detected 1 H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The 13 C-MAOSS results thus independently confirms previous findings from 15 N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution 13 C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates

  16. Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

    International Nuclear Information System (INIS)

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-01-01

    The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

  17. Detection of membrane-bound and soluble antigens by magnetic levitation

    DEFF Research Database (Denmark)

    Andersen, Mikkel Schou; Howard, Emily; Lu, Shulin

    2017-01-01

    blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its...

  18. Effect of zinc and calcium ions on the rat kidney membrane-bound ...

    Indian Academy of Sciences (India)

    2013-07-11

    Jul 11, 2013 ... 2009, Yilmaz et al. 2009). Strategies for inhibition of DPP-IV activity have been developed for the treatment of various diseases, in particular type 2 diabetes mellitus and cancer (Matteucci and. Giampietro 2009; Peters 2010; Stulc and Sedo 2010). In several of these pathologies, a deficiency of cations (e.g..

  19. An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

    Science.gov (United States)

    Pundir, C S; Narang, Jagriti; Chauhan, Nidhi; Sharma, Preety; Sharma, Renu

    2012-10-01

    The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. K m and I max for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4 ± 2.8 and 92.3 ± 3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential.

  20. Ficolins and FIBCD1: Soluble and membrane bound pattern recognition molecules with acetyl group selectivity

    DEFF Research Database (Denmark)

    Thomsen, Theresa; Schlosser, Anders; Holmskov, Uffe

    2011-01-01

    as pattern recognition molecules. Ficolins are soluble oligomeric proteins composed of trimeric collagen-like regions linked to fibrinogen-related domains (FReDs) that have the ability to sense molecular patterns on both pathogens and apoptotic cell surfaces and activate the complement system. The ficolins......D-containing molecules, and discusses structural resemblance but also diversity in recognition of acetylated ligands....

  1. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  2. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    Science.gov (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  3. Revealing the membrane-bound structure of neurokinin A using neutron diffraction

    Science.gov (United States)

    Darkes, Malcolm J. M.; Hauss, Thomas; Dante, Silvia; Bradshaw, Jeremy P.

    2000-03-01

    Neurokinin A (or substance K) belongs to the tachykinin family, a group of small amphipathic peptides that bind to specific membrane-embedded, G-protein coupled receptors. The agonist/receptor complex is quaternary in nature because the receptor binding sites are thought to be located within the lipid bilayer and because the role of water cannot be ignored. The cell membrane acts as a solvent to accumulate peptide and an inducer of peptide secondary structure. The three-dimensional shape that the peptide assumes when associated to the cell membrane will be an important parameter with regards to the receptor selectivity and affinity. Neutron diffraction measurements were carried out in order to define the location of the N-terminus of the peptide in synthetic phospholipid multi-bilayer stacks.

  4. Novel Fabrication of CA Membrane Bound Carbon Electrode for Bi-enzymatic Determination of Lactate

    Directory of Open Access Journals (Sweden)

    VIKAS

    2006-12-01

    Full Text Available Lactate oxidase from Pediococcus species has been immobilized onto cellulose acetate (CA membrane to form an enzymatic membrane. HRP has been incorporated into carbon paste electrode. Enzymatic membrane was mounted over the HRP-carbon paste electrode with the help of dialyses membrane, which acts as working electrode. Lactate biosensor was constructed by connecting this fabricated working electrode to Ag/AgCl reference electrode along with platinum wire auxiliary electrode through potentiostate. The biosensor showed an excellent performance with a linear response range between 5µM to 1mML-1 of lactate with a correlation coefficient (r of .94 for n=30, when compared to standard colorimetric methods. The optimum pH of the biosensor is 6.5 and incubation temperature is 25°C. This bi-enzyme electrode can be used for 150 determinants; over 45 days with out any considerable lose of activity, when stored at 4°C in 0.5M sodium phosphate buffer (pH 6.5. The response time was 1 second and no major metabolic interference was observed.

  5. Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts

    Directory of Open Access Journals (Sweden)

    P. Narducci

    2010-03-01

    Full Text Available Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor κB (RANKL and its decoy receptor osteoprotegerin (OPG. Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture.

  6. Raman Spectroscopy of Conformational Changes in Membrane-Bound Sodium Potassium ATPase

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus; Abdali, Salim; Lundbæk, Jens August

    2007-01-01

    In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins — in this case, the sodium potassium adenosine triphosphatase (Na+,K+-ATPase). Spectral analysis of protein-lipid complexes is complicated by the presence...

  7. SYNTHESIS, CHARACTERIZATION AND APPLICATION OF A POLYURETHANE-BASED SUPPORT FOR IMMOBILIZING MEMBRANE-BOUND LIPASE

    Directory of Open Access Journals (Sweden)

    M. S. Soares

    Full Text Available Abstract This study conducted an assessment of polyurethane foams that were synthesized by one-shot process and used as a low-cost support to immobilize Mucor circinelloides URM 4182 whole-cells presenting high lipolytic activity. Polyols with different molecular weights (1100 to 6000 g mol-1 were applied to synthesize the polymer matrix, and the agitation speed effect was used for controlling the average pore size of the investigated polyurethane foams. The physical and mechanical properties of the polymers were evaluated by standard test methods, and their morphology was identified by Scanning Electron Microscopy. The immobilization procedure efficiency was assessed by quantifying the capability of the matrices to attach the cells and the catalytic activity of the biocatalysts in both aqueous (olive oil hydrolysis and non-aqueous media (ethanolysis of babassu oil under single and consecutive batch runs. Although all synthesized matrices were suitable to immobilize the whole cells with high catalytic performance, a better set of parameters was attained when the polyol ether with molecular weight of 6000 g mol-1 and 1100 g mol-1 was used. Both matrices yielded immobilized biocatalysts with high hydrolysis and transesterification activities, and exhibited a satisfactory operational stability with 96% and 81% retention of their initial hydrolytic and transesterification activities after three consecutive batch runs.

  8. An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario; Cavaleiro, Mafalda; Christensen, Ulla

    2017-01-01

    tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed...

  9. Model of fission yeast cell shape driven by membrane-bound growth factors and the cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Tyler Drake

    Full Text Available Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular level in the future.

  10. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    USER

    in the level of lipid peroxidation in liver was also observed. The results of the present investigation have .... membrane phospholipids not only altered the lipid milieu and structural as well as functional integrity of cell .... uncoupling of the human erythrocyte Ca2+ pump. Eur. J. Biochem.,. 221: 1103-1110. FAO (1999).

  11. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    International Nuclear Information System (INIS)

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-01-01

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca 2+ -mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1

  12. Optical waveguide lightmode spectroscopic techniques for investigating membrane-bound ion channel activities.

    Directory of Open Access Journals (Sweden)

    Inna Székács

    Full Text Available Optical waveguide lightmode spectroscopic (OWLS techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+ and organic cations through gramicidin channels and detecting the Cl(--channel functions of the (α5β2γ2 GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.

  13. Effect of zinc and calcium ions on the rat kidney membrane-bound ...

    Indian Academy of Sciences (India)

    2013-07-11

    Jul 11, 2013 ... which a mixed inhibition mechanism was observed (Ki values of the competitive component: 0.15±0.01 mM and. 50.0±1.05 mM ... porcine DPP-IV with a Ki value near circulating concentra- tions in mammals while the effect ..... peptidase-IV: Is there a risk of oncological and immunological adverse effects?

  14. The membrane-bound form of gene 9 minor coat protein of bacteriophage M13

    NARCIS (Netherlands)

    Houbiers, M.C.

    2002-01-01

    Bacteriophage M13 is a virus that infects the bacteria Escherichia coli ( E. coli ), a single cell organism that resides in our intestines. It consists of the cytoplasm (contents) and a double membrane that keeps the

  15. Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.; Wagner, G.J.; Siegelman, H.W.; Hind, G.

    1977-01-01

    Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner, G. J. and Siegelman, H. W. (1975) Science 190, 1298 to 1299). The ATPase activity of fresh vacuole suspensions was found to be 2 to 3 times that of protoplasts from the same tissue. 70 to 80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 ..mu..g/10/sup 6/ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbondiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K/sup +/, Na/sup +/, Mg/sup 2 +/, Cl/sup -/, and Ca/sup 2 +/ respectively, which are about the same as those in protoplasts.

  16. Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

    NARCIS (Netherlands)

    Timmers, J.F.P.; Vernhettes, S.; Desprez, T.; Vincken, J.P.; Visser, R.G.F.; Trindade, L.M.

    2009-01-01

    It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the

  17. Effect of zinc and calcium ions on the rat kidney membrane-bound ...

    Indian Academy of Sciences (India)

    ... Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, Madrid E-28029, Spain; Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, ...

  18. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C → U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  19. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  20. DNA Chip

    Indian Academy of Sciences (India)

    Imagine a world without identity cards; no I-cards for the college or office or bank account or anything! All you are carrying is a small (say, 2cm x 2cm) 'DNA-chip', which has the whole of your genetic profile on it. Your identity cannot get more authentic than that. Imagine a world where marriages are not decided by matching ...

  1. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  2. Stool DNA Test

    Science.gov (United States)

    ... The stool DNA test is a noninvasive laboratory test that identifies DNA changes in the cells of a stool sample. ... the presence of cancer. If a stool DNA test detects abnormal DNA, additional testing may be used to investigate the ...

  3. Single and repeated moderate consumption of native or dealcoholized red wine show different effects on antioxidant parameters in blood and DNA strand breaks in peripheral leukocytes in healthy volunteers: a randomized controlled trial [ISRCTN68505294

    Directory of Open Access Journals (Sweden)

    Spengler Ulrich

    2005-11-01

    Full Text Available Abstract Background Red wine (RW is rich in antioxidant polyphenols that might protect from oxidative stress related diseases, such as cardiovascular disease and cancer. Antioxidant effects after single ingestion of RW or dealcoholized RW (DRW have been observed in several studies, but results after regular consumption are contradictory. Thus, we examined if single or repeated consumption of moderate amounts of RW or DRW exert antioxidant activity in vivo. Methods Total phenolic content and concentration of other antioxidants in plasma/serum, total antioxidant capacity (TEAC in plasma as well as DNA strand breaks in peripheral leukocytes were measured in healthy non-smokers A before, 90 and 360 min after ingestion of one glass of RW, DRW or water; B before and after consumption of one glass of RW or DRW daily for 6 weeks. DNA strand breaks (SB were determined by single cell gel electrophoresis (Comet Assay in untreated cells and after induction of oxidative stress ex vivo with H2O2 (300 μM, 20 min. Results Both RW and DRW transiently increased total phenolic content in plasma after single consumption, but only RW lead to a sustained increase if consumed regularly. Plasma antioxidant capacity was not affected by single or regular consumption of RW or DRW. Effects of RW and DRW on DNA SB were conflicting. DNA strand breaks in untreated cells increased after a single dose of RW and DRW, whereas H2O2 induced SB were reduced after DRW. In contrast, regular RW consumption reduced SB in untreated cells but did not affect H2O2 induced SB. Conclusion The results suggest that consumption of both RW and DRW leads to an accumulation of phenolic compounds in plasma without increasing plasma antioxidant capacity. Red wine and DRW seem to affect the occurrence of DNA strand breaks, but this cannot be referred to antioxidant effects.

  4. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  5. Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNA

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Nes, Ingolf F.; Wells, Robert D.

    1976-01-01

    Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100–200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced...

  6. Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs Variação genética entre moluscos susceptíveis e não susceptíveis à infecção pelo Schistosoma através da análise do DNA polimórfico amplificado aleatóriamente (RAPDs

    Directory of Open Access Journals (Sweden)

    Abdel-Hamid Zaki ABDEL-HAMID

    1999-09-01

    Full Text Available Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers were used to amplify the extracted DNA by the polymerase chain reaction (PCR followed by polyacrylamide gel electrophoresis (PAGE and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.A susceptibilidade de moluscos à infecção por certos trematódeos e a sua capacidade como hospedeiro para o contínuo desenvolvimento é o problema mais deslumbrante nas relações parasita hospedeiro. O presente trabalho, focaliza nosso interesse na genética dos moluscos para determinar quais genes ou produtos gênicos são especificamente responsáveis pela susceptibilidade do molusco à infecção. DNA de alto peso molecular, foi extraído de ambos moluscos susceptíveis e não susceptíveis da espécie Biomphalaria tenagophila. Iniciadores aleatórios com 10 pares de bases foram usados na amplificação aleatória (RAPD de ambos os DNAs e análise por eletroforese em gel de poliacrilamida e coloração com prata. Os resultados mostram que a amplificação aleatória do DNA representa um eficiente caminho para a comparação dos genomas desde que marcadores moleculares foram detectados como variantes genéticos entre os moluscos susceptíveis e não susceptíveis.

  7. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults.

    Directory of Open Access Journals (Sweden)

    Juliet Mpendo

    Full Text Available Strategies to enhance the immunogenicity of DNA vaccines in humans include i co-administration of molecular adjuvants, ii intramuscular administration followed by in vivo electroporation (IM/EP and/or iii boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG plasmid DNA (pDNA vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12 (GENEVAX IL-12 given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs were reported. T cell and antibody response rates after HIVMAG (x3 prime-Ad35 (x1 boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ ELISPOT responses was highest after HIVMAG (x3 without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3 prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.ClinicalTrials.gov NCT01496989.

  8. HPV DNA test

    Science.gov (United States)

    ... HPV testing in women; Cervical cancer - HPV DNA test; Cancer of cervix - HPV DNA test ... The HPV DNA test may be done during a Pap smear . You lie on a table and place your feet in stirrups. The ...

  9. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  10. Random magnetism

    International Nuclear Information System (INIS)

    Tsallis, C.

    1981-01-01

    The 'ingredients' which control a phase transition in well defined systems as well as in random ones (e.q. random magnetic systems) are listed and discussed within a somehow unifying perspective. Among these 'ingredients' the couplings and elements responsible for the cooperative phenomenon, the topological connectivity as well as possible topological incompatibilities, the influence of new degrees of freedom, the order parameter dimensionality, the ground state degeneracy and finally the 'quanticity' of the system are found. The general trends, though illustrated in magnetic systems, essentially hold for all phase transitions, and give a basis for connection of this area with Field theory, Theory of dynamical systems, etc. (Author) [pt

  11. RAPD analysis of alfalfa DNA mutation via N+ implantation

    International Nuclear Information System (INIS)

    Li Yufeng; Huang Qunce; Yu Zengliang; Liang Yunzhang

    2003-01-01

    Germination capacity of alfalfa seeds under low energy N + implantation manifests oscillations going down with dose strength. From analyzing alfalfa genome DNA under low energy N + implantation by RAPD (Random Amplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primers in total 100 primers, and fluorescence intensity of the identical DNA fragment amplified by RAPD is different between CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N + implantation manifests going up with dose strength

  12. Fractional Brownian Motion:. Theory and Application to DNA Walk

    Science.gov (United States)

    Lim, S. C.; Muniandy, S. V.

    2001-09-01

    This paper briefly reviews the theory of fractional Brownian motion (FBM) and its generalization to multifractional Brownian motion (MBM). FBM and MBM are applied to a biological system namely the DNA sequence. By considering a DNA sequence as a fractal random walk, it is possible to model the noncoding sequence of human retinoblastoma DNA as a discrete version of FBM. The average scaling exponent or Hurst exponent of the DNA walk is estimated to be H = 0.60 ± 0.05 using the monofractal R/S analysis. This implies that the mean square fluctuation of DNA walk belongs to anomalous superdiffusion type. We also show that the DNA landscape is not monofractal, instead one has multifractal DNA landscape. The empirical estimates of the Hurst exponent falls approximately within the range H ~ 0.62 - 0.72. We propose two multifractal models, namely the MBM and multiscale FBM to describe the existence of different Hurst exponents in DNA walk.

  13. Random dynamics

    International Nuclear Information System (INIS)

    Bennett, D.L.; Brene, N.; Nielsen, H.B.

    1986-06-01

    The goal of random dynamics is the derivation of the laws of Nature as we know them (standard model) from inessential assumptions. The inessential assumptions made here are expressed as sets of general models at extremely high energies: gauge glass and spacetime foam. Both sets of models lead tentatively to the standard model. (orig.)

  14. DNA profiling of commercial chilli pepper ( Capsicum annuum L ...

    African Journals Online (AJOL)

    . Gemini, G-334, Agnirekha, Pusa jwala, Mangala, Black diamond, Sindura, Pusa jyothi, Badiga-2 and Teja (branch), cultivated in the local area of Andhra Pradesh State, India, using random amplified polymorphic DNA (RAPD) markers was ...

  15. Enhancement of DNA-transfection frequency by X-rays

    International Nuclear Information System (INIS)

    Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi

    1997-01-01

    This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

  16. Strategies for optimizing DNA hybridization on surfaces.

    Science.gov (United States)

    Ravan, Hadi; Kashanian, Soheila; Sanadgol, Nima; Badoei-Dalfard, Arastoo; Karami, Zahra

    2014-01-01

    Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Markov chain for estimating human mitochondrial DNA mutation pattern

    Science.gov (United States)

    Vantika, Sandy; Pasaribu, Udjianna S.

    2015-12-01

    The Markov chain was proposed to estimate the human mitochondrial DNA mutation pattern. One DNA sequence was taken randomly from 100 sequences in Genbank. The nucleotide transition matrix and mutation transition matrix were estimated from this sequence. We determined whether the states (mutation/normal) are recurrent or transient. The results showed that both of them are recurrent.

  18. Investigation of DNA changes in wheat ( Triticum aestivum L ...

    African Journals Online (AJOL)

    The purpose of study was to evaluate the effect of different concentrations of cadmium on amounts of the soluble proteins, investigation of growth and possible changes to the structure of DNA using random amplified polymorphic DNA (RAPD) marker. In the present study, wheat seedlings were used for detection of ...

  19. Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

    Science.gov (United States)

    Takahashi, Hirokazu; Yamazaki, Hiroyuki; Akanuma, Satoshi; Kanahara, Hiroko; Saito, Toshiyuki; Chimuro, Tomoyuki; Kobayashi, Takayoshi; Ohtani, Toshio; Yamamoto, Kimiko; Sugiyama, Shigeru; Kobori, Toshiro

    2014-01-01

    We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents. PMID:24505243

  20. Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

    Directory of Open Access Journals (Sweden)

    Hirokazu Takahashi

    Full Text Available We previously reported that multiply-primed rolling circle amplification (MRPCA using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

  1. Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi Otimização da reação de amplificação aleatória do DNA polimórfico - reação em cadeia da polimerase para tipagem molecular de Salmonella enterica sorovar Typhi

    Directory of Open Access Journals (Sweden)

    Bianca Ramalho Quintaes

    2004-03-01

    Full Text Available Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.A otimização da reação de RAPD para a caracterização de cepas de Salmonella enterica sorovar Typhi foi estudada com o objetivo de assegurar a reprodutibilidade e o poder discriminatório desta técnica. Oito cepas de Salmonella sorovar Typhi isoladas de algumas regiões do Brasil foram usadas para examinar os padrões de fragmentação produzidos quando foram empregadas concentrações diferentes do DNA molde, do iniciador, do MgCl2 e da enzima Taq DNA polimerase. Com a utilização de dois diferentes perfis de ciclos termais de baixa estringência, foram comparados os padrões de bandeamento obtidos. Um conjunto de dezesseis iniciadores foi avaliado quanto à capacidade de produzir elevado número de fragmentos distintos. Observou-se que variações associadas a todos os parâmetros testados modificaram os padrões de bandeamento. Para as amostras de Salmonella enterica sorovar Typhi utilizadas neste experimento, definiu-se um conjunto de condições para a reação de RAPD-PCR que resultou num método de tipagem simples, rápido e

  2. Impact of Consuming Extra-Virgin Olive Oil or Nuts within a Mediterranean Diet on DNA Methylation in Peripheral White Blood Cells within the PREDIMED-Navarra Randomized Controlled Trial: A Role for Dietary Lipids.

    Science.gov (United States)

    Arpón, Ana; Milagro, Fermín I; Razquin, Cristina; Corella, Dolores; Estruch, Ramón; Fitó, Montserrat; Marti, Amelia; Martínez-González, Miguel A; Ros, Emilio; Salas-Salvadó, Jordi; Riezu-Boj, José-Ignacio; Martínez, J Alfredo

    2017-12-23

    DNA methylation could be reversible and mouldable by environmental factors, such as dietary exposures. The objective was to analyse whether an intervention with two Mediterranean diets, one rich in extra-virgin olive oil (MedDiet + EVOO) and the other one in nuts (MedDiet + nuts), was influencing the methylation status of peripheral white blood cells (PWBCs) genes. A subset of 36 representative individuals were selected within the PREvención con DIeta MEDiterránea (PREDIMED-Navarra) trial, with three intervention groups in high cardiovascular risk volunteers: MedDiet + EVOO, MedDiet + nuts, and a low-fat control group. Methylation was assessed at baseline and at five-year follow-up. Ingenuity pathway analysis showed routes with differentially methylated CpG sites (CpGs) related to intermediate metabolism, diabetes, inflammation, and signal transduction. Two CpGs were specifically selected: cg01081346- CPT1B / CHKB-CPT1B and cg17071192- GNAS/GNASAS , being associated with intermediate metabolism. Furthermore, cg01081346 was associated with PUFAs intake, showing a role for specific fatty acids on epigenetic modulation. Specific components of MedDiet, particularly nuts and EVOO, were able to induce methylation changes in several PWBCs genes. These changes may have potential benefits in health; especially those changes in genes related to intermediate metabolism, diabetes, inflammation and signal transduction, which may contribute to explain the role of MedDiet and fat quality on health outcomes.

  3. DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

    Science.gov (United States)

    Gupta, Sachin; Clark, Emily S; Termini, James M; Boucher, Justin; Kanagavelu, Saravana; LeBranche, Celia C; Abraham, Sakhi; Montefiori, David C; Khan, Wasif N; Stone, Geoffrey W

    2015-04-01

    Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of

  4. DNA Damage, DNA Repair, Aging, and Neurodegeneration

    Science.gov (United States)

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

    2015-01-01

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

  5. Random walk polynomials and random walk measures

    NARCIS (Netherlands)

    van Doorn, Erik A.; Schrijner, Pauline

    1993-01-01

    Random walk polynomials and random walk measures play a prominent role in the analysis of a class of Markov chains called random walks. Without any reference to random walks, however, a random walk polynomial sequence can be defined (and will be defined in this paper) as a polynomial sequence{Pn(x)}

  6. Random walks in a random environment

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Abstract. Random walks as well as diffusions in random media are considered. Methods are developed that allow one to establish large deviation results for both the 'quenched' and the 'averaged' case. Keywords. Large deviations; random walks in a random environment. 1. Introduction. A random walk on Zd is a stochastic ...

  7. DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

    Science.gov (United States)

    Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

    2018-01-01

    Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. DNA-Templated Introduction of an Aldehyde Handle in Proteins

    DEFF Research Database (Denmark)

    Kodal, Anne Louise Bank; Rosen, Christian Bech; Mortensen, Michael Rosholm

    2016-01-01

    Many medical and biotechnological applications rely on labeling of proteins, but one key challenge is the production of homogeneous and site-specific conjugates. This can rarely be achieved by mere residue-specific random labeling, but requires genetic engineering. Using site-selective DNA......-templated reductive amination we create DNA-protein conjugates with control over labeling stoichiometry without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing coupling of a second reactive DNA strand to the vicinity of a protein metal......-binding site. Here, we demonstrate DNA-templated reductive amination for His6-tagged proteins and native metal-binding proteins, including IgG1 antibodies. We also use a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde to proteins. This functions as a handle...

  9. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  10. Random tensors

    CERN Document Server

    Gurau, Razvan

    2017-01-01

    Written by the creator of the modern theory of random tensors, this book is the first self-contained introductory text to this rapidly developing theory. Starting from notions familiar to the average researcher or PhD student in mathematical or theoretical physics, the book presents in detail the theory and its applications to physics. The recent detections of the Higgs boson at the LHC and gravitational waves at LIGO mark new milestones in Physics confirming long standing predictions of Quantum Field Theory and General Relativity. These two experimental results only reinforce today the need to find an underlying common framework of the two: the elusive theory of Quantum Gravity. Over the past thirty years, several alternatives have been proposed as theories of Quantum Gravity, chief among them String Theory. While these theories are yet to be tested experimentally, key lessons have already been learned. Whatever the theory of Quantum Gravity may be, it must incorporate random geometry in one form or another....

  11. Random functions and turbulence

    CERN Document Server

    Panchev, S

    1971-01-01

    International Series of Monographs in Natural Philosophy, Volume 32: Random Functions and Turbulence focuses on the use of random functions as mathematical methods. The manuscript first offers information on the elements of the theory of random functions. Topics include determination of statistical moments by characteristic functions; functional transformations of random variables; multidimensional random variables with spherical symmetry; and random variables and distribution functions. The book then discusses random processes and random fields, including stationarity and ergodicity of random

  12. Mitochondrial DNA exhibits resistance to induced point and deletion mutations

    Science.gov (United States)

    Valente, William J.; Ericson, Nolan G.; Long, Alexandra S.; White, Paul A.; Marchetti, Francesco; Bielas, Jason H.

    2016-01-01

    The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaTMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure. PMID:27550180

  13. DNA fragmentation by charged particle tracks

    Science.gov (United States)

    Stenerlöw, B.; Höglund, E.; Carlsson, J.

    High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons ( 60Co) or 125 keV/μm nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.

  14. A DNA enzyme that cleaves RNA

    Science.gov (United States)

    Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

    1994-01-01

    BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

  15. A simple method of DNA extraction from coffee seeds suitable for ...

    African Journals Online (AJOL)

    Quality of DNA was confirmed by digestion using EcoRI, HindIII and PstI restriction endonucleases and complete digestion was observed. PCR with random decamer primers and consensus primers of mitochondria and chloroplast DNA and PCR-RFLP revealed the suitability of the DNA for PCR based marker techniques ...

  16. Study on genetic variability of Cassidula aurisfelis (snail) by random ...

    African Journals Online (AJOL)

    The genetic variability among individuals of Cassidula aurisfelis from Setiu Wetland, Terengganu Darul Iman was examined by using the random amplified polymorphic DNA (RAPD) technique. Ten oligonucleotide primers were screened and three primers were selected (OPA 02, OPA 04 and OPA 10) to amplify DNA from ...

  17. Genetic relationships among Rosa species based on random ...

    African Journals Online (AJOL)

    To investigate the genetic diversity of Rosa accessions, random amplified polymorphism DNA (RAPD) approach was employed. Nine of ten primers amplified 138 scorable RAPD loci with 111 polymorphic bands (80%). Percentages of polymorphic bands ranged from 75 to 100%. Sizes of amplified DNA fragments ranged ...

  18. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  19. Generating equilateral random polygons in confinement

    International Nuclear Information System (INIS)

    Diao, Y; Ernst, C; Montemayor, A; Ziegler, U

    2011-01-01

    One challenging problem in biology is to understand the mechanism of DNA packing in a confined volume such as a cell. It is known that confined circular DNA is often knotted and hence the topology of the extracted (and relaxed) circular DNA can be used as a probe of the DNA packing mechanism. However, in order to properly estimate the topological properties of the confined circular DNA structures using mathematical models, it is necessary to generate large ensembles of simulated closed chains (i.e. polygons) of equal edge lengths that are confined in a volume such as a sphere of certain fixed radius. Finding efficient algorithms that properly sample the space of such confined equilateral random polygons is a difficult problem. In this paper, we propose a method that generates confined equilateral random polygons based on their probability distribution. This method requires the creation of a large database initially. However, once the database has been created, a confined equilateral random polygon of length n can be generated in linear time in terms of n. The errors introduced by the method can be controlled and reduced by the refinement of the database. Furthermore, our numerical simulations indicate that these errors are unbiased and tend to cancel each other in a long polygon. (paper)

  20. Subcloning of DNA fragments.

    Science.gov (United States)

    Struhl, K

    2001-05-01

    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

  1. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  2. DNA computing models

    CERN Document Server

    Ignatova, Zoya; Zimmermann, Karl-Heinz

    2008-01-01

    In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

  3. DNA Repair Systems

    Indian Academy of Sciences (India)

    exogenous damage). Endogenous damage ... of spontaneous DNA-damage due to endogenous factors. He es- timated that around 10,000 potentially mutagenic .... 3 –5 direction is defined as. 'upstream'. A single DNA strand is synthesized in a.

  4. Animal Mitochondrial DNA Replication

    Science.gov (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

    2016-01-01

    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  5. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  6. Pig transgenesis by Sleeping Beauty DNA transposition

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E.; Li, Juan; Kragh, Peter M.

    2011-01-01

    disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA...... transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted...

  7. Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

    Science.gov (United States)

    Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

    2018-01-10

    Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

  8. Random pulse generator

    International Nuclear Information System (INIS)

    Guo Ya'nan; Jin Dapeng; Zhao Dixin; Liu Zhen'an; Qiao Qiao; Chinese Academy of Sciences, Beijing

    2007-01-01

    Due to the randomness of radioactive decay and nuclear reaction, the signals from detectors are random in time. But normal pulse generator generates periodical pulses. To measure the performances of nuclear electronic devices under random inputs, a random generator is necessary. Types of random pulse generator are reviewed, 2 digital random pulse generators are introduced. (authors)

  9. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...... for a long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two...

  10. Fast phylogenetic DNA barcoding

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Willerslev, Eske

    2008-01-01

    We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect...... DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria...... for determining the taxonomic level at which a particular DNA sequence can be assigned....

  11. DNA Sequencing apparatus

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  12. Random fixed points and random differential inclusions

    Directory of Open Access Journals (Sweden)

    Nikolaos S. Papageorgiou

    1988-01-01

    Full Text Available In this paper, first, we study random best approximations to random sets, using fixed point techniques, obtaining this way stochastic analogues of earlier deterministic results by Browder-Petryshyn, KyFan and Reich. Then we prove two fixed point theorems for random multifunctions with stochastic domain that satisfy certain tangential conditions. Finally we consider a random differential inclusion with upper semicontinuous orientor field and establish the existence of random solutions.

  13. The time course of repair of ultraviolet-induced DNA damage; implications for the structural organization of repair

    International Nuclear Information System (INIS)

    Collins, A.; Squires, S.

    1986-01-01

    Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the 'random collision' model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a 'processive' mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate until scanning is complete. The authors have examined the time course of DNA repair in human fibroblasts given low doses of UV light. Using 3 distinct assays, the authors find no sign of a constant repair rate after 4 J/m 2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. (Auth.)

  14. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  15. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  16. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  17. Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD analysis Caracterização genotípica de Haematobia irritans procedentes de diferentes regiões geográficas brasileiras baseada na análise do DNA polimórfico amplificado ao acaso (RAPD-PCR

    Directory of Open Access Journals (Sweden)

    Luciana G. Brito

    2007-01-01

    Full Text Available Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.Dípteras hematófagos são importantes parasitas dentro de sistemas de produção de bovinos, especialmente em confinamento. Haematobia irritans, a mosca-dos-chifres, é uma das espécies que maiores problemas causa em sistemas de produção de bovinos, dado ao intenso estresse que impõe aos animais. Um importante aspecto quando se estuda a variabilidade genética dentro das espécies é o estudo da estrutura geográfica destas populações. Buscando-se estimar a similaridade genotípica das diferentes populações brasileiras da mosca do chifre utilizou-se a técnica do DNA polimórfico amplificado ao acaso (RAPD-PCR, que mostrou-se eficiente para tal propósito. A utilização dos marcadores moleculares gerados através da técnica de RAPD-PCR tornou possível a identificação da origem geográfica das amostras das diferentes regiões geográficas brasileiras, assim como, estimar o fluxo genotípico entre as diferentes populações brasileiras da mosca-dos-chifres.

  18. Topics in random walks in random environment

    International Nuclear Information System (INIS)

    Sznitman, A.-S.

    2004-01-01

    Over the last twenty-five years random motions in random media have been intensively investigated and some new general methods and paradigms have by now emerged. Random walks in random environment constitute one of the canonical models of the field. However in dimension bigger than one they are still poorly understood and many of the basic issues remain to this day unresolved. The present series of lectures attempt to give an account of the progresses which have been made over the last few years, especially in the study of multi-dimensional random walks in random environment with ballistic behavior. (author)

  19. Beyond DNA repair: DNA-PK function in cancer

    OpenAIRE

    Goodwin, Jonathan F.; Knudsen, Karen E.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

  20. The effect of exercise and metformin treatment on circulating free DNA in pregnancy.

    Science.gov (United States)

    Christiansen, S C; Vanky, E; Klungland, H; Stafne, S N; Mørkved, S; Salvesen, K Å; Sæther, M; Carlsen, S M

    2014-12-01

    Some pregnancy complications are characterized by increased levels of cell-free fetal (cffDNA) and maternal DNA (cfmDNA), the latter may also be elevated during physical strain. This study aims at assessing the impact of exercise and metformin intervention in pregnancy, and to compare the levels of cell free DNA in pregnant women with or without PCOS diagnosis. Consecutive women from two previous randomized controlled trials in pregnancy were included. Women came from a trial with organized exercise vs. standard antenatal care in pregnancy and a trial of metformin vs. placebo in PCOS women. Levels of cffDNA, cfmDNA and cell-free total DNA (cftDNA) were measured by qPCR. Training in pregnancy did not affect the levels of cffDNA, cfmDNA or cftDNA. PCOS-women treated with metformin had lower levels of cfmDNA and cftDNA at week 32 (mean ± SD: 301 ± 162 versus 570 ± 337, p = 0.012, 345 ± 173 versus 635 ± 370, p = 0.019); otherwise the levels were comparable to PCOS-controls. Metformin-treated PCOS-women had higher cffDNA at inclusion, in the 1st trimester; later on in pregnancy the levels in the metformin and placebo groups were equal. A comparison of pregnant women in the exercise study (TRIP) to placebo-treated pregnant PCOS-women, showed the levels of cffDNA, cfmDNA or cftDNA during mid-pregnancy (weeks 18-36) to be equal. Training during pregnancy was not associated with altered levels of cffDNA cfmDNA or cftDNA, but metformin treatment may reduce cfmDNA and cftDNA in pregnant PCOS women. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Development of DNA probes for Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  2. Development of DNA probes for Candida albicans

    International Nuclear Information System (INIS)

    Cheung, L.L.; Hudson, J.B.

    1988-01-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32 P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis

  3. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  4. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  5. DNA repair and cancer

    International Nuclear Information System (INIS)

    Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

    2012-01-01

    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

  6. DNA-based machines.

    Science.gov (United States)

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

  7. MITOCHONDRIAL DNA- REVOLUTIONARY EVOLUTION

    Directory of Open Access Journals (Sweden)

    Vaidhehi Narayan Nayak

    2017-07-01

    Full Text Available BACKGROUND Mitochondrion, the sausage-shaped organelle residing in the cytoplasm of all eukaryotic cells, apart from being the power house, represents endosymbiotic evolution of a free living organism to intracellular structure. Anthropologically, mitochondrial DNA is the fossilised source to trace the human ancestry particularly of maternal lineage. This article attempts to highlight the various biological functions of mitochondrial DNA (mtDNA with a note on its forensic application.

  8. Fragmentation in DNA double-strand breaks

    International Nuclear Information System (INIS)

    Wei Zhiyong; Suzhou Univ., Suzhou; Zhang Lihui; Li Ming; Fan Wo; Xu Yujie

    2005-01-01

    DNA double strand breaks are important lesions induced by irradiations. Random breakage model or quantification supported by this concept is suitable to analyze DNA double strand break data induced by low LET radiation, but deviation from random breakage model is more evident in high LET radiation data analysis. In this work we develop a new method, statistical fragmentation model, to analyze the fragmentation process of DNA double strand breaks. After charged particles enter the biological cell, they produce ionizations along their tracks, and transfer their energies to the cells and break the cellular DNA strands into fragments. The probable distribution of the fragments is obtained under the condition in which the entropy is maximum. Under the approximation E≅E 0 + E 1 l + E 2 l 2 , the distribution functions are obtained as exp(αl + βl 2 ). There are two components, the one proportional to exp(βl 2 ), mainly contributes to the low mass fragment yields, the other component, proportional to exp(αl), decreases slowly as the mass of the fragments increases. Numerical solution of the constraint equations provides parameters α and β. Experimental data, especially when the energy deposition is higher, support the statistical fragmentation model. (authors)

  9. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J

    1992-01-01

    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...

  10. DNA ELECTROPHORESIS AT SURFACES

    Energy Technology Data Exchange (ETDEWEB)

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  11. The Knot Spectrum of Confined Random Equilateral Polygons

    Directory of Open Access Journals (Sweden)

    Diao Y.

    2014-01-01

    Full Text Available It is well known that genomic materials (long DNA chains of living organisms are often packed compactly under extreme confining conditions using macromolecular self-assembly processes but the general DNA packing mechanism remains an unsolved problem. It has been proposed that the topology of the packed DNA may be used to study the DNA packing mechanism. For example, in the case of (mutant bacteriophage P4, DNA molecules packed inside the bacteriophage head are considered to be circular since the two sticky ends of the DNA are close to each other. The DNAs extracted from the capsid without separating the two ends can thus preserve the topology of the (circular DNAs. It turns out that the circular DNAs extracted from bacteriophage P4 are non-trivially knotted with very high probability and with a bias toward chiral knots. In order to study this problem using a systematic approach based on mathematical modeling, one needs to introduce a DNA packing model under extreme volume confinement condition and test whether such a model can produce the kind of knot spectrum observed in the experiments. In this paper we introduce and study a model of equilateral random polygons con_ned in a sphere. This model is not meant to generate polygons that model DNA packed in a virus head directly. Instead, the average topological characteristics of this model may serve as benchmark data for totally randomly packed circular DNAs. The difference between the biologically observed topological characteristics and our benchmark data might reveal the bias of DNA packed in the viral capsids and possibly lead to a better understanding of the DNA packing mechanism, at least for the bacteriophage DNA. The purpose of this paper is to provide information about the knot spectrum of equilateral random polygons under such a spherical confinement with length and confinement ratios in a range comparable to circular DNAs packed inside bacteriophage heads.

  12. Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles.

    Science.gov (United States)

    Wan, Juan; Basu, Kaustuv; Mui, Jeannie; Vali, Hojatollah; Zheng, Huanquan; Laliberté, Jean-François

    2015-12-01

    Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. The membrane-bound aspartyl protease BACE1:Molecular and functional properties in Alzheimer’s disease and beyond

    Directory of Open Access Journals (Sweden)

    Bastian eDislich

    2012-02-01

    Full Text Available The β-site APP cleaving enzyme 1 (BACE1 is a transmembrane aspartyl protease involved in Alzheimer’s disease (AD pathogenesis and in myelination. BACE1 initiates the generation of the pathogenic amyloid β-peptide, which makes BACE1 a major drug target for AD. BACE1 also cleaves and activates neuregulin 1, thereby contributing to postnatal myelination, in particular in the peripheral nervous system. Additional proteins are also cleaved by BACE1, but less is known about the physiological consequences of their cleavage. Recently, new phenotypes were described in BACE1-deficient mice. Although it remains unclear through which BACE1 substrates they are mediated, the phenotypes suggest a versatile role of this protease for diverse physiological processes. This review summarizes the enzymatic and cellular properties of BACE1 as well as its regulation by lipids, by transcriptional and by translational mechanisms. The main focus will be on the recent progress in understanding BACE1 function and its implication for potential mechanism-based side effects upon therapeutic inhibition.

  14. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

    Science.gov (United States)

    Elani, Yuval

    2016-06-15

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. © 2016 The Author(s).

  15. Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.; Provost, J.J.

    1997-01-01

    In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 µM GTP¿S (6-14-fold) and by the small G...... placenta, which is stimulated by PIP, but not by oleate. Here we show that oleic acid and a-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important...... was devoid of endogenous RhoA and Arf and could not be stimulated by GTP¿S. However, mArf3 (1 µM) still activated this partially purified membrane PLD, whereas RhoA (0.37 nM) was not able to activate this PLD fraction. In concludion, our results suggest that the human placenta contains a PLD that is located...

  16. IRON UPTAKE BY LEAF MESOPHYLL-CELLS - THE ROLE OF THE PLASMA MEMBRANE-BOUND FERRIC-CHELATE REDUCTASE

    NARCIS (Netherlands)

    BRUGGEMANN, W; MAASKANTEL, K; MOOG, PR

    The uptake of Fe-59 from FeCl3, ferric (Fe3+) citrate (FeCitr) and Fe3+-EDTA (FeEDTA) was studied in leaf mesophyll of Vigna unguiculata (L.) Walp. Uptake rates decreased in the order FeCl3>FeCitr much greater than FeEDTA, and uptake depended on an obligatory reduction step of Fe3+ to Fe2+, after

  17. Multi-membrane-bound structures of Apicomplexa: II. the ovoid mitochondrial cytoplasmic (OMC) complex of Toxoplasma gondii tachyzoites.

    Science.gov (United States)

    Köhler, Sabine

    2006-03-01

    Apicomplexa including the causative agents of toxoplasmosis and malaria reportedly possess one or few tubular-shaped mitochondria that permeate, more or less branched, throughout these unicellular parasites. Electron micrographs generated herein from serial-sectioned Toxoplasma gondii tachyzoites demonstrated, however, a greater diversity regarding both the shape of the cultured parasite's single mitochondrion and its sub-structural organization. Moreover, a unique subcellular construction was detected that basically comprised a pouch-shaped subdivision of the tachyzoite mitochondrion plus a fraction of parasitic cytoplasm enclosed therein. This composite assembling, termed ovoid mitochondrial cytoplasmic (OMC) complex, characteristically displayed a highly reduced matrix lumen of its mitochondrial border construction, which furthermore often failed to possess any cristae or contained tightly pleated cristae, thus creating a pouch-shaped multi-laminar wall of four or more membranous layers, respectively. Given this architecture, cross-sectioned OMC complexes of T. gondii tachyzoites frequently mimicked in size and shape the parasites' plastid-like organelle (apicoplast). Moreover, like the apicoplast, the OMC complex was often found adjacent to the tachyzoite's single Golgi complex and constantly located in close proximity to the outer membrane of the parasite's nuclear envelope. The T. gondii OMC complex differed, however, from the apicoplast in its exact fine structural organization and a stage-restricted presence that was apparently linked to mitochondrial growth and/or division. Any special function(s) possibly performed by the T. gondii OMC complex remains, nevertheless, to be elucidated.

  18. Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints

    NARCIS (Netherlands)

    Vos, W.L.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2005-01-01

    M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein

  19. Substrate specificity of membrane-bound alcohol oxidase from the tobacco hornworm moth (Manduca sexta) female pheromone glands

    Czech Academy of Sciences Publication Activity Database

    Luxová, Anna; Svatoš, Aleš

    2006-01-01

    Roč. 38, č. 1 (2006), s. 37-42 ISSN 1381-1177 Institutional research plan: CEZ:AV0Z40550506 Keywords : alcohol oxidase * insects * aldehydes Subject RIV: CC - Organic Chemistry Impact factor: 2.149, year: 2006

  20. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html, can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence.

  1. A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts.

    Science.gov (United States)

    Galperin, Michael Y

    2005-06-14

    Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html, can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence.

  2. Aminophospholipid glycation causes lipid bilayer structure alterations and inhibition of membrane-bound Na+, K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Obšil, Tomáš; Amler, Evžen; Pavlíček, Z.

    1998-01-01

    Roč. 63, - (1998), s. 1060-1073 ISSN 0010-0765 R&D Projects: GA ČR GA204/95/0624; GA AV ČR IAA5011503 Grant - others:GA UK(CZ) 182/97/B Subject RIV: ED - Physiology Impact factor: 0.546, year: 1998

  3. Cigarette smokers develop altered erythrocyte membrane composition: an investigation unmasking the role of membrane bound integral protein GLUT 1.

    Science.gov (United States)

    Sikdar, Jyotirmoy; Seal, Paromita; Roy, Amartya; Haldar, Rajen

    2017-04-01

    Erythrocytes in cigarette smokers are prone to oxidative damage. Here, we sought to elucidate the facts behind modifications and possible defense system developed in erythrocyte of cigarette smokers. We observed significant increase in stomatocytes and spherocytes, and osmotic fragility of erythrocyte, along with reduced level of protein thiol and increased fluorescence anisotropy in isolated membrane. Denaturing gel electrophoresis indicated alterations in band 3, band 4.2 and band 4.5. Among those, Glut 1 (i.e. band 4.5), which transports glucose (insulin independent) and dehydroascorbate (DHA), was selectively chosen for its long history in reducing reactive oxygen species (ROS). The increased Glut 1 level in smokers was confirmed by immunoblotting and immunocytochemistry. Furthermore, smokers showed significantly higher glucose uptake in whole blood. The intracellular (Ic) ROS (as indicated by 2',7'-dichlorofluorescin) was significantly higher in smokers as evidenced by flow cytometric assay. Glucose and DHA alone or together significantly reduced IcROS at higher rate in smokers. However, in presence of Glut 1 specific blocker, phloretin, neither glucose nor DHA could reduce IcROS in both non-smokers and smokers. This confirms that Glut 1 by transporting glucose or DHA attenuates IcROS. Therefore, we conclude that erythrocytes, although altered morphologically, also develop a defense system by upregulating Glut 1 to combat with enhanced Ic oxidative insult in cigarette smokers.

  4. Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes.

    Science.gov (United States)

    Hicks, D B; Yocum, C F

    1986-02-15

    Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.

  5. Identification of structural DNA variations in human cell cultures after long-term passage.

    Science.gov (United States)

    Pavlova, G V; Vergun, A A; Rybalkina, E Y; Butovskaya, P R; Ryskov, A P

    2015-01-01

    Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8-12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.

  6. Integration of random amplification of polymorphic DNA-polymerase ...

    African Journals Online (AJOL)

    Echinococcus granulosus a parasite characterized with intra-species variability and genetic studies show existence of 10 genotypes (G1-G10). Host specificity and different susceptibility to intermediate hosts has also been demonstrated. Better understanding of this parasitosis can assist in designing appropriate control and ...

  7. The reproducibility of random amplified polymorphic DNA (RAPD ...

    African Journals Online (AJOL)

    RAPD) profiles of Streptococcus thermophilus strains by using the polymerase chain reaction (PCR). Several factors can cause the amplification of false and non reproducible bands in the RAPD profiles. We tested three primers, OPI-02 MOD, ...

  8. (SDS-PAGE) and random amplified polymorphic DNA

    African Journals Online (AJOL)

    bgunni

    2011-11-09

    Nov 9, 2011 ... Four strains of eri, Samia cynthia ricini Lepidoptera: Saturniidae that can be identified morphologically and maintained at North East Institute of Science and Technology, Jorhat were characterized based on their protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and.

  9. (Wormwood capillary) by random amplified polymorphic DNA (RAPD)

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-04

    May 4, 2009 ... Faculty of Agrotechnology and Food Science, Universiti Malaysia Terengganu, 21030 Mengabang Telipot, Kuala. Terengganu, Malaysia. Accepted ... purposes, such as medicine, food, spices, and ornament- tation (Lee et al., 2006). ... gels were photographing with Image Master VDS. Screening of RAPD ...

  10. A random amplified polymorphic DNA (RAPD) molecular marker ...

    African Journals Online (AJOL)

    Jane

    2011-08-03

    Aug 3, 2011 ... to analyze F2 individuals of P-27 × P-28 to screen and characterize the molecular marker linked to late- bolting gene in pak-choi ... 40 days low temperature treatment at 5 ~ 10°C, during the experi- ments. The parental lines ... Frequency of bolting time distribution of F2 population. cultivated in the field.

  11. Analysis of phage Mu DNA transposition by whole-genome ...

    Indian Academy of Sciences (India)

    labelled ChIP DNA and Cy3-labelled whole-genome DNA, both amplified by random primers. The 'ratio' value of each probe (fluorescence intensity of Cy5 over Cy3) is the relative enrichment of that probe sequence in the ChIP sample, referred to as the relative MuB binding preference, or BBP. A scatter plot of log2 BBP ...

  12. Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

    International Nuclear Information System (INIS)

    Butterfield, D. Allan; Colvin, Joshua; Liu Jiangling; Wang Jianquan; Bachas, Leonidas; Bhattacharrya, Dibakar

    2002-01-01

    The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane >> polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic

  13. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

  14. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  15. DNA Repair Systems

    Indian Academy of Sciences (India)

    D N Rao is a professor at the. Department of Biochemistry,. Indian Institute of Science,. Bengaluru. His research work primarily focuses on. DNA interacting proteins in prokaryotes. This includes restriction-modification systems, DNA repair proteins from pathogenic bacteria and and proteins involved in horizontal gene ...

  16. Human Mitochondrial DNA Replication

    Science.gov (United States)

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  17. Dna fingerprinting - review paper

    OpenAIRE

    Blundell, Renald

    2006-01-01

    Before the Polymerase Chain Reaction (PCR) was established, DNA fingerprinting technology has relied for years on Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandom Repeats (VNTR) analysis, a very efficient technique but quite laborious and not suitable for high throughput mapping. Since its, development, PCR has provided a new and powerful tool for DNA fingerprinting.

  18. Characterization of muntjac DNA

    International Nuclear Information System (INIS)

    Davis, R.C.

    1981-01-01

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange

  19. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  20. DNA Microarray Technology

    Science.gov (United States)

    ... this page. En Español: Tecnología de micromatriz de ADN DNA Microarray Technology What is a DNA microarray? ... this page. En Español: Tecnología de micromatriz de ADN Get Email Updates Privacy Copyright Contact Accessibility Plug- ...

  1. Model of Biological Quantum Logic in DNA

    Directory of Open Access Journals (Sweden)

    F. Matthew Mihelic

    2013-08-01

    Full Text Available The DNA molecule has properties that allow it to act as a quantum logic processor. It has been demonstrated that there is coherent conduction of electrons longitudinally along the DNA molecule through pi stacking interactions of the aromatic nucleotide bases, and it has also been demonstrated that electrons moving longitudinally along the DNA molecule are subject to a very efficient electron spin filtering effect as the helicity of the DNA molecule interacts with the spin of the electron. This means that, in DNA, electrons are coherently conducted along a very efficient spin filter. Coherent electron spin is held in a logically and thermodynamically reversible chiral symmetry between the C2-endo and C3-endo enantiomers of the deoxyribose moiety in each nucleotide, which enables each nucleotide to function as a quantum gate. The symmetry break that provides for quantum decision in the system is determined by the spin direction of an electron that has an orbital angular momentum that is sufficient to overcome the energy barrier of the double well potential separating the C2-endo and C3-endo enantiomers, and that enantiomeric energy barrier is appropriate to the Landauer limit of the energy necessary to randomize one bit of information.

  2. Development of SCAR Markers for the DNA-Based Detection of the Asian Long-Horned Beetle; Anoplophora glabripennis (Motschulsky)

    Science.gov (United States)

    Damodar R. Kethidi; David B. Roden; Tim R. Ladd; Peter J. Krell; Arthur Ratnakaran; Qili Feng

    2003-01-01

    DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence charaterized amplified regions (SCARS) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after...

  3. Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: oxidative stress and DNA damage.

    Science.gov (United States)

    Kumar, Manoj; Trivedi, Nitin; Reddy, C R K; Jha, Bhavanath

    2011-11-21

    The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3-methylimidazolium bromide [C(n)mim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C(12)mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O(2)(•-), H(2)O(2), and OH(•)), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC(50) concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (~80 kDa and ~55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C(12)mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC(50) concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (~85 kDa), APX (~125 and 45 kDa), and GR (~135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed

  4. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  5. Whose DNA is this?

    DEFF Research Database (Denmark)

    Taroni, Franco; Biedermann, Alex; Vuille, Joëlle

    2013-01-01

    This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist's report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during...... evoked during the international conference "The hidden side of DNA profiles. Artifacts, errors and uncertain evidence" held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world's leading forensic DNA specialists, it appeared clearly...... talk considerably different languages. It thus is fundamental to address this issue of communication about results of forensic DNA analyses, and open a dialogue with practicing non-scientists at large who need to make meaningful use of scientific results to approach and help solve judicial cases...

  6. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. DNA complexes with Ni nanoparticles: structural and functional properties

    International Nuclear Information System (INIS)

    Tatarinova, Olga N.; Smirnov, Igor P.; Safenkova, Irina V.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2012-01-01

    Supramolecular complexes of biopolymers based on magnetic nanoparticles play an important role in creation of biosensors, implementation of theragnostic and gene therapeutic methods and biosafety evaluation. We investigated the impact of DNA interactions with nanoparticles of nickel (nNi) on the integrity and functionality of DNA. Data obtained by mass spectrometry, electrophoresis, TEM and AFM microscopy techniques, bacterial transformation, and real-time PCR provide evidence that ssDNA and plasmid DNA (pDNA) efficiently form complexes with nNi. AFM data suggest that the complexes are necklace-type structures, in which nanoparticles are randomly distributed along the DNA chains, rather than highly entangled clot-type structures. After desorption, observed DNA characteristics in bioanalytical and biological systems remain unchanged. Only supercoiled pDNA was nicked, but remained, as well as a plasmid–nNi complex, active in expression vector assays. These results are very important for creation of new methods of DNA immobilization and controlled manipulation.

  8. Directed evolution of DNA polymerases: construction and screening of DNA polymerase mutant libraries.

    Science.gov (United States)

    Gloeckner, Christian; Kranaster, Ramon; Marx, Andreas

    2010-06-01

    The protocols in this article describe the construction of a mutant DNA polymerase library using error-prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N-terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identification of variants with (1) increased DNA lesion-bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double-stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384-well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended. Curr. Protoc. Chem Biol. 2:89-109. © 2010 by John Wiley & Sons, Inc.

  9. Probing protein: DNA interactions using a uniform monolayer of DNA and surface plasmon resonance

    Science.gov (United States)

    Shumaker-Parry, Jennifer S.; Campbell, Charles T.; Stormo, Gary D.; Silbaq, Fauzi S.; Aebersold, Rudolf H.

    2000-04-01

    A method is described for immobilizing double-stranded DNAs to a planar gold surface with high density and uniform spacing. This is accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin. This streptavidin monolayer, which offers approximately 5 X 1012 biotin sites per cm2, is prepared first by adsorbing it onto a mixed self-assembled monolayer on gold which contains biotin-terminated and oligo-terminated alkylthiolates in a 3/7 ratio. This DNA- functionalized surface resists non-specific protein adsorption and is useful for probing the kinetics and equilibrium binding of proteins to DNA with surface plasmon resonance. This is demonstrated with the Mnt protein, which is found to bind in 3.8:1 ratio to its immobilized DNA operator sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA. A sequence with a single base-pair mutation shows nearly as much Mnt binding, but a completely random DNA sequence shows only 5 percent of this binding. This proves that DNA-binding proteins bind sequence-specifically to double-stranded DNAs which are immobilized to gold with this streptavidin linker layer.

  10. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  11. Completely random signed measures

    DEFF Research Database (Denmark)

    Hellmund, Gunnar

    Completely random signed measures are defined, characterized and related to Lévy random measures and Lévy bases.......Completely random signed measures are defined, characterized and related to Lévy random measures and Lévy bases....

  12. Random broadcast on random geometric graphs

    Energy Technology Data Exchange (ETDEWEB)

    Bradonjic, Milan [Los Alamos National Laboratory; Elsasser, Robert [UNIV OF PADERBORN; Friedrich, Tobias [ICSI/BERKELEY; Sauerwald, Tomas [ICSI/BERKELEY

    2009-01-01

    In this work, we consider the random broadcast time on random geometric graphs (RGGs). The classic random broadcast model, also known as push algorithm, is defined as: starting with one informed node, in each succeeding round every informed node chooses one of its neighbors uniformly at random and informs it. We consider the random broadcast time on RGGs, when with high probability: (i) RGG is connected, (ii) when there exists the giant component in RGG. We show that the random broadcast time is bounded by {Omicron}({radical} n + diam(component)), where diam(component) is a diameter of the entire graph, or the giant component, for the regimes (i), or (ii), respectively. In other words, for both regimes, we derive the broadcast time to be {Theta}(diam(G)), which is asymptotically optimal.

  13. Regulating DNA Self-assembly by DNA-Surface Interactions.

    Science.gov (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

    2017-12-14

    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. DNA Bending elasticity

    Science.gov (United States)

    Sivak, David Alexander

    DNA bending elasticity on length scales of tens of basepairs is of critical importance in numerous biological contexts. Even the simplest models of DNA bending admit of few simple analytic results, thus there is a need for numerical methods to calculate experimental observables, such as distance distributions, forces, FRET efficiencies, and timescales of particular large-scale motions. We have implemented and helped develop a coarse-grained representation of DNA and various other covalently-linked groups that allows simple calculation of such observables for varied experimental systems. The simple freely-jointed chain (FJC) model and extremely coarse resolution proved useful in understanding DNA threading through nanopores, identifying steric occlusion by other parts of the chain as a prime culprit for slower capture as distance to the pore decreased. Enhanced sampling techniques of a finer resolution discrete wormlike chain (WLC) model permitted calculation of cyclization rates for small chains and identified the ramifications of a thermodynamically-sound treatment of thermal melts. Adding treatment of double-stranded DNA's helical nature and single-stranded DNA provided a model system that helped demonstrate the importance of statistical fluctuations in even highly-stressed DNA mini-loops, and allowed us to verify that even these constructs show no evidence of excitation-induced softening. Additional incorporation of salt-sensitivity to the model allowed us to calculate forces and FRET efficiencies for such mini-loops and their uncircularized precursors, thereby furthering the understanding of the nature of IHF binding and bending of its recognition sequence. Adding large volume-excluding spheres linked to the ends of the dsDNA permits calculation of distance distributions and thus small-angle X-ray scattering, whereby we demonstrated the validity of the WLC in describing bending fluctuations in DNA chains as short as 42 bp. We also make important connections

  15. DNA-PK assay

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  16. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  17. DNA Mismatch Repair

    Science.gov (United States)

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  18. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2012-01-01

    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  19. Apoptosis and DNA Methylation

    International Nuclear Information System (INIS)

    Meng, Huan X.; Hackett, James A.; Nestor, Colm; Dunican, Donncha S.; Madej, Monika; Reddington, James P.; Pennings, Sari; Harrison, David J.; Meehan, Richard R.

    2011-01-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG

  20. How random is a random vector?

    International Nuclear Information System (INIS)

    Eliazar, Iddo

    2015-01-01

    Over 80 years ago Samuel Wilks proposed that the “generalized variance” of a random vector is the determinant of its covariance matrix. To date, the notion and use of the generalized variance is confined only to very specific niches in statistics. In this paper we establish that the “Wilks standard deviation” –the square root of the generalized variance–is indeed the standard deviation of a random vector. We further establish that the “uncorrelation index” –a derivative of the Wilks standard deviation–is a measure of the overall correlation between the components of a random vector. Both the Wilks standard deviation and the uncorrelation index are, respectively, special cases of two general notions that we introduce: “randomness measures” and “independence indices” of random vectors. In turn, these general notions give rise to “randomness diagrams”—tangible planar visualizations that answer the question: How random is a random vector? The notion of “independence indices” yields a novel measure of correlation for Lévy laws. In general, the concepts and results presented in this paper are applicable to any field of science and engineering with random-vectors empirical data.

  1. Implementation of digital image encryption algorithm using logistic function and DNA encoding

    Science.gov (United States)

    Suryadi, MT; Satria, Yudi; Fauzi, Muhammad

    2018-03-01

    Cryptography is a method to secure information that might be in form of digital image. Based on past research, in order to increase security level of chaos based encryption algorithm and DNA based encryption algorithm, encryption algorithm using logistic function and DNA encoding was proposed. Digital image encryption algorithm using logistic function and DNA encoding use DNA encoding to scramble the pixel values into DNA base and scramble it in DNA addition, DNA complement, and XOR operation. The logistic function in this algorithm used as random number generator needed in DNA complement and XOR operation. The result of the test show that the PSNR values of cipher images are 7.98-7.99 bits, the entropy values are close to 8, the histogram of cipher images are uniformly distributed and the correlation coefficient of cipher images are near 0. Thus, the cipher image can be decrypted perfectly and the encryption algorithm has good resistance to entropy attack and statistical attack.

  2. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  3. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with these modifications, it is likely that the primary use of DNA vaccines may be as primers for viral-vectored vaccines, rather than as single agents. This review discusses the approaches used to enhance DNA vaccine immunogenicity, with a primary focus on fusion strategies that enhance antigen presentation....

  4. Close encounters with DNA

    Science.gov (United States)

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  5. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  6. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  7. Gomphid DNA sequence data

    Data.gov (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  8. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    Stelow, R.B.

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

  9. Experimental DNA computing

    NARCIS (Netherlands)

    Henkel, Christiaan

    2005-01-01

    Because of their information storing and processing capabilities, nucleic acids are interesting building blocks for molecular scale computers. Potential applications of such DNA computers range from massively parallel computation to computational gene therapy. In this thesis, several implementations

  10. DNA Vaccines Against Anthrax

    National Research Council Canada - National Science Library

    Galloway, Darrell R; Baillie, Les

    2004-01-01

    DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development...

  11. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  12. Retroviral DNA Integration

    Science.gov (United States)

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  13. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.

    1978-01-01

    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

  14. DNA-Origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Gothelf, Kurt Vesterager

    2010-01-01

    DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau....

  15. Patterning nanocrystals using DNA

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)

    2003-01-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

  16. Celebrating DNA's Repair Crew.

    Science.gov (United States)

    Kunkel, Thomas A

    2015-12-03

    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Das DNA-Puzzle

    Science.gov (United States)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  18. PDA: Pooled DNA analyzer

    Directory of Open Access Journals (Sweden)

    Lin Chin-Yu

    2006-04-01

    Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at http://www.ibms.sinica.edu.tw/%7Ecsjfann/first%20flow/pda.htm.

  19. Variations in brain DNA

    Directory of Open Access Journals (Sweden)

    Jesus eAvila

    2014-11-01

    Full Text Available It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.

  20. Introduction to DNA methods

    International Nuclear Information System (INIS)

    Delincee, H.

    1991-01-01

    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey