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Sample records for addition specific molecular

  1. An Additive Definition of Molecular Complexity.

    PubMed

    Böttcher, Thomas

    2016-03-28

    A framework for molecular complexity is established that is based on information theory and consistent with chemical knowledge. The resulting complexity index Cm is derived from abstracting the information content of a molecule by the degrees of freedom in the microenvironments on a per-atom basis, allowing the molecular complexity to be calculated in a simple and additive way. This index allows the complexity of any molecule to be universally assessed and is sensitive to stereochemistry, heteroatoms, and symmetry. The performance of this complexity index is evaluated and compared against the current state of the art. Its additive character gives consistent values also for very large molecules and supports direct comparisons of chemical reactions. Finally, this approach may provide a useful tool for medicinal chemistry in drug design and lead selection, as demonstrated by correlating molecular complexities of antibiotics with compound-specific parameters.

  2. An Additive Definition of Molecular Complexity.

    PubMed

    Böttcher, Thomas

    2016-03-28

    A framework for molecular complexity is established that is based on information theory and consistent with chemical knowledge. The resulting complexity index Cm is derived from abstracting the information content of a molecule by the degrees of freedom in the microenvironments on a per-atom basis, allowing the molecular complexity to be calculated in a simple and additive way. This index allows the complexity of any molecule to be universally assessed and is sensitive to stereochemistry, heteroatoms, and symmetry. The performance of this complexity index is evaluated and compared against the current state of the art. Its additive character gives consistent values also for very large molecules and supports direct comparisons of chemical reactions. Finally, this approach may provide a useful tool for medicinal chemistry in drug design and lead selection, as demonstrated by correlating molecular complexities of antibiotics with compound-specific parameters. PMID:26857537

  3. Molecular Mechanisms of Synaptic Specificity

    PubMed Central

    Margeta, Milica A.; Shen, Kang

    2011-01-01

    Synapses are specialized junctions that mediate information flow between neurons and their targets. A striking feature of the nervous system is the specificity of its synaptic connections: an individual neuron will form synapses only with a small subset of available presynaptic and postsynaptic partners. Synaptic specificity has been classically thought to arise from homophilic or heterophilic interactions between adhesive molecules acting across the synaptic cleft. Over the past decade, many new mechanisms giving rise to synaptic specificity have been identified. Synapses can be specified by secreted molecules that promote or inhibit synaptogenesis, and their source can be a neighboring guidepost cell, not just presynaptic and postsynaptic neurons. Furthermore, lineage, fate, and timing of development can also play critical roles in shaping neural circuits. Future work utilizing large-scale screens will aim to elucidate the full scope of cellular mechanisms and molecular players that can give rise to synaptic specificity. PMID:19969086

  4. Theory of atomic additivity in molecular hyperpolizabilities

    NASA Technical Reports Server (NTRS)

    Baird, James K.

    1987-01-01

    Hyperpolarizability is a function of frequency. This is called dispersion. Because of the Kramers-Kronig relations, researchers expect that a material that is dispersing light is also absorbing it. Where there is both dispersion and absorption, the molecular polarizabilities are complex functions of the frequency. This led researchers to consider atomic additivity in both the real and imaginary parts of the ordinary and hyperpolarizabilities. This effort is desirable not only from a theoretical point of view, but also because of the existence of a large body of complex refractive index data, which may be used to test the additivity principle with the complex valued ordinary dipole polarizability.

  5. Molecular-specific urokinase antibodies

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  6. Molecular specific optoacoustic imaging with plasmonic nanoparticles

    NASA Astrophysics Data System (ADS)

    Mallidi, Srivalleesha; Larson, Timothy; Aaron, Jesse; Sokolov, Konstantin; Emelianov, Stanislav

    2007-05-01

    Gold nanoparticles functionalized with antibodies can specifically bind to molecular biomarkers such as epithelial growth factor receptor (EGFR). The molecule specific nature of the antibody-functionalized gold nanoparticles forms the basis for the developed optoacoustic imaging technique to detect cancer at an asymptotic stage. Optoacoustic imaging was performed with 532 nm and 680 nm pulsed laser irradiation on three-dimensional tissue phantoms prepared using a human keratinocyte cell line. The results of our study demonstrate that the combination of anti-EGFR gold ioconjugates and optoacoustic imaging can allow highly sensitive and selective detection of human epithelial cancer cells.

  7. 45 CFR 156.285 - Additional standards specific to SHOP.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Additional standards specific to SHOP. 156.285 Section 156.285 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES REQUIREMENTS RELATING TO HEALTH CARE ACCESS HEALTH INSURANCE ISSUER STANDARDS UNDER THE AFFORDABLE CARE ACT, INCLUDING...

  8. Molecular and cellular limits to somatosensory specificity

    PubMed Central

    Belmonte, Carlos; Viana, Félix

    2008-01-01

    Animals detect environmental changes through sensory neural mechanisms that enable them to differentiate the quality, intensity and temporal characteristics of stimuli. The 'doctrine of specific nervous energies' postulates that the different sensory modalities experienced by humans result of the activation of specific nervous pathways. Identification of functional classes of sensory receptors provided scientific support to the concept that somatosensory modalities (touch, pain, temperature, kinesthesis) are subserved by separate populations of sensory receptor neurons specialized in detecting innocuous and injurious stimuli of different quality (mechanical forces, temperature, chemical compounds). The identification of receptor proteins activated by different physicochemical stimuli, in particular ion channels of the Transient Receptor Potential (TRP) superfamily, has put forward the concept that specificity of peripheral sensory receptor neurons is determined by their expression of a particular "molecular sensor" that confers to each functional type its selectivity to respond with a discharge of nerve impulses to stimuli of a given quality. Nonetheless, recent experimental data suggest that the various molecular sensors proposed as specific transducer molecules for stimuli of different quality are not as neatly associated with the distinct functional types of sensory receptors as originally proposed. First, many ion channel molecules initially associated to the transduction of only one particular form of energy are also activated by stimuli of different quality, implying a limited degree of specificity in their transducing capacities. Second, molecular sensors associated with a stimulus quality and hence to a sensory receptor type and ultimately to a sensory modality may be concomitantly expressed in sensory receptor neurons functionally defined as specific for another stimulus quality. Finally, activation of voltage gated channels involved primarily in nerve

  9. Non-additive model for specific heat of electrons

    NASA Astrophysics Data System (ADS)

    Anselmo, D. H. A. L.; Vasconcelos, M. S.; Silva, R.; Mello, V. D.

    2016-10-01

    By using non-additive Tsallis entropy we demonstrate numerically that one-dimensional quasicrystals, whose energy spectra are multifractal Cantor sets, are characterized by an entropic parameter, and calculate the electronic specific heat, where we consider a non-additive entropy Sq. In our method we consider an energy spectra calculated using the one-dimensional tight binding Schrödinger equation, and their bands (or levels) are scaled onto the [ 0 , 1 ] interval. The Tsallis' formalism is applied to the energy spectra of Fibonacci and double-period one-dimensional quasiperiodic lattices. We analytically obtain an expression for the specific heat that we consider to be more appropriate to calculate this quantity in those quasiperiodic structures.

  10. Molecular Aluminum Additive for Burn Enhancement of Hydrocarbon Fuels.

    PubMed

    Guerieri, Philip M; DeCarlo, Samantha; Eichhorn, Bryan; Connell, Terrence; Yetter, Richard A; Tang, Xin; Hicks, Zachary; Bowen, Kit H; Zachariah, Michael R

    2015-11-12

    Additives to hydrocarbon fuels are commonly explored to change the combustion dynamics, chemical distribution, and/or product integrity. Here we employ a novel aluminum-based molecular additive, Al(I) tetrameric cluster [AlBrNEt3]4 (Et = C2H5), to a hydrocarbon fuel and evaluate the resultant single-droplet combustion properties. This Al4 cluster offers a soluble alternative to nanoscale particulate additives that have recently been explored and may mitigate the observed problems of particle aggregation. Results show the [AlBrNEt3]4 additive to increase the burn rate constant of a toluene-diethyl ether fuel mixture by ∼20% in a room temperature oxygen environment with only 39 mM of active aluminum additive (0.16 wt % limited by additive solubility). In comparison, a roughly similar addition of nano-aluminum particulate shows no discernible difference in burn properties of the hydrocarbon fuel. High speed video shows the [AlBrNEt3]4 to induce microexplosive gas release events during the last ∼30% of the droplet combustion time. We attribute this to HBr gas release based on results of temperature-programmed reaction (TPR) experiments of the [AlBrNEt3]4 dosed with O2 and D2O. A possible mechanism of burn rate enhancement is presented that is consistent with microexplosion observations and TPR results. PMID:26488461

  11. The specific molecular identification of life experiment ( SMILE)

    NASA Astrophysics Data System (ADS)

    Sims, M. R.; Cullen, D. C.; Bannister, N. P.; Grant, W. D.; Henry, O.; Jones, R.; McKnight, D.; Thompson, D. P.; Wilson, P. K.

    2005-07-01

    We describe a compact, highly integrated instrument concept for the detection and identification of a wide range of molecules associated with extinct/extant life or potential life processes. The Specific Molecular Identification of Life Experiment ( SMILE) will be sensitive to the presence of a range of target molecules using both electrical and optical transduction techniques, and incorporates molecular imprinted polymers in addition to traditional biological receptors such as antibodies. A number of versions of the concept are possible depending on available resources e.g. mass, volume, etc. The full concept utilises a novel imaging interferometer where a large number of molecular receptors are deposited on the measurement plane of an imaging interferometer and read out by an imaging detector, enabling multiple targets - biomarkers - within a sample to be measured simultaneously. The optics can also form the basis of an UV-NIR imaging Fourier spectrometer allowing basic mineralogy studies to be conducted using optical properties to assist in the determination of the geological context of the samples. By incorporating micro-fabricated transducer arrays, micro-fluidics and artificial molecular recognition systems, as well as recombinant antibody technology with appropriate integration methods, SMILE forms a compact and robust "Life Marker Chip" which has been proposed for future planetary missions including ESA's ExoMars mission, where the instrument offers the possibility of conducting a direct in situ search for signs of past or present biological activity on Mars. In addition to its role in planetary exploration, derivatives of SMILE have multiple terrestrial applications in fields such as forensic analysis and environmental monitoring.

  12. FPGA-specific decimal sign-magnitude addition and subtraction

    NASA Astrophysics Data System (ADS)

    Vázquez, Martín; Todorovich, Elías

    2016-07-01

    The interest in sign-magnitude (SM) representation in decimal numbers lies in the IEEE 754-2008 standard, where the significand in floating-point numbers is coded as SM. However, software implementations do not meet performance constraints in some applications and more development is required in programmable logic, a key technology for hardware acceleration. Thus, in this work, two strategies for SM decimal adder/subtractors are studied and six new Field Programmable Gate Array (FPGA)-specific circuits are derived from these strategies. The first strategy is based on ten's complement (C10) adder/subtractors and the second one is based on parallel computation of an unsigned adder and an unsigned subtractor. Four of these alternative circuits are useful for at least one area-time-trade-off and specific operand size. For example, the fastest SM adder/subtractor for operand sizes of 7 and 16 decimal digits is based on the second proposed strategy with delays of 3.43 and 4.33 ns, respectively, but the fastest circuit for 34-digit operands is one of the three specific implementations based on C10 adder/subtractors with a delay of 4.65 ns.

  13. The specification of personalised insoles using additive manufacturing.

    PubMed

    Salles, André S; Gyi, Diane E

    2012-01-01

    Research has been conducted to explore a process that delivers insoles for personalised footwear for the high street using additive manufacturing (AM) and to evaluate the use of such insoles in terms of discomfort. Therefore, the footwear personalisation process was first identified: (1) foot capture; (2) anthropometric measurements; (3) insole design; and (4) additive manufacturing. In order to explore and evaluate this process, recreational runners were recruited. They had both feet scanned and 15 anthropometric measurements taken. Personalised insoles were designed from the scans and manufactured using AM. Participants were fitted with footwear under two experimental conditions: personalised and control, which were compared in terms of discomfort. The mean ratings for discomfort variables were generally low for both conditions and no significant differences were detected between conditions. In general, the personalisation process showed promise in terms of the scan data, although the foot capture position may not be considered 'gold standard'. Polyamide, the material used for the insoles, demonstrated positive attributes: visual inspection revealed no signs of breaking. The footwear personalisation process described and explored in this study shows potential and can be considered a good starting point for designer and researchers.

  14. Additive manufacturing of patient-specific tubular continuum manipulators

    NASA Astrophysics Data System (ADS)

    Amanov, Ernar; Nguyen, Thien-Dang; Burgner-Kahrs, Jessica

    2015-03-01

    Tubular continuum robots, which are composed of multiple concentric, precurved, elastic tubes, provide more dexterity than traditional surgical instruments at the same diameter. The tubes can be precurved such that the resulting manipulator fulfills surgical task requirements. Up to now the only material used for the component tubes of those manipulators is NiTi, a super-elastic shape-memory alloy of nickel and titan. NiTi is a cost-intensive material and fabrication processes are complex, requiring (proprietary) technology, e.g. for shape setting. In this paper, we evaluate component tubes made of 3 different thermoplastic materials (PLA, PCL and nylon) using fused filament fabrication technology (3D printing). This enables quick and cost-effective production of custom, patient-specific continuum manipulators, produced on site on demand. Stress-strain and deformation characteristics are evaluated experimentally for 16 fabricated tubes of each thermoplastic with diameters and shapes equivalent to those of NiTi tubes. Tubes made of PCL and nylon exhibit properties comparable to those made of NiTi. We further demonstrate a tubular continuum manipulator composed of 3 nylon tubes in a transnasal, transsphenoidal skull base surgery scenario in vitro.

  15. Molecular basis of host specificity in human pathogenic bacteria

    PubMed Central

    Pan, Xiaolei; Yang, Yang; Zhang, Jing-Ren

    2014-01-01

    Pathogenic bacteria display various levels of host specificity or tropism. While many bacteria can infect a wide range of hosts, certain bacteria have strict host selectivity for humans as obligate human pathogens. Understanding the genetic and molecular basis of host specificity in pathogenic bacteria is important for understanding pathogenic mechanisms, developing better animal models and designing new strategies and therapeutics for the control of microbial diseases. The molecular mechanisms of bacterial host specificity are much less understood than those of viral pathogens, in part due to the complexity of the molecular composition and cellular structure of bacterial cells. However, important progress has been made in identifying and characterizing molecular determinants of bacterial host specificity in the last two decades. It is now clear that the host specificity of bacterial pathogens is determined by multiple molecular interactions between the pathogens and their hosts. Furthermore, certain basic principles regarding the host specificity of bacterial pathogens have emerged from the existing literature. This review focuses on selected human pathogenic bacteria and our current understanding of their host specificity. PMID:26038515

  16. The morpholino molecular beacon for specific RNA visualization in vivo.

    PubMed

    Chen, Jianbin; Wu, Jikui; Hong, Yunhan

    2016-02-21

    A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research.

  17. The morpholino molecular beacon for specific RNA visualization in vivo.

    PubMed

    Chen, Jianbin; Wu, Jikui; Hong, Yunhan

    2016-02-21

    A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research. PMID:26810703

  18. Molecular logic of neocortical projection neuron specification, development and diversity

    PubMed Central

    Galazo, MJ; Padmanabhan, H; Macklis, JD

    2013-01-01

    The sophisticated circuitry of the neocortex is assembled from a diverse repertoire of neuronal subtypes generated during development under precise molecular regulation. In recent years, several key controls over the specification and differentiation of neocortical projection neurons have been identified. This work provides substantial insight into the “molecular logic” underlying cortical development, increasingly supporting a model in which individual progenitor-stage and postmitotic regulators are embedded within highly-interconnected networks that gate sequential developmental decisions. Here, we provide an integrative account of the molecular controls that direct the progressive development and delineation of subtype and area identity of neocortical projection neurons. PMID:24105342

  19. Addition of molecular methods to mutation studies with Drosophila melanogaster

    SciTech Connect

    Lee, W.R. )

    1989-01-01

    For 80 years, Drosophila melanogaster has been used as a major tool in analyzing Mendelian genetics. By using chromosome inversions that suppress crossing over, geneticists have developed a large number of stocks for mutation analysis. These stocks permit numerous tests for specific locus mutations, lethals at multiple loci on any chromosome, chromosome exchanges, insertions, and deletions. The entire genome can be manipulated for a degree of genetic control not found in other germ-line systems. Recombinant DNA techniques now permit analysis of mutations to the nucleotide level. By combining classical genetic analysis with recombinant DNA techniques, it is possible to analyze mutations that range from chromosome aberrations and multilocus deficiencies to single nucleotide transitions.

  20. System among the corticosteroids: specificity and molecular dynamics

    PubMed Central

    Brookes, Jennifer C.; Galigniana, Mario D.; Harker, Anthony H.; Stoneham, A. Marshall; Vinson, Gavin P.

    2012-01-01

    Understanding how structural features determine specific biological activities has often proved elusive. With over 161 000 steroid structures described, an algorithm able to predict activity from structural attributes would provide manifest benefits. Molecular simulations of a range of 35 corticosteroids show striking correlations between conformational mobility and biological specificity. Thus steroid ring A is important for glucocorticoid action, and is rigid in the most specific (and potent) examples, such as dexamethasone. By contrast, ring C conformation is important for the mineralocorticoids, and is rigid in aldosterone. Other steroids that are less specific, or have mixed functions, or none at all, are more flexible. One unexpected example is 11-deoxycorticosterone, which the methods predict (and our activity studies confirm) is not only a specific mineralocorticoid, but also has significant glucocorticoid activity. These methods may guide the design of new corticosteroid agonists and antagonists. They will also have application in other examples of ligand–receptor interactions. PMID:21613285

  1. Site-specific recombinases: molecular machines for the Genetic Revolution.

    PubMed

    Olorunniji, Femi J; Rosser, Susan J; Stark, W Marshall

    2016-03-15

    The fields of molecular genetics, biotechnology and synthetic biology are demanding ever more sophisticated molecular tools for programmed precise modification of cell genomic DNA and other DNA sequences. This review presents the current state of knowledge and development of one important group of DNA-modifying enzymes, the site-specific recombinases (SSRs). SSRs are Nature's 'molecular machines' for cut-and-paste editing of DNA molecules by inserting, deleting or inverting precisely defined DNA segments. We survey the SSRs that have been put to use, and the types of applications for which they are suitable. We also discuss problems associated with uses of SSRs, how these problems can be minimized, and how recombinases are being re-engineered for improved performance and novel applications.

  2. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  3. 29 CFR 1926.452 - Additional requirements applicable to specific types of scaffolds.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... scaffolds. 1926.452 Section 1926.452 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Scaffolds § 1926.452 Additional requirements applicable to specific types of scaffolds. In addition to the applicable requirements of § 1926.451, the following requirements apply to the specific types of...

  4. 29 CFR 1926.452 - Additional requirements applicable to specific types of scaffolds.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... scaffolds. 1926.452 Section 1926.452 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Scaffolds § 1926.452 Additional requirements applicable to specific types of scaffolds. In addition to the applicable requirements of § 1926.451, the following requirements apply to the specific types of...

  5. 29 CFR 1926.452 - Additional requirements applicable to specific types of scaffolds.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... scaffolds. 1926.452 Section 1926.452 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Scaffolds § 1926.452 Additional requirements applicable to specific types of scaffolds. In addition to the applicable requirements of § 1926.451, the following requirements apply to the specific types of...

  6. 29 CFR 1926.452 - Additional requirements applicable to specific types of scaffolds.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... scaffolds. 1926.452 Section 1926.452 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Scaffolds § 1926.452 Additional requirements applicable to specific types of scaffolds. In addition to the applicable requirements of § 1926.451, the following requirements apply to the specific types of...

  7. 29 CFR 1926.452 - Additional requirements applicable to specific types of scaffolds.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... scaffolds. 1926.452 Section 1926.452 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Scaffolds § 1926.452 Additional requirements applicable to specific types of scaffolds. In addition to the applicable requirements of § 1926.451, the following requirements apply to the specific types of...

  8. Luminescent Nanomaterials for Molecular-Specific Cellular Imaging

    NASA Astrophysics Data System (ADS)

    Zvyagin, Andrei Vasilyevich; Song, Zhen; Nadort, Annemarie; Sreenivasan, Varun Kumaraswamy Annayya; Deyev, Sergey Mikhailovich

    Imaging of molecular trafficking in cells and biological tissue aided by molecular-specific fluorescent labeling is very attractive, since it affords capturing the key processes in comprehensive biological context. Several shortcomings of the existing organic dye labeling technology, however, call for development of alternative molecular reporters, with improved photostability, reduced cytotoxicity, and an increased number of controllable surface moieties. Such alternative molecular reporters are represented by inorganic luminescent nanoparticles (NP) whose optical, physical, and chemical properties are discussed on the examples of luminescent nanodiamonds (LND) and upconversion nanoparticles (UCNP). The emission origins of these nanomaterials differ markedly. LND emission results from individual nitrogen-vacancy color-centers in a biocompatible nanodiamond host whose properties can be controlled via size and surface groups. Photophysics of UCNP is governed by the collective, nonlinear excitation transfer processes, resulting in conversion of longer-wavelength excitation to the shorter-wavelength emission. The emission/excitation spectral properties of UCNP falling within the biological tissue transparency window open new opportunities of almost complete suppression of the cell/tissue autofluorescence background. The developed surface of these nanoparticles represents a flexible platform populated with biocompatible surface moieties onto which cargo and targeting biomolecules can be firmly docked through a process called bioconjugation. These bioconjugated modules, e.g., nanodiamond-antibody, (quantum dot)-somatostatin, or (upconversion nanoparticle)-(mini-antibody) can gain admission into the cells by initiating the cell-specific, cell-recognized communication protocol. In this chapter, we aim to demonstrate the whole bottom-up bio-nano-optics approach for optical biological imaging capturing luminescent nanoparticle design, surface activation, and bioconjugation

  9. Structure, molecular evolution, and hydrolytic specificities of largemouth bass pepsins.

    PubMed

    Miura, Yoko; Suzuki-Matsubara, Mieko; Kageyama, Takashi; Moriyama, Akihiko

    2016-02-01

    The nucleotide sequences of largemouth bass pepsinogens (PG1, 2 and 3) were determined after molecular cloning of the respective cDNAs. Encoded PG1, 2 and 3 were classified as fish pepsinogens A1, A2 and C, respectively. Molecular evolutionary analyses show that vertebrate pepsinogens are classified into seven monophyletic groups, i.e. pepsinogens A, F, Y (prochymosins), C, B, and fish pepsinogens A and C. Regarding the primary structures, extensive deletion was obvious in S'1 loop residues in fish pepsin A as well as tetrapod pepsin Y. This deletion resulted in a decrease in hydrophobic residues in the S'1 site. Hydrolytic specificities of bass pepsins A1 and A2 were investigated with a pepsin substrate and its variants. Bass pepsins preferred both hydrophobic/aromatic residues and charged residues at the P'1 sites of substrates, showing the dual character of S'1 sites. Thermodynamic analyses of bass pepsin A2 showed that its activation Gibbs energy change (∆G(‡)) was lower than that of porcine pepsin A. Several sites of bass pepsin A2 moiety were found to be under positive selection, and most of them are located on the surface of the molecule, where they are involved in conformational flexibility. The broad S'1 specificity and flexible structure of bass pepsin A2 are thought to cause its high proteolytic activity. PMID:26627128

  10. Molecular evidence for host-symbiont specificity in soritid foraminifera.

    PubMed

    Garcia-Cuetos, Lydia; Pochon, Xavier; Pawlowski, Jan

    2005-12-01

    Symbiosis between the dinoflagellate genus Symbiodinium and various invertebrates and protists is an ubiquitous phenomenon in shallow tropical and subtropical waters. Molecular studies undertaken on cnidarian symbionts revealed the presence of several distinctive lineages or subgeneric clades of Symbiodinium whose taxonomic level provides limited information about the specificity between invertebrate hosts and their symbionts. This contrasts with the finding of several Symbiodinium clades being present almost exclusively in foraminifera and belonging to the subfamily Soritinae. To test whether such specificity also exists at a lower taxonomic level within Soritinae, we obtained the SSU rDNA sequences from 159 soritid individuals collected in nine localities worldwide and representing all known morphospecies of this subfamily. For each individual, the symbionts were determined either by sequencing or by RFLP analysis. We distinguished 22 phylotypes of Soritinae in relation with a number of symbiont "groups" corresponding to 3 clades and 5 subclades of Symbiodinium. Among the 22 soritid phylotypes, 14 show strict symbiont specificity and only one was found to be a host for more than two "groups" of Symbiodinium. It is suggested that the strong host-symbiont specificity observed in Soritinae is a combined effect of a selective recognition mechanism, vertical transmission of symbionts, and biogeographical isolation.

  11. Real-time PCR detection of telomerase activity using specific molecular beacon probes.

    PubMed

    Kong, Deming; Jin, Yawei; Yin, Yuji; Mi, Huaifeng; Shen, Hanxi

    2007-06-01

    Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes.

  12. Molecular Specificity of Multiple Hippocampal Processes Governing Fear Extinction

    PubMed Central

    Radulovic, Jelena; Tronson, Natalie C.

    2010-01-01

    SYNOPSIS Over many years, fear extinction has been conceptualized as one dominant process, new inhibitory learning, which serves to dampen previously acquired fear. Here we present an alternative view, that brain region-specific processing of representations, expectations and emotional attributes of the fear-provoking event, recruits unique mechanisms that interdependently contribute to the conditioning and extinction of fear. The co-occurrence of these mechanisms within the fear circuit can thus be tracked and differentiated at a molecular and cellular level. Among others, the transcriptional regulators cFos, cAMP-dependent response element binding protein (CREB), Zif268, and extracellular signal-regulated kinases (Erk) stand out as hippocampal nuclear markers signaling novelty, arousal, retrieval, and prediction error, respectively. Consistent with evidence from human studies, these findings indicate that, beyond inhibitory learning, fear extinction requires modification of the emotional attributes and expectations that define the threatening context. Given the likely dysregulation of one or more of these processes in anxiety disorders, a key research challenge for the future is the identification and enhancement of individual extinction mechanisms to target the specific components of fear. Environmental stimuli lacking affective properties (conditioned stimuli, CS) rapidly become threatening if presented with stressful events (unconditioned stimuli, US). Consequently, based on a CS-US association, the presentation of the CS triggers species-specific fear responses until the US consistently stops occurring. At that point, new learning takes place and the fear response declines, a phenomenon termed extinction. The view that extinction occurs because a new, inhibitory CS-noUS association gains control over behavior 106, has remained dominant in the field (reviewed by 20,33,35,100). The implications of impaired fear regulation in the development of anxiety disorders

  13. Specific effects of Ca2+ ions and molecular structure of β-lactoglobulin interfacial layers that drive macroscopic foam stability† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sm00636a Click here for additional data file.

    PubMed Central

    Schulze-Zachau, Felix; Nagel, Eva; Engelhardt, Kathrin; Stoyanov, Stefan; Gochev, Georgi; Khristov, Khr.; Mileva, Elena; Exerowa, Dotchi; Miller, Reinhard; Peukert, Wolfgang

    2016-01-01

    β-Lactoglobulin (BLG) adsorption layers at air–water interfaces were studied in situ with vibrational sum-frequency generation (SFG), tensiometry, surface dilatational rheology and ellipsometry as a function of bulk Ca2+ concentration. The relation between the interfacial molecular structure of adsorbed BLG and the interactions with the supporting electrolyte is additionally addressed on higher length scales along the foam hierarchy – from the ubiquitous air–water interface through thin foam films to macroscopic foam. For concentrations <1 mM, a strong decrease in SFG intensity from O–H stretching bands and a slight increase in layer thickness and surface pressure are observed. A further increase in Ca2+ concentrations above 1 mM causes an apparent change in the polarity of aromatic C–H stretching vibrations from interfacial BLG which we associate to a charge reversal at the interface. Foam film measurements show formation of common black films at Ca2+ concentrations above 1 mM due to considerable decrease of the stabilizing electrostatic disjoining pressure. These observations also correlate with a minimum in macroscopic foam stability. For concentrations >30 mM Ca2+, micrographs of foam films show clear signatures of aggregates which tend to increase the stability of foam films. Here, the interfacial layers have a higher surface dilatational elasticity. In fact, macroscopic foams formed from BLG dilutions with high Ca2+ concentrations where aggregates and interfacial layers with higher elasticity are found, showed the highest stability with much smaller bubble sizes. PMID:27337699

  14. Molecular Insights for Optimizing T Cell Receptor Specificity Against Cancer

    PubMed Central

    Hebeisen, Michael; Oberle, Susanne G.; Presotto, Danilo; Speiser, Daniel E.; Zehn, Dietmar; Rufer, Nathalie

    2013-01-01

    Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses. PMID:23801991

  15. Dissecting the molecular origins of specific protein-nucleic acid recognition: hydrostatic pressure and molecular dynamics.

    PubMed Central

    Lynch, Thomas W; Kosztin, Dorina; McLean, Mark A; Schulten, Klaus; Sligar, Stephen G

    2002-01-01

    The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability. PMID:11751298

  16. Properties and Microstructural Characteristic of Kaolin Geopolymer Ceramics with Addition of Ultra High Molecular Weight Polyethylene

    NASA Astrophysics Data System (ADS)

    Ahmad, Romisuhani; Bakri Abdullah, Mohd Mustafa Al; Hussin, Kamarudin; Sandu, Andrei Victor; Binhussain, Mohammed; Ain Jaya, Nur

    2016-06-01

    In this paper, the mechanical properties and microstructure of kaolin geopolymer ceramics with addition of Ultra High Molecular Weight Polyethylene were studied. Inorganic polymers based on alumina and silica polysialate units were synthesized at room temperature from kaolin and sodium silicate in a highly alkaline medium, followed by curing and drying at 80 °C. Alkaline activator was formed by mixing the 12 M NaOH solution with sodium silicate at a ratio of 0.24. Addition of Ultra High Molecular Weight Polyethylene to the kaolin geopolymer are fabricated with Ultra High Molecular Weight Polyethylene content of 2, 4, 6 and 8 (wt. %) by using powder metallurgy method. The samples were heated at 1200 °C and the strength and morphological were tested. It was found that the flexural strength for the kaolin geopolymer ceramics with addition of UHMWPE were improved and generally increased with the increasing of UHMWPE loading. The result revealed that the optimum flexural strength was obtained at UHMWPE loading of 4 wt. % (92.1 MPa) and the flexural strength started to decrease. Microstructural analysis showed the samples appeared to have more number of pores and connected of pores increased with the increasing of UHMWPE content.

  17. Effects of molecular architectures and solvophobic additives on the aggregative properties of polymeric surfactants

    NASA Astrophysics Data System (ADS)

    Lin, Yung-Lung; Wu, Ming-Zher; Sheng, Yu-Jane; Tsao, Heng-Kwong

    2012-03-01

    The aggregative behavior of the polymeric surfactants with various molecular architectures in dilute solutions is studied by dissipative particle dynamics. The effects of the solvophobic/solvophilic length, polymeric architecture (linear, star, dendritic, and cyclic type), chain rigidity, and solvophobic additives on the critical micelle concentration (CMC) and the aggregative patterns are systematically investigated. It is found that molecular architectures have a noteworthy impact on the aggregative properties. For linear diblock copolymers, the CMC declines with increasing solvophobic length but rises with increasing solvophilic length. Nonetheless, the solvophobic group has comparatively greater influence on the CMC. Imposition of the star, dendritic, or cyclic structures onto the solvophobic or solvophilic parts of the polymeric surfactant leads to an increase in the CMC. On the contrary, polymers imposed with the greater degree of the rigidity on the solvophobic or solvophilic block have lower CMC. The addition of solvophobic additives results in a decrease of CMC as well. The effects of the concentration and length of the additives on the aggregative behaviors of polymer surfactants were investigated. Interesting supramolecular structures such as caterpillar and worm-like micelles were observed.

  18. Conjugation of antibodies to gold nanorods through Fc portion: synthesis and molecular specific imaging

    PubMed Central

    Joshi, Pratixa P.; Yoon, Soon Joon; Hardin, William G.; Emelianov, Stanislav; Sokolov, Konstantin V.

    2013-01-01

    Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross-sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection. PMID:23631707

  19. The effect of the molecular weight of additive on the properties of antimisting fuels

    SciTech Connect

    Hadermann, A.F.; Trippe, J.C.; Waters, P.F.

    1983-09-01

    Antimisting aircraft fuels, when ignited, do not produce the roaring fireball which often accompanies aircraft crashes. This result is attributable to the suppression of the aerosolization of the fuel by added macromolecules which alter the structure of the droplets of fuel emanating from rent fuel tanks after the crash. The first studies of the antimisting effect of macromolecules on aviation fuel were carried out in Great Britain in 1968. In that early work it was established that there was a qualitative relationship between the suppression of the atomization of the fuel and the molecular weight of the additive above a certain critical concentration; the latter being inverse to the molecular weight of the additive. Subsequent investigations have demonstrated a dependence of the antimisting effectiveness of polyisobutylene in diesel fuel on the viscosity average molecular weight to a power exceeding 2, and in jet-A fuel to the 2..cap alpha.. + 1 power, where ..cap alpha.. is the exponent in the Mark-Houwink equation. In their study Chao et al, were able to demonstrate a strong correlation between the extent of antimisting effectiveness and flammability reduction with the maximum ductless siphon height supported by the solution. They introduced the ductless siphon to the study of antimisting fuels as a measure of the elongational viscosity impaired by the macromolecules to the fuel.

  20. In Vitro Selection of Cancer Cell-Specific Molecular Recognition Elements from Amino Acid Libraries

    PubMed Central

    Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Differential cell systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro selection method for obtaining molecular recognition elements (MREs) that specifically bind to individual cell types with high affinity. MREs are selected from initial large libraries of different nucleic or amino acids. This review outlines the construction of peptide and antibody fragment libraries as well as their different host types. Common methods of selection are also reviewed. Additionally, examples of cancer cell MREs are discussed, as well as their potential applications. PMID:26436100

  1. Molecular Determinants of Substrate Specificity in Plant 5-Methylthioadenosine Nucleosidases

    SciTech Connect

    Siu,K.; Lee, J.; Sufrin, J.; Moffatt, B.; McMillan, M.; Cornell, K.; Isom, C.; Howell, L.

    2008-01-01

    5?-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5?-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5?-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Angstroms resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5?-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5?-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pKa of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and

  2. Polydimethylsiloxane as a Macromolecular Additive for Enhanced Performance of Molecular Bulk Heterojunction Organic Solar Cells

    SciTech Connect

    Graham, Kenneth R.; Mei, Jianguo; Stalder, Romain; Shim, Jae Won; Cheun, Hyeunseok; Steffy, Fred; So, Franky; Kippelen, Bernard; Reynolds, John R.

    2011-03-15

    The effect of the macromolecular additive, polydimethylsiloxane (PDMS), on the performance of solution processed molecular bulk heterojunction solar cells is investigated, and the addition of PDMS is shown to improve device power conversion efficiency by ~70% and significantly reduce cell-to-cell variation, from a power conversion efficiency of 1.25 ± 0.37% with no PDMS to 2.16 ± 0.09% upon the addition of 0.1 mg/mL PDMS to the casting solution. The cells are based on a thiophene and isoindigo containing oligomer as the electron donor and [6,6]-phenyl-C61 butyric acid methyl ester (PC61BM) as the electron acceptor. PDMS is shown to have a strong influence on film morphology, with a significant decrease in film roughness and feature size observed. The morphology change leads to improved performance parameters, most notably an increase in the short circuit current density from 4.3 to 6.8 mA/cm2 upon addition of 0.1 mg/mL PDMS. The use of PDMS is of particular interest, as this additive appears frequently as a lubricant in plastic syringes commonly used in device fabrication; therefore, PDMS may unintentionally be incorporated into device active layers.

  3. High-Flow, High-Molecular-Weight, Addition-Curing Polyimides

    NASA Technical Reports Server (NTRS)

    Chuang, Kathy C.; Vannucci, Raymond D.

    1993-01-01

    In developed series of high-flow PMR-type polyimide resins, 2, 2'-bis(trifluoromethyl)-4, 4'-diaminobiphenyl (BTDB) substituted for 1, 4-pheylenediamine in PMR-II formulation. Polyimides designated either as PMR-12F when nadic ester (NE) end caps used, or as V-CAP-12F when p-aminostyrene end caps used. High-molecular-weight, addition-curing polyimides based on BTBD and HFDE highly processable high-temperature matrix resins used to make composite materials with excellent retention of properties during long-term exposure to air at 650 degrees F or higher temperature. Furthermore, 12F addition-curing polyimides useful for electronic applications; fluorinated rigid-rod polyimides known to exhibit low thermal expansion coefficients as well as low absorption of moisture.

  4. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties....

  5. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties....

  6. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties....

  7. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties....

  8. 21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties....

  9. FADB: a food additive molecular database for in silico screening in food toxicology.

    PubMed

    Ginex, Tiziana; Spyrakis, Francesca; Cozzini, Pietro

    2014-01-01

    A crucial limit to in silico preliminary toxicological evaluations in the "food safety" area is the lack of a specific, efficient and available free dataset of 3D small molecules. In this direction, we present the first version of FADB (Food Additives Data Base), a suitable and freely available food additives dataset. FADB is the 3D version of the EAFUS (Everything Added to Food in the United States) list, a sum of WHO, FAO food additive databases and could be a useful starting material in preliminary stages of toxicological assessments. Molecules in FADB are represented through several chemical and 1D identifies, physical properties and 3D (SD and Mol2 file) file formats. FADB also contains important information about functional uses of chemicals as food additives. The aim of the work is to put together substances potentially relevant to food into a "computational" library for virtual screening and docking studies with interesting scenarios for toxicology.

  10. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

    PubMed

    Ortega, Davi R; Zhulin, Igor B

    2016-02-01

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  11. Evolutionary genomics suggests that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex

    DOE PAGES

    Ortega, Davi R.; Zhulin, Igor B.; Punta, Marco

    2016-02-04

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linkingmore » the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Altogether, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.« less

  12. Molecular-scale evidence of aerosol particle formation via sequential addition of HIO3

    NASA Astrophysics Data System (ADS)

    Sipilä, Mikko; Sarnela, Nina; Jokinen, Tuija; Henschel, Henning; Junninen, Heikki; Kontkanen, Jenni; Richters, Stefanie; Kangasluoma, Juha; Franchin, Alessandro; Peräkylä, Otso; Rissanen, Matti P.; Ehn, Mikael; Vehkamäki, Hanna; Kurten, Theo; Berndt, Torsten; Petäjä, Tuukka; Worsnop, Douglas; Ceburnis, Darius; Kerminen, Veli-Matti; Kulmala, Markku; O'Dowd, Colin

    2016-09-01

    Homogeneous nucleation and subsequent cluster growth leads to the formation of new aerosol particles in the atmosphere. The nucleation of sulfuric acid and organic vapours is thought to be responsible for the formation of new particles over continents, whereas iodine oxide vapours have been implicated in particle formation over coastal regions. The molecular clustering pathways that are involved in atmospheric particle formation have been elucidated in controlled laboratory studies of chemically simple systems, but direct molecular-level observations of nucleation in atmospheric field conditions that involve sulfuric acid, organic or iodine oxide vapours have yet to be reported. Here we present field data from Mace Head, Ireland, and supporting data from northern Greenland and Queen Maud Land, Antarctica, that enable us to identify the molecular steps involved in new particle formation in an iodine-rich, coastal atmospheric environment. We find that the formation and initial growth process is almost exclusively driven by iodine oxoacids and iodine oxide vapours, with average oxygen-to-iodine ratios of 2.4 found in the clusters. On the basis of this high ratio, together with the high concentrations of iodic acid (HIO3) observed, we suggest that cluster formation primarily proceeds by sequential addition of HIO3, followed by intracluster restructuring to I2O5 and recycling of water either in the atmosphere or on dehydration. Our study provides ambient atmospheric molecular-level observations of nucleation, supporting the previously suggested role of iodine-containing species in the formation of new aerosol particles, and identifies the key nucleating compound.

  13. Towards the design of new and improved drilling fluid additives using molecular dynamics simulations.

    PubMed

    Anderson, Richard L; Greenwel, H Christopher; Suter, James L; Jarvis, Rebecca M; Coveney, Peter V

    2010-03-01

    During exploration for oil and gas, a technical drilling fluid is used to lubricate the drill bit, maintain hydrostatic pressure, transmit sensor readings, remove rock cuttings and inhibit swelling of unstable clay based reactive shale formations. Increasing environmental awareness and resulting legislation has led to the search for new, improved biodegradable drilling fluid components. In the case of additives for clay swelling inhibition, an understanding of how existing effective additives interact with clays must be gained to allow the design of improved molecules. Owing to the disordered nature and nanoscopic dimension of the interlayer pores of clay minerals, computer simulations have become an increasingly useful tool for studying clay-swelling inhibitor interactions. In this work we briefly review the history of the development of technical drilling fluids, the environmental impact of drilling fluids and the use of computer simulations to study the interactions between clay minerals and swelling inhibitors. We report on results from some recent large-scale molecular dynamics simulation studies on low molecular weight water-soluble macromolecular inhibitor molecules. The structure and interactions of poly(propylene oxide)-diamine, poly(ethylene glycol) and poly(ethylene oxide)-diacrylate inhibitor molecules with montmorillonite clay are studied.

  14. Just-in-time Design and Additive Manufacture of Patient-specific Medical Implants

    NASA Astrophysics Data System (ADS)

    Shidid, Darpan; Leary, Martin; Choong, Peter; Brandt, Milan

    Recent advances in medical imaging and manufacturing science have enabled the design and production of complex, patient-specific orthopaedic implants. Additive Manufacture (AM) generates three-dimensional structures layer by layer, and is not subject to the constraints associated with traditional manufacturing methods. AM provides significant opportunities for the design of novel geometries and complex lattice structures with enhanced functional performance. However, the design and manufacture of patient-specific AM implant structures requires unique expertise in handling various optimization platforms. Furthermore, the design process for complex structures is computationally intensive. The primary aim of this research is to enable the just-in-time customisation of AM prosthesis; whereby AM implant design and manufacture be completed within the time constraints of a single surgical procedure, while minimising prosthesis mass and optimising the lattice structure to match the stiffness of the surrounding bone tissue. In this research, a design approach using raw CT scan data is applied to the AM manufacture of femoral prosthesis. Using the proposed just-in-time concept, the mass of the prosthesis was rapidly designed and manufactured while satisfying the associated structural requirements. Compressive testing of lattice structures manufactured using proposed method shows that the load carrying capacity of the resected composite bone can be recovered by up to 85% and the compressive stiffness of the AM prosthesis is statistically indistinguishable from the stiffness of the initial bone.

  15. Requirement for additional treatment for dogs with atopic dermatitis undergoing allergen-specific immunotherapy.

    PubMed

    Colombo, S; Hill, P B; Shaw, D J; Thoday, K L

    2007-06-23

    Allergen-specific immunotherapy (ASIT) is one of the main treatments for atopic dermatitis in dogs, but it often requires additional treatments such as antibacterial and antifungal therapy for secondary bacterial and yeast infections, or antipruritic drugs to control the clinical signs or treat the adverse effects of the immunotherapy. Twenty-seven dogs enrolled in a study of ASIT were clinically assessed four times over a period of nine months; their requirement for treatment for secondary bacterial and yeast infections, for the administration of glucocorticoids as additional antipruritic therapy, and for the treatment of any adverse effects of the ASIT were evaluated. Twenty (74 per cent) of the dogs were treated for superficial bacterial pyoderma, 18 (66.6 per cent) required treatment for Malassezia species dermatitis on one or more occasions, eight (29.6 per cent) required treatment for otitis externa due to Malassezia species or bacteria, and eight required glucocorticoids to control their clinical signs. Five (18.5 per cent) of the dogs experienced adverse effects due to the ASIT and two required treatment with antihistamines (H1 receptor antagonists) in order to continue with the ASIT. PMID:17586789

  16. Genetic rearrangements of six wheat-agropyron cristatum 6P addition lines revealed by molecular markers.

    PubMed

    Han, Haiming; Bai, Li; Su, Junji; Zhang, Jinpeng; Song, Liqiang; Gao, Ainong; Yang, Xinming; Li, Xiuquan; Liu, Weihua; Li, Lihui

    2014-01-01

    Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) not only is cultivated as pasture fodder but also could provide many desirable genes for wheat improvement. It is critical to obtain common wheat-A. cristatum alien disomic addition lines to locate the desired genes on the P genome chromosomes. Comparative analysis of the homoeologous relationships between the P genome chromosome and wheat genome chromosomes is a key step in transferring different desirable genes into common wheat and producing the desired alien translocation line while compensating for the loss of wheat chromatin. In this study, six common wheat-A. cristatum disomic addition lines were produced and analyzed by phenotypic examination, genomic in situ hybridization (GISH), SSR markers from the ABD genomes and STS markers from the P genome. Comparative maps, six in total, were generated and demonstrated that all six addition lines belonged to homoeologous group 6. However, chromosome 6P had undergone obvious rearrangements in different addition lines compared with the wheat chromosome, indicating that to obtain a genetic compensating alien translocation line, one should recombine alien chromosomal regions with homoeologous wheat chromosomes. Indeed, these addition lines were classified into four types based on the comparative mapping: 6PI, 6PII, 6PIII, and 6PIV. The different types of chromosome 6P possessed different desirable genes. For example, the 6PI type, containing three addition lines, carried genes conferring high numbers of kernels per spike and resistance to powdery mildew, important traits for wheat improvement. These results may prove valuable for promoting the development of conventional chromosome engineering techniques toward molecular chromosome engineering. PMID:24595330

  17. Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

    PubMed

    Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

    2013-11-01

    Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

  18. Molecular Imprinting of Silica Nanoparticle Surfaces via Reversible Addition-Fragmentation Polymerization for Optical Biosensing Applications

    NASA Astrophysics Data System (ADS)

    Oluz, Zehra; Nayab, Sana; Kursun, Talya Tugana; Caykara, Tuncer; Yameen, Basit; Duran, Hatice

    Azo initiator modified surface of silica nanoparticles were coated via reversible addition-fragmentation polymerization (RAFT) of methacrylic acid and ethylene glycol dimethacrylate using 2-phenylprop 2-yl dithobenzoate as chain transfer agent. Using L-phenylalanine anilide as template during polymerization led molecularly imprinted nanoparticles. RAFT polymerization offers an efficient control of grafting process, while molecularly imprinted polymers shows enhanced capacity as sensor. L-phenylalanine anilide imprinted silica particles were characterized by X-Ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM). Performances of the particles were followed by surface plasmon resonance spectroscopy (SPR) after coating the final product on gold deposited glass substrate against four different analogous of analyte molecules: D-henylalanine anilide, L-tyrosine, L-tryptophan and L-phenylalanine. Characterizations indicated that silica particles coated with polymer layer do contain binding sites for L-phenylalanine anilide, and are highly selective for the molecule of interest. This project was supported by TUBITAK (Project No:112M804).

  19. Power conversion efficiency enhancement in OPV devices using spin 1/2 molecular additives

    NASA Astrophysics Data System (ADS)

    Basel, Tek; Vardeny, Valy; Yu, Luping

    2014-03-01

    We investigated the power conversion efficiency of bulk heterojunction OPV cells based on the low bandgap polymer PTB7, blend with C61-PCBM. We also employed the technique of photo-induced absorption, PA; electrical and magneto-PA (MPA) techniques to understand the details of the photocurrent generation process in this blend. We found that spin 1/2 molecular additives, such as Galvinoxyl (Gxl) radicals dramatically enhance the cell efficiency; we obtained 20% increase in photocurrent upon Gxl doping with 2% weight. We explain our finding by the ability of the spin 1/2 radicals to interfere with the known major loss mechanism in the cell due to recombination of charge transfer exciton at the D-A interface via triplet excitons in the polymer donors. Supported by National Science Foundation-Material Science & Engineering Center (NSF-MRSEC), University of Utah.

  20. Optimal receiver design for diffusive molecular communication with flow and additive noise.

    PubMed

    Noel, Adam; Cheung, Karen C; Schober, Robert

    2014-09-01

    In this paper, we perform receiver design for a diffusive molecular communication environment. Our model includes flow in any direction, sources of information molecules in addition to the transmitter, and enzymes in the propagation environment to mitigate intersymbol interference. We characterize the mutual information between receiver observations to show how often independent observations can be made. We derive the maximum likelihood sequence detector to provide a lower bound on the bit error probability. We propose the family of weighted sum detectors for more practical implementation and derive their expected bit error probability. Under certain conditions, the performance of the optimal weighted sum detector is shown to be equivalent to a matched filter. Receiver simulation results show the tradeoff in detector complexity versus achievable bit error probability, and that a slow flow in any direction can improve the performance of a weighted sum detector.

  1. Molecular Features of Subtype-Specific Progression from Ductal Carcinoma In Situ to Invasive Breast Cancer.

    PubMed

    Lesurf, Robert; Aure, Miriam Ragle; Mørk, Hanne Håberg; Vitelli, Valeria; Lundgren, Steinar; Børresen-Dale, Anne-Lise; Kristensen, Vessela; Wärnberg, Fredrik; Hallett, Michael; Sørlie, Therese

    2016-07-26

    Breast cancer consists of at least five main molecular "intrinsic" subtypes that are reflected in both pre-invasive and invasive disease. Although previous studies have suggested that many of the molecular features of invasive breast cancer are established early, it is unclear what mechanisms drive progression and whether the mechanisms of progression are dependent or independent of subtype. We have generated mRNA, miRNA, and DNA copy-number profiles from a total of 59 in situ lesions and 85 invasive tumors in order to comprehensively identify those genes, signaling pathways, processes, and cell types that are involved in breast cancer progression. Our work provides evidence that there are molecular features associated with disease progression that are unique to the intrinsic subtypes. We additionally establish subtype-specific signatures that are able to identify a small proportion of pre-invasive tumors with expression profiles that resemble invasive carcinoma, indicating a higher likelihood of future disease progression. PMID:27396337

  2. Molecular phylogenies reveal host-specific divergence of Ophiocordyceps unilateralis sensu lato following its host ants.

    PubMed

    Kobmoo, N; Mongkolsamrit, S; Tasanathai, K; Thanakitpipattana, D; Luangsa-Ard, J J

    2012-06-01

    Ophiocordyceps unilateralis (Hypocreales, Ascomycetes) is an entomopathogenic fungus specific to formicine ants (Formicinae, Hymenoptera). Previous works have shown that the carpenter ant Camponotus leonardi acts as the principal host with occasional infections of ants from the genus Polyrhachis (sister genus of Camponotus). Observations were made on the permanent plots of Mo Singto, Khao Yai National Park of Thailand according to which O. unilateralis was found to occur predominantly on three host species: C. leonardi, C. saundersi and P. furcata. Molecular phylogenies of the elongation factor 1-α and β-Tubulin genes indicate a separation of O. unilateralis samples into three clades, reflecting specificity to each of the three different ant species. Samples collected from P. furcata and from C. leonardi were found to form sister groups with samples from C. saundersi forming an outgroup to the latter. Additional samples collected from unidentified ant species of Camponotus and Polyrhachis were positioned as outgroups to those samples on identified species. These results demonstrate that O. unilateralis is clearly not a single phylogenetic species and comprises at least three species that are specific to different host ant species. These cryptic species may arise through recent events of speciation driven by their specificity to host ant species.

  3. Molecular phylogenies reveal host-specific divergence of Ophiocordyceps unilateralis sensu lato following its host ants.

    PubMed

    Kobmoo, N; Mongkolsamrit, S; Tasanathai, K; Thanakitpipattana, D; Luangsa-Ard, J J

    2012-06-01

    Ophiocordyceps unilateralis (Hypocreales, Ascomycetes) is an entomopathogenic fungus specific to formicine ants (Formicinae, Hymenoptera). Previous works have shown that the carpenter ant Camponotus leonardi acts as the principal host with occasional infections of ants from the genus Polyrhachis (sister genus of Camponotus). Observations were made on the permanent plots of Mo Singto, Khao Yai National Park of Thailand according to which O. unilateralis was found to occur predominantly on three host species: C. leonardi, C. saundersi and P. furcata. Molecular phylogenies of the elongation factor 1-α and β-Tubulin genes indicate a separation of O. unilateralis samples into three clades, reflecting specificity to each of the three different ant species. Samples collected from P. furcata and from C. leonardi were found to form sister groups with samples from C. saundersi forming an outgroup to the latter. Additional samples collected from unidentified ant species of Camponotus and Polyrhachis were positioned as outgroups to those samples on identified species. These results demonstrate that O. unilateralis is clearly not a single phylogenetic species and comprises at least three species that are specific to different host ant species. These cryptic species may arise through recent events of speciation driven by their specificity to host ant species. PMID:22494010

  4. Generation of an antibody specific to erythritol, a non-immunogenic food additive.

    PubMed

    Sreenath, K; Prabhasankar, P; Venkatesh, Y P

    2006-09-01

    Erythritol, a simple sugar alcohol, is widely used as a food and drug additive owing to its chemical inertness, sweetness and non-toxicity. Adverse reactions to erythritol are rare and only three cases of allergic reactions to foods containing erythritol have been reported. Being inert, erythritol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of erythritol, a method to obtain erythritol epitopes on a carrier protein, which can serve as an immunogen to develop antibodies against erythritol, is described. D-Erythrose was conjugated to bovine serum albumin at pH 8 by reductive amination. The reduction product of the Schiff base of D-erythrose-bovine serum albumin conjugate creates erythritoyl groups. Rabbits immunized with erythritol-bovine serum albumin conjugate (29 haptens/molecule) showed good antibody response (detection of 1 microg antigen, erythritol-keyhole limpet haemocyanin conjugate possessing 50% modified amino groups, at 1 : 50,000 dilution). Anti-erythritol immunoglobulin-G antibodies were purified from the immune serum using hapten-affinity chromatography on an erythritol-keyhole limpet haemocyanin-Sepharose CL-6B affinity matrix. The yield of erythritol-specific antibody was approximately 40 microg ml-1 of rabbit antiserum. Enzyme-linked immunobsorbant assay inhibition studies using sugars, sugar alcohols and L-lysine showed minimal cross-reactivity (approximately 4%) when compared with erythritol; only dithioerythritol showed a cross-reactivity of approximately 33%. D-Threitol and L-threitol (isomers of erythritol) had cross-reactivities of 15 and 11%, respectively. The inhibition studies confirmed the haptenic nature of erythritol and indicated that the erythritoyl group is a single epitope. The reaction scheme outlined here for the generation of erythritol epitopes appears to provide a basis for the immunogenicity of erythritol.

  5. Face-specific molecular adhesion and binding to calcium oxalate monohydrate: Implication for kidney stone formation

    NASA Astrophysics Data System (ADS)

    Sheng, Xiaoxia

    This thesis focuses on the face-specific molecular adhesion to calcium oxalate monohydrate (COM) crystals, the principal crystalline in kidney stones. The primary technique used is atomic force microscopy (AFM), which allows visualizing the structure and growth of crystals, measuring the adhesion force between functional groups and crystal faces, and examining adhesion and binding of the molecules to crystals. The microscopic events associated with crystal growth on the {100}, {12-1}, and {010} faces have been investigated. Each face exhibits hillocks with step sites that can be assigned to specific crystal planes, enabling direct determination of growth rates along specific crystallographic directions. The growth rates are found to depend on the degree of supersaturation. The addition of macromolecules with anionic side chains results in inhibition of hillock growth. The magnitude of this effect depends on the macromolecule structure & concentration, and the identity of the step site. The different profiles observed for three synthetic macromolecules, which have similar backbones but different side chains, argues that local binding of anionic side chains to crystal surface sites governs growth inhibition rather than any secondary polymer structure. The dependence of adhesion force on the functional group-COM crystal face combinations has been identified. Tip-immobilized carboxylate and amidinium groups display the largest adhesion forces among all the functional groups examined, and the adhesive strength decreases as (100) > (12-1) > (010). The more adherent surface of COM, compared with its dihydrate form COD, corroborates the critical role of COM in stone formation. The influence of small molecules, synthetic polymers and native proteins on adhesion was examined. The introduction of these molecular additives, except osteopontin, result in a reduction in the adhesion force measured for all three faces. The extent of suppression, however, varies for molecule

  6. Low-temperature specific heat of molecular glasses and crystals

    NASA Astrophysics Data System (ADS)

    Talón, César; Ramos, Miguel A.; Vieira, Sebastián

    2000-07-01

    We have measured the low-temperature specific heat Cp of several glass-forming alcohols. Special attention has been paid to ethanol, either hydrogenated or deuterated, in its different solid phases (fully ordered crystal, disordered crystal or orientational glass, and true structural glass). New measurements have been carried out on 1- and 2-propanol (for crystal and glass states) and on the glass phase of glycerol. The comparison of the isotopic effects in ethanol, as well as that with two isomers of propanol, provide a unique benchmark to study the relative importance of different kinds of disorder. In this work, the whole set of maxima in Cp/ T3 is discussed in the framework of the scaling procedure of Liu and Löhneysen [Europhys. Lett. 33 (1996) 617].

  7. Quantitative determination of protein molecular weight with an acoustic sensor; significance of specific versus non-specific binding.

    PubMed

    Mitsakakis, Konstantinos; Tsortos, Achilleas; Gizeli, Electra

    2014-08-21

    Surface acoustic wave sensors with integrated microfluidics for multi-sample sensing have been implemented in this work towards the quantitative correlation of the acoustic signal with the molecular weight of surface bound proteins investigating different interaction/binding conditions. The results are presented for: (i) four different biotinylated molecules (30 ≤ Mw ≤ 150 kDa) specifically binding to neutravidin; (ii) the same four non-biotinylated molecules, as well as neutravidin, adsorbing onto gold; and (iii) four cardiac marker proteins (86 ≤ Mw ≤ 540 kDa) specifically binding to their homologous antibodies. Surface plasmon resonance was employed as an independent optical mass sensor. A linear relationship was found to exist between the phase change of the acoustic signal and the molecular weight of the proteins in both cases of specific binding. In contrast, non-specific binding of proteins directly onto gold exhibited no such linear relationship. In all three cases phase change was correlated with the bound mass per area. The underlying mechanism behind the different behavior between specific and non-specific binding is discussed by taking into account the geometrical restrictions imposed by the size of the specific biorecognition molecule and the corresponding bound protein. Our results emphasize the quantitative nature of the phase of the acoustic signal in determining the Mw (in the case of specific binding) with a resolution of 15% and the mass of the bound proteins (in all cases), as well as the significance of the biorecognition molecules in deriving the molecular weight from acoustic or optical detectors.

  8. Modified oleic cottonseeds show altered content, composition and tissue-specific distribution of triacylglycerol molecular species.

    PubMed

    Horn, Patrick J; Sturtevant, Drew; Chapman, Kent D

    2014-01-01

    Targeted increases in monounsaturated (oleic acid) fatty acid content of refined cottonseed oil could support improved human nutrition and cardiovascular health. Genetic modifications of cottonseed fatty acid composition have been accomplished using several different molecular strategies. Modification of oleic acid content in cottonseed embryos using a dominant-negative protein approach, while successful in effecting change in the desired fatty acid composition, resulted in reduced oil content and seed viability. Here these changes in fatty acid composition were associated with changes in dominant molecular species of triacylglycerols (TAGs) and their spatial distributions within embryo tissues. A combination of mass spectrometry (MS)-based lipidomics approaches, including MS imaging of seed cryo-sections, revealed that cotton embryos expressing a non-functional allele of a Brassica napus delta-12 desaturase showed altered accumulation of TAG species, especially within cotyledonary tissues. While lipid analysis of seed extracts could demonstrate detailed quantitative changes in TAG species in transgenics, the spatial contribution of metabolite compartmentation could only be visualized by MS imaging. Our results suggest tissue-specific differences in TAG biosynthetic pathways within cotton embryos, and indicate the importance of considering the location of metabolites in tissues in addition to their identification and quantification when developing a detailed view of cellular metabolism.

  9. 77 FR 58499 - Substitution of Term in a Definition; Addition and Adoption of the Use of Specific...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-21

    ... 51-1 continues to read as follows: Authority: 56 FR 48976, Sept. 26, 1991, unless otherwise noted. 0...; Addition and Adoption of the Use of Specific Interchangeable or Synonymous Terms AGENCY: Committee...

  10. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  11. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    SciTech Connect

    Matsushita, Y. Murakawa, T. Shimamura, K. Oishi, M. Ohyama, T. Kurita, N.

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  12. Novel therapeutic approaches for pulmonary arterial hypertension: Unique molecular targets to site-specific drug delivery.

    PubMed

    Vaidya, Bhuvaneshwar; Gupta, Vivek

    2015-08-10

    Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by increased blood pressure in the small arterioles supplying blood to lungs for oxygenation. Advances in understanding of molecular and cellular biology techniques have led to the findings that PAH is indeed a cascade of diseases exploiting multi-faceted complex pathophysiology, with cellular proliferation and vascular remodeling being the key pathogenic events along with several cellular pathways involved. While current therapies for PAH do provide for amelioration of disease symptoms and acute survival benefits, their full therapeutic potential is hindered by patient incompliance and off-target side effects. To overcome the issues related with current therapy and to devise a more selective therapy, various novel pathways are being investigated for PAH treatment. In addition, inability to deliver anti-PAH drugs to the disease site i.e., distal pulmonary arterioles has been one of the major challenges in achieving improved patient outcomes and improved therapeutic efficacy. Several novel carriers have been explored to increase the selectivity of currently approved anti-PAH drugs and to act as suitable carriers for the delivery of investigational drugs. In the present review, we have discussed potential of various novel molecular pathways/targets including RhoA/Rho kinase, tyrosine kinase, endothelial progenitor cells, vasoactive intestinal peptide, and miRNA in PAH therapeutics. We have also discussed various techniques for site-specific drug delivery of anti-PAH therapeutics so as to improve the efficacy of approved and investigational drugs. This review will provide gainful insights into current advances in PAH therapeutics with an emphasis on site-specific drug payload delivery.

  13. 49 CFR 173.301a - Additional general requirements for shipment of specification cylinders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... temperature of 55 °C (131 °F) that is greater than permitted. (d) Cylinder pressure at 55 °C (131 °F). The... gases in specification cylinders. (b) Authorized cylinders not marked with a service pressure. For... marked or designated, except as provided in § 173.302a(b). For certain liquefied gases, the pressure...

  14. 49 CFR 173.301a - Additional general requirements for shipment of specification cylinders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... temperature of 55 °C (131 °F) that is greater than permitted. (d) Cylinder pressure at 55 °C (131 °F). The... gases in specification cylinders. (b) Authorized cylinders not marked with a service pressure. For... marked or designated, except as provided in § 173.302a(b). For certain liquefied gases, the pressure...

  15. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    PubMed

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-01

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. PMID:26785049

  16. The PP1 binding code: a molecular-lego strategy that governs specificity.

    PubMed

    Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

    2013-01-01

    Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo.

  17. All-organic microelectromechanical systems integrating specific molecular recognition--a new generation of chemical sensors.

    PubMed

    Ayela, Cédric; Dubourg, Georges; Pellet, Claude; Haupt, Karsten

    2014-09-01

    Cantilever-type all-organic microelectromechanical systems based on molecularly imprinted polymers for specific analyte recognition are used as chemical sensors. They are produced by a simple spray-coating-shadow-masking process. Analyte binding to the cantilever generates a measurable change in its resonance frequency. This allows label-free detection by direct mass sensing of low-molecular-weight analytes at nanomolar concentrations.

  18. Effect of Operating Parameters and Chemical Additives on Crystal Habit and Specific Cake Resistance of Zinc Hydroxide Precipitates

    SciTech Connect

    Alwin, Jennifer Louise

    1999-08-01

    The effect of process parameters and chemical additives on the specific cake resistance of zinc hydroxide precipitates was investigated. The ability of a slurry to be filtered is dependent upon the particle habit of the solid and the particle habit is influenced by certain process variables. The process variables studied include neutralization temperature, agitation type, and alkalinity source used for neutralization. Several commercially available chemical additives advertised to aid in solid/liquid separation were also examined in conjunction with hydroxide precipitation. A statistical analysis revealed that the neutralization temperature and the source of alkalinity were statistically significant in influencing the specific cake resistance of zinc hydroxide precipitates in this study. The type of agitation did not significantly effect the specific cake resistance of zinc hydroxide precipitates. The use of chemical additives in conjunction with hydroxide precipitation had a favorable effect on the filterability. The morphology of the hydroxide precipitates was analyzed using scanning electron microscopy.

  19. Telomere Capping Proteins are Structurally Related to RPA with an additional Telomere-Specific Domain

    SciTech Connect

    Gelinas, A.; Paschini, M; Reyes, F; Heroux, A; Batey, R; Lundblad, V; Wuttke, D

    2009-01-01

    Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

  20. Ab initio molecular simulations on specific interactions between amyloid beta and monosaccharides

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuya; Okamoto, Akisumi; Yano, Atsushi; Higai, Shin'ichi; Kondo, Takashi; Kamba, Seiji; Kurita, Noriyuki

    2012-09-01

    Aggregation of amyloid β (Aβ) peptides, which is a key pathogenetic event in Alzheimer's disease, can be caused by cell-surface saccharides. We here investigated stable structures of the solvated complexes of Aβ with some types of monosaccharides using molecular simulations based on protein-ligand docking and classical molecular mechanics methods. Moreover, the specific interactions between Aβ and the monosaccharides were elucidated at an electronic level by ab initio fragment molecular orbital calculations. Based on the results, we proposed which type of monosaccharide prefers to have large binding affinity to Aβ and inhibit the Aβ aggregation.

  1. Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.

    PubMed

    Wile, Brian M; Ban, Kiwon; Yoon, Young-Sup; Bao, Gang

    2014-10-01

    Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

  2. Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A.

    PubMed

    Turner, Nicholas W; Piletska, Elena V; Karim, Khalku; Whitcombe, Michael; Malecha, Michael; Magan, Naresh; Baggiani, Claudio; Piletsky, Sergey A

    2004-12-15

    A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.

  3. Investigation of effect of particle size and rumen fluid addition on specific methane yields of high lignocellulose grass silage.

    PubMed

    Wall, D M; Straccialini, B; Allen, E; Nolan, P; Herrmann, C; O'Kiely, P; Murphy, J D

    2015-09-01

    This work examines the digestion of advanced growth stage grass silage. Two variables were investigated: particle size (greater than 3 cm and less than 1cm) and rumen fluid addition. Batch studies indicated particle size and rumen fluid addition had little effect on specific methane yields (SMYs). In continuous digestion of 3 cm silage the SMY was 342 and 343 L CH4 kg(-1)VS, respectively, with and without rumen fluid addition. However, digester operation was significantly affected through silage floating on the liquor surface and its entanglement in the mixing system. Digestion of 1cm silage with no rumen fluid addition struggled; volatile fatty acid concentrations rose and SMYs dropped. The best case was 1cm silage with rumen fluid addition, offering higher SMYs of 371 L CH4 kg(-1)VS and stable operation throughout. Thus, physical and biological treatments benefited continuous digestion of high fibre grass silage.

  4. Tuning protein-protein interactions using cosolvents: specific effects of ionic and non-ionic additives on protein phase behavior.

    PubMed

    Hansen, Jan; Platten, Florian; Wagner, Dana; Egelhaaf, Stefan U

    2016-04-21

    Cosolvents are routinely used to modulate the (thermal) stability of proteins and, hence, their interactions with proteins have been studied intensely. However, less is known about their specific effects on protein-protein interactions, which we characterize in terms of the protein phase behavior. We analyze the phase behavior of lysozyme solutions in the presence of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), glycerol, and dimethyl sulfoxide (DMSO). We experimentally determined the crystallization boundary (XB) and, in combination with data on the cloud-point temperatures (CPTs), the crystallization gap. In agreement with other studies, our data indicate that the additives might affect the protein phase behavior through electrostatic screening and additive-specific contributions. At high salt concentrations, where electrostatic interactions are screened, both the CPT and the XB are found to be linear functions of the additive concentration. Their slopes quantify the additive-specific changes of the phase behavior and thus of the protein-protein interactions. While the specific effect of NaCl is to induce attractions between proteins, DMSO, glycerol and GuHCl (with increasing strength) weaken attractions and/or induce repulsions. Except for DMSO, changes of the CPT are stronger than those of the XB. Furthermore, the crystallization gap widens in the case of GuHCl and glycerol and narrows in the case of NaCl. We relate these changes to colloidal interaction models, namely square-well and patchy interactions. PMID:27020538

  5. Development and molecular characterization of wheat--Aegilops kotschyi addition and substitution lines with high grain protein, iron, and zinc.

    PubMed

    Rawat, Nidhi; Neelam, Kumari; Tiwari, Vijay K; Randhawa, Gursharn S; Friebe, Bernd; Gill, Bikram S; Dhaliwal, Harcharan S

    2011-11-01

    Over two billion people, depending largely on staple foods, suffer from deficiencies in protein and some micronutrients such as iron and zinc. Among various approaches to overcome protein and micronutrient deficiencies, biofortification through a combination of conventional and molecular breeding methods is the most feasible, cheapest, and sustainable approach. An interspecific cross was made between the wheat cultivar 'Chinese Spring' and Aegilops kotschyi Boiss. accession 396, which has a threefold higher grain iron and zinc concentrations and about 33% higher protein concentration than wheat cultivars. Recurrent backcrossing and selection for the micronutrient content was performed at each generation. Thirteen derivatives with high grain iron and zinc concentrations and contents, ash and ash micronutrients, and protein were analyzed for alien introgression. Morphological markers, high molecular weight glutenin subunit profiles, anchored wheat microsatellite markers, and GISH showed that addition and substitution of homoeologous groups 1, 2, and 7 chromosomes of Ae. kotschyi possess gene(s) for high grain micronutrients. The addition of 1U/1S had high molecular weight glutenin subunits with higher molecular weight than those of wheat, and the addition of 2S in most of the derivatives also enhanced grain protein content by over 20%. Low grain protein content in a derivative with a 2S-wheat translocation, waxy leaves, and absence of the gdm148 marker strongly suggests that the gene for higher grain protein content on chromosome 2S is orthologous to the grain protein QTL on the short arm of group 2 chromosomes.

  6. A molecular-gap device for specific determination of mercury ions

    NASA Astrophysics Data System (ADS)

    Guo, Zheng; Liu, Zhong-Gang; Yao, Xian-Zhi; Zhang, Kai-Sheng; Chen, Xing; Liu, Jin-Huai; Huang, Xing-Jiu

    2013-11-01

    Specific determination/monitoring of trace mercury ions (Hg2+) in environmental water is of significant importance for drinking safety. Complementarily to conventional inductively coupled plasma mass spectrometry and atomic emission/absorption spectroscopy, several methods, i.e., electrochemical, fluorescent, colorimetric, and surface enhanced Raman scattering approaches, have been developed recently. Despite great success, many inevitably encounter the interferences from other metal ions besides the complicated procedures and sophisticated equipments. Here we present a molecular-gap device for specific determination of trace Hg2+ in both standardized solutions and environmental samples based on conductivity-modulated glutathione dimer. Through a self-assembling technique, a thin film of glutathione monolayer capped Au nanoparticles is introduced into 2.5 μm-gap-electrodes, forming numerous double molecular layer gaps. Notably, the fabricated molecular-gap device shows a specific response toward Hg2+ with a low detection limit actually measured down to 1 nM. Theoretical calculations demonstrate that the specific sensing mechanism greatly depends on the electron transport ability of glutathione dimer bridged by heavy metal ions, which is determined by its frontier molecular orbital, not the binding energy.

  7. A molecular-gap device for specific determination of mercury ions

    PubMed Central

    Guo, Zheng; Liu, Zhong-Gang; Yao, Xian-Zhi; Zhang, Kai-Sheng; Chen, Xing; Liu, Jin-Huai; Huang, Xing-Jiu

    2013-01-01

    Specific determination/monitoring of trace mercury ions (Hg2+) in environmental water is of significant importance for drinking safety. Complementarily to conventional inductively coupled plasma mass spectrometry and atomic emission/absorption spectroscopy, several methods, i.e., electrochemical, fluorescent, colorimetric, and surface enhanced Raman scattering approaches, have been developed recently. Despite great success, many inevitably encounter the interferences from other metal ions besides the complicated procedures and sophisticated equipments. Here we present a molecular-gap device for specific determination of trace Hg2+ in both standardized solutions and environmental samples based on conductivity-modulated glutathione dimer. Through a self-assembling technique, a thin film of glutathione monolayer capped Au nanoparticles is introduced into 2.5 μm-gap-electrodes, forming numerous double molecular layer gaps. Notably, the fabricated molecular-gap device shows a specific response toward Hg2+ with a low detection limit actually measured down to 1 nM. Theoretical calculations demonstrate that the specific sensing mechanism greatly depends on the electron transport ability of glutathione dimer bridged by heavy metal ions, which is determined by its frontier molecular orbital, not the binding energy. PMID:24178058

  8. Alignment of absolute and relative molecular size specifications for a polyvalent pneumococcal polysaccharide vaccine (PNEUMOVAX 23).

    PubMed

    MacNair, John E; Desai, Tejal; Teyral, Jennifer; Abeygunawardana, Chitrananda; Hennessey, John P

    2005-03-01

    An approach was developed to align release and end-expiry specifications for molecular size for the polyvalent pneumococcal polysaccharide vaccine (PNEUMOVAX 23). Each of the 23 polysaccharide components of the vaccine was separately subjected to ultrasonication to produce a series of preparations of decreasing weight-average molecular mass (Mw). These size-reduced polysaccharides were analysed as monovalent solutions by high-performance size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and refractive index (RI) detection to measure their Mw. These samples were also analysed by HPSEC with rate nephelometry (RN) detection to measure their relative molecular size (r-MS). The data from the two molecular size measurements established a correlation between Mw and r-MS. For each polysaccharide component of the vaccine, this correlation permits the direct alignment of the r-MS specification in the final formulated product with the Mw specification for the monovalent polysaccharide preparation. The alignment of specifications provides a high level of assurance that the quality control of the final vaccine product is consistent with that of the polysaccharide starting materials.

  9. Enhancing Specific Energy and Power in Asymmetric Supercapacitors - A Synergetic Strategy based on the Use of Redox Additive Electrolytes

    NASA Astrophysics Data System (ADS)

    Singh, Arvinder; Chandra, Amreesh

    2016-05-01

    The strategy of using redox additive electrolyte in combination with multiwall carbon nanotubes/metal oxide composites leads to a substantial improvements in the specific energy and power of asymmetric supercapacitors (ASCs). When the pure electrolyte is optimally modified with a redox additive viz., KI, ~105% increase in the specific energy is obtained with good cyclic stability over 3,000 charge-discharge cycles and ~14.7% capacitance fade. This increase is a direct consequence of the iodine/iodide redox pairs that strongly modifies the faradaic and non-faradaic type reactions occurring on the surface of the electrodes. Contrary to what is shown in few earlier reports, it is established that indiscriminate increase in the concentration of redox additives will leads to performance loss. Suitable explanations are given based on theoretical laws. The specific energy or power values being reported in the fabricated ASCs are comparable or higher than those reported in ASCs based on toxic acetonitrile or expensive ionic liquids. The paper shows that the use of redox additive is economically favorable strategy for obtaining cost effective and environmentally friendly ASCs.

  10. Enhancing Specific Energy and Power in Asymmetric Supercapacitors - A Synergetic Strategy based on the Use of Redox Additive Electrolytes.

    PubMed

    Singh, Arvinder; Chandra, Amreesh

    2016-01-01

    The strategy of using redox additive electrolyte in combination with multiwall carbon nanotubes/metal oxide composites leads to a substantial improvements in the specific energy and power of asymmetric supercapacitors (ASCs). When the pure electrolyte is optimally modified with a redox additive viz., KI, ~105% increase in the specific energy is obtained with good cyclic stability over 3,000 charge-discharge cycles and ~14.7% capacitance fade. This increase is a direct consequence of the iodine/iodide redox pairs that strongly modifies the faradaic and non-faradaic type reactions occurring on the surface of the electrodes. Contrary to what is shown in few earlier reports, it is established that indiscriminate increase in the concentration of redox additives will leads to performance loss. Suitable explanations are given based on theoretical laws. The specific energy or power values being reported in the fabricated ASCs are comparable or higher than those reported in ASCs based on toxic acetonitrile or expensive ionic liquids. The paper shows that the use of redox additive is economically favorable strategy for obtaining cost effective and environmentally friendly ASCs. PMID:27184260

  11. Enhancing Specific Energy and Power in Asymmetric Supercapacitors - A Synergetic Strategy based on the Use of Redox Additive Electrolytes

    PubMed Central

    Singh, Arvinder; Chandra, Amreesh

    2016-01-01

    The strategy of using redox additive electrolyte in combination with multiwall carbon nanotubes/metal oxide composites leads to a substantial improvements in the specific energy and power of asymmetric supercapacitors (ASCs). When the pure electrolyte is optimally modified with a redox additive viz., KI, ~105% increase in the specific energy is obtained with good cyclic stability over 3,000 charge-discharge cycles and ~14.7% capacitance fade. This increase is a direct consequence of the iodine/iodide redox pairs that strongly modifies the faradaic and non-faradaic type reactions occurring on the surface of the electrodes. Contrary to what is shown in few earlier reports, it is established that indiscriminate increase in the concentration of redox additives will leads to performance loss. Suitable explanations are given based on theoretical laws. The specific energy or power values being reported in the fabricated ASCs are comparable or higher than those reported in ASCs based on toxic acetonitrile or expensive ionic liquids. The paper shows that the use of redox additive is economically favorable strategy for obtaining cost effective and environmentally friendly ASCs. PMID:27184260

  12. Molecular aspects of aromatic C additions to soils: Implications of biochar quality for ecosystem functionality

    EPA Science Inventory

    Solid residues of incomplete combustion (biochar or char) are continuously being added to soils due to natural vegetation fires in many ecosystems. However, new strategies for carbon sequestration in soils are likely to include the active addition of biochar to soils. Since bioc...

  13. Specific binding of molecularly targeted agents to pancreas tumors and impact on observed optical contrast

    NASA Astrophysics Data System (ADS)

    Samkoe, Kimberley S.; Hextrum, Shannon K.; Pardesi, Omar; O'Hara, Julia A.; Hasan, Tayyaba; Pogue, Brian W.

    2010-02-01

    In optical imaging it is thought that optimum tumor contrast can be achieved with the use of small-labeled molecular tracers that have high affinity to their targets and fast clearance rates from the blood stream and healthy tissues. An example of this is fluorescently tagged EGF to monitor the molecular activity of tumors, such as pancreatic cancer. Extensive fluorescence contrast analysis for fluorescence molecular tomography has been performed on the AsPC-1 pancreas tumor, grown orthotopically in mice; yet, the binding dynamics of the EGF-fluorescent agent in vivo is not completely known. The bulk pancreatic tumor displays 3:1 contrast relative to the normal pancreas at long times after injection; however, even higher levels of fluorescence in the liver, kidney and intestine suggest that molecular specificity for the tumor may be low. Mice were administered a fluorescently labeled EGF agent and were sacrificed at various time points post-injection. To analyze the amount of specific binding at each time point frozen tissue samples were fluorescently imaged, washed with saline to remove the interstitially distributed contrast agent, and then imaged again. This technique demonstrated that approximately ~10% of the molecular target was firmly bound to the cell, while 90% was mobile or unbound. This low binding ratio suggests that the contrast observed is from inherent properties of the tumor (i.e. enhanced permeability and retention effect) and not from specific bound contrast as previously anticipated. The use of EGF contrast agents in MRI-guided fluorescence tomography and the impact of low binding specificity are discussed.

  14. Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines.

    PubMed

    Hasterok, Robert; Wolny, Elzbieta; Kulak, Sylwia; Zdziechiewicz, Aleksandra; Maluszynska, Jolanta; Heneen, Waheeb K

    2005-07-01

    Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.

  15. Host specificity of North American Rhabdias spp. (Nematoda: Rhabdiasidae): combining field data and experimental infections with a molecular phylogeny.

    PubMed

    Langford, Gabriel J; Janovy, John

    2013-04-01

    Lungworms of the cosmopolitan genus Rhabdias are among the most common parasites of amphibians and squamate reptiles. The present study used experimental infections, field studies, and a molecular phylogeny to determine the host specificity of 6 Rhabdias spp. that infect snakes and anurans from North America. The molecular phylogeny suggests Rhabdias ranae from Nebraska and Mississippi may represent separate, cryptic species. In addition, the phylogeny strongly supports separate clades for anuran and snake lungworms. Field studies and experimental infections indicate that snake lungworms are generalist snake parasites; however, laboratory experiments also suggest that lizards can be infected under some environmental conditions. Lungworms from anurans were found not to infect salamanders or reptiles, in nature or in the laboratory; anuran lungworm species ranged from strict host specificity, e.g., R. ranae from Nebraska, to relative generalist, e.g., Rhabdias joaquinensis from Nebraska. Overall, host specificity for species of Rhabdias does not provide support for the evolution of progressive specialization over time. For most species of lungworms, host specificity in nature appears to be limited by both ecological and physiological factors, which vary between species and their hosts. Furthermore, some lungworms, e.g., Rhabdias bakeri from Missouri, appear to be tracking host resources instead of host phylogenies, an example of ecological fitting. PMID:22988815

  16. Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies

    PubMed Central

    Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

    2015-01-01

    Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage. PMID:26605008

  17. Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members.

    PubMed

    Rigoldi, Federica; Spero, Ludovica; Dalle Vedove, Andrea; Redaelli, Alberto; Parisini, Emilio; Gautieri, Alfonso

    2016-07-19

    Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels. PMID:27327839

  18. Quantum state specific reactant preparation in a molecular beam by rapid adiabatic passage.

    PubMed

    Chadwick, Helen; Hundt, P Morten; van Reijzen, Maarten E; Yoder, Bruce L; Beck, Rainer D

    2014-01-21

    Highly efficient preparation of molecules in a specific rovibrationally excited state for gas/surface reactivity measurements is achieved in a molecular beam using tunable infrared (IR) radiation from a single mode continuous wave optical parametric oscillator (cw-OPO). We demonstrate that with appropriate focusing of the IR radiation, molecules in the molecular beam crossing the fixed frequency IR field experience a Doppler tuning that can be adjusted to achieve complete population inversion of a two-level system by rapid adiabatic passage (RAP). A room temperature pyroelectric detector is used to monitor the excited fraction in the molecular beam and the population inversion is detected and quantified using IR bleaching by a second IR-OPO. The second OPO is also used for complete population transfer to an overtone or combination vibration via double resonance excitation using two spatially separated RAP processes.

  19. Quantum state specific reactant preparation in a molecular beam by rapid adiabatic passage.

    PubMed

    Chadwick, Helen; Hundt, P Morten; van Reijzen, Maarten E; Yoder, Bruce L; Beck, Rainer D

    2014-01-21

    Highly efficient preparation of molecules in a specific rovibrationally excited state for gas/surface reactivity measurements is achieved in a molecular beam using tunable infrared (IR) radiation from a single mode continuous wave optical parametric oscillator (cw-OPO). We demonstrate that with appropriate focusing of the IR radiation, molecules in the molecular beam crossing the fixed frequency IR field experience a Doppler tuning that can be adjusted to achieve complete population inversion of a two-level system by rapid adiabatic passage (RAP). A room temperature pyroelectric detector is used to monitor the excited fraction in the molecular beam and the population inversion is detected and quantified using IR bleaching by a second IR-OPO. The second OPO is also used for complete population transfer to an overtone or combination vibration via double resonance excitation using two spatially separated RAP processes. PMID:25669393

  20. Quantum state specific reactant preparation in a molecular beam by rapid adiabatic passage

    SciTech Connect

    Chadwick, Helen Hundt, P. Morten; Reijzen, Maarten E. van; Yoder, Bruce L.; Beck, Rainer D.

    2014-01-21

    Highly efficient preparation of molecules in a specific rovibrationally excited state for gas/surface reactivity measurements is achieved in a molecular beam using tunable infrared (IR) radiation from a single mode continuous wave optical parametric oscillator (cw-OPO). We demonstrate that with appropriate focusing of the IR radiation, molecules in the molecular beam crossing the fixed frequency IR field experience a Doppler tuning that can be adjusted to achieve complete population inversion of a two-level system by rapid adiabatic passage (RAP). A room temperature pyroelectric detector is used to monitor the excited fraction in the molecular beam and the population inversion is detected and quantified using IR bleaching by a second IR-OPO. The second OPO is also used for complete population transfer to an overtone or combination vibration via double resonance excitation using two spatially separated RAP processes.

  1. Quantum state specific reactant preparation in a molecular beam by rapid adiabatic passage

    NASA Astrophysics Data System (ADS)

    Chadwick, Helen; Hundt, P. Morten; van Reijzen, Maarten E.; Yoder, Bruce L.; Beck, Rainer D.

    2014-01-01

    Highly efficient preparation of molecules in a specific rovibrationally excited state for gas/surface reactivity measurements is achieved in a molecular beam using tunable infrared (IR) radiation from a single mode continuous wave optical parametric oscillator (cw-OPO). We demonstrate that with appropriate focusing of the IR radiation, molecules in the molecular beam crossing the fixed frequency IR field experience a Doppler tuning that can be adjusted to achieve complete population inversion of a two-level system by rapid adiabatic passage (RAP). A room temperature pyroelectric detector is used to monitor the excited fraction in the molecular beam and the population inversion is detected and quantified using IR bleaching by a second IR-OPO. The second OPO is also used for complete population transfer to an overtone or combination vibration via double resonance excitation using two spatially separated RAP processes.

  2. Specificity of Furanoside–Protein Recognition through Antibody Engineering and Molecular Modeling

    PubMed Central

    Lak, Parnian; Makeneni, Spandana; Woods, Robert J.

    2014-01-01

    Recognition of furanosides (five-membered ring sugars) by proteins plays important roles in host–pathogen interactions. In comparison to their six-membered ring counterparts (pyranosides), detailed studies of the molecular motifs involved in the recognition of furanosides by proteins are scarce. Here the first in-depth molecular characterization of a furanoside–protein interaction system, between an antibody (CS-35) and cell wall polysaccharides of mycobacteria, including the organism responsible for tuberculosis is reported. The approach was centered on the generation of the single chain variable fragment of CS-35 and a rational library of its mutants. Investigating the interaction from various aspects revealed the structural motifs that govern the interaction, as well as the relative contribution of molecular forces involved in the recognition. The specificity of the recognition was shown to originate mainly from multiple CH–π interactions and, to a lesser degree, hydrogen bonds formed in critical distances and geometries. PMID:25413161

  3. Fiber-specific molecular features of tumors induced in rat peritoneum.

    PubMed Central

    Unfried, K; Roller, M; Pott, F; Friemann, J; Dehnen, W

    1997-01-01

    Molecular markers such as mutational spectra or mRNA expression patterns may give some indication of the mechanisms of carcinogenesis induced by fibers and other carcinogens. In our study, tumors were induced by application of crocidolite asbestos or benzo[a]pyrene (B[a]P) to rat peritoneum. DNA and RNA of these tumors were subjected to analysis of point mutations and to investigation of mRNA expression patterns. With both assays we found typical features depending on the type of carcinogen applied. The analysis of point mutations in the tumor suppressor gene p53 revealed mutations in the B[a]P-induced tumors. However, in the tumors induced by crocidolite asbestos that were of the same tumor type as those induced by B[a]P, mutations in p53 were not detectable. Every mutation detected on the DNA level causes an amino acid substitution within one of the functional domains of the tumor suppressor protein. Therefore, these mutations seem to be of biological relevance for tumor progression and indicate a difference in the carcinogenesis regarding the type of the carcinogenic substance. An additional specificity of crocidolite-induced tumors was detectable by analyzing the mRNA expression of the tumor suppressor gene WT1, which is known to be expressed in human mesothelial and mesothelioma cells. A relatively high amount of WT1 mRNA was measured by quantitative competitive reverse transcription-polymerase using RNA extracted from crocidolite-induced tumors. However, WT1 seems to be expressed on a rather low level in tumors induced by B[a]P. Images Figure 1. Figure 2. Figure 2. Figure 2. Figure 2. PMID:9400707

  4. Rapid identification of Candida dubliniensis using a species-specific molecular beacon.

    PubMed

    Park, S; Wong, M; Marras, S A; Cross, E W; Kiehn, T E; Chaturvedi, V; Tyagi, S; Perlin, D S

    2000-08-01

    Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference Candida strains was used to develop molecular beacon probes for rapid, high-fidelity identification of C. dubliniensis as well as C. albicans. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely contained C. albicans and a smaller amount of C. dubliniensis, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes BsmAI and NspBII, which cleave recognition sequences within the ITS2 regions of C. dubliniensis and C. albicans, respectively. Molecular beacons are promising new probes for the rapid detection of Candida species.

  5. Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

    PubMed

    Popp, Maximilian Wei-Lin

    2015-01-01

    Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant. PMID:25560076

  6. Communication: High pressure specific heat spectroscopy reveals simple relaxation behavior of glass forming molecular liquid.

    PubMed

    Roed, Lisa Anita; Niss, Kristine; Jakobsen, Bo

    2015-12-14

    The frequency dependent specific heat has been measured under pressure for the molecular glass forming liquid 5-polyphenyl-4-ether in the viscous regime close to the glass transition. The temperature and pressure dependences of the characteristic time scale associated with the specific heat is compared to the equivalent time scale from dielectric spectroscopy performed under identical conditions. It is shown that the ratio between the two time scales is independent of both temperature and pressure. This observation is non-trivial and demonstrates the existence of specially simple molecular liquids in which different physical relaxation processes are both as function of temperature and pressure/density governed by the same underlying "inner clock." Furthermore, the results are discussed in terms of the recent conjecture that van der Waals liquids, like the measured liquid, comply to the isomorph theory.

  7. In vitro quantification of specific microRNA using molecular beacons.

    PubMed

    Baker, Meredith B; Bao, Gang; Searles, Charles D

    2012-01-01

    MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)-DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.

  8. Polyfunctional epoxies - Different molecular weights of brominated polymeric additives as flame retardants in graphite composites

    NASA Technical Reports Server (NTRS)

    Nir, Z.; Gilwee, W. J.; Kourtides, D. A.; Parker, J. A.

    1983-01-01

    The imparting of flame retardancy to graphite-reinforced composites without incurring mechanical property deterioration is investigated for the case of an experimental, trifunctional epoxy resin incorporating brominated polymeric additives (BPAs) of the diglycidyl type. Such mechanical properties as flexural strength and modulus, and short beam shear strength, were measured in dry and in hot/wet conditions, and the glass transition temperature, flammability, and water absorption were measured and compared with nonbromilated systems. Another comparison was made with a tetrafunctional epoxy system. The results obtained are explained in terms of differences in the polymeric backbone length of the bromine carrier polymer. BPAs are found to be a reliable bromine source for fire inhibition in carbon-reinforced composites without compromise of mechanical properties.

  9. Boronate Affinity-Molecularly Imprinted Biocompatible Probe: An Alternative for Specific Glucose Monitoring.

    PubMed

    Chen, Guosheng; Qiu, Junlang; Fang, Xu'an; Xu, Jianqiao; Cai, Siying; Chen, Qing; Liu, Yan; Zhu, Fang; Ouyang, Gangfeng

    2016-08-19

    A biocompatible probe for specific glucose recognition is based on photoinitiated boronate affinity-molecular imprinted polymers (BA-MIPs). The unique pre-self-assembly between glucose and boronic acids creates glucose-specific memory cavities in the BA-MIPs coating. As a result, the binding constant toward glucose was enhanced by three orders of magnitude. The BA-MIPs probe was applied to glucose determination in serum and urine and implanted into plant tissues for low-destructive and long-term in vivo continuous glucose monitoring. PMID:27411946

  10. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

    PubMed Central

    Williams, Ryan M.; Kulick, Amanda R.; Yedlapalli, Srilakshmi; Battistella, Louisa; Hajiran, Cyrus J.; Sooter, Letha J.

    2014-01-01

    Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd = 9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device. PMID:25400940

  11. A specific molecular beacon probe for the detection of human prostate cancer cells.

    PubMed

    Jiang, Yu Lin; McGoldrick, Christopher A; Yin, Deling; Zhao, Jing; Patel, Vini; Brannon, Marianne F; Lightner, Janet W; Krishnan, Koyamangalath; Stone, William L

    2012-06-01

    The small-molecule, water-soluble molecular beacon probe 1 is hydrolyzed by the lysate and living cells of human prostate cancer cell lines (LNCaP), resulting in strong green fluorescence. In contrast, probe 1 does not undergo significant hydrolysis in either the lysate or living cells of human nontumorigenic prostate cells (RWPE-1). These results, corroborated by UV-Vis spectroscopy and fluorescent microscopy, reveal that probe 1 is a sensitive and specific fluorogenic and chromogenic sensor for the detection of human prostate cancer cells among nontumorigenic prostate cells and that carboxylesterase activity is a specific biomarker for human prostate cancer cells.

  12. Fusarium diversity in soil using a specific molecular approach and a cultural approach.

    PubMed

    Edel-Hermann, Véronique; Gautheron, Nadine; Mounier, Arnaud; Steinberg, Christian

    2015-04-01

    Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples.

  13. Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides.

    PubMed

    Du, Yuzhe; Nomura, Yoshiko; Luo, Ningguang; Liu, Zhiqi; Lee, Jung-Eun; Khambay, Bhupinder; Dong, Ke

    2009-01-15

    Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an alpha-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report the identification of a residue G(1111) and two positively charged lysines immediately downstream of G(1111) in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G(1111), a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G(1111) had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.

  14. Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides

    SciTech Connect

    Du Yuzhe; Nomura, Yoshiko; Luo Ningguang; Liu Zhiqi; Lee, Jung-Eun; Khambay, Bhupinder; Dong Ke

    2009-01-15

    Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an {alpha}-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report the identification of a residue G{sup 1111} and two positively charged lysines immediately downstream of G{sup 1111} in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G{sup 1111}, a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G{sup 1111} had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.

  15. Fusarium diversity in soil using a specific molecular approach and a cultural approach.

    PubMed

    Edel-Hermann, Véronique; Gautheron, Nadine; Mounier, Arnaud; Steinberg, Christian

    2015-04-01

    Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples. PMID:25655778

  16. An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone.

    PubMed

    An, Byung Chull; Lee, Seung Sik; Jung, Hyun Suk; Kim, Jin Young; Lee, Yuno; Lee, Keun Woo; Lee, Sang Yeol; Tripathi, Bhumi Nath; Chung, Byung Yeoup

    2015-09-14

    Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone. PMID:26278368

  17. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  18. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    PubMed

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-01

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer.

  19. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    PubMed

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-01

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer. PMID:27152751

  20. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

    PubMed Central

    Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B.

    2015-01-01

    Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors. PMID:27013913

  1. Single molecule vibrationally mediated chemistry. Towards state-specific strategies for molecular handling

    NASA Astrophysics Data System (ADS)

    Pascual, J. I.

    2005-08-01

    Tunnelling electrons may scatter inelastically with an adsorbate, releasing part of their energy through the excitation of molecular vibrations. The resolution of inelastic processes with a low temperature scanning tunnelling microscope (STM) provides a valuable tool to chemically characterize single adsorbates and their adsorption mechanisms. Here, we present a molecular scale picture of single molecule vibrational chemistry, as resolved by STM. To understand the way a reaction proceed it is needed knowledge about both the excitation and damping of a molecular vibration. The excitation is mediated by the specific coupling between electronic molecular resonances present at the Fermi level and vibrational states of the adsorbate. Thus, the two-dimensional mapping of the inelastic signal with an STM provides the spatial distribution of the adsorbate electronic states (near the Fermi level) which are predominantly coupled to the particular vibrational mode observed. The damping of the vibration follows a competition between different mechanisms, mediated via the creation of electron-hole pairs or via anharmonic coupling between vibrational states. This latter case give rise to effective energy transfer mechanisms which eventually may focus vibrational energy in a specific reaction coordinate. In this single-molecule work-bench, STM provides alternative tools to understand reactivity in the limit of low excitation rate, which demonstrate the existence of state-specific excitation strategies which may lead to selectivity in the product of a reaction. The author acknowledges his co-workers in the work presented here, H. Conrad, N. Lorente, H.-P. Rust, and Z. Song, as well as collaborations with J. Gómez Herrero, J.J. Jackiw, D. Sánchez-Portal and P.S. Weiss.

  2. Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    NASA Astrophysics Data System (ADS)

    Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

  3. Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    PubMed Central

    Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    2010-01-01

    Summary Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity. PMID:19488728

  4. Evolvability of individual traits in a multivariate context: partitioning the additive genetic variance into common and specific components.

    PubMed

    McGuigan, Katrina; Blows, Mark W

    2010-07-01

    Genetic covariation among multiple traits will bias the direction of evolution. Although a trait's phenotypic context is crucial for understanding evolutionary constraints, the evolutionary potential of one (focal) trait, rather than the whole phenotype, is often of interest. The extent to which a focal trait can evolve independently depends on how much of the genetic variance in that trait is unique. Here, we present a hypothesis-testing framework for estimating the genetic variance in a focal trait that is independent of variance in other traits. We illustrate our analytical approach using two Drosophila bunnanda trait sets: a contact pheromone system comprised of cuticular hydrocarbons (CHCs), and wing shape, characterized by relative warps of vein position coordinates. Only 9% of the additive genetic variation in CHCs was trait specific, suggesting individual traits are unlikely to evolve independently. In contrast, most (72%) of the additive genetic variance in wing shape was trait specific, suggesting relative warp representations of wing shape could evolve independently. The identification of genetic variance in focal traits that is independent of other traits provides a way of studying the evolvability of individual traits within the broader context of the multivariate phenotype.

  5. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.

    PubMed

    Jin, Peng; Kang, Zhen; Yuan, Panhong; Du, Guocheng; Chen, Jian

    2016-05-01

    Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides. PMID:26851304

  6. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.

    PubMed

    Jin, Peng; Kang, Zhen; Yuan, Panhong; Du, Guocheng; Chen, Jian

    2016-05-01

    Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.

  7. A Telomerase-Specific Doxorubicin-Releasing Molecular Beacon for Cancer Theranostics.

    PubMed

    Ma, Yi; Wang, Zhaohui; Zhang, Min; Han, Zhihao; Chen, Dan; Zhu, Qiuyun; Gao, Weidong; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    A molecular beacon-based drug delivery system was designed for both detection of telomerase activity in living cells and telomerase-triggered drug release for precise cancer treatment. This system is composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences hybridized with telomerase primers. Molecules of the anticancer drug doxorubicin were intercalated into the stem region of the DNA sequence. The presence of telomerase will elongate the primers, leading to inner chain substitution followed by the release of the FITC fluorescence and the trapped doxorubicin. This molecular beacon could specifically distinguish tumor cells and normal cells based on telomerase activity, precisely release doxorubicin in response to telomerase activity in the tumor cells, and prevent toxicity to normal organs. PMID:26848056

  8. Molecular basis for species-specific sensitivity to "hot" chili peppers.

    PubMed

    Jordt, Sven-Eric; Julius, David

    2002-02-01

    Chili peppers produce the pungent vanilloid compound capsaicin, which offers protection from predatory mammals. Birds are indifferent to the pain-producing effects of capsaicin and therefore serve as vectors for seed dispersal. Here, we determine the molecular basis for this species-specific behavioral response by identifying a domain of the rat vanilloid receptor that confers sensitivity to capsaicin to the normally insensitive chicken ortholog. Like its mammalian counterpart, the chicken receptor is activated by heat or protons, consistent with the fact that both mammals and birds detect noxious heat and experience thermal hypersensitivity. Our findings provide a molecular basis for the ecological phenomenon of directed deterence and suggest that the capacity to detect capsaicin-like inflammatory substances is a recent acquisition of mammalian vanilloid receptors.

  9. A Telomerase-Specific Doxorubicin-Releasing Molecular Beacon for Cancer Theranostics.

    PubMed

    Ma, Yi; Wang, Zhaohui; Zhang, Min; Han, Zhihao; Chen, Dan; Zhu, Qiuyun; Gao, Weidong; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    A molecular beacon-based drug delivery system was designed for both detection of telomerase activity in living cells and telomerase-triggered drug release for precise cancer treatment. This system is composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences hybridized with telomerase primers. Molecules of the anticancer drug doxorubicin were intercalated into the stem region of the DNA sequence. The presence of telomerase will elongate the primers, leading to inner chain substitution followed by the release of the FITC fluorescence and the trapped doxorubicin. This molecular beacon could specifically distinguish tumor cells and normal cells based on telomerase activity, precisely release doxorubicin in response to telomerase activity in the tumor cells, and prevent toxicity to normal organs.

  10. Molecular basis for species-specific sensitivity to "hot" chili peppers.

    PubMed

    Jordt, Sven-Eric; Julius, David

    2002-02-01

    Chili peppers produce the pungent vanilloid compound capsaicin, which offers protection from predatory mammals. Birds are indifferent to the pain-producing effects of capsaicin and therefore serve as vectors for seed dispersal. Here, we determine the molecular basis for this species-specific behavioral response by identifying a domain of the rat vanilloid receptor that confers sensitivity to capsaicin to the normally insensitive chicken ortholog. Like its mammalian counterpart, the chicken receptor is activated by heat or protons, consistent with the fact that both mammals and birds detect noxious heat and experience thermal hypersensitivity. Our findings provide a molecular basis for the ecological phenomenon of directed deterence and suggest that the capacity to detect capsaicin-like inflammatory substances is a recent acquisition of mammalian vanilloid receptors. PMID:11853675

  11. Molecular cytogenetic identification of a wheat-rye 1R addition line with multiple spikelets and resistance to powdery mildew.

    PubMed

    Yang, Wujuan; Wang, Changyou; Chen, Chunhuan; Wang, Yajuan; Zhang, Hong; Liu, Xinlun; Ji, Wanquan

    2016-04-01

    Alien addition lines are important for transferring useful genes from alien species into common wheat. Rye is an important and valuable gene resource for improving wheat disease resistance, yield, and environment adaptation. A new wheat-rye addition line, N9436B, was developed from the progeny of the cross of common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) cultivar Shaanmai 611 and rye (Secale cereal L., 2n = 2x = 14, RR) accession Austrian rye. We characterized this new line by cytology, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), molecular markers, and disease resistance screening. N9436B was stable in morphology and cytology, with a chromosome composition of 2n = 42 + 2t = 22II. GISH investigations showed that this line contained two rye chromosomes. GISH, FISH, and molecular maker identification suggested that the introduced R chromosome and the missing wheat chromosome arms were 1R chromosome and 2DL chromosome arm, respectively. N9436B exhibited 30-37 spikelets per spike and a high level of resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) isolate E09 at the seedling stage. N9436B was cytologically stable, had the trait of multiple spikelets, and was resistant to powdery mildew; this line should thus be useful in wheat improvement.

  12. Environmental Molecular Sciences Laboratory Operations System: Version 4.0 - system requirements specification

    SciTech Connect

    Kashporenko, D.

    1996-07-01

    This document is intended to provide an operations standard for the Environmental Molecular Sciences Laboratory OPerations System (EMSL OPS). It is directed toward three primary audiences: (1) Environmental Molecular Sciences Laboratory (EMSL) facility and operations personnel; (2) laboratory line managers and staff; and (3) researchers, equipment operators, and laboratory users. It is also a statement of system requirements for software developers of EMSL OPS. The need for a finely tuned, superior research environment as provided by the US Department of Energy`s (DOE) Environmental Molecular Sciences Laboratory has never been greater. The abrupt end of the Cold War and the realignment of national priorities caused major US and competing overseas laboratories to reposition themselves in a highly competitive research marketplace. For a new laboratory such as the EMSL, this means coming into existence in a rapidly changing external environment. For any major laboratory, these changes create funding uncertainties and increasing global competition along with concomitant demands for higher standards of research product quality and innovation. While more laboratories are chasing fewer funding dollars, research ideas and proposals, especially for molecular-level research in the materials and biological sciences, are burgeoning. In such an economically constrained atmosphere, reduced costs, improved productivity, and strategic research project portfolio building become essential to establish and maintain any distinct competitive advantage. For EMSL, this environment and these demands require clear operational objectives, specific goals, and a well-crafted strategy. Specific goals will evolve and change with the evolution of the nature and definition of DOE`s environmental research needs. Hence, EMSL OPS is designed to facilitate migration of these changes with ease into every pertinent job function, creating a facile {open_quotes}learning organization.{close_quotes}

  13. Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

    PubMed Central

    Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

    2015-01-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

  14. Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

    PubMed

    Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

    2015-05-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

  15. Molecular determinants of humoral immune specificity for the occupational allergen, methylene diphenyl diisocyanate.

    PubMed

    Wisnewski, Adam V; Liu, Jian

    2013-06-01

    Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents.

  16. A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose.

    PubMed

    Ng, Sim-Kun; Huang, Yu-Tsyr; Lee, Yuan-Chuan; Low, Ee-Ling; Chiu, Cheng-Hsun; Chen, Shiu-Ling; Mao, Liang-Chi; Chang, Margaret Dah-Tsyr

    2014-01-01

    Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens. PMID:25541995

  17. A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose.

    PubMed

    Ng, Sim-Kun; Huang, Yu-Tsyr; Lee, Yuan-Chuan; Low, Ee-Ling; Chiu, Cheng-Hsun; Chen, Shiu-Ling; Mao, Liang-Chi; Chang, Margaret Dah-Tsyr

    2014-01-01

    Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.

  18. A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen-Associated Molecular Patterns of Bacteria through Rhamnose

    PubMed Central

    Ng, Sim-Kun; Huang, Yu-Tsyr; Lee, Yuan-Chuan; Low, Ee-Ling; Chiu, Cheng-Hsun; Chen, Shiu-Ling; Mao, Liang-Chi; Chang, Margaret Dah-Tsyr

    2014-01-01

    Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens. PMID:25541995

  19. Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator

    PubMed Central

    Severinov, Konstantin; Minakhin, Leonid; Sekine, Shun-ichi; Lopatina, Anna; Yokoyama, Shigeyuki

    2014-01-01

    Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue. PMID:25105059

  20. Specific interactions between amyloid-β peptide and curcumin derivatives: Ab initio molecular simulations

    NASA Astrophysics Data System (ADS)

    Ishimura, Hiromi; Kadoya, Ryushi; Suzuki, Tomoya; Murakawa, Takeru; Shulga, Sergiy; Kurita, Noriyuki

    2015-07-01

    Alzheimer's disease is caused by accumulation of amyloid-β (Aβ) peptides in a brain. To suppress the production of Aβ peptides, it is effective to inhibit the cleavage of amyloid precursor protein (APP) by secretases. However, because the secretases also play important roles to produce vital proteins for human body, inhibitors for the secretases may have side effects. To propose new agents for protecting the cleavage site of APP from the attacking of the γ-secretase, we have investigated here the specific interactions between a short APP peptide and curcumin derivatives, using protein-ligand docking as well as ab initio molecular simulations.

  1. Molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from Calystegia sepium.

    PubMed

    Van Damme, E J; Barre, A; Verhaert, P; Rougé, P; Peumans, W J

    1996-11-18

    cDNA clones encoding the mitogenic mannose/maltose-specific lectin from the rhizomes of hedge bindweed (Calystegia sepium) have been isolated and sequenced. Comparison of the deduced amino acid sequence and the molecular weight of the lectin subunit as determined by mass spectrometry indicated that the mature protein comprises the entire open reading frame of the cDNA, which implies that the primary translation product contains no signal peptide and is not proteolytically processed. Searches in the databases revealed sequence homology with the previously described lectins from the taxonomically unrelated Moraceae species Artocarpus integrifolia and Maclura pomifera.

  2. An accurate metalloprotein-specific scoring function and molecular docking program devised by a dynamic sampling and iteration optimization strategy.

    PubMed

    Bai, Fang; Liao, Sha; Gu, Junfeng; Jiang, Hualiang; Wang, Xicheng; Li, Honglin

    2015-04-27

    Metalloproteins, particularly zinc metalloproteins, are promising therapeutic targets, and recent efforts have focused on the identification of potent and selective inhibitors of these proteins. However, the ability of current drug discovery and design technologies, such as molecular docking and molecular dynamics simulations, to probe metal-ligand interactions remains limited because of their complicated coordination geometries and rough treatment in current force fields. Herein we introduce a robust, multiobjective optimization algorithm-driven metalloprotein-specific docking program named MpSDock, which runs on a scheme similar to consensus scoring consisting of a force-field-based scoring function and a knowledge-based scoring function. For this purpose, in this study, an effective knowledge-based zinc metalloprotein-specific scoring function based on the inverse Boltzmann law was designed and optimized using a dynamic sampling and iteration optimization strategy. This optimization strategy can dynamically sample and regenerate decoy poses used in each iteration step of refining the scoring function, thus dramatically improving both the effectiveness of the exploration of the binding conformational space and the sensitivity of the ranking of the native binding poses. To validate the zinc metalloprotein-specific scoring function and its special built-in docking program, denoted MpSDockZn, an extensive comparison was performed against six universal, popular docking programs: Glide XP mode, Glide SP mode, Gold, AutoDock, AutoDock4Zn, and EADock DSS. The zinc metalloprotein-specific knowledge-based scoring function exhibited prominent performance in accurately describing the geometries and interactions of the coordination bonds between the zinc ions and chelating agents of the ligands. In addition, MpSDockZn had a competitive ability to sample and identify native binding poses with a higher success rate than the other six docking programs.

  3. Molecularly imprinted polymers on a silica surface for the adsorption of tobacco-specific nitrosamines in mainstream cigarette smoke.

    PubMed

    Li, Min-ting; Zhu, Yong-yan; Li, Li; Wang, Wen-na; Yin, Yong-guan; Zhu, Quan-hong

    2015-07-01

    Tobacco-specific nitrosamines are one of the most important groups of carcinogens in tobacco products. Using adsorbents as filter additives is an effective way to reduce tobacco-specific nitrosamines in cigarette smoke. Molecularly imprinted polymers (MIPs) using nicotinamide as template were grafted on the silica gel surface to obtain MIP@SiO2 and employed as filter additives to absorb tobacco-specific nitrosamines in mainstream cigarette smoke. Four milligrams of MIP@SiO2 per cigarette was added to the interface between filter and tobacco rod to prepare a binary filter system. The mainstream smoke was collected on an industry-standard Cambridge filter pad and extracted with ammonium acetate aqueous solution before analysis. Compared to the cigarette smoke of the control group, the levels of tobacco-specific nitrosamines with silica gel and with MIP@SiO2 were both reduced, and the adsorption rates of N-nitrosonornicotine, N-nitrosoanabasine, N-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridine)-1-butanone with silica gel and with MIP@SiO2 were 20.76, 15.32, 18.79, and 18.01%, and 41.33, 34.04, 37.86, and 35.53%, respectively. Furthermore the content of total particle materials in cigarette smoke with silica gel was decreased evidently but showed no observable change with MIP@SiO2 . It indicated MIP@SiO2 could selectively reduce tobacco-specific nitrosamines in the mainstream cigarette smoke with no change to the cigarette flavor.

  4. Molecularly imprinted polymers on a silica surface for the adsorption of tobacco-specific nitrosamines in mainstream cigarette smoke.

    PubMed

    Li, Min-ting; Zhu, Yong-yan; Li, Li; Wang, Wen-na; Yin, Yong-guan; Zhu, Quan-hong

    2015-07-01

    Tobacco-specific nitrosamines are one of the most important groups of carcinogens in tobacco products. Using adsorbents as filter additives is an effective way to reduce tobacco-specific nitrosamines in cigarette smoke. Molecularly imprinted polymers (MIPs) using nicotinamide as template were grafted on the silica gel surface to obtain MIP@SiO2 and employed as filter additives to absorb tobacco-specific nitrosamines in mainstream cigarette smoke. Four milligrams of MIP@SiO2 per cigarette was added to the interface between filter and tobacco rod to prepare a binary filter system. The mainstream smoke was collected on an industry-standard Cambridge filter pad and extracted with ammonium acetate aqueous solution before analysis. Compared to the cigarette smoke of the control group, the levels of tobacco-specific nitrosamines with silica gel and with MIP@SiO2 were both reduced, and the adsorption rates of N-nitrosonornicotine, N-nitrosoanabasine, N-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridine)-1-butanone with silica gel and with MIP@SiO2 were 20.76, 15.32, 18.79, and 18.01%, and 41.33, 34.04, 37.86, and 35.53%, respectively. Furthermore the content of total particle materials in cigarette smoke with silica gel was decreased evidently but showed no observable change with MIP@SiO2 . It indicated MIP@SiO2 could selectively reduce tobacco-specific nitrosamines in the mainstream cigarette smoke with no change to the cigarette flavor. PMID:25914259

  5. A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity.

    PubMed

    Li, Xiaomin; Huang, Yong; Guan, Yuan; Zhao, Meiping; Li, Yuanzong

    2007-02-12

    We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3' (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the "wrong" genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3' exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3'-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3'-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.

  6. The DNase of gammaherpesviruses impairs recognition by virus-specific CD8+ T cells through an additional host shutoff function.

    PubMed

    Zuo, Jianmin; Thomas, Wendy; van Leeuwen, Daphne; Middeldorp, Jaap M; Wiertz, Emmanuel J H J; Ressing, Maaike E; Rowe, Martin

    2008-03-01

    The DNase/alkaline exonuclease (AE) genes are well conserved in all herpesvirus families, but recent studies have shown that the AE proteins of gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) exhibit an additional function which shuts down host protein synthesis. One correlate of this additional shutoff function is that levels of cell surface HLA molecules are downregulated, raising the possibility that shutoff/AE genes of gammaherpesviruses might contribute to viral immune evasion. In this study, we show that both BGLF5 (EBV) and SOX (KSHV) shutoff/AE proteins do indeed impair the ability of virus-specific CD8+ T-cell clones to recognize endogenous antigen via HLA class I. Random mutagenesis of the BGLF5 gene enabled us to genetically separate the shutoff and AE functions and to demonstrate that the shutoff function was the critical factor determining whether BGLF5 mutants can impair T-cell recognition. These data provide further evidence that EBV has multiple mechanisms to modulate HLA class I-restricted T-cell responses, thus enabling the virus to replicate and persist in the immune-competent host.

  7. Optimization of the thermophilic anaerobic co-digestion of pig manure, agriculture waste and inorganic additive through specific methanogenic activity.

    PubMed

    Jiménez, J; Cisneros-Ortiz, M E; Guardia-Puebla, Y; Morgan-Sagastume, J M; Noyola, A

    2014-01-01

    The anaerobic co-digestion of three wastes (manure, rice straw and clay residue, an inorganic additive) at different concentration levels and their interactive effects on methanogenic activity were investigated in this work at thermophilic conditions in order to enhance hydrolytic activity and methane production. A central composite design and the response surface methodology were applied for the optimization of specific methanogenic activity (SMA) by assessing their interaction effects with a reduced number of experiments. The results showed a significant interaction among the wastes on the SMA and confirmed that co-digestion enhances methane production. Rice straw apparently did not supply a significant amount of substrate to make a difference in SMA or methane yield. On the other hand, clay residue had a positive effect as an inorganic additive for stimulating the anaerobic process, based on its mineral content and its adsorbent properties for ammonia. Finally, the optimal conditions for achieving a thermophilic SMA value close to 1.4 g CH4-COD/g VSS · d(-1) were 20.3 gVSS/L of manure, 9.8 gVSS/L of rice straw and 3.3 gTSS/L of clay. PMID:24959998

  8. Configuration control on the shape memory stiffness of molecularly imprinted polymer for specific uptake of creatinine

    NASA Astrophysics Data System (ADS)

    Ang, Qian Yee; Zolkeflay, Muhammad Helmi; Low, Siew Chun

    2016-04-01

    In this study, sol-gel processing was proposed to prepare a creatinine (Cre)-imprinted molecularly imprinted polymer (MIP). The intermolecular interaction constituted by the cross-linkers, i.e., 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) and aluminium ion (Al3+), was studied and compared in order to form a confined matrix that promises the effectiveness of molecular imprinting. In view of the shape recognition, the hydrogen bonded Cre-AMPS did not demonstrate good recognition of Cre, with Cre binding found only at 5.70 ± 0.15 mg g-1 of MIP. Whilst, MIP cross-linked using Al3+ was able to attain an excellent Cre adsorption capacity of 19.48 ± 0.64 mg g-1 of MIP via the stronger ionic interaction of Cre-Al3+. Based on the Scatchard analysis, a higher Cre concentration in testing solution required greater driving force to resolve the binding resistance of Cre molecules, so as to have a precise Cre binding with shape factor. The molecular recognition ability of Cre-MIP in present work was shape-specific for Cre as compared to its structural analogue, 2-pyrrolidinone (2-pyr), by an ideal selectivity coefficient of 6.57 ± 0.10. In overall, this study has come up with a practical approach on the preparation of MIP for the detection of renal dysfunction by point-of-care Cre testing.

  9. Molecular dynamics of spermine-DNA interactions: sequence specificity and DNA bending for a simple ligand.

    PubMed Central

    Feuerstein, B G; Pattabiraman, N; Marton, L J

    1989-01-01

    We used molecular dynamics to model interactions between the physiologically important polyamine spermine and two B-DNA oligomers, the homopolymer (dG)10-(dC)10 and the heteropolymer (dGdC)5-(dGdC)5. Water and counterions were included in the simulation. Starting coordinates for spermine-DNA complexes were structures obtained by molecular mechanics modeling of spermine with the two oligomers; in these models, spermine binding induced a bend in the heteropolymer but not in the homopolymer. During approximately 40 psec of molecular dynamics simulation, spermine moves away from the floor of the major groove and interacts nospecifically with d(G)10-d(C)10. In contrast, a spermine-induced bend in the helix of (dGdC)5-(dGdC)5 is maintained throughout the simulation and spermine remains closely associated with the major groove. These results provide further evidence that the binding of spermine to nucleic acids can be sequence specific and that bending of alternating purine-pyrimidine sequences may be a physiologically important result of spermine binding. PMID:2780313

  10. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA.

    PubMed

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping

    2016-02-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction.

  11. Correlating Molecular Character of NIR Imaging Agents with Tissue-Specific Uptake

    PubMed Central

    Owens, Eric A.; Hyun, Hoon; Tawney, Joseph G.; Choi, Hak Soo; Henary, Maged

    2015-01-01

    Near-infrared (NIR) fluorescent contrast agents are emerging in optical imaging as sensitive, cost-effective, and nonharmful alternatives to current agents that emit harmful ionizing radiation. Developing spectrally distinct NIR fluorophores to visualize sensitive vital tissues to selectively avoid them during surgical resection of diseased tissue is of great significance. Herein, we report the synthetic variation of pentamethine cyanine fluorophores with modifications of physicochemical properties toward prompting tissue-specific uptake into sensitive tissues (i.e., endocrine glands). Tissue-specific targeting and biodistribution studies revealed localization of contrast agents in the adrenal and pituitary glands, pancreas, and lymph nodes with dependence on molecular characteristics. Incorporation of hydrophobic heterocyclic rings, alkyl groups, and halogens allowed a fine-tuning capability to the hydrophobic character and dipole moment for observing perturbation in biological activity in response to minor structural alterations. These NIR contrast agents have potential for clinical translation for intraoperative imaging in the delineation of delicate glands. PMID:25923454

  12. Membranes for specific adsorption: immobilizing molecularly imprinted polymer microspheres using electrospun nanofibers.

    PubMed

    Büttiker, Roman; Ebert, Jürgen; Hinderling, Christian; Adlhart, Christian

    2011-01-01

    Molecularly imprinted polymer microspheres were immobilized within a polymer nanofiber membrane by electrospinning. Such membranes simplify the handling of functional microspheres and provide specific recognition capabilities for solid-phase extraction and filtration applications. In this study, microspheres were prepared by precipitation polymerization of methacrylic acid and divinylbenzene as a cross-linker with the target molecule (-)-cinchonidine and then, they were electrospun into a non-woven polyacrylonitrile nanofiber membrane. The composite membrane showed specific affinity for (-)-cinchonidine which was attributed to the functional microspheres as confirmed by Raman microscopy. The target molecule capturing capacity of the composite membrane was 5 mg/g or 25 mg/g immobilized functional microsphere. No difference in target affinity was observed between the immobilized microspheres and the free microspheres. These results reveal that electrospun composite membranes are a feasible approach to immobilizing functional microspheres.

  13. Membranes for specific adsorption: immobilizing molecularly imprinted polymer microspheres using electrospun nanofibers.

    PubMed

    Büttiker, Roman; Ebert, Jürgen; Hinderling, Christian; Adlhart, Christian

    2011-01-01

    Molecularly imprinted polymer microspheres were immobilized within a polymer nanofiber membrane by electrospinning. Such membranes simplify the handling of functional microspheres and provide specific recognition capabilities for solid-phase extraction and filtration applications. In this study, microspheres were prepared by precipitation polymerization of methacrylic acid and divinylbenzene as a cross-linker with the target molecule (-)-cinchonidine and then, they were electrospun into a non-woven polyacrylonitrile nanofiber membrane. The composite membrane showed specific affinity for (-)-cinchonidine which was attributed to the functional microspheres as confirmed by Raman microscopy. The target molecule capturing capacity of the composite membrane was 5 mg/g or 25 mg/g immobilized functional microsphere. No difference in target affinity was observed between the immobilized microspheres and the free microspheres. These results reveal that electrospun composite membranes are a feasible approach to immobilizing functional microspheres. PMID:21528654

  14. Whole Cell-SELEX Aptamers for Highly Specific Fluorescence Molecular Imaging of Carcinomas In Vivo

    PubMed Central

    He, Xiaoxiao; Guo, Qiuping; Wang, Kemin; Ye, Xiaosheng; Tang, Jinlu

    2013-01-01

    Background Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. PMID:23950940

  15. Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

    PubMed

    Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

    2016-12-01

    Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies. PMID:27497873

  16. Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

    PubMed

    Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

    2016-12-01

    Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies.

  17. Rational molecular dynamics scheme for predicting optimum concentration loading of nano-additive in phase change materials

    NASA Astrophysics Data System (ADS)

    Rastogi, Monisha; Vaish, Rahul; Madhar, Niyaz Ahamad; Shaikh, Hamid; Al-Zahrani, S. M.

    2015-10-01

    The present study deals with the diffusion and phase transition behaviour of paraffin reinforced with carbon nano-additives namely graphene oxide (GO) and surface functionalized single walled carbon nanotubes (SWCNT). Bulk disordered systems of paraffin hydrocarbons impregnated with carbon nano-additives have been generated in realistic equilibrium conformations for potential application as latent heat storage systems. Ab initio molecular dynamics(MD) in conjugation with COMPASS forcefield has been implemented using periodic boundary conditions. The proposed scheme allows determination of optimum nano-additive loading for improving thermo-physical properties through analysis of mass, thermal and transport properties; and assists in determination of composite behaviour and related performance from microscopic point of view. It was observed that nanocomposites containing 7.8 % surface functionalised SWCNT and 55% GO loading corresponds to best latent heat storage system. The propounded methodology could serve as a by-pass route for economically taxing and iterative experimental procedures required to attain the optimum composition for best performance. The results also hint at the large unexplored potential of ab-initio classical MD techniques for predicting performance of new nanocomposites for potential phase change material applications.

  18. Molecular and Merrifield supported chiral diamines for enantioselective addition of ZnR2 (R = Me, Et) to ketones.

    PubMed

    Calvillo-Barahona, Mercedes; Cordovilla, Carlos; Genov, Miroslav N; Martínez-Ilarduya, Jesús M; Espinet, Pablo

    2013-10-28

    Chiral 1,2-ethylenediamines have been previously reported as active catalysts in the enantioselective addition reactions of ZnR2 to either methyl- or trifluoromethyl-ketones. Subtle changes in the molecular structure of different catalysts are described herein and lead to a dramatic effect in their catalytic activity. From these findings, we demonstrate the selective reactivity of the ligands used in the addition of ZnR2 (R = Me, Et) to methyl- and trifluoromethyl-ketones offering an enantioselective access either to chiral non-fluorinated alcohols or to chiral fluorinated tertiary alcohols. Considering the importance of the chiral trifluoromethyl carbinol fragment in several biologically active compounds, we have extended the scope of the addition reaction of ZnEt2 to several trifluoromethylketones catalyzed by (R,R)-1,2-diphenylethylenediamine derivatives. This work explores a homogeneous approach that provides excellent yields and very high ee and the use of a heterogenized tail-tied ligand affording moderate ee, high yields and allowing an easier handling and recycling.

  19. Overdispersion of the molecular clock: temporal variation of gene-specific substitution rates in Drosophila.

    PubMed

    Bedford, Trevor; Hartl, Daniel L

    2008-08-01

    Simple models of molecular evolution assume that sequences evolve by a Poisson process in which nucleotide or amino acid substitutions occur as rare independent events. In these models, the expected ratio of the variance to the mean of substitution counts equals 1, and substitution processes with a ratio greater than 1 are called overdispersed. Comparing the genomes of 10 closely related species of Drosophila, we extend earlier evidence for overdispersion in amino acid replacements as well as in four-fold synonymous substitutions. The observed deviation from the Poisson expectation can be described as a linear function of the rate at which substitutions occur on a phylogeny, which implies that deviations from the Poisson expectation arise from gene-specific temporal variation in substitution rates. Amino acid sequences show greater temporal variation in substitution rates than do four-fold synonymous sequences. Our findings provide a general phenomenological framework for understanding overdispersion in the molecular clock. Also, the presence of substantial variation in gene-specific substitution rates has broad implications for work in phylogeny reconstruction and evolutionary rate estimation.

  20. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers.

    PubMed

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-10-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.

  1. Exploration of Deinococcus-Thermus molecular diversity by novel group-specific PCR primers

    PubMed Central

    Theodorakopoulos, Nicolas; Bachar, Dipankar; Christen, Richard; Alain, Karine; Chapon, Virginie

    2013-01-01

    The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly, Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deinococcus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum. PMID:23996915

  2. Biokinetics and dosimetry of target-specific radiopharmaceuticals for molecular imaging and therapy

    NASA Astrophysics Data System (ADS)

    Ferro-Flores, Guillermina; Torres-García, Eugenio; Gonz&Ález-v&Ázquez, Armando; de Murphy, Consuelo Arteaga

    Molecular imaging techniques directly or indirectly monitor and record the spatiotemporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic or therapeutic applications. 99mTc-HYNIC-TOC has shown high stability both in vitro and in vivo and rapid detection of somatostatin receptor-positive tumors. Therapies using radiolabeled anti-CD20 have demonstrated their efficacy in patients with B-cell non-Hodgkin's lymphoma (NHL). The aim of this study was to establish biokinetic models for 99mTc-HYNIC-TOC and 188Re-anti-CD20 and to evaluate their dosimetry as target-specific radiopharmaceuticals. The OLINDA/EXM code was used to calculate patient-specific internal radiation dose estimates. 99mTc-HYNIC-TOC images showed an average tumor/blood ratio of 4.3±0.7 in receptor-positive tumors with an average effective dose of 4.4 mSv. Dosimetric studies indicated that after administration of 5.8 to 7.5 GBq of 188Re-anti-CD20 the absorbed dose to total body would be 0.75 Gy which corresponds to the recommended dose for NHL therapies.

  3. Molecular basis for specificity in the druggable kinome: sequence-based analysis

    PubMed Central

    Chen, Jianping; Zhang, Xi

    2007-01-01

    Motivation Rational design of kinase inhibitors remains a challenge partly because there is no clear delineation of the molecular features that direct the pharmacological impact towards clinically relevant targets. Standard factors governing ligand affinity, such as potential for intermolecular hydrophobic interactions or for intermolecular hydrogen bonding do not provide good markers to assess cross reactivity. Thus, a core question in the informatics of drug design is what type of molecular similarity among targets promotes promiscuity and what type of molecular difference governs specificity. This work answers the question for a sizable screened sample of the human pharmacokinome including targets with unreported structure. Results We show that drug design aimed at promoting pairwise interactions between ligand and kinase target actually fosters promiscuity because of the high conservation of the partner groups on or around the ATP-binding site of the kinase. Alternatively, we focus on a structural marker that may be reliably determined from sequence and measures dehydration propensities mostly localized on the loopy regions of kinases. Based on this marker, we construct a sequence-based kinase classifier that enables the accurate prediction of pharmacological differences. Our indicator is a micro-environmental descriptor that quantifies the propensity for water exclusion around preformed polar pairs. The results suggest that targeting polar dehydration patterns heralds a new generation of drugs that enable a tighter control of specificity than designs aimed at promoting ligand–kinase pairwise interactions. Availability The predictor of polar hot spots for dehydration propensity, or solvent-accessible hydrogen bonds in soluble proteins, named YAPView, may be freely downloaded from the University of Chicago website http://protlib.uchicago.edu/dloads.html PMID:17255140

  4. A microfluidic chamber to study the dynamics of muscle-contraction-specific molecular interactions.

    PubMed

    Roman, Horia Nicolae; Juncker, David; Lauzon, Anne-Marie

    2015-03-01

    In vitro motility and laser trap assays are commonly used for molecular mechanics measurements. However, chemicals cannot be added during these measurements, because they create flows that alter the molecular mechanics. Thus, we designed a microfluidic device that allows the addition of chemicals without creating bulk flows. Biocompatibility of the components of this device was tested. A microchannel chamber was created by photolithography with the patterns transferred to polydimethylsiloxane (PDMS). The PDMS chamber was bound to a polycarbonate membrane, which itself was bound to a molecular mechanics chamber. The microchannels ensured rapid distribution of the chemicals over the membrane, whereas the membrane ensured efficient delivery to the mechanics chamber while preventing bulk flow. The biocompatibility of the materials was tested by comparing the velocity (ν(max)) of propulsion by myosin of fluorescently labeled actin filaments to that of the conventional assay; no difference in ν(max) was observed. To estimate total chemical delivery time, labeled bovine serum albumin was injected in the channel chamber and TIRF was used to determine the time to reach the assay surface (2.7 ± 0.1 s). Furthermore, the standard distance of a trapped microsphere calculated during buffer diffusion using the microfluidic device (14.9 ± 3.2 nm) was not different from that calculated using the conventional assay (15.6 ± 5.3 nm, p = 0.922). Finally, ν(max) obtained by injecting adenosine triphosphate (ATP) in the microchannel chamber (2.37 ± 0.48 μm/s) was not different from that obtained when ATP was delivered directly to the mechanics chamber (2.52 ± 0.42 μm/s, p = 0.822). This microfluidic prototype validates the design for molecular mechanics measurements. PMID:25629255

  5. Linearity and additivity in cluster-induced sputtering: A molecular-dynamics study of van der Waals bonded systems

    SciTech Connect

    Anders, Christian; Urbassek, Herbert M.; Johnson, Robert E.

    2004-10-15

    Using molecular-dynamics simulation, we study sputtering of a condensed-gas solid induced by the impact of atomic clusters with sizes 1{<=}n{<=}10{sup 4}. Above a nonlinear onset regime, we find a linear increase of the sputter yield Y with the total energy E of the bombarding cluster. The fitting coefficients in the linear regime depend only on the cluster size n such that for fixed bombardment energy, sputtering decreases with increasing cluster size n. We find that to a good approximation the sputter yield in this regime obeys an additivity rule in cluster size n such that doubling the cluster size at the same cluster velocity amounts to doubling the sputter yield. The sputter-limiting energy {epsilon}{sub s} is introduced which separates erosion ({epsilon}>{epsilon}{sub s}) from growth ({epsilon}<{epsilon}{sub s}) under cluster impact.

  6. Species-specific size expansion and molecular evolution of the oleosins in angiosperms.

    PubMed

    Liu, Qi; Sun, Yepeng; Su, Wujie; Yang, Jing; Liu, Xiuming; Wang, Yanfang; Wang, Fawei; Li, Haiyan; Li, Xiaokun

    2012-11-10

    Oleosins are hydrophobic plant proteins thought to be important for the formation of oil bodies, which supply energy for seed germination and subsequent seedling growth. To better understand the evolutionary history and diversity of the oleosin gene family in plants, especially angiosperms, we systematically investigated the molecular evolution of this family using eight representative angiosperm species. A total of 73 oleosin members were identified, with six members in each of four monocot species and a greater but variable number in the four eudicots. A phylogenetic analysis revealed that the angiosperm oleosin genes belonged to three monophyletic lineages. Species-specific gene duplications, caused mainly by segmental duplication, led to the great expansion of oleosin genes and occurred frequently in eudicots after the monocot-eudicot divergence. Functional divergence analyses indicate that significant amino acid site-specific selective constraints acted on the different clades of oleosins. Adaptive evolution analyses demonstrate that oleosin genes were subject to strong purifying selection after their species-specific duplications and that rapid evolution occurred with a high degree of evolutionary dynamics in the pollen-specific oleosin genes. In conclusion, this study serves as a foundation for genome-wide analyses of the oleosins. These findings provide insight into the function and evolution of this gene family in angiosperms and pave the way for studies in other plants.

  7. Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

    PubMed

    Tonelli, C; Consonni, G; Dolfini, S F; Dellaporta, S L; Viotti, A; Gavazzi, G

    1991-03-01

    The Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

  8. Species-specific size expansion and molecular evolution of the oleosins in angiosperms.

    PubMed

    Liu, Qi; Sun, Yepeng; Su, Wujie; Yang, Jing; Liu, Xiuming; Wang, Yanfang; Wang, Fawei; Li, Haiyan; Li, Xiaokun

    2012-11-10

    Oleosins are hydrophobic plant proteins thought to be important for the formation of oil bodies, which supply energy for seed germination and subsequent seedling growth. To better understand the evolutionary history and diversity of the oleosin gene family in plants, especially angiosperms, we systematically investigated the molecular evolution of this family using eight representative angiosperm species. A total of 73 oleosin members were identified, with six members in each of four monocot species and a greater but variable number in the four eudicots. A phylogenetic analysis revealed that the angiosperm oleosin genes belonged to three monophyletic lineages. Species-specific gene duplications, caused mainly by segmental duplication, led to the great expansion of oleosin genes and occurred frequently in eudicots after the monocot-eudicot divergence. Functional divergence analyses indicate that significant amino acid site-specific selective constraints acted on the different clades of oleosins. Adaptive evolution analyses demonstrate that oleosin genes were subject to strong purifying selection after their species-specific duplications and that rapid evolution occurred with a high degree of evolutionary dynamics in the pollen-specific oleosin genes. In conclusion, this study serves as a foundation for genome-wide analyses of the oleosins. These findings provide insight into the function and evolution of this gene family in angiosperms and pave the way for studies in other plants. PMID:22951805

  9. Evaluation of swine-specific PCR assays used for fecal source tracking and analysis of molecular diversity of swine-specific "bacteroidales" populations.

    PubMed

    Lamendella, Regina; Santo Domingo, Jorge W; Yannarell, Anthony C; Ghosh, Shreya; Di Giovanni, George; Mackie, Roderick I; Oerther, Daniel B

    2009-09-01

    In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 "Bacteroidales" 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.

  10. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities. PMID:26754790

  11. ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations.

    PubMed

    Williams, Benfeard; Convertino, Marino; Das, Jhuma; Dokholyan, Nikolay V

    2015-10-01

    The increased risk of developing Alzheimer's disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.

  12. ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations

    PubMed Central

    Williams II, Benfeard; Convertino, Marino; Das, Jhuma; Dokholyan, Nikolay V.

    2015-01-01

    The increased risk of developing Alzheimer’s disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD. PMID:26506597

  13. Molecular Characterization of Arabidopsis GAL4/UAS Enhancer Trap Lines Identifies Novel Cell-Type-Specific Promoters1[OPEN

    PubMed Central

    Radoeva, Tatyana; Saiga, Shunsuke

    2016-01-01

    Cell-type-specific gene expression is essential to distinguish between the numerous cell types of multicellular organism. Therefore, cell-type-specific gene expression is tightly regulated and for most genes RNA transcription is the central point of control. Thus, transcriptional reporters are broadly used markers for cell identity. In Arabidopsis (Arabidopsis thaliana), a recognized standard for cell identities is a collection of GAL4/UAS enhancer trap lines. Yet, while greatly used, very few of them have been molecularly characterized. Here, we have selected a set of 21 frequently used GAL4/UAS enhancer trap lines for detailed characterization of expression pattern and genomic insertion position. We studied their embryonic and postembryonic expression domains and grouped them into three groups (early embryo development, late embryo development, and embryonic root apical meristem lines) based on their dominant expression. We show that some of the analyzed lines are expressed in a domain often broader than the one that is reported. Additionally, we present an overview of the location of the T-DNA inserts of all lines, with one exception. Finally, we demonstrate how the obtained information can be used for generating novel cell-type-specific marker lines and for genotyping enhancer trap lines. The knowledge could therefore support the extensive use of these valuable lines. PMID:27208300

  14. Molecular Characterization of Arabidopsis GAL4/UAS Enhancer Trap Lines Identifies Novel Cell-Type-Specific Promoters.

    PubMed

    Radoeva, Tatyana; Ten Hove, Colette A; Saiga, Shunsuke; Weijers, Dolf

    2016-06-01

    Cell-type-specific gene expression is essential to distinguish between the numerous cell types of multicellular organism. Therefore, cell-type-specific gene expression is tightly regulated and for most genes RNA transcription is the central point of control. Thus, transcriptional reporters are broadly used markers for cell identity. In Arabidopsis (Arabidopsis thaliana), a recognized standard for cell identities is a collection of GAL4/UAS enhancer trap lines. Yet, while greatly used, very few of them have been molecularly characterized. Here, we have selected a set of 21 frequently used GAL4/UAS enhancer trap lines for detailed characterization of expression pattern and genomic insertion position. We studied their embryonic and postembryonic expression domains and grouped them into three groups (early embryo development, late embryo development, and embryonic root apical meristem lines) based on their dominant expression. We show that some of the analyzed lines are expressed in a domain often broader than the one that is reported. Additionally, we present an overview of the location of the T-DNA inserts of all lines, with one exception. Finally, we demonstrate how the obtained information can be used for generating novel cell-type-specific marker lines and for genotyping enhancer trap lines. The knowledge could therefore support the extensive use of these valuable lines. PMID:27208300

  15. Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation.

    PubMed

    Parthasarathy, Ranganath; Subramanian, Shyamsundar; Boder, Eric T

    2007-01-01

    The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.

  16. Molecular-Scale Structural Controls on Nanoscale Growth Processes: Step-Specific Regulation of Biomineral Morphology

    NASA Astrophysics Data System (ADS)

    Dove, P. M.; Davis, K. J.; De Yoreo, J. J.; Orme, C. A.

    2001-12-01

    Deciphering the complex strategies by which organisms produce nanocrystalline materials with exquisite morphologies is central to understanding biomineralizing systems. One control on the morphology of biogenic nanoparticles is the specific interactions of their surfaces with the organic functional groups provided by the organism and the various inorganic species present in the ambient environment. It is now possible to directly probe the microscopic structural controls on crystal morphology by making quantitative measurements of the dynamic processes occurring at the mineral-water interface. These observations can provide crucial information concerning the actual mechanisms of growth that is otherwise unobtainable through macroscopic techniques. Here we use in situ molecular-scale observations of step dynamics and growth hillock morphology to directly resolve roles of principal impurities in regulating calcite surface morphologies. We show that the interactions of certain inorganic as well as organic impurities with the calcite surface are dependent upon the molecular-scale structures of step-edges. These interactions can assume a primary role in directing crystal morphology. In calcite growth experiments containing magnesium, we show that growth hillock structures become modified owing to the preferential inhibition of step motion along directions approximately parallel to the [010]. Compositional analyses have shown that Mg incorporates at different levels into the two types of nonequivalent steps, which meet at the hillock corner parallel to [010]. A simple calculation of the strain caused by this difference indicates that we should expect a significant retardation at this corner, in agreement with the observed development of [010] steps. If the low-energy step-risers produced by these [010] steps is perpendicular to the c-axis as seems likely from crystallographic considerations, this effect provides a plausible mechanism for the elongated calcite crystal

  17. Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes.

    PubMed

    Tenthorey, Jeannette L; Kofoed, Eric M; Daugherty, Matthew D; Malik, Harmit S; Vance, Russell E

    2014-04-10

    NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes. PMID:24657167

  18. Measurement of State-Specific Association Constants in Allosteric Sensors through Molecular Stapling and NMR.

    PubMed

    Moleschi, Kody J; Akimoto, Madoka; Melacini, Giuseppe

    2015-08-26

    Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e., state-selective molecular stapling through disulfide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA-RIα), for which the structures of the inactive and active states are available, as needed to design the state-selective disulfide bridges. Surprisingly, the PKA-RIα state-specific association constants are comparable to those of a structurally homologous domain with ∼10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.

  19. Carbon sources in the Beaufort Sea revealed by molecular lipid biomarkers and compound specific isotope analysis

    NASA Astrophysics Data System (ADS)

    Tolosa, I.; Fiorini, S.; Gasser, B.; Martín, J.; Miquel, J. C.

    2012-10-01

    Molecular lipid biomarkers (hydrocarbons, alcohols, sterols and fatty acids) and compound specific isotope analysis of suspended particulate organic matter (SPM) and surface sediments of the Mackenzie Shelf and slope (Southeast Beaufort Sea, Arctic Ocean), were studied in summer 2009. The concentrations of the molecular lipid markers, characteristic of known organic matter sources, were grouped and used as proxies to evaluate the relative importance of fresh algal, detrital algal, fossil, C3 terrestrial plants, bacterial and zooplankton material in the sedimentary organic matter (OM). Fossil and detrital algal contributions were the major fractions of the freshwater SPM from the Mackenzie River with ~34% each of the total molecular biomarkers. Fresh algal, C3 terrestrial, bacterial and zooplanktonic components represented much lower percentages, 17, 10, 4 and < 1%, respectively. In marine SPM from the Mackenzie slope, the major contributions were fresh and detrital algal components (> 80%) with a minor contribution of fossil and C3 terrestrial biomarkers. Characterization of the sediments revealed a major sink of refractory algal material mixed with some fresh algal material, fossil hydrocarbons and a small input of C3 terrestrial sources. In particular, the sediments from the shelf and at the mouth of the Amundsen Gulf presented the highest contribution of detrital algal material (60-75%) whereas those from the slope contained the highest proportion of fossil (40%) and C3 terrestrial plant material (10%). Overall, considering that the detrital algal material is marine derived, autochthonous sources contributed more than allochthonous sources to the OM lipid pool. Using the ratio of an allochthonous biomarker (normalized to total organic carbon, TOC) found in the sediments to those measured at the river mouth water, we estimated that the fraction of terrestrial material preserved in the sediments accounted for 30-40% of the total carbon in the inner shelf sediments

  20. Intraindividual genome expression analysis reveals a specific molecular signature of psoriasis and eczema.

    PubMed

    Quaranta, Maria; Knapp, Bettina; Garzorz, Natalie; Mattii, Martina; Pullabhatla, Venu; Pennino, Davide; Andres, Christian; Traidl-Hoffmann, Claudia; Cavani, Andrea; Theis, Fabian J; Ring, Johannes; Schmidt-Weber, Carsten B; Eyerich, Stefanie; Eyerich, Kilian

    2014-07-01

    Previous attempts to gain insight into the pathogenesis of psoriasis and eczema by comparing their molecular signatures were hampered by the high interindividual variability of those complex diseases. In patients affected by both psoriasis and nonatopic or atopic eczema simultaneously (n = 24), an intraindividual comparison of the molecular signatures of psoriasis and eczema identified genes and signaling pathways regulated in common and exclusive for each disease across all patients. Psoriasis-specific genes were important regulators of glucose and lipid metabolism, epidermal differentiation, as well as immune mediators of T helper 17 (TH17) responses, interleukin-10 (IL-10) family cytokines, and IL-36. Genes in eczema related to epidermal barrier, reduced innate immunity, increased IL-6, and a TH2 signature. Within eczema subtypes, a mutually exclusive regulation of epidermal differentiation genes was observed. Furthermore, only contact eczema was driven by inflammasome activation, apoptosis, and cellular adhesion. On the basis of this comprehensive picture of the pathogenesis of psoriasis and eczema, a disease classifier consisting of NOS2 and CCL27 was created. In an independent cohort of eczema (n = 28) and psoriasis patients (n = 25), respectively, this classifier diagnosed all patients correctly and also identified initially misdiagnosed or clinically undifferentiated patients.

  1. Disease-specific target gene expression profiling of molecular imaging probes: database development and clinical validation.

    PubMed

    Chan, Lawrence Wing-Chi; Ngo, Connie Hiu-Ching; Wang, Fengfeng; Zhao, Moss Y; Zhao, Mengying; Law, Helen Ka-Wai; Wong, Sze Chuen Cesar; Yung, Benjamin Yat-Ming

    2014-01-01

    Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2-deoxy-2-d-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/. PMID:25022454

  2. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. PMID:27174700

  3. Structural specifics of light-induced metastable states in copper(II)-nitroxide molecular magnets.

    PubMed

    Barskaya, I Yu; Veber, S L; Fokin, S V; Tretyakov, E V; Bagryanskaya, E G; Ovcharenko, V I; Fedin, M V

    2015-12-28

    Although light-induced magnetostructural switching in copper(II)-nitroxide molecular magnets Cu(hfac)2L(R) has been known for several years, structural characterization of metastable photoinduced states has not yet been accomplished due to significant technical demands. In this work we apply, for the first time, variable-temperature FTIR spectroscopy with photoexcitation to investigate the structural specifics of light-induced states in the Cu(hfac)2L(R) family represented by (i) Cu(hfac)2L(Me) comprising two-spin copper(II)-nitroxide clusters, and (ii) Cu(hfac)2L(Pr) comprising three-spin nitroxide-copper(II)-nitroxide clusters. The light-induced state of Cu(hfac)2L(Me) manifests the same set of vibrational bands as the corresponding thermally-induced state, implying their similar structures. For the second compound Cu(hfac)2L(Pr), the coordination environment of copper(II) is similar in light- and thermally-induced states, but distinct differences are found for packing of the peripheral n-propyl substituent of nitroxide. Thus, generally the structures of the corresponding thermally- and light-induced states in molecular magnets Cu(hfac)2L(R) might differ, and FTIR spectroscopy provides a useful approach for revealing and elucidating such differences.

  4. Molecular Basis of Hydroperoxide Specificity in Peroxiredoxins: The Case of AhpE from Mycobacterium tuberculosis.

    PubMed

    Zeida, Ari; Reyes, Aníbal M; Lichtig, Pablo; Hugo, Martín; Vazquez, Diego S; Santos, Javier; González Flecha, F Luis; Radi, Rafael; Estrin, Dario A; Trujillo, Madia

    2015-12-15

    Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzyme's dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.

  5. Methanol Oxidative Dehydrogenation on Oxide Catalysts: Molecular and Dissociative Routes and Hydrogen Addition Energies as Descriptors of Reactivity

    SciTech Connect

    Deshlahra, Prashant; Iglesia, Enrique

    2014-11-13

    The oxidative dehydrogenation (ODH) of alkanols on oxide catalysts is generally described as involving H-abstraction from alkoxy species formed via O–H dissociation. Kinetic and isotopic data cannot discern between such routes and those involving kinetically-relevant H-abstraction from undissociated alkanols. Here, we combine such experiments with theoretical estimates of activation energies and entropies to show that the latter molecular routes prevail over dissociative routes for methanol reactions on polyoxometalate (POM) clusters at all practical reaction temperatures. The stability of the late transition states that mediate H-abstraction depend predominantly on the stability of the O–H bond formed, making H-addition energies (HAE) accurate and single-valued descriptors of reactivity. Density functional theory-derived activation energies depend linearly on HAE values at each O-atom location on clusters with a range of composition (H3PMo12, H4SiMo12, H3PW12, H4PV1Mo11, and H4PV1W11); both barriers and HAE values reflect the lowest unoccupied molecular orbital energy of metal centers that accept the electron and the protonation energy of O-atoms that accept the proton involved in the H-atom transfer. Bridging O-atoms form O–H bonds that are stronger than those of terminal atoms and therefore exhibit more negative HAE values and higher ODH reactivity on all POM clusters. For each cluster composition, ODH turnover rates reflect the reactivity-averaged HAE of all accessible O-atoms, which can be evaluated for each cluster composition to provide a rigorous and accurate predictor of ODH reactivity for catalysts with known structure. These relations together with oxidation reactivity measurements can then be used to estimate HAE values and to infer plausible structures for catalysts with uncertain active site structures.

  6. Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp.

    PubMed

    Rai, Varun; Nyine, Yin Thu; Hapuarachchi, Hapuarachchige C; Yap, Hooi Ming; Ng, Lee Ching; Toh, Chee-Seng

    2012-02-15

    An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from "on" to "off" states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10(-14)M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.

  7. Microscopic Analysis of Corn Fiber Using Corn Starch- and Cellulose-Specific Molecular Probes

    SciTech Connect

    Porter, S. E.; Donohoe, B. S.; Beery, K. E.; Xu, Q.; Ding, S.-Y.; Vinzant, T. B.; Abbas, C. A.; Himmel, M. E.

    2007-09-01

    Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.

  8. Major histocompatibility complex class II molecules can protect from diabetes by positively selecting T cells with additional specificities.

    PubMed

    Lühder, F; Katz, J; Benoist, C; Mathis, D

    1998-02-01

    Insulin-dependent diabetes is heavily influenced by genes encoded within the major histocompatibility complex (MHC), positively by some class II alleles and negatively by others. We have explored the mechanism of MHC class II-mediated protection from diabetes using a mouse model carrying the rearranged T cell receptor (TCR) transgenes from a diabetogenic T cell clone derived from a nonobese diabetic mouse. BDC2.5 TCR transgenics with C57Bl/6 background genes and two doses of the H-2(g7) allele exhibited strong insulitis at approximately 3 wk of age and most developed diabetes a few weeks later. When one of the H-2(g7) alleles was replaced by H-2(b), insulitis was still severe and only slightly delayed, but diabetes was markedly inhibited in both its penetrance and time of onset. The protective effect was mediated by the Abetab gene, and did not merely reflect haplozygosity of the Abetag7 gene. The only differences we observed in the T cell compartments of g7/g7 and g7/b mice were a decrease in CD4(+) cells displaying the transgene-encoded TCR and an increase in cells expressing endogenously encoded TCR alpha-chains. When the synthesis of endogenously encoded alpha-chains was prevented, the g7/b animals were no longer protected from diabetes. g7/b mice did not have a general defect in the production of Ag7-restricted T cells, and antigen-presenting cells from g7/b animals were as effective as those from g7/g7 mice in stimulating Ag7-restricted T cell hybridomas. These results argue against mechanisms of protection involving clonal deletion or anergization of diabetogenic T cells, or one depending on capture of potentially pathogenic Ag7-restricted epitopes by Ab molecules. Rather, they support a mechanism based on MHC class II-mediated positive selection of T cells expressing additional specificities. PMID:9449718

  9. Determination of specific molecular markers of biomass burning in lake sediments

    NASA Astrophysics Data System (ADS)

    Kirchgeorg, Torben; Schüpbach, Simon; Kehrwald, Natalie; McWethy, David; Barbante, Carlo

    2014-05-01

    Fire influences regional to global atmospheric chemistry and climate. Molecular markers of biomass burning archived in lake sediments are becoming increasingly important in paleoenvironmental reconstruction and may help determine interactions between climate and fire activity. One group of these molecular markers is the monosaccharide anhydrides levoglucosan, mannosan and galactosan. Several aerosol studies and recent ice core research use these compounds as a marker for biomass burning, but studies from lake sediment cores are rare. Previous sediment methods used gas chromatography - mass spectrometry and required derivatization of samples. Here, we present a high performance anion exchange chromatography-mass spectrometry method to allow separation and detection of the three monosaccharide anhydrides in lake sediments with implications for reconstructing past biomass burning events. We validated the method by quantifying levoglucosan, mannosan and galactosan in selected sediment core samples from Lake Kirkpatrick, New Zealand. The freeze-dried, milled and homogenized sediment samples were first extracted with methanol by pressurized solvent extraction, pre-concentrated and finally separated and analyzed by high performance anion exchange chromatography-mass spectrometry. We compared these isomers with macroscopic charcoal concentrations, as charcoal is a well-known proxy for biomass burning. In addition, we applied the method to a sediment core from Lake Petén Itzá, Guatemala to prove the suitability of these markers for reconstructing biomass burning history over the entire Holocene. In the Lake Kirkpatrick samples, levoglucosan, mannosan and galactosan concentrations significantly correlate with macroscopic charcoal concentrations. The three isomers are present in samples without any macroscopic charcoal, and may reflect the presence of microscopic charcoal. Levoglucosan/mannosan and levoglucosan/(mannosan+galactosan) ratios differ between samples with high

  10. DrugLogit: logistic discrimination between drugs and nondrugs including disease-specificity by assigning probabilities based on molecular properties.

    PubMed

    García-Sosa, Alfonso T; Oja, Mare; Hetényi, Csaba; Maran, Uko

    2012-08-27

    The increasing knowledge of both structure and activity of compounds provides a good basis for enhancing the pharmacological characterization of chemical libraries. In addition, pharmacology can be seen as incorporating both advances from molecular biology as well as chemical sciences, with innovative insight provided from studying target-ligand data from a ligand molecular point of view. Predictions and profiling of libraries of drug candidates have previously focused mainly on certain cases of oral bioavailability. Inclusion of other administration routes and disease-specificity would improve the precision of drug profiling. In this work, recent data are extended, and a probability-based approach is introduced for quantitative and gradual classification of compounds into categories of drugs/nondrugs, as well as for disease- or organ-specificity. Using experimental data of over 1067 compounds and multivariate logistic regressions, the classification shows good performance in training and independent test cases. The regressions have high statistical significance in terms of the robustness of coefficients and 95% confidence intervals provided by a 1000-fold bootstrapping resampling. Besides their good predictive power, the classification functions remain chemically interpretable, containing only one to five variables in total, and the physicochemical terms involved can be easily calculated. The present approach is useful for an improved description and filtering of compound libraries. It can also be applied sequentially or in combinations of filters, as well as adapted to particular use cases. The scores and equations may be able to suggest possible routes for compound or library modification. The data is made available for reuse by others, and the equations are freely accessible at http://hermes.chem.ut.ee/~alfx/druglogit.html.

  11. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

    PubMed Central

    Wissenbach, Dirk K.; Pfeiffer, Susanne E. M.; Baumann, Sven; Hofbauer, Lorenz C.; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

    2016-01-01

    Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis. PMID:27441377

  12. Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity.

    PubMed

    Carsia, R V; Macdonald, G J; Malamed, S

    1983-06-01

    The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.

  13. Electrochemical molecular beacon biosensor for sequence-specific recognition of double-stranded DNA.

    PubMed

    Miao, Xiangmin; Guo, Xiaoting; Xiao, Zhiyou; Ling, Liansheng

    2014-09-15

    Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au-S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350 pM to 25 nM, with a detection limit of 275 pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches.

  14. Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase*

    PubMed Central

    Liu, Chunhui; Sheng, Juzheng; Krahn, Juno M.; Perera, Lalith; Xu, Yongmei; Hsieh, Po-Hung; Dou, Wenfang; Liu, Jian; Pedersen, Lars C.

    2014-01-01

    Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway. PMID:24652287

  15. Hydrogen-bonding molecular ruler surfactants as probes of specific solvation at liquid/liquid interfaces.

    PubMed

    Siler, A Renee; Brindza, Michael R; Walker, Robert A

    2009-10-01

    Resonance-enhanced, second harmonic generation (SHG) is used to measure the electronic structure of solutes sensitive to specific solvation adsorbed to liquid/liquid and liquid/solid interfaces. Here, specific solvation refers to solvent-solute interactions that are directional and localized. N-methyl-p-methoxyaniline (NMMA) is a solute whose first allowed electronic transition wavelength remains almost constant (approximately 315 nm) in non-hydrogen-bonding solvents regardless of solvent polarity. However, in hydrogen-bond-accepting solvents such as dimethylsulfoxide, NMMA's absorbance shifts to longer wavelengths (320 nm), whereas in hydrogen-bond-donating solvents (e.g., water), the absorbance shifts to shorter wavelengths (approximately 300 nm). SHG experiments show that at alkane/silica interfaces, surface silanol groups serve as moderately strong hydrogen-bond donors as evidenced by NMMA's absorbance of 307 nm. At the carbon tetrachloride/water interface, NMMA absorbance also shifts to slightly shorter wavelengths (298 nm) implying that water molecules at this liquid/liquid interface are donating strong hydrogen bonds to the adsorbed NMMA solutes. In contrast, experiments using newly developed molecular ruler surfactants with NMMA as a model hydrophobic solute and a hydrophilic, cationic headgroup imply that, as NMMA migrates across an aqueous/alkane interface, it carries with it water that functions as a hydrogen-bond-accepting partner.

  16. Predictive Bioinformatic Assignment of Methyl-Bearing Stereocenters, Total Synthesis, and an Additional Molecular Target of Ajudazol B.

    PubMed

    Essig, Sebastian; Schmalzbauer, Björn; Bretzke, Sebastian; Scherer, Olga; Koeberle, Andreas; Werz, Oliver; Müller, Rolf; Menche, Dirk

    2016-02-19

    Full details on the evaluation and application of an easily feasible and generally useful method for configurational assignments of isolated methyl-bearing stereocenters are reported. The analytical tool relies on a bioinformatic gene cluster analysis and utilizes a predictive enoylreductase alignment, and its feasibility was demonstrated by the full stereochemical determination of the ajudazols, highly potent inhibitors of the mitochondrial respiratory chain. Furthermore, a full account of our strategies and tactics that culminated in the total synthesis of ajudazol B, the most potent and least abundant of these structurally unique class of myxobacterial natural products, is presented. Key features include an application of an asymmetric ortholithiation strategy for synthesis of the characteristic anti-configured hydroxyisochromanone core bearing three contiguous stereocenters, a modular oxazole formation, a flexible cross-metathesis approach for terminal allyl amide synthesis, and a late-stage Z,Z-selective Suzuki coupling. This total synthesis unambiguously proves the correct stereochemistry, which was further corroborated by comparison with reisolated natural material. Finally, 5-lipoxygenase was discovered as an additional molecular target of ajudazol B. Activities against this clinically validated key enzyme of the biosynthesis of proinflammatory leukotrienes were in the range of the approved drug zileuton, which further underlines the biological importance of this unique natural product.

  17. Predictive Bioinformatic Assignment of Methyl-Bearing Stereocenters, Total Synthesis, and an Additional Molecular Target of Ajudazol B.

    PubMed

    Essig, Sebastian; Schmalzbauer, Björn; Bretzke, Sebastian; Scherer, Olga; Koeberle, Andreas; Werz, Oliver; Müller, Rolf; Menche, Dirk

    2016-02-19

    Full details on the evaluation and application of an easily feasible and generally useful method for configurational assignments of isolated methyl-bearing stereocenters are reported. The analytical tool relies on a bioinformatic gene cluster analysis and utilizes a predictive enoylreductase alignment, and its feasibility was demonstrated by the full stereochemical determination of the ajudazols, highly potent inhibitors of the mitochondrial respiratory chain. Furthermore, a full account of our strategies and tactics that culminated in the total synthesis of ajudazol B, the most potent and least abundant of these structurally unique class of myxobacterial natural products, is presented. Key features include an application of an asymmetric ortholithiation strategy for synthesis of the characteristic anti-configured hydroxyisochromanone core bearing three contiguous stereocenters, a modular oxazole formation, a flexible cross-metathesis approach for terminal allyl amide synthesis, and a late-stage Z,Z-selective Suzuki coupling. This total synthesis unambiguously proves the correct stereochemistry, which was further corroborated by comparison with reisolated natural material. Finally, 5-lipoxygenase was discovered as an additional molecular target of ajudazol B. Activities against this clinically validated key enzyme of the biosynthesis of proinflammatory leukotrienes were in the range of the approved drug zileuton, which further underlines the biological importance of this unique natural product. PMID:26796481

  18. Novel glycol chitosan-based polymeric gene carrier synthesized by a Michael addition reaction with low molecular weight polyethylenimine.

    PubMed

    Lee, Young Hwa; Park, Hae In; Choi, Joon Sig

    2016-02-10

    A glycol chitosan-based polymer that spontaneously assembles with plasmid DNA into nanorods was evaluated as a non-viral vector for gene delivery. Glycol chitosan-methyl acrylate-polyethylenimine (GMP) was synthesized by grafting polyethylenimine onto glycol chitosan via amidation after Michael addition using methyl acrylate. Gel retardation and PicoGreen assay experiments showed complete complex formation with plasmid DNA. GMP/pDNA complexes were characterized using biophysical techniques and were found to be positively charged rod-shape structures with widths in the nanometer scale and lengths in the micrometer scale. Transfection efficiency and cytotoxicity of GMP polymer was evaluated in human epithelial ovary carcinoma (HeLa) cells, human embryonic kidney 293 (HEK293) cells, and human hepatocellular liver carcinoma (HepG2) cells, in comparison to high molecular weight polyethylenimine, a commonly used transfection reagent. Intracellular polymer uptake was compared and confirmed by confocal microscopy. The results demonstrate that GMP, a hybrid polymer of glycol chitosan grafted with branched polyethylenimine, may serve as a promising vehicle for efficient gene delivery.

  19. Electrical and Nonlinear Optical Studies of Specific Organic Molecular and Nonconjugated Conductive Polymeric Systems

    NASA Astrophysics Data System (ADS)

    Narayanan, Ananthakrishnan

    In this research, structural, electrical and nonlinear optical characteristics of: (a) single crystal films involving a noncentrosymmetric molecule DAST and a laser dye IR125 and (b) specific nonconjugated conducting polymers including poly(beta-pinene) and polynorbornene have been studied. 4'-dimethylamino-N-methyl-4-stilbazolium tosylate (DAST) is a well known second order nonlinear optical material. This material has exceptionally high electro-optic coefficients, high thermal stability and ultrafast response time. In this work single crystal films involving a combination of DAST and IR125 have been prepared using modified shear method and the films have been characterized using polarized optical microscopy, X-ray diffraction, polarization dependent optical absorption and photoluminescence spectroscopy. The electro-optic coefficient of these films measured at 633nm was found to be 300pm/V. Since IR-125 has a strong absorption band from 500nm to 800nm, these films are promising for various applications in nonlinear optics at longer wavelength and for light emission. Nonconjugated conducting polymers are a class of polymers that have at least one double bond in their repeat units. 1,4-cis polyisoprene, polyalloocimene, styrene butadiene rubber, poly(ethylenepyrrolediyl) derivatives, and poly(beta-pinene) are some of the well known examples of nonconjugated conducting polymers. In this work, polynorborne, a new addition to the class of nonconjugated conducting polymers is discussed. Like other polymers in this class, polynorbornene exhibits increase in electrical conductivity by many orders of magnitude upon doping with iodine. The maximum electrical conductivity of this material is 0.01 S/cm. As shown by using FTIR microscopy, the C=C bonds are transformed into cation radicals when polynorborne is doped. This is due to the charge-transfer from the double bond to the dopant (iodine). These materials like other nonconjugated conducting polymers have significant

  20. Recent advances in Employing Molecular Modelling to Determine the Specificity of Glycan-Binding Proteins

    PubMed Central

    Grant, Oliver C.; Woods, Robert J.

    2014-01-01

    Impressive improvements in docking performance can be achieved by applying energy bonuses to poses in which glycan hydroxyl groups occupy positions otherwise preferred by bound waters. In addition, inclusion of glycosidic conformational energies allows unlikely glycan conformations to be appropriately penalized. A method for predicting the binding specificity of glycan-binding proteins has been developed, which is based on grafting glycan branches onto a minimal binding determinant in the binding site. Grafting can be used either to screen virtual libraries of glycans, such as the known glycome, or to identify docked poses of minimal binding determinants that are consistent with specificity data. The reviewed advances allow accurate modelling of carbohydrate-protein 3D co-complexes, but challenges remain in ranking the affinity of congeners. PMID:25108191

  1. Specific plant DNA adducts as molecular biomarkers of genotoxic atmospheric environments.

    PubMed

    Weber-Lotfi, F; Obrecht-Pflumio, S; Guillemaut, P; Kleinpeter, J; Dietrich, A

    2005-03-01

    The general purpose of this study was to determine whether the formation of DNA addition products ('adducts') in plants could be a valuable biomarker of genotoxic air pollution. Plants from several species were exposed to ambient atmosphere at urban and suburban sites representative of different environmental conditions. The levels of NO2 and of the quantitatively major genotoxic air pollutants benzene, toluene, and xylene were monitored in parallel with plant exposure. DNA adducts were measured in bean (Phaseolus vulgaris), rye-grass (Lolium perenne), and tobacco (Nicotiana tabacum) seedlings by means of the [32P]-postlabeling method. Whereas, no correlation was found between the levels of the major genotoxic air pollutants and the total amounts of DNA adducts, individual analyses revealed site-specific and plant species-specific adduct responses, both at the qualitative and quantitative level. Among these, the amount of a specific rye-grass DNA adduct (rgs1) correlated with benzene/toluene/xylene levels above a threshold. For further characterization, rye-grass seedlings were treated in controlled conditions with benzene, toluene, xylene or their derivatives. On the other hand, in vitro DNA adduct formation assays were developed involving benzene, toluene, xylene, or their derivatives, and plant microsomes or purified peroxidase. Although in some cases, these approaches produced specific adduct responses, they failed to generate the rgs1 DNA adduct, which appeared to be characteristic for on-site test-plant exposure. Our studies have thus identified an interesting candidate for further analysis of environmental biomarkers of genotoxicity.

  2. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

    2015-01-01

    The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

  3. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    PubMed Central

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways. PMID:25275011

  4. Molecular targets of (-)-epigallocatechin-3-gallate (EGCG): specificity and interaction with membrane lipid rafts.

    PubMed

    Patra, S K; Rizzi, F; Silva, A; Rugina, D O; Bettuzzi, S

    2008-12-01

    Proteomic studies on anticancer activity of Green Tea Catechins (specifically EGCG) are suggesting a large set of protein targets that may directly interact with EGCG and alter the physiology of diseased cells, including cancer. Of notice, benign cells are usually left untouched. Lipid rafts have been recently recognized as signal processing hubs and suggested to be involved in drug uptake by means of endocytosis. These findings are suggesting new insights on the molecular mechanisms of anticancer drugs action. In the membrane, EGCG is hijacked by the laminin receptor (LamR), a lipid raft protein. Similar to aplidin and edelfosin, EGCG alters membrane domains composition also preventing EGF binding to EGFR, imerization of EGFR and relocation of phosphorylated EGFR to lipid rafts. In vitro studies have recently shown that EGCG also binds both DNA and RNA in GpC-rich regions. This event may importantly affect genes function. Moreover, EGCG was shown to inhibit telomerase, topoisomerase II and DNA methyltransferase 1 (DNMT1), thus ultimately affecting chromatin maintenance and remodeling. But another important alternative pathway besides interaction with specific proteins may play an important role in EGCG action: direct targeting of bioactive membrane platforms, lipid rafts. Structural alteration of the platforms deeply impact (and often inactivates) important pathways involving MAP kinases. The key issue is that, important and specific differences in lipid rafts composition have been found in transformed versus benign cells and apoptotic versus non-apoptotic cells. We suggest here that the anticancer activity of Green Tea Catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts by EGCG. PMID:19261982

  5. Molecular genetics of race-specific resistance of cowpea to Striga gesnerioides (Willd.).

    PubMed

    Li, Jianxiong; Lis, Karolina E; Timko, Michael P

    2009-05-01

    Cowpea, Vigna unguiculata (L.) Walp., is an important warm-season legume grown primarily in the semi-arid tropics. The majority of cowpea is grown by subsistence farmers in West and Central sub-Saharan Africa, where its grain and leaves are valued as human food and its stover is used for animal forage. Like all crop plants, cowpea is subject to yield losses resulting from a variety of biotic and abiotic stresses. Among the important biotic constraints to cowpea production is parasitism by the root hemiparasitic weeds Striga gesnerioides (Willd.) [witchweed] and Alectra vogelii (Benth.). At least seven races of S. gesnerioides have been identified within the cowpea-growing regions of West Africa, based on host differential response and genetic diversity analysis. Several race-specific resistance genes have been identified and located to one of two linkage groups (LG1 and LG6) of the current cowpea genetic map. Molecular markers associated with race-specific resistance genes have been identified, and several sequence-confirmed amplified regions (SCARs) have been developed for use in marker-assisted selection and breeding strategies for rapid cowpea improvement. The mechanism of race-specific resistance in the cowpea-Striga interaction has also been examined, with several genes involved in phytohormone and general disease resistance signaling transduction observed to be differentially expressed in resistant and susceptible phenotypes. Results suggest that PR5 expression may be a useful marker of Striga infection, and that salicylic acid signaling appears to play a role in the cowpea-Striga interaction.

  6. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

    NASA Astrophysics Data System (ADS)

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

    2016-06-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

  7. Molecular location of a species-specific epitope on the hamster scrapie agent protein.

    PubMed Central

    Bolton, D C; Seligman, S J; Bablanian, G; Windsor, D; Scala, L J; Kim, K S; Chen, C M; Kascsak, R J; Bendheim, P E

    1991-01-01

    Scrapie is a transmissible neurodegenerative disease of sheep and goats. An abnormal host protein, Sp33-37, is the major protein component of the scrapie agent and the only known disease- or agent-specific macromolecule. Two monoclonal antibodies (MAbs), 4H8 (immunoglobulin G2b [IgG2b]) and 6B11 (IgG1), produced by immunizing mice with the intact hamster 263K scrapie agent protein, Sp33-37Ha, were found to have species specificity similar to that reported previously for MAb 3F4 (IgG2a), which was produced by using PrP-27-30 as the immunogen (R. J. Kascsak, R. Rubenstein, P. A. Merz, M. Tonna-DeMasi, R. Fersko, R. I. Carp, H. M. Wisniewski, and H. Diringer, J. Virol. 61:3688-3693, 1987). These antibodies all bound to Sp33-37 derived from hamster but not from mouse cells. Competitive binding assays demonstrated that all three MAbs bound to the same or overlapping sites on Sp33-37Ha. The molecular location of the epitope for these antibodies was determined to within 10 residues by using an antigen competition enzyme-linked immunosorbent assay in which synthetic peptides spanning Sp33-37Ha residues 79 to 93 or 84 to 93 specifically inhibited binding of these antibodies to plates coated with purified Sp33-37Ha. A synthetic peptide with the mouse-specific sequence (83 to 92) that differed from the hamster sequence by substitution at two positions (MetHa-87----LeuMo-86 and MetHa-90----ValMo-89) did not inhibit antibody binding to Sp33-37Ha. MAb 3F4 binding to hamster Sp33-37 was eliminated by chemical modification of Sp33-37Ha with diethylpyrocarbonate or succinic anhydride and by cleavage with CNBr or trypsin. The effect of diethylpyrocarbonate on MAb 3F4 binding was not reversed by hydroxylamine treatment. MAb 3F4 binding was not affected by prolonged exposure of Sp33-37Ha to 70% formic acid or by boiling in sodium dodecyl sulfate. We conclude that the epitope for these MAbs is a linear determinant that includes Met-87, Lys-88, and Met-90 and that Met-90 is probably the

  8. RSV molecular characterization and specific antibody response in young children with acute lower respiratory infection.

    PubMed

    Baumeister, Elsa G; Hünicken, Diego S; Savy, Vilma L

    2003-05-01

    The presence of respiratory syncytial virus (RSV) in nasopharyngeal aspirates (NPA) were studied in 254 hospitalized Argentinean children with acute lower respiratory infection tract (ALRI). The specific humoral immune response and partial sequences of the G protein gene were studied in a subset of 22 children with RSV confirmed infection. The RSV IgM detection and the RSV IgG titration were made by immunofluorescence assay (IFA) in pairs of sera. The partial RSV G gene sequences were obtained by an RT-PCR amplification directly from de NPAs. RSV was present in 44.5% of the children. The RSV IgM was detected in 22.7 and 68.8% of the first and second sera, respectively. The IgG geometric mean titers of the acute and convalescent sera were 8 and 589. The RSV IgG titration was able to define 86.4% of the RSV confirmed cases. The percentage of coincidence between RSV IgM detection in the second sera and diagnosis by RSV IgG titration was 72.7% and no significant differences were observed. The nucleotide sequence of one group A and three group B viruses were identified. The first one was related with circulating viruses in Madrid, Montevideo and Mozambique during 1992, 1989 and 1999, respectively. The three sequences identified as group B viruses were closely related with circulating viruses in 1998 from South Africa and Canada during 1999 and 2000. The data obtained in our study provide the first approach at the molecular level (nucleotide) of the RSV circulating strains in Argentina and the lack of genotype patterns previously determined make necessary a continuous molecular surveillance in order to contribute to the understanding of the behavior of this virus in our community.

  9. Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates

    PubMed Central

    Cole, Graham B.; Keum, Gyochang; Liu, Jie; Small, Gary W.; Satyamurthy, Nagichettiar; Kepe, Vladimir; Barrio, Jorge R.

    2010-01-01

    This work focuses on the development of specific substrates for estrogen sulfotransferase (SULT1E1) to produce molecular imaging probes for this enzyme. SULT1E1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after β-naphthol (βN), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULT1E1 over SULT1A1*1, SULT1A1*2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity—Vmax/Km ratios—and specificity for SULT1E1. Km values ranged from 0.12–2.36 μM. A strong correlation was observed between polarity of the 4′-sustituent on the 2-aryl moiety (Hammett σp) and the log(Vmax/Km) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its 1H NMR chemical shift (δOH) with the log(Vmax/Km) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4′-substituent outlined by the correlation of the C-2 13C NMR chemical shift (δC2) with the log(Vmax/Km) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 (11C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled 11C-(2b)-6-O-sulfate and the SULT1E1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans. PMID:20304798

  10. Specific age-related molecular alterations in the cerebellum of Down syndrome mouse models.

    PubMed

    Créau, Nicole; Cabet, Eva; Daubigney, Fabrice; Souchet, Benoit; Bennaï, Soumia; Delabar, Jean

    2016-09-01

    Down syndrome, or trisomy 21, has been modeled with various trisomic and transgenic mice to help understand the consequences of an altered gene dosage in brain development and function. Though Down syndrome has been associated with premature aging, little is known about the molecular and cellular alterations that target brain function. To help identify alterations at specific ages, we analyzed the cerebellum of Ts1Cje mice, trisomic for 77 HSA21 orthologs, at three ages-young (4 months), middle-age (12 months), and old (17 months)-compared to age-matched controls. Quantification of neuronal and glial markers (n=11) revealed increases in GFAP, with an age effect, and S100B, with age and genotype effects. The genotype effect on S100B with age was unexpected as Ts1Cje has only two copies of the S100b gene. Interestingly, the different increase in GFAP observed between Ts1Cje (trisomic segment includes Pcp4 gene) and controls was magnified in TgPCP4 mice (1 extra copy of the human PCP4 gene) at the same age. S100B increase was not found in the TgPCP4 confirming a difference of regulation with aging for GFAP and S100B and excluding the calcium signaling regulator, Pcp4, as a potential candidate for increase of S100B in the Ts1Cje. To understand these differences, comparison of GFAP and S100B immunostainings at young and middle-age were performed. Immunohistochemical detection of differences in GFAP and S100B localization with aging implicate S100B+ oligodendrocytes as a new phenotypic target in this specific aging process. PMID:27297494

  11. Revealing species-specific trophic links in soil food webs: molecular identification of scarab predators.

    PubMed

    Juen, A; Traugott, M

    2007-04-01

    Soil food webs are particularly important in terrestrial systems, but studying them is difficult. Here we report on the first study to apply a molecular approach to identify species-specific trophic interactions in below-ground food webs. To identify the invertebrate predator guild of the garden chafer Phyllopertha horticola (Coleoptera, Scarabaeidae) whose root-feeding larvae can be highly abundant in grasslands, a specific DNA marker was developed. It allowed detection of P. horticola egg and white grub meals within the gut content of Poecilus versicolor (Coleoptera, Carabidae) larvae for up to 24 h post-feeding. Soil samples from an alpine grassland revealed a diverse below-ground macro-invertebrate community with earthworms, P. horticola larvae, and centipedes as well as beetle larvae as the most abundant detritivores, herbivores, and predators, respectively. Garden chafer DNA was detected in 18.6%, 4.1%, and 4.4% of field-collected Geophilidae (n = 124), beetle larvae (n = 159), and Lithobiidae (n = 49), respectively. We conclude that most of the investigated predators actively preyed on P. horticola, as secondary predation is unlikely to be detected in below-ground systems. Moreover, scavenging most likely contributes only to a small percentage of the revealed trophic links due to the low availability of carrion. Sampling date did not influence prey detection rates, indicating that both P. horticola eggs and larvae were preyed on. Only 2.7% of the below-ground predators tested positive for earthworms, an alternative, highly abundant prey, suggesting that P. horticola represents an important prey source for centipedes and predatory beetle larvae during summer within the soil food web.

  12. Revealing species-specific trophic links in soil food webs: molecular identification of scarab predators.

    PubMed

    Juen, A; Traugott, M

    2007-04-01

    Soil food webs are particularly important in terrestrial systems, but studying them is difficult. Here we report on the first study to apply a molecular approach to identify species-specific trophic interactions in below-ground food webs. To identify the invertebrate predator guild of the garden chafer Phyllopertha horticola (Coleoptera, Scarabaeidae) whose root-feeding larvae can be highly abundant in grasslands, a specific DNA marker was developed. It allowed detection of P. horticola egg and white grub meals within the gut content of Poecilus versicolor (Coleoptera, Carabidae) larvae for up to 24 h post-feeding. Soil samples from an alpine grassland revealed a diverse below-ground macro-invertebrate community with earthworms, P. horticola larvae, and centipedes as well as beetle larvae as the most abundant detritivores, herbivores, and predators, respectively. Garden chafer DNA was detected in 18.6%, 4.1%, and 4.4% of field-collected Geophilidae (n = 124), beetle larvae (n = 159), and Lithobiidae (n = 49), respectively. We conclude that most of the investigated predators actively preyed on P. horticola, as secondary predation is unlikely to be detected in below-ground systems. Moreover, scavenging most likely contributes only to a small percentage of the revealed trophic links due to the low availability of carrion. Sampling date did not influence prey detection rates, indicating that both P. horticola eggs and larvae were preyed on. Only 2.7% of the below-ground predators tested positive for earthworms, an alternative, highly abundant prey, suggesting that P. horticola represents an important prey source for centipedes and predatory beetle larvae during summer within the soil food web. PMID:17391275

  13. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

  14. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism. PMID:24664316

  15. 49 CFR 173.304a - Additional requirements for shipment of liquefied compressed gases in specification cylinders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... authorized only when transported by motor vehicle, rail car, or cargo-only aircraft. (ii) Additional... dangerous projection of a closing device when exposed to fire. (4) Verification of content. A cylinder...

  16. A thymidine-terminated molecular beacon for selective Hg 2+ or sequence-specific DNA assay

    NASA Astrophysics Data System (ADS)

    Hou, Peng; Long, Yunfei; Zhao, Jin; Wang, Jianxiu; Zhou, Feimeng

    2012-02-01

    A new molecular beacon (MB) in which fluorescein (FAM) attached to its 3' end acts as a fluorophore and a bridged thymidine-Hg-thymidine (T-Hg-T) complex acts as a quencher is designed. The fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher results in annihilation of the FAM fluorescence. Experimental conditions that govern the fluorescence quenching, such as number of thymidine bases, pH value, and salt concentration, have been optimized. The MB was found to be highly selective for Hg 2+ among a number of metal ions investigated. In the presence of single-stranded (ss-) target oligonucleotides (ODNs) with bases complementary to those in the loop of MB, the FAM fluorescence can be largely restored due to DNA duplex formation. The present method for DNA assay is also sequence-specific and can determine target ODN concentration at a nanomolar level. The substitution of the quencher group in a conventional MB molecule with simple thymidine bases affords an inexpensive ODN that retains the unique property of the MB molecule.

  17. Unraveling the molecular mechanism governing the tissue specific expression of IFNλR1.

    PubMed

    Akhtar, Hashaam; Hamming, Ole Jensen; Jan, Syed Umer; Akhtar, Samar; Terczyn' ska-Dyla, Ewa; Siupka, Piotr; Shafique, Adeena; Hartmann, Rune; Sadia, Hajra

    2016-05-01

    The functional receptor for type III interferons (IFNs) is a heterodimer of IFNLR1 and IL10R2. IFNLR1 is expressed in a highly tissue specific manner, with epithelial and liver tissue as the prime expressing tissues in humans. However, knowledge about the molecular pathways responsible for regulating the expression of IFNLR1 is yet unknown. In this study, various bioinformatics tools were used to predict the scores of signal peptides of IFNλR1 and IFNαR1, which was considered as an important difference in the expression of both receptors or participation in regulating the IFNLR1 gene. In silico study revealed that the signal peptide of IFNαR1 had more potential than the signal peptide of IFNλR1 but changing the signal peptide of wild type IFNλR1 with the signal peptide of IFNαR1 in wet lab had barely shown any differences. Selective expression of IFNλR1 was considered to be a plus point towards the targeted anti-viral activity of IFNλs but artificial control on its expression will surely make IFNλs a better drug with enhanced activity. The results of this study may help us in contributing some understanding towards the mechanisms involved in the selective expression of IFNLR1 and exceptionalities involved. PMID:27166550

  18. Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics.

    PubMed

    Kumar, Rajesh; Grønhaug, Kirsten M; Romijn, Elisabeth I; Finnøy, Andreas; Davies, Catharina L; Drogset, Jon O; Lilledahl, Magnus B

    2015-09-01

    Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.

  19. A thymidine-terminated molecular beacon for selective Hg2+ or sequence-specific DNA assay.

    PubMed

    Hou, Peng; Long, Yunfei; Zhao, Jin; Wang, Jianxiu; Zhou, Feimeng

    2012-02-01

    A new molecular beacon (MB) in which fluorescein (FAM) attached to its 3' end acts as a fluorophore and a bridged thymidine-Hg-thymidine (T-Hg-T) complex acts as a quencher is designed. The fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher results in annihilation of the FAM fluorescence. Experimental conditions that govern the fluorescence quenching, such as number of thymidine bases, pH value, and salt concentration, have been optimized. The MB was found to be highly selective for Hg(2+) among a number of metal ions investigated. In the presence of single-stranded (ss-) target oligonucleotides (ODNs) with bases complementary to those in the loop of MB, the FAM fluorescence can be largely restored due to DNA duplex formation. The present method for DNA assay is also sequence-specific and can determine target ODN concentration at a nanomolar level. The substitution of the quencher group in a conventional MB molecule with simple thymidine bases affords an inexpensive ODN that retains the unique property of the MB molecule.

  20. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1). PMID:26196069

  1. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1).

  2. Magnetic molecularly imprinted polymers synthesized by surface-initiated reversible addition-fragmentation chain transfer polymerization for the enrichment and determination of synthetic estrogens in aqueous solution.

    PubMed

    Chen, Fangfang; Zhang, Jingjing; Wang, Minjun; Kong, Jie

    2015-08-01

    Magnetic molecularly imprinted polymers have attracted significant interest because of their multifunctionality of selective recognition of target molecules and rapid magnetic response. In this contribution, magnetic molecularly imprinted polymers were synthesized via surface-initiated reversible addition addition-fragmentation chain transfer polymerization using diethylstilbestrol as the template for the enrichment of synthetic estrogens. The uniform imprinted surface layer and the magnetic property of the magnetic molecularly imprinted polymers favored a fast binding kinetics and rapid analysis of target molecules. The static and selective binding experiments demonstrated a desirable adsorption capacity and good selectivity of the magnetic molecularly imprinted polymers in comparison to magnetic non-molecularly imprinted polymers. Accordingly, a corresponding analytical method was developed in which magnetic molecularly imprinted polymers were employed as magnetic solid-phase extraction materials for the concentration and determination of four synthetic estrogens (diethylstilbestrol, hexestrol, dienestrol, and bisphenol A) in fish pond water. The recoveries of these synthetic estrogens in spiked fish pond water samples ranged from 61.2 to 99.1% with a relative standard deviation of lower than 6.3%. This study provides a versatile approach to prepare well-defined magnetic molecularly imprinted polymers sorbents for the analysis of synthetic estrogens in water solution. PMID:25989155

  3. Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

    PubMed Central

    Talesa, V; Grauso, M; Giovannini, E; Rosi, G; Toutant, J P

    1995-01-01

    Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M. Images Figure 2 Figure 3 Figure 4 PMID:7702560

  4. Molecular and biochemical characterization of a Coccidioides immitis-specific antigen.

    PubMed Central

    Pan, S; Cole, G T

    1995-01-01

    Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture

  5. Phylogenomic Analyses and Comparative Studies on Genomes of the Bifidobacteriales: Identification of Molecular Signatures Specific for the Order Bifidobacteriales and Its Different Subclades

    PubMed Central

    Zhang, Grace; Gao, Beile; Adeolu, Mobolaji; Khadka, Bijendra; Gupta, Radhey S.

    2016-01-01

    The order Bifidobacteriales comprises a diverse variety of species found in the gastrointestinal tract of humans and other animals, some of which are opportunistic pathogens, whereas a number of others exhibit health-promoting effects. However, currently very few biochemical or molecular characteristics are known which are specific for the order Bifidobacteriales, or specific clades within this order, which distinguish them from other bacteria. This study reports the results of detailed comparative genomic and phylogenetic studies on 62 genome-sequenced species/strains from the order Bifidobacteriales. In a robust phylogenetic tree for the Bifidobacteriales constructed based on 614 core proteins, a number of well-resolved clades were observed including a clade separating the Scarodvia-related genera (Scardovia clade) from the genera Bifidobacterium and Gardnerella, as well as a number of previously reported clusters of Bifidobacterium spp. In parallel, our comparative analyses of protein sequences from the Bifidobacteriales genomes have identified numerous molecular markers that are specific for this group of bacteria. Of these markers, 32 conserved signature indels (CSIs) in widely distributed proteins and 10 signature proteins are distinctive characteristics of all sequenced Bifidobacteriales species and provide novel and highly specific means for distinguishing these bacteria. In addition, multiple other molecular signatures are specific for the following clades of Bifidobacteriales: (i) 5 CSIs specific for a clade comprising of the Scardovia-related genera; (ii) 3 CSIs and 2 CSPs specific for a clade consisting of the Bifidobacterium and Gardnerella spp.; (iii) multiple other signatures demarcating a number of clusters of the B. asteroides-and B. longum- related species. The described molecular markers provide novel and reliable means for distinguishing the Bifidobacteriales and a number of their clades in molecular terms and for the classification of these

  6. Phylogenomic Analyses and Comparative Studies on Genomes of the Bifidobacteriales: Identification of Molecular Signatures Specific for the Order Bifidobacteriales and Its Different Subclades.

    PubMed

    Zhang, Grace; Gao, Beile; Adeolu, Mobolaji; Khadka, Bijendra; Gupta, Radhey S

    2016-01-01

    The order Bifidobacteriales comprises a diverse variety of species found in the gastrointestinal tract of humans and other animals, some of which are opportunistic pathogens, whereas a number of others exhibit health-promoting effects. However, currently very few biochemical or molecular characteristics are known which are specific for the order Bifidobacteriales, or specific clades within this order, which distinguish them from other bacteria. This study reports the results of detailed comparative genomic and phylogenetic studies on 62 genome-sequenced species/strains from the order Bifidobacteriales. In a robust phylogenetic tree for the Bifidobacteriales constructed based on 614 core proteins, a number of well-resolved clades were observed including a clade separating the Scarodvia-related genera (Scardovia clade) from the genera Bifidobacterium and Gardnerella, as well as a number of previously reported clusters of Bifidobacterium spp. In parallel, our comparative analyses of protein sequences from the Bifidobacteriales genomes have identified numerous molecular markers that are specific for this group of bacteria. Of these markers, 32 conserved signature indels (CSIs) in widely distributed proteins and 10 signature proteins are distinctive characteristics of all sequenced Bifidobacteriales species and provide novel and highly specific means for distinguishing these bacteria. In addition, multiple other molecular signatures are specific for the following clades of Bifidobacteriales: (i) 5 CSIs specific for a clade comprising of the Scardovia-related genera; (ii) 3 CSIs and 2 CSPs specific for a clade consisting of the Bifidobacterium and Gardnerella spp.; (iii) multiple other signatures demarcating a number of clusters of the B. asteroides-and B. longum- related species. The described molecular markers provide novel and reliable means for distinguishing the Bifidobacteriales and a number of their clades in molecular terms and for the classification of these

  7. Phylogenomic Analyses and Comparative Studies on Genomes of the Bifidobacteriales: Identification of Molecular Signatures Specific for the Order Bifidobacteriales and Its Different Subclades.

    PubMed

    Zhang, Grace; Gao, Beile; Adeolu, Mobolaji; Khadka, Bijendra; Gupta, Radhey S

    2016-01-01

    The order Bifidobacteriales comprises a diverse variety of species found in the gastrointestinal tract of humans and other animals, some of which are opportunistic pathogens, whereas a number of others exhibit health-promoting effects. However, currently very few biochemical or molecular characteristics are known which are specific for the order Bifidobacteriales, or specific clades within this order, which distinguish them from other bacteria. This study reports the results of detailed comparative genomic and phylogenetic studies on 62 genome-sequenced species/strains from the order Bifidobacteriales. In a robust phylogenetic tree for the Bifidobacteriales constructed based on 614 core proteins, a number of well-resolved clades were observed including a clade separating the Scarodvia-related genera (Scardovia clade) from the genera Bifidobacterium and Gardnerella, as well as a number of previously reported clusters of Bifidobacterium spp. In parallel, our comparative analyses of protein sequences from the Bifidobacteriales genomes have identified numerous molecular markers that are specific for this group of bacteria. Of these markers, 32 conserved signature indels (CSIs) in widely distributed proteins and 10 signature proteins are distinctive characteristics of all sequenced Bifidobacteriales species and provide novel and highly specific means for distinguishing these bacteria. In addition, multiple other molecular signatures are specific for the following clades of Bifidobacteriales: (i) 5 CSIs specific for a clade comprising of the Scardovia-related genera; (ii) 3 CSIs and 2 CSPs specific for a clade consisting of the Bifidobacterium and Gardnerella spp.; (iii) multiple other signatures demarcating a number of clusters of the B. asteroides-and B. longum- related species. The described molecular markers provide novel and reliable means for distinguishing the Bifidobacteriales and a number of their clades in molecular terms and for the classification of these

  8. Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR).

    PubMed

    Strachan, Ryan T; Sun, Jin-peng; Rominger, David H; Violin, Jonathan D; Ahn, Seungkirl; Rojas Bie Thomsen, Alex; Zhu, Xiao; Kleist, Andrew; Costa, Tommaso; Lefkowitz, Robert J

    2014-05-16

    The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.

  9. Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR).

    PubMed

    Strachan, Ryan T; Sun, Jin-peng; Rominger, David H; Violin, Jonathan D; Ahn, Seungkirl; Rojas Bie Thomsen, Alex; Zhu, Xiao; Kleist, Andrew; Costa, Tommaso; Lefkowitz, Robert J

    2014-05-16

    The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR. PMID:24668815

  10. Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)*

    PubMed Central

    Strachan, Ryan T.; Sun, Jin-peng; Rominger, David H.; Violin, Jonathan D.; Ahn, Seungkirl; Rojas Bie Thomsen, Alex; Zhu, Xiao; Kleist, Andrew; Costa, Tommaso; Lefkowitz, Robert J.

    2014-01-01

    The concept of “biased agonism” arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. “efficacy”) can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR. PMID:24668815

  11. Low Molecular Weight Amidoximes that Act as Potent Inhibitors of Lysine-Specific Demethylase 1

    PubMed Central

    Hazeldine, Stuart; Pachaiyappan, Boobalan; Steinbergs, Nora; Nowotarski, Shannon; Hanson, Allison S.; Casero, Robert A.; Woster, Patrick M.

    2012-01-01

    The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 dysregulation is thought to contribute to the development of cancer. We reported that (bis)guanidines, (bis)biguanides and their urea- and thiourea isosteres are potent inhibitors of LSD1, and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of small molecule amidoximes that are moderate inhibitors of recombinant LSD1, but that produce dramatic changes in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2, H-cadherin (HCAD) and transcription factor GATA4. These compounds represent leads for an important new series of drug-like epigenetic modulators with the potential for use as antitumor agents. PMID:22876979

  12. Molecular Characterization of the Toxic Cyanobacterium Cylindrospermopsis raciborskii and Design of a Species-Specific PCR

    PubMed Central

    Wilson, Kim M.; Schembri, Mark A.; Baker, Peter D.; Saint, Christopher P.

    2000-01-01

    Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii. PMID:10618244

  13. Molecular recognition of malachite green by hemoglobin and their specific interactions: insights from in silico docking and molecular spectroscopy.

    PubMed

    Peng, Wei; Ding, Fei; Peng, Yu-Kui; Sun, Ying

    2014-01-01

    Malachite green is an organic compound that can be widely used as a dyestuff for various materials; it has also emerged as a controversial agent in aquaculture. Since malachite green is proven to be carcinogenic and mutagenic, it may become a hazard to public health. For this reason, it is urgently required to analyze this controversial dye in more detail. In our current research, the interaction between malachite green and hemoglobin under physiological conditions was investigated by the methods of molecular modeling, fluorescence spectroscopy, circular dichroism (CD) as well as hydrophobic ANS displacement experiments. From the molecular docking, the central cavity of hemoglobin was assigned to possess high-affinity for malachite green, this result was corroborated by time-resolved fluorescence and hydrophobic ANS probe results. The recognition mechanism was found to be of static type, or rather the hemoglobin-malachite green complex formation occurred via noncovalent interactions such as π-π interactions, hydrogen bonds and hydrophobic interactions with an association constant of 10(4) M(-1). Moreover, the results also show that the spatial structure of the biopolymer was changed in the presence of malachite green with a decrease of the α-helix and increase of the β-sheet, turn and random coil suggesting protein damage, as derived from far-UV CD and three-dimensional fluorescence. Results of this work will help to further comprehend the molecular recognition of malachite green by the receptor protein and the possible toxicological profiles of other compounds, which are the metabolites and ramifications of malachite green.

  14. Resolving Non-Specific and Specific Adhesive Interactions of Catechols at Solid/Liquid Interfaces at the Molecular Scale.

    PubMed

    Utzig, Thomas; Stock, Philipp; Valtiner, Markus

    2016-08-01

    The adhesive system of mussels evolved into a powerful and adaptive system with affinity to a wide range of surfaces. It is widely known that thereby 3,4-dihydroxyphenylalanine (Dopa) plays a central role. However underlying binding energies remain unknown at the single molecular scale. Here, we use single-molecule force spectroscopy to estimate binding energies of single catechols with a large range of opposing chemical functionalities. Our data demonstrate significant interactions of Dopa with all functionalities, yet most interactions fall within the medium-strong range of 10-20 kB T. Only bidentate binding to TiO2 surfaces exhibits a higher binding energy of 29 kB T. Our data also demonstrate at the single-molecule level that oxidized Dopa and amines exhibit interaction energies in the range of covalent bonds, confirming the important role of Dopa for cross-linking in the bulk mussel adhesive. We anticipate that our approach and data will further advance the understanding of biologic and technologic adhesives. PMID:27374053

  15. M118--a rationally engineered low-molecular-weight heparin designed specifically for the treatment of acute coronary syndromes.

    PubMed

    Kishimoto, Takashi Kei; Qi, Yi Wei; Long, Alison; Capila, Ishan; Sasisekharan, Ram; Guerrero, Luis; Fier, Ian; Roach, James; Venkataraman, Ganesh

    2009-11-01

    The initial choice of anticoagulant therapy administered in emergency departments for acute coronary syndromes (ACS) has important consequences for subsequent patient care, as neither unfractionated heparin (UFH) nor low-molecular-weight heparin (LMWH) are ideally suited for all potential clinical treatment pathways. UFH remains widely used for surgical interventions because of the ability to rapidly reverse its anticoagulant activity. However, the unpredictable pharmacokinetic profile of UFH presents safety issues, and the low subcutaneous bioavailability limits the utility of UFH for patients who are medically managed. LMWH has superior pharmacokinetic properties, but its anticoagulant activity cannot be effectively monitored or reversed during surgery. There is an unmet medical need for a baseline anticoagulant therapy that addresses these shortcomings while retaining the beneficial properties of both UFH and LMWH. We describe here M118, a novel LMWH designed specifically for use in the treatment of ACS. M118 shows broad anticoagulant activity, including potent activity against both factor Xa (~240 IU/mg) and thrombin (factor IIa; ~170 IU/mg), low polydispersity, high (78%) subcutaneous bioavailability in rabbits, and predictable subcutaneous and intravenous pharmacokinetics. Additionally, the anticoagulant activity of M118 is monitorable by standard coagulation assays and is reversible with protamine. M118 demonstrates superior activity to conventional LMWH in a rabbit model of abdominal arterial thrombosis without increasing bleeding risk, and is currently being evaluated in a phase II clinical trial evaluating efficacy and safety in patients undergoing percutaneous coronary intervention. PMID:19888526

  16. Pomegranate juice and specific components inhibit cell and molecular processes critical for metastasis of breast cancer.

    PubMed

    Rocha, Ana; Wang, Lei; Penichet, Manuel; Martins-Green, Manuela

    2012-12-01

    Breast cancer is the most common cancer and the second leading cause of cancer death and morbidity among women in the western world. Pomegranate juice (PJ) and three of its specific components have been shown to inhibit processes involved in prostate cancer metastasis. If this also proves to be true for breast cancer, these natural treatments will be promising agents against breast cancer that can serve as potentially effective and nontoxic alternatives or adjuncts to the use of conventional selective estrogen receptor modulators for breast cancer prevention and treatment. To test this possibility, we have used two breast cancer cell lines, MDA-MB-231 cells (ER(-)) and MCF7 (ER(+)), and the non-neoplastic cell line MCF10A. We show that, in addition to inhibiting growth of the breast cancer cells, PJ or a combination of its components luteolin (L) + ellagic acid (E) + punicic acid (P) increase cancer cell adhesion and decrease cancer cell migration but do not affect normal cells. These treatments also inhibit chemotaxis of the cancer cells to SDF1α, a chemokine that attracts breast cancer cells to the bone. We hypothesized that PJ and L + E + P stimulate expression of genes that increase adhesion and inhibit genes that stimulate cell migration and inhibit chemotaxis to SDF1α. Using qPCR, we confirmed these proposed effects on gene expression and in addition we found that a gene important in epithelial-to-meshenchymal transitions is decreased. We also found that pro-inflammatory cytokines/chemokines are significantly reduced by these treatments, thereby having the potential to decrease inflammation and its impact on cancer progression. Discovery that PJ and L + E + P are inhibitory of metastatic processes in breast cancer cells in addition to prostate cancer cells indicate that they are potentially a very effective treatment to prevent cancer progression in general.

  17. Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Kanofsky, Konstantin; Hanko, Erik K R; Ahrendt, Sarah; Wehrs, Maren; Machens, Fabian; Hehl, Reinhard

    2016-01-01

    Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.

  18. Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Kanofsky, Konstantin; Hanko, Erik K R; Ahrendt, Sarah; Wehrs, Maren; Machens, Fabian; Hehl, Reinhard

    2016-01-01

    Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA. PMID:25819608

  19. Molecular evolution of a chordate specific family of G protein-coupled receptors

    PubMed Central

    2011-01-01

    Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become

  20. Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS.

    PubMed

    Albi, Tomás; Serrano, Aurelio

    2014-09-01

    The genome of the thermophilic green-sulfur bacterium Chlorobium tepidum TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppx genes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K(+)-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg(2+). Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world.

  1. Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS.

    PubMed

    Albi, Tomás; Serrano, Aurelio

    2014-09-01

    The genome of the thermophilic green-sulfur bacterium Chlorobium tepidum TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppx genes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K(+)-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg(2+). Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world. PMID:24969471

  2. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

    PubMed

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2016-07-01

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. PMID:27320834

  3. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

    PubMed

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2016-07-01

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

  4. Evaluation of molecular markers for Phytophthora ramorum detection and identification; testing for specificity using a standardized library of isolates.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A number of molecular techniques have been developed for detection of Phytophthora ramorum from infected tissue. These have been based on spacer regions (the rDNA ITS region, the spacer region between the cox I and II gene) or specific genes (beta tubulin, elicitin) and have been configured for use ...

  5. Effects of specific domains of high-molecular-weight glutenin subunits’ on dough properties by an in vitro assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An in vitro system for incorporating bacterially produced high-molecular-weight glutenin subunits (HMW-GS) into doughs was used to study the effects of specific domains of the HMW-GS. Synergistic effects of incorporating into doughs both the Dx5 and Dy10 subunits are localized to the N-terminal do...

  6. Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs.

    PubMed

    Nakamura, Yoriko; Iwasaki, Takehiro; Umei, Yosuke; Saotome, Kazuhiro; Nakajima, Yukiko; Kitahara, Shoichi; Uno, Yoshinobu; Matsuda, Yoichi; Oike, Akira; Kodama, Maho; Nakamura, Masahisa

    2015-10-01

    The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 μm in diameter), and absent from small germ cells (<50 μm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa.

  7. Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid.

    PubMed

    Tárraga, Susana; Lisón, Purificación; López-Gresa, María Pilar; Torres, Cristina; Rodrigo, Ismael; Bellés, José María; Conejero, Vicente

    2010-10-01

    The importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many incompatible plant-pathogen interactions, but less is known about signalling in compatible interactions. In this type of interaction, tomato plants have been found to accumulate high levels of 2,5-dihydroxybenzoic acid (gentisic acid, GA), a metabolic derivative of SA. Exogenous GA treatments induce in tomato plants a set of PR proteins that differ from those induced by salicylic acid. While SA accumulates in tomato plants mainly as 2-O-β-D-glucoside, GA has only been found as 5-O-β-D-xyloside. To characterize this step of the GA signalling pathway further, the present work focuses on the study of the GA-conjugating activity in tomato plants. A gentisate glycosyltransferase (GAGT) cDNA has been isolated and overexpressed in Pichia pastoris, and GA-conjugating activity was confirmed by detecting the xylosylated GA. The purified plant protein is highly specific for GA, showing no activity toward many other phenolic compounds, including SA. In addition, it shows an outstanding selectivity for UDP-xylose as the sugar donor, which differentiates this enzyme from most glycosyltransferases. Both the GA-conjugating activity and the corresponding mRNA show a strong, rapid, and transient induction upon treatment of tomato plants with GA or SA. Furthermore, its expression is rapidly induced by compatible infections. However, neither the gene nor the activity seems to respond to incompatible infections or wounding. The unique properties of this new glycosyltransferase suggest a specific role in regulating the free GA levels in compatible plant-pathogen interactions.

  8. SELECTIVE OXIDATION OF ALCOHOLS BY MOLECULAR OXYGEN OVER A PD/MGO CATALYST IN THE ABSENCE OF ANY ADDITIVES

    EPA Science Inventory

    Selective oxidation of alcohols to the corresponding carbonyl products using molecular oxygen is achieved over a simple and easily recyclable 1% Pd/MgO impregnated heterogeneous catalyst in the presence of trifluorotoluene. A variety of activated and non-activated alcohols are ef...

  9. Ultra-low-molecular-weight heparins: precise structural features impacting specific anticoagulant activities.

    PubMed

    Lima, Marcelo A; Viskov, Christian; Herman, Frederic; Gray, Angel L; de Farias, Eduardo H C; Cavalheiro, Renan P; Sassaki, Guilherme L; Hoppensteadt, Debra; Fareed, Jawed; Nader, Helena B

    2013-03-01

    Ultra-low-molecular-weight heparins (ULMWHs) with better efficacy and safety ratios are under development; however, there are few structural data available. The main structural features and molecular weight of ULMWHs were studied and compared to enoxaparin. Their monosaccharide composition and average molecular weights were determined and preparations studied by nuclear magnetic resonance spectroscopy, scanning ultraviolet spectroscopy, circular dichroism and gel permeation chromatography. In general, ULMWHs presented higher 3-O-sulphated glucosamine and unsaturated uronic acid residues, the latter being comparable with their higher degree of depolymerisation. The analysis showed that ULMWHs are structurally related to LMWHs; however, their monosaccharide/oligosaccharide compositions and average molecular weights differed considerably explaining their different anticoagulant activities. The results relate structural features to activity, assisting the development of new and improved therapeutic agents, based on depolymerised heparin, for the prophylaxis and treatment of thrombotic disorders.

  10. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

    SciTech Connect

    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  11. Fast molecular beacon hybridization in organic solvents with improved target specificity.

    PubMed

    Dave, Neeshma; Liu, Juewen

    2010-12-01

    DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics. Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison with detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared with water. For example, in 56% ethanol, the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single-base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentration-dependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.

  12. Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid.

    PubMed Central

    Parker, J S; Parrish, C R

    1997-01-01

    We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range. PMID:9371580

  13. Quantum ring-polymer contraction method: Including nuclear quantum effects at no additional computational cost in comparison to ab initio molecular dynamics.

    PubMed

    John, Christopher; Spura, Thomas; Habershon, Scott; Kühne, Thomas D

    2016-04-01

    We present a simple and accurate computational method which facilitates ab initio path-integral molecular dynamics simulations, where the quantum-mechanical nature of the nuclei is explicitly taken into account, at essentially no additional computational cost in comparison to the corresponding calculation using classical nuclei. The predictive power of the proposed quantum ring-polymer contraction method is demonstrated by computing various static and dynamic properties of liquid water at ambient conditions using density functional theory. This development will enable routine inclusion of nuclear quantum effects in ab initio molecular dynamics simulations of condensed-phase systems. PMID:27176426

  14. Quantum ring-polymer contraction method: Including nuclear quantum effects at no additional computational cost in comparison to ab initio molecular dynamics

    NASA Astrophysics Data System (ADS)

    John, Christopher; Spura, Thomas; Habershon, Scott; Kühne, Thomas D.

    2016-04-01

    We present a simple and accurate computational method which facilitates ab initio path-integral molecular dynamics simulations, where the quantum-mechanical nature of the nuclei is explicitly taken into account, at essentially no additional computational cost in comparison to the corresponding calculation using classical nuclei. The predictive power of the proposed quantum ring-polymer contraction method is demonstrated by computing various static and dynamic properties of liquid water at ambient conditions using density functional theory. This development will enable routine inclusion of nuclear quantum effects in ab initio molecular dynamics simulations of condensed-phase systems.

  15. Counter-ion specificity explored in abnormal expansion of supra-molecular aggregates in aqueous solution of alkaline metal salts.

    PubMed

    Huang, Ningdong; Tao, Jiaojiao; Wei, Shenghui; Chen, Mingming; Wei, Chengsha; Li, Liangbin

    2015-09-21

    Ionic effects in aqueous solution of macro-ions showing specificity and unconventional characters, respectively, receive a lot of interests recently; however, the complexity of specific ion effects in unconventional phenomena remains ambiguous. In this study, the effects of univalent ions on aggregation of supra-molecular nano-fibrils with charged carboxylate groups on the surface as a prototype of macro-ions are investigated by Small Angle X-ray Scattering (SAXS) in aqueous solutions of alkaline metal chlorides. It is found that the columnar bundles of charged fibrils are expanded in certain salt concentration range contradicting the conventional screening effects of salts. The degree of expansion is dominated by cations as Na(+) induces drastic effects in comparison to rather gentle changes from K(+) and Cs(+). The specific cations effects observed by SAXS correlate with the pH behavior of the solutions, an indicator of surface charge, or number of carboxylate groups along the supra-molecular fibrils. It is postulated that while Na(+) with stronger affinity to carboxylates apparently reduces the surface charge, K(+) and Cs(+) only weakly interact with carboxylates and induce minor changes, accounting for the cation-sensitive aggregation behavior of fibrils observed by SAXS. By probing the bundling aggregation of charged supra-molecular nano-fibrils in salty water, we provide direct evidence of specific counter-ion effects in unusual expansion caused by univalent salts. PMID:26395732

  16. Nanosilica-based molecularly imprinted polymer nanoshell for specific recognition and determination of rhodamine B in red wine and beverages.

    PubMed

    Long, Zerong; Xu, Weiwei; Lu, Yi; Qiu, Hongdeng

    2016-09-01

    A new and facile rhodamine B (RhB)-imprinted polymer nanoshell coating for SiO2 nanoparticles was readily prepared by a combination of silica gel modification and molecular surface imprinting. The RhB-imprinted polymers (RhB-MIPs) were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and UV-vis spectroscopy; the binding properties and selectivity of these MIPs were investigated in detail. The uniformly imprinted nanoparticles displayed a rather thin shell thickness (23nm) with highly effective recognition sites, showing homogenous distribution and monolayer adsorption. The maximum MIP adsorption capacity (Qm) was as high as 45.2mgg(-1), with an adsorption equilibrium time of about 15min at ambient temperature. Dynamic rebinding experiments showed that chemical adsorption is crucial for RhB binding to RhB-MIPs. The adsorption isotherm for RhB-MIPs binding could also be described by the Langmuir equation at different temperatures and pH values. Increasing temperature led to an enhanced Qm, a decreased dissociation constant (K'd), and a more negative free energy (ΔG), indicating that adsorption is favored at higher temperatures. Moreover, the adsorption capacity of RhB was remarkably affected by pH. At pH>7, the adsorption of RhB was driven by hydrogen bonding interactions, while at pH<7 electrostatic forces were dominant. Additionally, the MIPs also showed specific recognition of RhB from the standard mixture solution containing five structurally analogs. This method was also successfully employed to determine RhB content in red wine and beverages using three levels of spiking, with recoveries in the range of 91.6-93.1% and relative standard deviations lower than 4.1%. PMID:27372912

  17. Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF.

    PubMed

    Bielnicki, Jakub A; Shkumatov, Alexander V; Derewenda, Urszula; Somlyo, Avril V; Svergun, Dmitri I; Derewenda, Zygmunt S

    2011-10-01

    PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

  18. Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel.

    PubMed

    Okubo, K; Suetake, H; Usami, T; Aida, K

    2000-08-01

    Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

  19. A Fluorescent Molecular Probe for the Detection of Hydrogen Based on Oxidative Addition Reactions with Crabtree-Type Hydrogenation Catalysts.

    PubMed

    Kos, Pavlo; Plenio, Herbert

    2015-11-01

    A Crabtree-type Ir(I) complex tagged with a fluorescent dye (bodipy) was synthesized. The oxidative addition of H2 converts the weakly fluorescent Ir(I) complex (Φ=0.038) into a highly fluorescent Ir(III) species (Φ=0.51). This fluorogenic reaction can be utilized for the detection of H2 and to probe the oxidative addition step in the catalytic hydrogenation of olefins.

  20. Molecular Form Differences Between Prostate-Specific Antigen (PSA) Standards Create Quantitative Discordances in PSA ELISA Measurements

    NASA Astrophysics Data System (ADS)

    McJimpsey, Erica L.

    2016-02-01

    The prostate-specific antigen (PSA) assays currently employed for the detection of prostate cancer (PCa) lack the specificity needed to differentiate PCa from benign prostatic hyperplasia and have high false positive rates. The PSA calibrants used to create calibration curves in these assays are typically purified from seminal plasma and contain many molecular forms (intact PSA and cleaved subforms). The purpose of this study was to determine if the composition of the PSA molecular forms found in these PSA standards contribute to the lack of PSA test reliability. To this end, seminal plasma purified PSA standards from different commercial sources were investigated by western blot (WB) and in multiple research grade PSA ELISAs. The WB results revealed that all of the PSA standards contained different mass concentrations of intact and cleaved molecular forms. Increased mass concentrations of intact PSA yielded higher immunoassay absorbance values, even between lots from the same manufacturer. Standardization of seminal plasma derived PSA calibrant molecular form mass concentrations and purification methods will assist in closing the gaps in PCa testing measurements that require the use of PSA values, such as the % free PSA and Prostate Health Index by increasing the accuracy of the calibration curves.

  1. Molecular Models Underlying the Sensitive and Specific Determination of Copy Number Changes in the Human Genome and Transcriptome

    NASA Astrophysics Data System (ADS)

    Laderman, Stephen

    2004-03-01

    DNA microarrays enable the measurement of relative abundances of messenger RNA (mRNA) molecules across biological conditions on a gene-by-gene basis for tens of thousands of genes at a time, encompassing essentially all the mRNA molecules present in the sample's cells. These highly multiplexed molecular measurements are greatly accelerating basic and applied research in disease progression and developmental biology. In the study of cancer and certain genetic disorders, similar snapshots of copy numbers of genomic DNA are also of great interest. For example, tumor suppressor genes that protect against improper cell growth are lost from the genome and oncogenes that accelerate cell proliferation are multiplied within the genome as cancer cells evolve. Microarray measurements of mRNA and DNA are themselves an aggregate of molecular level phenomena. Quantitative models of in vitro behavior at the molecular scale are essential for creating effective measurement systems that have sufficient sensitivity and specificity to realize the highest utility. Such models are actively used in critical areas such as designing the means of incorporating fluorophores into the sample molecules, building high quality DNA sequences onto the microarrays, and developing assay parameters that balance high sensitivity with an ability to distinguish genes from each other. Trends in the applications of the technology ensure continued development towards even more precise, reproducible, sensitive and specific measurement systems, encouraging the further development and application of relevant molecular models.

  2. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology

    PubMed Central

    Cyphert, Jaime M.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects. PMID:26448754

  3. Universality and specificity in molecular orientation in anisotropic gels prepared by diffusion method.

    PubMed

    Maki, Yasuyuki; Furusawa, Kazuya; Yasuraoka, Sho; Okamura, Hideki; Hosoya, Natsuki; Sunaga, Mari; Dobashi, Toshiaki; Sugimoto, Yasunobu; Wakabayashi, Katsuzo

    2014-08-01

    Molecular orientation in anisotropic gels of chitosan, Curdlan and DNA obtained by dialysis of those aqueous solutions in gelation-inducing solutions was investigated. In this diffusion method (or dialysis method), the gel formation was induced by letting small molecules diffuse in or out of the polymer solutions through the surface. For the gels of DNA and chitosan, the polymer chains aligned perpendicular to the diffusion direction. The same direction of molecular orientation was observed for the Curdlan gel prepared in the dialysis cell. On the other hand, a peculiar nature was observed for the Curdlan gel prepared in the dialysis tube: the molecular orientation was perpendicular to the diffusion direction in the outermost layer of the gel, while the orientation was parallel to the diffusion direction in the inner translucent layer. The orientation parallel to the diffusion direction is attributed to a small deformation of the inner translucent layer caused by a slight shrinkage of the central region after the gel formation. At least near the surface of the gel, the molecular orientation perpendicular to the diffusion direction is a universal characteristic for the gels prepared by the diffusion method. PMID:24751255

  4. Molecular species specificity of phospholipid breakdown in microsomal membranes of senescing carnation flowers

    SciTech Connect

    Brown, J.H.; Lynch, D.V.; Thompson, J.E.

    1987-11-01

    During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0/sup *//16:0/sup */; 16:0/18:2/sup */, and 18:1/sup *//18:1/sup */ phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, (U-/sup 14/C)phosphatidylcholine, which comprises various molecular species including those containing polyunsaturated acyl chains, and 18:0/20:4/sup */ phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes.

  5. Molecular species specificity of phospholipid breakdown in microsomal membranes of senescing carnation flowers.

    PubMed

    Brown, J H; Lynch, D V; Thompson, J E

    1987-11-01

    During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0(*)/16:0(*), 16:0/18:2(*), and 18:1(*)/18:1(*) phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, [U-(14)C]phosphatidylcholine, which comprises various molecular species including those containing polyunsaturated acyl chains, and 18:0/20:4(*) phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes.

  6. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines.

    PubMed

    Ori, Alessandro; Banterle, Niccolò; Iskar, Murat; Andrés-Pons, Amparo; Escher, Claudia; Khanh Bui, Huy; Sparks, Lenore; Solis-Mezarino, Victor; Rinner, Oliver; Bork, Peer; Lemke, Edward A; Beck, Martin

    2013-01-01

    To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.

  7. Effect of minimizing amount of template by addition of macromolecular crowding agent on preparation of molecularly imprinted monolith.

    PubMed

    Sun, Guang-Ying; Zhong, Dan-Dan; Li, Xiang-Jie; Luo, Yu-Qing; Ba, Hang; Liu, Zhao-Sheng; Aisa, Haji Akber

    2015-09-01

    One of the main challenges in the preparation of molecularly imprinted polymers (MIPs) is the substantial initial amount of template needed because of the requirement of high load capacities for most applications. A new strategy of macromolecular crowding was suggested to solve this problem by reducing the amount of template in the polymerization recipe. In a ternary porogenic system of polystyrene (PS) (crowding agent), tetrahydrofuran, and toluene, an imprinted monolithic column with high porosity and good permeability was synthesized using a mixture of ellagic acid (template), acrylamide, and ethylene glycol dimethacrylate. The effect of polymerization factors, including monomer-template molar ratio and the molecular weight and concentration of PS, on the imprinting effect of the resulting MIP monoliths was systematically investigated. At a high ratio of monomer-template (120:1), the greatest imprinting factor of 32.4 was obtained on the MIP monolith with the aid of macromolecular crowding agent. The PS-based imprinted monolith had imprinting even at the extremely high ratio of functional monomer to template of 1510:1. Furthermore, an off-line solid-phase extraction based on the ground MIP was conducted, and the purification recovery of ellagic acid from pomegranate-rind extract was up to 80 %. In conclusion, this approach based on macromolecular crowding is simple, and is especially valuable for those applications of MIP preparation for which a rare template is used.

  8. Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon.

    PubMed

    Wu, Sheng-Mei; Tian, Zhi-Quan; Zhang, Zhi-Ling; Huang, Bi-Hai; Jiang, Peng; Xie, Zhi-Xiong; Pang, Dai-Wen

    2010-10-15

    Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.

  9. Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones.

    PubMed

    Tóth, Gergő; Baska, Ferenc; Schretner, András; Rácz, Akos; Noszál, Béla

    2013-09-01

    Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications. PMID:23907234

  10. atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

    PubMed Central

    2013-01-01

    Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

  11. Monoclonal antibodies with equal specificity to D-dimer and high-molecular-weight fibrin degradation products

    PubMed Central

    Kogan, Alexander E.; Mukharyamova, Kadriya S.; Bereznikova, Anastasia V.; Filatov, Vladimir L.; Koshkina, Ekaterina V.; Bloshchitsyna, Marina N.; Katrukha, Alexey G.

    2016-01-01

    Fibrin degradation results in the formation of fibrin degradation products (FDPs) of different molecular weights, which include D-dimer. Commercial D-dimer assays recognize multiple forms of FDP with different specificity. As a result, the absence of an international D-dimer standard and the marked discrepancy in the D-dimer values in the same samples measured by assays from different manufacturers have become the primary problems that clinicians face in the D-dimer determination. We consider that an assay with equal specificity to all FDP forms regardless of their molecular weights could help to solve these problems. We aimed to produce mAbs that could equally recognize high-molecular-weight FDP (HMW FDP) and D-dimer. mAbs against D-dimer were produced. The HMW FDP/D-dimer ratios in plasma samples were analyzed following protein separation by gel filtration using the developed fluoroimmunoassay. A sandwich immunoassay with equal specificity to HMW FDP and D-dimer was developed and applied to determine HMW FDP/D-dimer ratios in patients with different diseases. Although the HMW FDP levels prevailed in thrombotic patients, the FDP and D-dimer levels were comparable in septic patients. Meanwhile, the D-dimer levels often exceeded the HMW FDP levels in patients who had undergone surgery. The ‘D-dimer’ levels that were detected by different assays also varied greatly depending on the assay specificities to FDP and D-dimer. Our findings show that the introduction of assays with equal specificities to FDP and D-dimer in clinical practice is a possible way of standardizing D-dimer measurements. PMID:26656897

  12. Cancer in silico drug discovery: a systems biology tool for identifying candidate drugs to target specific molecular tumor subtypes.

    PubMed

    San Lucas, F Anthony; Fowler, Jerry; Chang, Kyle; Kopetz, Scott; Vilar, Eduardo; Scheet, Paul

    2014-12-01

    Large-scale cancer datasets such as The Cancer Genome Atlas (TCGA) allow researchers to profile tumors based on a wide range of clinical and molecular characteristics. Subsequently, TCGA-derived gene expression profiles can be analyzed with the Connectivity Map (CMap) to find candidate drugs to target tumors with specific clinical phenotypes or molecular characteristics. This represents a powerful computational approach for candidate drug identification, but due to the complexity of TCGA and technology differences between CMap and TCGA experiments, such analyses are challenging to conduct and reproduce. We present Cancer in silico Drug Discovery (CiDD; scheet.org/software), a computational drug discovery platform that addresses these challenges. CiDD integrates data from TCGA, CMap, and Cancer Cell Line Encyclopedia (CCLE) to perform computational drug discovery experiments, generating hypotheses for the following three general problems: (i) determining whether specific clinical phenotypes or molecular characteristics are associated with unique gene expression signatures; (ii) finding candidate drugs to repress these expression signatures; and (iii) identifying cell lines that resemble the tumors being studied for subsequent in vitro experiments. The primary input to CiDD is a clinical or molecular characteristic. The output is a biologically annotated list of candidate drugs and a list of cell lines for in vitro experimentation. We applied CiDD to identify candidate drugs to treat colorectal cancers harboring mutations in BRAF. CiDD identified EGFR and proteasome inhibitors, while proposing five cell lines for in vitro testing. CiDD facilitates phenotype-driven, systematic drug discovery based on clinical and molecular data from TCGA.

  13. Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon.

    PubMed

    Munakata, Ryosuke; Inoue, Tsuyoshi; Koeduka, Takao; Karamat, Fazeelat; Olry, Alexandre; Sugiyama, Akifumi; Takanashi, Kojiro; Dugrand, Audray; Froelicher, Yann; Tanaka, Ryo; Uto, Yoshihiro; Hori, Hitoshi; Azuma, Jun-Ichi; Hehn, Alain; Bourgaud, Frédéric; Yazaki, Kazufumi

    2014-09-01

    Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.

  14. Molecular dynamics simulations for the examination of mechanical properties of hydroxyapatite/ poly α-n-butyl cyanoacrylate under additive manufacturing.

    PubMed

    Wang, Yanen; Wei, Qinghua; Pan, Feilong; Yang, Mingming; Wei, Shengmin

    2014-01-01

    Molecular dynamics (MD) simulations emerged to be a helpful tool in the field of material science. In rapid prototyping artificial bone scaffolds process, the binder spraying volume and mechanism are very important for bone scaffolds mechanical properties. In this study, we applied MD simulations to investigating the binding energy of α-n-butyl cyanoacrylate (NBCA) on Hydroxyapatite (HA) crystallographic planes (001, 100 and 110), and to calculating and analyzing the mechanical properties and radial distribution function of the HA(110)/NBCA mixed system. The simulation results suggested that HA (110) has the highest binding energy with NBCA owing to the high planar atom density, and the mechanical properties of HA(110)/NBCA mixed system is stronger than pure HA system. Therefore, the multi-grade strength bone scaffold could be fabricated through spraying various volume NBCA binders during 3D printing process. By calculating the radial distribution function of HA(110)/NBCA, the essence of the interface interaction were successfully elucidated. The forming situation parameters can be referred to calculation results. There exists a strong interaction between HA crystallographic plane (110) and NBCA, it is mainly derived from the hydrogen bonds between O atoms which connect with C atoms of NBCA and H atoms in HA crystal. Furthermore, a strong adsorption effect can be demonstrated between HA and NBCA.

  15. A dumbbell molecular beacon for the specific recognition of nucleic acids.

    PubMed

    Lv, Cong; Yu, Lili; Wang, Jie; Tang, Xinjing

    2010-11-15

    A dumbbell molecular beacon (DMB) was designed and synthesized with the attachment of a fluorophore and a quencher at two ends. This DMB probe can be used to detect single mismatch of a 20mer oligodeoxynucleotide in two different buffers and discrimination factors were as high as 60 at 37°C. Statistics of single substitutions of analytes showed that both substituted positions and substituted nucleotides have important contributions for this probe to efficiently distinguish the true analyte from mismatched ones. Hybridization kinetics of DMB with the target oligonucleotide was also studied.

  16. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    PubMed

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. PMID:21328406

  17. Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma

    PubMed Central

    Kervoëlen, Charlotte; Ménoret, Emmanuelle; Gomez-Bougie, Patricia; Bataille, Régis; Godon, Catherine; Marionneau-Lambot, Séverine; Moreau, Philippe; Pellat-Deceunynck, Catherine; Amiot, Martine

    2015-01-01

    Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects. PMID:26323097

  18. Addition of Molecular Adsorbent Recirculating System (MARS®) Albumin Dialysis for the Preoperative Management of Jaundiced Patients with Hilar Cholangiocarcinoma

    PubMed Central

    Regimbeau, Jean-Marc; Fuks, David; Chapuis-Roux, Emilie; Yzet, Thierry; Cosse, Cyril; Bartoli, Eric; N'Guyen-Khac, Eric; Robert, Brice; Lobjoie, Eric

    2013-01-01

    The preoperative management of hilar cholangiocarcinoma (HC) with jaundice focuses on decreasing the total serum bilirubin level (SBL) by performing preoperative biliary drainage (PBD). However, it takes about 6–8 weeks for the SBL to fall at a sufficient extent. The objective of this preliminary study was to evaluate the impact of Molecular Adsorbent Recirculating System (MARS®) dialysis (in association with PBD) on SBL decrease. From January 2010 to January 2011, we prospectively selected all jaundiced patients admitted to our university hospital for resectable HC and requiring PBD prior to major hepatectomy. The PBD was followed by 3 sessions of MARS dialysis over a period of 72 h. A total of 10 patients with HC were screened and two of them were included (Bismuth-Corlette stage IIIa, gender ratio 1, median age 68 years). The initial SBL in the two patients was 328 and 242 μmol/l, respectively. After three MARS dialysis sessions, the SBL had fallen by 30 and 52%, respectively. After the end of each session, there was a SBL rebound of about 10 μmol/l. The MARS decreased the serum creatinine level, the platelet count and the prothrombin index, but did not modify the serum albumin level. Pruritus disappeared after one and two sessions, respectively. MARS-related morbidity included hypotension (n = 1), tachycardia (n = 1), thrombocytopenia (n = 2) and anaemia (n = 1). When combined with PBD, MARS dialysis appears to accelerate the decrease in SBL and thus may enable earlier surgery. This hypothesis must be validated in a larger study. PMID:24163652

  19. Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages.

    PubMed

    Dietrich, Jens-Erik; Panavaite, Laura; Gunther, Stefan; Wennekamp, Sebastian; Groner, Anna C; Pigge, Anton; Salvenmoser, Stefanie; Trono, Didier; Hufnagel, Lars; Hiiragi, Takashi

    2015-08-01

    Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability. PMID:26142281

  20. Molecular basis of sidekick-mediated cell-cell adhesion and specificity

    PubMed Central

    Goodman, Kerry M; Yamagata, Masahito; Jin, Xiangshu; Mannepalli, Seetha; Katsamba, Phinikoula S; Ahlsén, Göran; Sergeeva, Alina P; Honig, Barry; Sanes, Joshua R; Shapiro, Lawrence

    2016-01-01

    Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions. DOI: http://dx.doi.org/10.7554/eLife.19058.001 PMID:27644106

  1. Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages.

    PubMed

    Dietrich, Jens-Erik; Panavaite, Laura; Gunther, Stefan; Wennekamp, Sebastian; Groner, Anna C; Pigge, Anton; Salvenmoser, Stefanie; Trono, Didier; Hufnagel, Lars; Hiiragi, Takashi

    2015-08-01

    Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability.

  2. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

  3. Molecular profiling of tumor-specific TH1 cells activated in vivo

    PubMed Central

    Lorvik, Kristina Berg; Haabeth, Ole Audun Werner; Clancy, Trevor; Bogen, Bjarne; Corthay, Alexandre

    2013-01-01

    The central role of tumor-specific TH1 cells in anticancer immune responses is becoming increasingly appreciated. However, little is known about how these cells are generated in vivo. Here, we used flow cytometry and gene expression microarrays to characterize the primary activation and TH1 differentiation of naïve tumor-specific CD4+ T cells in a mouse model of cancer immunosurveillance. We took advantage of T-cell receptor-transgenic mice in which CD4+ T cells recognize a tumor-specific antigen secreted by MHC class II-negative MOPC315 myeloma cells. Cancer cells were injected subcutaneously and T-cell activation was analyzed in draining lymph nodes and at the incipient tumor site 8 d later. Upon activation and migration to incipient tumor sites, tumor-specific CD4+ T cells exhibited the upregulation of 29 cell-surface molecules (CD2, CD5, CD11a, CD18, CD25, CD28, CD44, CD45, CD49d, CD51, CD54, CD69, CD71, CD83, CD86, CD90, CD95, CD102, CD122, CD153, CD166, CD200, CD249, CD254, CD274, CD279, Ly6C, MHC class I and CCR7) and the downregulation of five (CD27, CD31, CD45RB, CD62L and CD126). Activated CD4+ T cells produced interferon γ, a cytokine consistent with a TH1-polarized response, tumor necrosis factor α as well as interleukin (IL)-2, IL-3 and IL-10. The activation of naïve tumor-specific CD4+ T cells in draining lymph nodes resulted in the upregulation of 609 genes and the downregulation of 284 genes. The bioinformatic analysis of differentially expressed genes identified functional pathways related to tumor-specific TH1 cell activation. This study may represent a useful resource to guide the development of TH1-based immunotherapies against cancer. PMID:23762808

  4. Molecular Basis of Signaling Specificity of Insulin and IGF Receptors: Neglected Corners and Recent Advances

    PubMed Central

    Siddle, Kenneth

    2011-01-01

    Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears

  5. Additives Effects on Crystal Morphology of Dihydroxylammonium 5,5ʹ-Bistetrazole-1,1ʹ-diolate by Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Xiong, Shu-Ling; Chen, Shu-Sen; Jin, Shao-Hua; Li, Li-Jie

    2016-10-01

    Dihydroxylammonium 5,5‧-bistetrazole-1,1‧-diolate (TKX-50) is a newly synthesized explosive with excellent comprehensive properties: high energy storage, low impact sensitivity, and low toxicity. To understand and improve the crystal morphology of TKX-50, we reported the polymer consistent force field to simulate the crystal morphology of TKX-50 by growth morphology (GM) method. We then used this force field in molecular dynamics (MD) simulations to predict the influences of additives on crystal facets of TKX-50. The calculated results indicate that ethanol, ethylene glycol, and acetic acid are more favorable to the spheroidization of TKX-50, which provides a theoretical support for the additive selection of crystalline system. Furthermore, we added the selected additives in the recrystallization system of TKX-50. The recrystallized samples possessed a small aspect ratio and were close to spherical in shape, which indicates that the experimental results are consistent with the simulated results.

  6. Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance.

    PubMed

    An, Diaoguo; Zheng, Qi; Luo, Qiaoling; Ma, Pengtao; Zhang, Hongxia; Li, Lihui; Han, Fangpu; Xu, Hongxing; Xu, Yunfeng; Zhang, Xiaotian; Zhou, Yilin

    2015-01-01

    Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.

  7. Molecular cloning and functional expression of a brain-specific somatostatin receptor.

    PubMed Central

    Bruno, J F; Xu, Y; Song, J; Berelowitz, M

    1992-01-01

    The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor. Images PMID:1360663

  8. Specific Lipid Binding of Membrane Proteins in Detergent Micelles Characterized by NMR and Molecular Dynamics.

    PubMed

    Zhao, Linlin; Wang, Shuqing; Run, Changqing; OuYang, Bo; Chou, James J

    2016-09-27

    Many membrane proteins bind specifically to lipids as an integral component of their structures. The ability of detergents to support lipid binding is thus an important consideration when solubilizing membrane proteins for structural studies. In particular, the zwitterionic phosphocholine (PC)-based detergents, which have been widely used in solution NMR studies of channels and transporters, are controversial because of their strong solubilization power and thus perceived as more denaturing than nonionic detergents such as the maltosides. Here, we investigate the ability of the mitochondrial ADP/ATP carrier (AAC) to specifically bind cardiolipin, a mitochondrial lipid important for the carrier function, in dodecylphosphocholine (DPC) micelles. We found that in DPC, the AAC specifically binds cardiolipin in a manner consistent with the bound cardiolipins found in the crystal structures of the AAC determined in n-decyl β-d-maltoside. Our results suggest that PC detergent is compatible with specific lipid binding and that PC detergent mixed with the relevant lipid represents a viable solubilization system for NMR studies of membrane proteins. PMID:27625145

  9. Molecular and Behavioral Changes Associated with Adult Hippocampus-Specific SynGAP1 Knockout

    ERIC Educational Resources Information Center

    Muhia, Mary; Willadt, Silvia; Yee, Benjamin K.; Feldon, Joram; Paterna, Jean-Charles; Schwendener, Severin; Vogt, Kaspar; Kennedy, Mary B.; Knuesel, Irene

    2012-01-01

    The synaptic Ras/Rap-GTPase-activating protein (SynGAP1) plays a unique role in regulating specific downstream intracellular events in response to N-methyl-D-aspartate receptor (NMDAR) activation. Constitutive heterozygous loss of SynGAP1 disrupts NMDAR-mediated physiological and behavioral processes, but the disruptions might be of developmental…

  10. Model-specific selection of molecular targets for heart failure gene therapy

    PubMed Central

    Katz, Michael G.; Fargnoli, Anthony S.; Tomasulo, Catherine E.; Pritchette, Louella A.; Bridges, Charles R.

    2013-01-01

    Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of β-adrenergic receptor signaling, Ca2+ handling proteins and angiogenesis in the most common extrinsic models of HF. PMID:21954055

  11. Model-specific selection of molecular targets for heart failure gene therapy.

    PubMed

    Katz, Michael G; Fargnoli, Anthony S; Tomasulo, Catherine E; Pritchette, Louella A; Bridges, Charles R

    2011-10-01

    Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of β-adrenergic receptor signaling, Ca(2+) handling proteins and angiogenesis in the most common extrinsic models of HF.

  12. [Multivariate analysis as a means of access to optimal pharmacochemical specificity and to molecular archetypes].

    PubMed

    Doré, J C; Viel, C; Lacroix, R; Lacroix, J

    1990-01-01

    For complex works as studies relationships structure-activity, in heterogeneous therapeutic families we have selected mathematical methods founded upon systemic approach rather analytic one, appealing to bibliographical data, taking into consideration a plurality of biological targets and envisaging structural extrapolations rather than interpolations. Compared with classical QSAR, multivariate analysis (factorial analysis and multidimentional data reduction) intend from structuration of whole complex items to definite spheres of correlations between structural parameters and biological ones to issue then on a symetric typology of this two groups of parameters. These approaches have not only a descriptive character but lead to operational conclusions through an interactive dialogue with data bank; for example: --to explore acting potentiality of others molecular families or/and particular sub-structures --to find chemical sequences of molecules synthesized for other aims but not again experimented for this property. The case of antiparasitic agents is here developed.

  13. Structural and Functional Analysis of JMJD2D Reveals Molecular Basis for Site-Specific Demethylation among JMJD2 Demethylases

    SciTech Connect

    Krishnan, Swathi; Trievel, Raymond C.

    2013-01-08

    We found that JMJD2 lysine demethylases (KDMs) participate in diverse genomic processes. Most JMJD2 homologs display dual selectivity toward H3K9me3 and H3K36me3, with the exception of JMJD2D, which is specific for H3K9me3. Here, we report the crystal structures of the JMJD2D•2-oxoglutarate•H3K9me3 ternary complex and JMJD2D apoenzyme. Utilizing structural alignments with JMJD2A, molecular docking, and kinetic analysis with an array of histone peptide substrates, we elucidate the specific signatures that permit efficient recognition of H3K9me3 by JMJD2A and JMJD2D, and the residues in JMJD2D that occlude H3K36me3 demethylation. Surprisingly, these results reveal that JMJD2A and JMJD2D exhibit subtle yet important differences in H3K9me3 recognition, despite the overall similarity in the substrate-binding conformation. Further, we show that H3T11 phosphorylation abrogates demethylation by JMJD2 KDMs. These studies reveal the molecular basis for JMJD2 site specificity and provide a framework for structure-based design of selective inhibitors of JMJD2 KDMs implicated in disease.

  14. TNNI3K, a novel cardiac-specific kinase, emerging as a molecular target for the treatment of cardiac disease

    PubMed Central

    Lal, Hind; Ahmad, Firdos; Parikh, Shan; Force, Thomas

    2014-01-01

    Coronary heart disease (AHD) is the leading cause of death and disability worldwide. In patients with acute coronary syndromes (ACS), timely and effective myocardial reperfusion by percutaneous coronary intervention (PCI) is the primary treatment of choice to minimize the ischemic injury and limit MI size. However, reperfusion can itself promote cardiomyocyte death which leads to cardiac dysfunction via reperfusion injury. The molecular mechanisms of ischemia/reperfusion (I/R) injury are not completely understood and new drug targets are needed. Recently we reported that cardiac troponin I-interacting protein kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes I/R injury via profound oxidative stress, thereby promoting cardiomyocyte death. By using novel genetic animal models and newly developed small-molecule TNNI3K inhibitors, we demonstrate that TNNI3K-mediated I/R injury occurs through impaired mitochondrial function and is in part dependent on p38 MAPK. Herein we discuss the emerging role of TNNI3K as a promising new drug target to limit the I/R-induced myocardial injury. We will also examine the underlying mechanisms that drive the profoundly reduced infarct size in mice in which TNNI3K is specifically deleted in cardiomyocytes. Since TNNI3K is a cardiac-specific kinase, it could be an ideal molecular target since inhibiting it would have little or no effect on other organ systems, a serious problem associated with the use of kinase inhibitors targeting kinases that are more widely expressed. PMID:24899531

  15. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging

    PubMed Central

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-01-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo. PMID:27446643

  16. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging.

    PubMed

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-04-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo. PMID:27446643

  17. Molecular Specificities of R Factor-Determined Beta-Lactamases: Correlation with Plasmid Compatibility

    PubMed Central

    Hedges, R. W.; Datta, Naomi; Kontomichalou, Polyxeni; Smith, J. T.

    1974-01-01

    Beta (β)-lactamases determined by 29 ampicillin resistance plasmids could be divided into two types. One, TEM-type, was very uniform with respect to substrate specificity but heterogeneous in absolute levels of β-lactamase activity. The TEM-type β-lactamase was determined by R factors of compatibility groups FII, Iα, Iε, N, C, A, T, W, P, L, and X, and by prophage φ Amp. The other type, characterized by the ability to hydrolyze oxacillin, was less common, showed lower absolute levels of activity, and was heterogeneous as regards substrate specificities. Oxacillin-hydrolyzing β-lactamases were determined by R factors of compatibility groups FI, Iα, N, C, and O. PMID:4587613

  18. Molecular Mechanisms of Substrate Recognition and Specificity of New Delhi Metallo-β-Lactamase

    PubMed Central

    Chiou, Jiachi; Leung, Thomas Yun-Chung

    2014-01-01

    Carbapenems are one of the last lines of defense for Gram-negative pathogens, such as members of the Enterobacteriaceae. Despite the fact that most carbapenems are resistant to extended-spectrum β-lactamase (ESBL), emerging metallo-β-lactamases (MBLs), including New Delhi metallo-β-lactamase 1 (NDM-1), that can hydrolyze carbapenems have become prevalent and are frequently associated with the so-called “superbugs,” for which treatments are extremely limited. Crystallographic study sheds light on the modes of antibiotic binding to NDM-1, yet the mechanisms governing substrate recognition and specificity are largely unclear. This study provides a connection between crystallographic study and the functional significance of NDM-1, with an emphasis on the substrate specificity and catalysis of various β-lactams. L1 loop residues L59, V67, and W87 were important for the activity of NDM-1, most likely through maintaining the partial folding of the L1 loop or active site conformation through hydrophobic interaction with the R groups of β-lactams or the β-lactam ring. Substitution of alanine for L59 showed greater reduction of MICs to ampicillin and selected cephalosporins, whereas substitutions of alanine for V67 had more impact on the MICs of carbapenems. K224 and N233 on the L3 loop played important roles in the recognition of substrate and contributed to substrate hydrolysis. These data together with the structure comparison of the B1 and B2 subclasses of MBLs revealed that the broad substrate specificity of NDM-1 could be due to the ability of its wide active site cavity to accommodate a wide range of β-lactams. This study provides insights into the development of efficient inhibitors for NDM-1 and offers an efficient tactic with which to study the substrate specificities of other β-lactamases. PMID:24982075

  19. Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations

    PubMed Central

    Förster, Frank; Beisser, Daniela; Grohme, Markus A.; Liang, Chunguang; Mali, Brahim; Siegl, Alexander Matthias; Engelmann, Julia C.; Shkumatov, Alexander V.; Schokraie, Elham; Müller, Tobias; Schnölzer, Martina; Schill, Ralph O.; Frohme, Marcus; Dandekar, Thomas

    2012-01-01

    Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant. PMID:22563243

  20. Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.

    PubMed

    Zhao, Yongqian; Moreland, Nicole J; Tay, Moon Y F; Lee, Chin Chin; Swaminathan, Kunchithapadam; Vasudevan, Subhash G

    2014-01-22

    The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex.

  1. Molecular Dipole Moments within the Incremental Scheme Using the Domain-Specific Basis-Set Approach.

    PubMed

    Fiedler, Benjamin; Coriani, Sonia; Friedrich, Joachim

    2016-07-12

    We present the first implementation of the fully automated incremental scheme for CCSD unrelaxed dipole moments using the domain-specific basis-set approach. Truncation parameters are varied, and the accuracy of the method is statistically analyzed for a test set of 20 molecules. The local approximations introduce small errors at second order and negligible ones at third order. For a third-order incremental CCSD expansion with a CC2 error correction, a cc-pVDZ/SV domain-specific basis set (tmain = 3.5 Bohr), and the truncation parameter f = 30 Bohr, we obtain a mean error of 0.00 mau (-0.20 mau) and a standard deviation of 1.95 mau (2.17 mau) for the total dipole moments (Cartesian components of the dipole vectors). By analyzing incremental CCSD energies, we demonstrate that the MP2 and CC2 error correction schemes are an exclusive correction for the domain-specific basis-set error. Our implementation of the incremental scheme provides fully automated computations of highly accurate dipole moments at reduced computational cost and is fully parallelized in terms of the calculation of the increments. Therefore, one can utilize the incremental scheme, on the same hardware, to extend the basis set in comparison to standard CCSD and thus obtain a better total accuracy. PMID:27300371

  2. Transcriptome analysis in tardigrade species reveals specific molecular pathways for stress adaptations.

    PubMed

    Förster, Frank; Beisser, Daniela; Grohme, Markus A; Liang, Chunguang; Mali, Brahim; Siegl, Alexander Matthias; Engelmann, Julia C; Shkumatov, Alexander V; Schokraie, Elham; Müller, Tobias; Schnölzer, Martina; Schill, Ralph O; Frohme, Marcus; Dandekar, Thomas

    2012-01-01

    Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.

  3. The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases.

    PubMed

    Pruneda, Jonathan N; Durkin, Charlotte H; Geurink, Paul P; Ovaa, Huib; Santhanam, Balaji; Holden, David W; Komander, David

    2016-07-21

    Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins. PMID:27425412

  4. Transcriptome analysis in tardigrade species reveals specific molecular pathways for stress adaptations.

    PubMed

    Förster, Frank; Beisser, Daniela; Grohme, Markus A; Liang, Chunguang; Mali, Brahim; Siegl, Alexander Matthias; Engelmann, Julia C; Shkumatov, Alexander V; Schokraie, Elham; Müller, Tobias; Schnölzer, Martina; Schill, Ralph O; Frohme, Marcus; Dandekar, Thomas

    2012-01-01

    Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant. PMID:22563243

  5. Screening targeted testis-specific genes for molecular assessment of aberrant sperm quality

    PubMed Central

    Liu, Xue Xia; Shen, Xiao Fang; Liu, Fu-Jun

    2016-01-01

    Teratospermia is a heterogeneous and complex disorder, which is closely associated with male fertility. Genes and gene products associated with teratospermia may serve as targeted biomarkers that help understand the underlying mechanisms of male infertility; however, systematic information on the subject remains to be elucidated. The present study performed a comparative bioinformatics analysis to identify biomarkers associated with sperm quality, particular focusing on testis-specific biomarkers. A stepwise screening approach identified 1,085 testis/epididymis-specific genes and 3,406 teratospermia-associated genes, resulting in 348 testis-specific genes associated with aberrant sperm quality. These genes were functionally associated with the reproduction process. Gene products corresponding to heat shock protein family A (Hsp70) member 4 like (HSPA4L) and phosphoglycerate kinase 2 were characterized at the cellular level in human testes and ejaculated spermatozoa. HSPA4L expression in sperm was revealed to be associated with sperm quality. The present study provided a novel insight into the understanding of sperm quality, and a potential method for the diagnosis and assessment of sperm quality in the event of male infertility. PMID:27356588

  6. RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus

    PubMed Central

    2011-01-01

    Background In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. Results Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. Conclusions For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance

  7. Molecular Phylogeny, Homology Modeling, and Molecular Dynamics Simulation of Race-Specific Bacterial Blight Disease Resistance Protein (xa5) of Rice: A Comparative Agriproteomics Approach

    PubMed Central

    Dehury, Budheswar; Sahu, Mousumi; Sarma, Kishore; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Sharma, Gauri Dutta; Choudhury, Manabendra Dutta

    2013-01-01

    Abstract Rice (Oryza sativa L.), a model plant belonging to the family Poaceae, is a staple food for a majority of the people worldwide. Grown in the tropical and subtropical regions of the world, this important cereal crop is under constant and serious threat from both biotic and abiotic stresses. Among the biotic threats, Xanthomonas oryzae pv. oryzae, causing the damaging bacterial blight disease in rice, is a prominent pathogen. The xa5 gene in the host plant rice confers race-specific resistance to this pathogen. This recessive gene belongs to the Xa gene family of rice and encodes a gamma subunit of transcription factor IIA (TFIIAγ). In view of the importance of this gene in conferring resistance to the devastating disease, we reconstructed the phylogenetic relationship of this gene, developed a three-dimensional protein model, followed by long-term molecular dynamics simulation studies to gain a better understanding of the evolution, structure, and function of xa5. The modeled structure was found to fit well with the small subunit of TFIIA from human, suggesting that it may also act as a small subunit of TFIIA in rice. The model had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 20 nano second in aqueous solution. Further structural analysis of xa5 indicated that the protein retained its basic transcription factor function, suggesting that it might govern a novel pathway responsible for bacterial blight resistance. Future molecular docking studies of xa5 underway with its corresponding avirulence gene is expected to shed more direct light into plant–pathogen interactions at the molecular level and thus pave the way for richer agriproteomic insights. PMID:23758479

  8. New molecular quantitative PCR assay for detection of host-specific Bifidobacteriaceae suitable for microbial source tracking.

    PubMed

    Gómez-Doñate, Marta; Ballesté, Elisenda; Muniesa, Maite; Blanch, Anicet R

    2012-08-01

    Bifidobacterium spp. belong to the commensal intestinal microbiota of warm-blooded animals. Some strains of Bifidobacterium show host specificity and have thus been proposed as host-specific targets to determine the origin of fecal pollution. Most strains have been used in microbial-source-tracking (MST) studies based on culture-dependent methods. Although some of these approaches have proved very useful, the low prevalence of culturable Bifidobacterium strains in the environment means that molecular culture-independent procedures could provide practical applications for MST. Reported here is a set of common primers and four Bifidobacterium sp. host-associated (human, cattle, pig, and poultry) probes for quantitative-PCR (qPCR) assessment of fecal source tracking. This set was tested using 25 water samples of diverse origin: urban sewage samples, wastewater from four abattoirs (porcine, bovine, and poultry), and water from a river with a low pollution load. The selected sequences showed a high degree of host specificity. There were no cross-reactions between the qPCR assays specific for each origin and samples from different fecal origins. On the basis of the findings, it was concluded that the host-specific qPCRs are sufficiently robust to be applied in environmental MST studies.

  9. Specifics of solvation of sulfonated polyelectrolytes in water, dimethylmethylphosphonate, and their mixture: A molecular simulation study

    NASA Astrophysics Data System (ADS)

    Vishnyakov, Aleksey; Neimark, Alexander V.

    2008-04-01

    Sulfonated polyelectrolyte membranes (PEMs), such as Nafion and styrene-olefin block copolymers, are explored as permselective membranes for fuel cells as well as suitable barrier materials against chemical agents. The permselective properties of PEM are determined by their microphase segregation into hydrophilic and hydrophobic domains. We performed classical molecular dynamics simulations of solvation of the hydrophilic fragments of PEM exemplified on sulfonated polystyrene (sPS) with potassium, calcium, and aluminum as counterions, in water, phosphor-organic nerve agent simulant dimethylmethylphosphonate (DMMP), and their binary mixture. The force field for the sulfonate group has been developed by optimizing the potential parameters to fit the benzenesulfonate conformations obtained from the density functional theory. For a comparison, we considered perfluorosulfonate oligomers representing fragments of Nafion polymer. We found a noticeable difference between the geometries of the polymer backbone in different solvents. The polymer backbone is stiffer in DMMP for both sPS and Nafion. An anisotropic structuring of the solvent around the phenylsulfonate group is substantially stronger than around the Nafion sidechain due to the rigidity and the anisotropy of the phenylsulfonate group. The counterion significantly affects the conformations of solvated sPS: the rigidity of the backbone increases when potassium or calcium ions are replaced by trivalent aluminum ions.

  10. Multimodal molecular imaging system for pathway-specific reporter gene expression.

    PubMed

    Rossi, Marco; Massai, Luisa; Diamanti, Daniela; Fiengo, Pasquale; De Rosa, Antonella; Magrini, Roberta; Magnoni, Letizia; Chellini, Sara; Coniglio, Silvia; Diodato, Enrica; Pilli, Elena; Caradonna, Nicola Pasquale; Sardone, Gianluca; Monti, Martina; Roggeri, Riccardo; Lionetti, Vincenzo; Recchia, Fabio; Tunici, Patrizia; Valensin, Silvia; Scali, Carla; Pollio, Giuseppe; Porcari, Valentina

    2016-04-30

    Preclinical imaging modalities represent an essential tool to develop a modern and translational biomedical research. To date, Optical Imaging (OI) and Magnetic Resonance Imaging (MRI) are used principally in separate studies for molecular imaging studies. We decided to combine OI and MRI together through the development of a lentiviral vector to monitor the Wnt pathway response to Lithium Chloride (LiCl) treatment. The construct was stably infected in glioblastoma cells and, after intracranial transplantation in mice, serial MRI and OI imaging sessions were performed to detect human ferritin heavy chain protein (hFTH) and firefly luciferase enzyme (FLuc) respectively. The system allowed also ex vivo analysis using a constitutive fluorescence protein expression. In mice, LiCl administration has shown significantly increment of luminescence signal and a lower signal of T2 values (P<0.05), recorded noninvasively with OI and a 7 Tesla MRI scanner. This study indicates that OI and MRI can be performed in a single in vivo experiment, providing an in vivo proof-of-concept for drug discovery projects in preclinical phase. PMID:26987608

  11. Tissue-specific molecular immune response to lipopolysaccharide challenge in emaciated anadromous Arctic charr.

    PubMed

    Philip, Anju M; Jørgensen, Even H; Maule, Alec G; Vijayan, Mathilakath M

    2014-07-01

    Anadromous Arctic charr (Salvelinus alpinus) undergo voluntary winter fasting for months in the Arctic. We tested the hypothesis that extended fasting will compromise the ability of this species to evoke an immune response. Charr were either fed or fasted for 85 days and challenged with lipopolysaccharide (LPS), and the molecular immune response in the liver and spleen assessed at 8 and 96 h post-injection. LPS increased IL-1β, IL-8, and serum amyloid protein A (SAA) mRNA levels in both groups, but the liver IL-1β and IL-8, and spleen IL-8 responses were reduced in the fasted group. Fasting upregulated SOCS-1 and SOCS-2 mRNA abundance, while LPS stimulated SOCS-3 mRNA abundance and this response was higher in the fasted liver. Collectively, extended fasting and emaciation does not curtail the capacity of charr to evoke an immune response, whereas upregulation of SOCS may be a key adaptation to conserve energy by restricting the inflammatory response.

  12. Molecular interactions and trafficking of influenza A virus polymerase proteins analyzed by specific monoclonal antibodies

    SciTech Connect

    MacDonald, Leslie A.; Aggarwal, Shilpa; Bussey, Kendra A.; Desmet, Emily A.; Kim, Baek; Takimoto, Toru

    2012-04-25

    The influenza polymerase complex composed of PA, PB1 and PB2, plays a key role in viral replication and pathogenicity. Newly synthesized components must be translocated to the nucleus, where replication and transcription of viral genomes take place. Previous studies suggest that while PB2 is translocated to the nucleus independently, PA and PB1 subunits could not localize to the nucleus unless in a PA-PB1 complex. To further determine the molecular interactions between the components, we created a panel of 16 hybridoma cell lines, which produce monoclonal antibodies (mAbs) against each polymerase component. We showed that, although PB1 interacts with both PA and PB2 individually, nuclear localization of PB1 is enhanced only when co-expressed with PA. Interestingly, one of the anti-PA mAbs reacted much more strongly with PA when co-expressed with PB1. These results suggest that PA-PB1 interactions induce a conformational change in PA, which could be required for its nuclear translocation.

  13. The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

    PubMed

    Aluvila, Sreevidya; Kotaria, Rusudan; Sun, Jiakang; Mayor, June A; Walters, D Eric; Harrison, David H T; Kaplan, Ronald S

    2010-08-27

    The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

  14. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    DOE PAGES

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test thismore » strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.« less

  15. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    SciTech Connect

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  16. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  17. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. PMID:25313039

  18. Molecular Epidemiology of Helicobacter pylori Infection in Nepal: Specific Ancestor Root

    PubMed Central

    Miftahussurur, Muhammad; Sharma, Rabi Prakash; Shrestha, Pradeep Krishna; Suzuki, Rumiko; Uchida, Tomohisa; Yamaoka, Yoshio

    2015-01-01

    Prevalence of Helicobacter pylori infection in Nepal, a low-risk country for gastric cancer, is debatable. To our knowledge, no studies have examined H. pylori virulence factors in Nepal. We determined the prevalence of H. pylori infection by using three different tests, and the genotypes of virulence factors were determined by PCR followed by sequencing. Multilocus sequence typing was used to analyze the population structure of the Nepalese strains. The prevalence of H. pylori infection in dyspeptic patients was 38.4% (56/146), and was significantly related with source of drinking water. In total, 51 strains were isolated and all were cagA-positive. Western-type-cagA (94.1%), cagA pre-EPIYA type with no deletion (92.2%), vacA s1a (74.5%), and m1c (54.9%) were the predominant genotypes. Antral mucosal atrophy levels were significantly higher in patients infected with vacA s1 than in those infected with s2 genotypes (P = 0.03). Several Nepalese strains were H. pylori recombinants with genetic features of South Asian and East Asian genotypes. These included all East-Asian-type-cagA strains, with significantly lesser activity and inflammation in the corpus than the strains of the specific South Asian genotype (P = 0.03 and P = 0.005, respectively). Although the population structure confirmed that most Nepalese strains belonged to the hpAsia2 population, some strains shared hpEurope- and Nepalese-specific components. Nepalese patients infected with strains belonging to hpEurope showed higher inflammation in the antrum than strains from the Nepalese specific population (P = 0.05). These results support that ancestor roots of Kathmandu`s people not only connected with India alone. PMID:26226153

  19. Molecular mechanisms that govern the specificity of Sushi peptides for Gram-negative bacterial membrane lipids.

    PubMed

    Li, Peng; Sun, Miao; Wohland, Thorsten; Yang, Daiwen; Ho, Bow; Ding, Jeak Ling

    2006-09-01

    Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.

  20. Of arrows and flows. Causality, determination, and specificity in the Central Dogma of molecular biology.

    PubMed

    Fantini, Bernardino

    2006-01-01

    From its first proposal, the Central Dogma had a graphical form, complete with arrows of different types, and this form quickly became its standard presentation. In different scientific contexts, arrows have different meanings and in this particular case the arrows indicated the flow of information among different macromolecules. A deeper analysis illustrates that the arrows also imply a causal statement, directly connected to the causal role of genetic information. The author suggests a distinction between two different kinds of causal links, defined as 'physical causality' and 'biological determination', both implied in the production of biological specificity. PMID:18351053

  1. Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP

    PubMed Central

    Guerra-Pérez, Natalia; Ramos, Edurne; García-Hernández, Marta; Pinto, Celia; Soto, Manuel; Martín, M. Elena; González, Víctor M.

    2015-01-01

    Summary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. Results We have selected a ssDNA aptamer population against a recombinant 6xHIS–LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. Conclusion Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be

  2. Molecular cloning and characterization of an achene-seed-specific promoter from motherwort (Leonurus japonicus Houtt).

    PubMed

    Xie, Chengjian; Zhang, Beibei; Wang, De; Kou, Fei; Zhao, Xupeng; Yang, Xingyong

    2011-01-01

    LJAMP1 is a small antimicrobial protein purified previously from the seeds of motherwort, and it is expressed preferentially in seeds. A 794-bp upstream sequence of the ATG start codon was isolated using a genome walking method and cloned into the upstream of the β-glucuronidase (GUS) reporter gene to determine the GUS tissue-specific expression pattern. The transgenic tobacco showed that pLJAMP1 promoter derived GUS reporter gene special expression in pollen, achene and seed. The analysis of cis-acting elements also revealed pLJAMP1 promoter contained pollen and seed related transcriptional control elements.

  3. Additive manufactured Ti-6Al-4V using welding wire: comparison of laser and arc beam deposition and evaluation with respect to aerospace material specifications

    NASA Astrophysics Data System (ADS)

    Brandl, E.; Baufeld, B.; Leyens, C.; Gault, R.

    In this paper, the results of two different wire based additive-layer-manufacturing systems are compared: in one system Ti-6Al4V is deposited by a Nd:YAG laser beam, in the other by an arc beam (tungsten inert gas process). Mechanical properties of the deposits and of plate material are presented and evaluated with respect to aerospace material specifications. The mechanical tests including static tension and high cycle fatigue were performed in as-built, stress-relieved and annealed conditions. Generally, the mechanical properties of the components are competitive to cast and even wrought material properties and can attain properties suitable for space or aerospace applications.

  4. Molecular evolution and species-specific expansion of the NAP members in plants.

    PubMed

    Fan, Kai; Shen, Hao; Bibi, Noreen; Li, Feng; Yuan, Shuna; Wang, Ming; Wang, Xuede

    2015-08-01

    The NAP (NAC-Like, Activated by AP3 /PI) subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants, but no NAP members were found in eight non-vascular plants. All NAP proteins were phylogenetically classified into two groups (NAP I and NAP II), and the origin time of the NAP I group might be relatively later than that of the NAP II group. Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns. Besides, functional divergence was limited after the gene duplication. Abscisic acid (ABA) might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

  5. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

    PubMed Central

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  6. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

    PubMed

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T; Texier, Yves; van Beersum, Sylvia E C; Horn, Nicola; Willer, Jason R; Mans, Dorus A; Dougherty, Gerard; Lamers, Ideke J C; Coene, Karlien L M; Arts, Heleen H; Betts, Matthew J; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J F; Marcelis, Carlo L; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A; Toedt, Grischa; van Dam, Teunis J P; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L; Blacque, Oliver E; Gibson, Toby J; Huynen, Martijn A; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E; Franco, Brunella; Giles, Rachel H; Ueffing, Marius; Russell, Robert B; Roepman, Ronald

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  7. The rotational specific heat of molecular hydrogen in the old quantum theory

    NASA Astrophysics Data System (ADS)

    Gearhart, Clayton

    2005-04-01

    ``Astonishing successes'' and ``bitter disappointment'': Thus did the German physicist Fritz Reiche portray the state of quantum theory in his 1921 text. His words apply in miniature to early descriptions of the fall in the specific heat of hydrogen gas at low temperatures---among the first systems studied in the old quantum theory. The earliest measurements were made in 1912 by Arnold Eucken in Walther Nernst's laboratory in Berlin. The possibility of applying a theory of quantized rotators to diatomic gases had emerged even earlier, at the first Solvay conference in 1911. Eucken's experiment was the first of many. Paul Ehrenfest, Erwin Schrödinger, Edwin C. Kemble, and John Van Vleck, among others, attempted theoretical descriptions of the rotational specific heat, as did Reiche himself in a widely cited 1919 paper. Despite these efforts, the problem proved intractable---its explanation involves identical particles in ways unsuspected before modern quantum mechanics. Nevertheless, the older theory worked reasonably well to describe the infrared spectra of other diatomic molecules. I will sketch the history of this intriguing problem in early quantum theory.

  8. Assembly, molecular organization, and membrane-binding properties of development-specific septins

    PubMed Central

    Garcia, Galo; Finnigan, Gregory C.; Heasley, Lydia R.; Sterling, Sarah M.; Aggarwal, Adeeti; Pearson, Chad G.

    2016-01-01

    Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28–Spr3–Cdc3–Cdc10–Cdc10–Cdc3–Spr3–Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3–capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties. PMID:26929450

  9. Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

    PubMed Central

    Hartikainen, Hanna; Ashford, Oliver S; Berney, Cédric; Okamura, Beth; Feist, Stephen W; Baker-Austin, Craig; Stentiford, Grant D; Bass, David

    2014-01-01

    Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk. PMID:23966100

  10. Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters.

    PubMed

    Silverstein, Nechama; Ewers, David; Forrest, Lucy R; Fahlke, Christoph; Kanner, Baruch I

    2015-11-27

    Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

  11. Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

    PubMed

    Mata-Garrido, Jorge; Casafont, Iñigo; Tapia, Olga; Berciano, Maria T; Lafarga, Miguel

    2016-04-22

    There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of

  12. Kawasaki Disease-Specific Molecules in the Sera Are Linked to Microbe-Associated Molecular Patterns in the Biofilms

    PubMed Central

    Murata, Kenji; Kanno, Shunsuke; Nishio, Hisanori; Saito, Mitsumasa; Tanaka, Tamami; Yamamura, Kenichiro; Sakai, Yasunari; Takada, Hidetoshi; Miyamoto, Tomofumi; Mizuno, Yumi; Ouchi, Kazunobu; Waki, Kenji; Hara, Toshiro

    2014-01-01

    Background Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. The innate immune system is involved in its pathophysiology at the acute phase. We have recently established a novel murine model of KD coronary arteritis by oral administration of a synthetic microbe-associated molecular pattern (MAMP). On the hypothesis that specific MAMPs exist in KD sera, we have searched them to identify KD-specific molecules and to assess the pathogenesis. Methods We performed liquid chromatography-mass spectrometry (LC-MS) analysis of fractionated serum samples from 117 patients with KD and 106 controls. Microbiological and LC-MS evaluation of biofilm samples were also performed. Results KD samples elicited proinflammatory cytokine responses from human coronary artery endothelial cells (HCAECs). By LC-MS analysis of KD serum samples collected at 3 different periods, we detected a variety of KD-specific molecules in the lipophilic fractions that showed distinct m/z and MS/MS fragmentation patterns in each cluster. Serum KD-specific molecules showed m/z and MS/MS fragmentation patterns almost identical to those of MAMPs obtained from the biofilms formed in vitro (common MAMPs from Bacillus cereus, Yersinia pseudotuberculosis and Staphylococcus aureus) at the 1st study period, and from the biofilms formed in vivo (common MAMPs from Bacillus cereus, Bacillus subtilis/Bacillus cereus/Yersinia pseudotuberculosis and Staphylococcus aureus) at the 2nd and 3rd periods. The biofilm extracts from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus aureus also induced proinflammatory cytokines by HCAECs. By the experiments with IgG affinity chromatography, some of these serum KD-specific molecules bound to IgG. Conclusions We herein conclude that serum KD-specific molecules were mostly derived from biofilms and possessed molecular structures common to MAMPs from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus

  13. Characteristics of Sucrose Transport through the Sucrose-Specific Porin ScrY Studied by Molecular Dynamics Simulations

    PubMed Central

    Sun, Liping; Bertelshofer, Franziska; Greiner, Günther; Böckmann, Rainer A.

    2016-01-01

    Sucrose-specific porin (ScrY) is a transmembrane protein that allows for the uptake of sucrose under growth-limiting conditions. The crystal structure of ScrY was resolved before by X-ray crystallography, both in its uncomplexed form and with bound sucrose. However, little is known about the molecular characteristics of the transport mechanism of ScrY. To date, there has not yet been any clear demonstration for sucrose transport through the ScrY. Here, the dynamics of the ScrY trimer embedded in a phospholipid bilayer as well as the characteristics of sucrose translocation were investigated by means of atomistic molecular dynamics (MD) simulations. The potential of mean force (PMF) for sucrose translocation through the pore showed two main energy barriers within the constriction region of ScrY. Energy decomposition allowed to pinpoint three aspartic acids as key residues opposing the passage of sucrose, all located within the L3 loop. Mutation of two aspartic acids to uncharged residues resulted in an accordingly modified electrostatics and decreased PMF barrier. The chosen methodology and results will aid in the design of porins with modified transport specificities. PMID:26913282

  14. Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.

    PubMed

    Wu, Wei-Ming; Tsai, Hsiang-Ju; Pang, Jong-Hwei S; Wang, Tzu-Hao; Wang, Hsin-Shih; Hong, Hong-Shang; Lee, Yun-Shien

    2005-10-01

    Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

  15. RNA sequencing reveals region-specific molecular mechanisms associated with epileptogenesis in a model of classical hippocampal sclerosis

    PubMed Central

    Vieira, A. S.; de Matos, A. H.; do Canto, A. M.; Rocha, C. S.; Carvalho, B. S.; Pascoal, V. D. B.; Norwood, B.; Bauer, S.; Rosenow, F.; Gilioli, R.; Cendes, F.; Lopes-Cendes, I.

    2016-01-01

    We report here the first complete transcriptome analysis of the dorsal (dDG) and ventral dentate gyrus (vDG) of a rat epilepsy model presenting a hippocampal lesion with a strict resemblance to classical hippocampal sclerosis (HS). We collected the dDG and vDG by laser microdissection 15 days after electrical stimulation and performed high-throughput RNA-sequencing. There were many differentially regulated genes, some of which were specific to either of the two sub-regions in stimulated animals. Gene ontology analysis indicated an enrichment of inflammation-related processes in both sub-regions and of axonal guidance and calcium signaling processes exclusively in the vDG. There was also a differential regulation of genes encoding molecules involved in synaptic function, neural electrical activity and neuropeptides in stimulated rats. The data presented here suggests, in the time point analyzed, a remarkable interaction among several molecular components which takes place in the damaged hippocampi. Furthermore, even though similar mechanisms may function in different regions of the DG, the molecular components involved seem to be region specific. PMID:26935982

  16. Protein-specific force field derived from the fragment molecular orbital method can improve protein-ligand binding interactions.

    PubMed

    Chang, Le; Ishikawa, Takeshi; Kuwata, Kazuo; Takada, Shoji

    2013-05-30

    Accurate computational estimate of the protein-ligand binding affinity is of central importance in rational drug design. To improve accuracy of the molecular mechanics (MM) force field (FF) for protein-ligand simulations, we use a protein-specific FF derived by the fragment molecular orbital (FMO) method and by the restrained electrostatic potential (RESP) method. Applying this FMO-RESP method to two proteins, dodecin, and lysozyme, we found that protein-specific partial charges tend to differ more significantly from the standard AMBER charges for isolated charged atoms. We did not see the dependence of partial charges on the secondary structure. Computing the binding affinities of dodecin with five ligands by MM PBSA protocol with the FMO-RESP charge set as well as with the standard AMBER charges, we found that the former gives better correlation with experimental affinities than the latter. While, for lysozyme with five ligands, both charge sets gave similar and relatively accurate estimates of binding affinities.

  17. Dipeptidase 1: a candidate tumor-specific molecular marker in colorectal carcinoma.

    PubMed

    McIver, C M; Lloyd, J M; Hewett, P J; Hardingham, J E

    2004-06-01

    The aim of this study was to identify tumor-specific markers for the detection of rare disseminated colorectal tumor cells in peripheral venous blood and in intra-peritoneal saline lavage samples collected before and after resection of colorectal tumors. Using cDNA micro-array screening, we found dipeptidase 1 (DPEP1) to be highly expressed in colon tumors compared to matched normal mucosa. Relative reverse transcriptase (RT)-PCR showed that DPEP1 was over-expressed by >/=2 fold in colon tumor compared to normal colonic mucosal tissue in 56/68 (82%) patients. Using immunobead RT-PCR, a technique that first enriches for epithelial cells, we found DPEP1 positive cells in intra-peritoneal lavage and venous blood samples from 15/38 (39%) colorectal cancer cases. This is the first report of DPEP1 as a marker for disseminated colon tumor cells.

  18. Morphological and molecular analyses support the existence of host-specific Peronospora species infecting Chenopodium.

    PubMed

    Choi, Young-Joon; Denchev, Cvetomir M; Shin, Hyeon-Dong

    2008-03-01

    About 20 species of Peronospora have been reported to cause downy mildew on Chenopodium, but, particularly in plant pathology literature, only one species, P. farinosa, is considered to be involved. We performed sequence analysis of the ITS rDNA to reveal the phylogenetic relationships of Peronospora specimens from five species of Chenopodium, viz. C. album, C. ambrosioides, C. bonus-henricus, C. hybridum, and C. polyspermum. The five clades corresponded to particular Chenopodium species, and showed a high level of sequence divergence. Differences in the morphology of the conidia and ultimate branchlets also supported the separation of the five groups at the host species level. These results suggest that the names P. variabilis, P. boni-henrici, P. chenopodii, and P. chenopodii-polyspermi should be used for the four downy mildew pathogens specific to C. album, C. bonus-henricus, C. hybridum, and C. polyspermum, respectively. The Peronospora on C. ambrosioides was found to be an independent species.

  19. Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

    PubMed

    Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

    2012-08-15

    Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration.

  20. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    PubMed Central

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  1. Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.

    PubMed

    Rhee, Won Jong; Santangelo, Philip J; Jo, Hanjoong; Bao, Gang

    2008-03-01

    The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.

  2. Significant enhancement of fluorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-specific DNA nickase.

    PubMed

    Zheleznaya, Ludmila A; Kopein, Damir S; Rogulin, Evgeniy A; Gubanov, Sergey I; Matvienko, Nikolay I

    2006-01-01

    We have developed a simple isothermal (55 degrees C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase-molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.

  3. Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa

    PubMed Central

    2012-01-01

    Background The routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs). Results Headspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole). Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml]. Conclusions The results obtained provide strong evidence that the detection and perhaps even identification of bacteria

  4. Functional cloning of recurrence-specific antigens identifies molecular targets to treat tumor relapse.

    PubMed

    Boisgerault, Nicolas; Kottke, Timothy; Pulido, Jose; Thompson, Jill; Diaz, Rosa Maria; Rommelfanger-Konkol, Diana; Embry, Addie; Saenz, Dyana; Poeschla, Eric; Pandha, Hardev; Harrington, Kevin; Melcher, Alan; Selby, Peter; Vile, Richard

    2013-08-01

    Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expressed cDNA library derived from recurrent tumor cells. By screening the protective, recurrence-derived VSV-cDNA library, here we identify topoisomerase-IIα (TOPO-IIα) as a recurrence-specific tumor antigen against which tolerance can be broken. Tumor recurrences, in two different types of tumor (prostate and melanoma), which had evaded two different frontline treatments (immunotherapy or chemotherapy), significantly overexpressed TOPO-IIα compared with their primary tumor counterparts, which conferred a novel sensitivity to doxorubicin (DOX) chemotherapy upon the recurrent tumors. This was exploited in vivo using combination therapies to cure mice, which would otherwise have relapsed, after suboptimal primary therapy in both models. Our data show that recurrent tumors-across histologies and primary treatments-express distinct antigens compared with the primary tumor which can be identified using the VSV-cDNA library technology. These results suggest that it may be possible to design a few common second-line therapies against a variety of tumor recurrences, in some cases using agents with no obvious activity against the primary tumor. PMID:23752316

  5. Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae.

    PubMed

    Liu, Shao-Yan; Chen, Jie-Yin; Wang, Jin-Long; Li, Lei; Xiao, Hong-Li; Adam, Sami M; Dai, Xiao-Feng

    2013-10-25

    Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.

  6. Molecular studies of Ssa1, a serotype-specific antigen of Pasteurella haemolytica A1.

    PubMed Central

    Lo, R Y; Strathdee, C A; Shewen, P E; Cooney, B J

    1991-01-01

    A serotype-specific antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pSSA1 is characterized. Nucleotide sequence analysis of the insert DNA in pSSA1 identified the gene ssaI, which codes for a protein of approximately 100 kDa. In vivo labeling of pSSA1-encoded protein in Escherichia coli maxicells showed the expression of a 100-kDa protein from the insert DNA on the recombinant plasmid. Northern blot and primer extension analyses were used to identify the mRNA transcript in P. haemolytica A1 and the putative promoter of ssaI. The antigen (designated Ssa1) could be localized to the outer membrane of P. haemolytica A1 and E. coli clones carrying pSSA1. A rabbit serum against Ssa1 was produced by using whole cells of E. coli expressing Ssa1 on the surface as the immunogen, demonstrating that Ssa1 is immunogenic in rabbits. The results from colony immunoblot analysis with calf serum from animals that were resistant to P. haemolytica A1-induced pneumonia suggest indirectly that Ssa1 is also immunogenic in the animals. Images PMID:1840576

  7. Molecular analysis of the relationship between specific vaginal bacteria and bacterial vaginosis metronidazole therapy failure.

    PubMed

    Wang, B; Xiao, B B; Shang, C G; Wang, K; Na, R S; Nu, X X; Liao, Q

    2014-10-01

    Bacterial vaginosis frequently persists, even after treatment. The role of some strains of bacteria associated with bacterial vaginosis treatment failure remains poorly defined. The aim of our study was to define the risk of bacterial vaginosis treatment failure, including pre-treatment detection of specific vaginal bacteria. Bacterial vaginosis is present when the Nugent score is ≥7 and the modified Amsel criteria is positive. Women with bacterial vaginosis were treated with intravaginal metronidazole gel nightly for 5 nights. The 454 pyrosequencing method was used to detect bacteria in vaginal fluid. By univariate analysis, a history of bacterial vaginosis, intrauterine device use and the presence of Facklamia, Corynebacterium and Veillonella were significantly associated with bacterial vaginosis treatment failure. Lactobacillus crispatus, Lactobacillus pentosus and Megasphaera were significantly associated with curing bacterial vaginosis. After logistic regression analysis and detection of these bacteria for test-of-cure, we found that women who had a history of bacterial vaginosis had a higher incidence of bacterial vaginosis treatment failure, whereas women with L. crispatus had a lower incidence of treatment failure. Post-treatment sexual activity was not associated with the treatment effect. Our data suggested that treatment failure may be not caused by drug resistance. Rather, it has a closer relationship with the failed restoration of lactobacilli.

  8. Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues.

    PubMed

    Geiger, Johannes; Aneja, Manish K; Hasenpusch, Günther; Yüksekdag, Gülnihal; Kummerlöwe, Grit; Luy, Burkhard; Romer, Tina; Rothbauer, Ulrich; Rudolph, Carsten

    2010-04-01

    Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2-4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F(ILO) (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F(ILO) = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F(ILO) = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer.

  9. Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies.

    PubMed

    Sato, Ken-ichi; Iwasaki, Tetsushi; Hirahara, Shino; Nishihira, Yusuke; Fukami, Yasuo

    2004-03-11

    Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.

  10. Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues.

    PubMed

    Geiger, Johannes; Aneja, Manish K; Hasenpusch, Günther; Yüksekdag, Gülnihal; Kummerlöwe, Grit; Luy, Burkhard; Romer, Tina; Rothbauer, Ulrich; Rudolph, Carsten

    2010-04-01

    Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2-4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F(ILO) (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F(ILO) = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F(ILO) = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer. PMID:20045181

  11. Sequence-specific recognition of double-stranded DNA with molecular beacon with the aid of Ag(+) under neutral pH environment.

    PubMed

    Xiao, Zhiyou; Guo, Xiaoting; Ling, Liansheng

    2013-05-01

    A sensitive fluorescent sensor for sequence-specific recognition of dsDNA was established with a molecular beacon (MB) based upon the formation of parallel triplex DNA in the presence of Ag(+) under neutral pH environment.

  12. Addition of β-mercaptoethanol is a prerequisite for high-quality RNA isolation using QIAsymphony technology as demonstrated by detection of molecular aberrations in hematologic malignancies.

    PubMed

    van der Poel-van de Luytgaarde, Sonja C P A M; Geertsma-Kleinekoort, Wendy M C; Goudswaard, Chantal S; Hogenbirk-Hupkes, Pauline E; van Hoven-Beijen, M Antoinette; van de Werf, Marloes; Chu, Isabel W T; van Kapel, Jan; Valk, Peter J M

    2013-06-01

    The isolation of high-quality RNA and DNA from various specimens is essential to perform reliable molecular diagnostic assays. In routine diagnostics of hematologic malignancies isolation of high-quality RNA is a prerequisite. We used QIAsymphony technology (QST) using a customized RNA CT 800 V6 protocol for automated semi-high-throughput isolation of RNA from human specimens and compared the results for breakpoint cluster region-c-abl oncogene 1 (BCR-ABL1) quantification by real-time quantitative polymerase chain reaction (RQ-PCR) and detection of JAK2 V617F mutations by reverse-transcriptase PCR (RT-PCR) on QST RNA with RNA isolation performed with our routine manual method using RNA-Bee (RB). QST RNA was isolated with and without the addition of β-mercaptoethanol (BME). Addition of BME to the lysis buffer RLT Plus resulted in consistently lower Ct values in analyses of the reference gene porphobilinogen deaminase (PBGD). Further, the BCR-ABL1 mRNA levels of the QST RNA isolation were highly consistent with RB RNA isolation, only when the lysis buffer RLT Plus in addition contained BME. Moreover, cases of myeloproliferative neoplasms (MPN) with low levels of JAK2 V617F mRNA were even missed in QST when lysis buffer RLT Plus was used, but they were readily detected after addition of BME.

  13. Molecular characterization of fruit-specific class III peroxidase genes in tomato (Solanum lycopersicum).

    PubMed

    Wang, Chii-Jeng; Chan, Yuan-Li; Shien, Chin Hui; Yeh, Kai-Wun

    2015-04-01

    In this study, expression of four peroxidase genes, LePrx09, LePrx17, LePrx35 and LePrxA, was identified in immature tomato fruits, and the function in the regulation of fruit growth was characterized. Analysis of amino acid sequences revealed that these genes code for class III peroxidases, containing B, D and F conserved domains, which bind heme groups, and a buried salt bridge motif. LePrx35 and LePrxA were identified as novel peroxidase genes in Solanum lycopersicum (L.). The temporal expression patterns at various fruit growth stages revealed that LePrx35 and LePrxA were expressed only in immature green (IMG) fruits, whereas LePrx17 and LePrx09 were expressed in both immature and mature green fruits. Tissue-specific expression profiles indicated that only LePrx09 was expressed in the mesocarp but not the inner tissue of immature fruits. The effects of hormone treatments and stresses on the four genes were examined; only the expression levels of LePrx17 and LePrx09 were altered. Transcription of LePrx17 was up-regulated by jasmonic acid (JA) and pathogen infection and expression of LePrx09 was induced by ethephon, salicylic acid (SA) and JA, in particular, as well as wounding, pathogen infection and H2O2 stress. Tomato plants over-expressing LePrx09 displayed enhanced resistance to H2O2 stress, suggesting that LePrx09 may participate in the H2O2 signaling pathway to regulate fruit growth and disease resistance in tomato fruits.

  14. High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.

    PubMed

    Rahman, Mukhlesur; Sun, Zudong; McVetty, Peter B E; Li, Genyi

    2008-10-01

    A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

  15. Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase

    PubMed Central

    2016-01-01

    Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to

  16. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  17. Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

    PubMed Central

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M.

    2016-01-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  18. Molecular Engineering of Thiazole Orange Dye: Change of Fluorescent Signaling from Universal to Specific upon Binding with Nucleic Acids in Bioassay.

    PubMed

    Lu, Yu-Jing; Deng, Qiang; Hou, Jin-Qiang; Hu, Dong-Ping; Wang, Zheng-Ya; Zhang, Kun; Luyt, Leonard G; Wong, Wing-Leung; Chow, Cheuk-Fai

    2016-04-15

    The universal fluorescent staining property of thiazole orange (TO) dye was adapted in order to be specific for G-quadruplex DNA structures, through the introduction of a styrene-like substituent at the ortho-position of the TO scaffold. This extraordinary outcome was determined from experimental studies and further explored through molecular docking studies. The molecular docking studies help understand how such a small substituent leads to remarkable fluorescent signal discrimination between G-quadruplex DNA and other types of nucleic acids. The results reveal that the modified dyes bind to the G-quadruplex or duplex DNA in a similar fashion as TO, but exhibit either enhanced or quenched fluorescent signal, which is determined by the spatial length and orientation of the substituent and has never been known. The new fluorescent dye modified with a p-(dimethylamino)styryl substituent offers 10-fold more selectivity toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based fluorescent probes in bioassays and cell imaging.

  19. Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes.

    PubMed

    Finetti-Sialer, M M; Ciancio, A

    2005-03-01

    ABSTRACT Tests with a real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed on specimens of Xiphinema index collected from the rhizosphere of Grapevine fanleaf virus (GFLV)-infected grapevines at Palagiano, Italy. A 1,157-bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and sequenced. A fluorescent Scorpion probe was designed to detect a highly conserved CP region. A second region with isolate-specific multiple nucleotide polymorphisms was used to detect GFLV isolates using molecular beacons (MB). The Scorpion probe allowed quantitative estimation of GFLV RNA-2 in single nematodes, using a dilution series of a 692-nucleotide transcript of the CP gene. The assay allowed detection of GFLV RNA-2 in individual X. index, with a minimum template threshold of 800 fg or 2.8 x 10(6) RNA-2 molecules per nematode. The CP fragment used for GFLV detection with the Scorpion probe appeared highly conserved among isolates. The probes were tested against other GFLV isolates, which were recognized by the species-specific Scorpion probe and by the corresponding MB specific to the particular isolate. Both tests appeared useful as diagnostic tools or for studies on GFLV in acquisition, retention, and transmission experiments.

  20. Design of molecular beacons for AmpliDet RNA assay--characterization of binding stability and probe specificity.

    PubMed

    Szemes, Marianna; Schoen, Cor D

    2003-04-15

    AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe-target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these deltaG(0) parameters, which was compared to experimental analysis. The average absolute discrepancies for deltaG(0)(41) and for the melting temperatures of MB secondary structures were 0.30 +/- 0.26 kcal/mol and 2.15 +/- 1.5 degrees C, respectively. deltaG(0)(41) of probe-target interaction was predicted with a discrepancy of 1.2 +/- 1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.

  1. Effects of eye movement desensitization and reprocessing (EMDR) on non-specific chronic back pain: a randomized controlled trial with additional exploration of the underlying mechanisms

    PubMed Central

    2013-01-01

    Background Non-specific chronic back pain (CBP) is often accompanied by psychological trauma, but treatment for this associated condition is often insufficient. Nevertheless, despite the common co-occurrence of pain and psychological trauma, a specific trauma-focused approach for treating CBP has been neglected to date. Accordingly, eye movement desensitization and reprocessing (EMDR), originally developed as a treatment approach for posttraumatic stress disorders, is a promising approach for treating CBP in patients who have experienced psychological trauma. Thus, the aim of this study is to determine whether a standardized, short-term EMDR intervention added to treatment as usual (TAU) reduces pain intensity in CBP patients with psychological trauma vs. TAU alone. Methods/design The study will recruit 40 non-specific CBP patients who have experienced psychological trauma. After a baseline assessment, the patients will be randomized to either an intervention group (n = 20) or a control group (n = 20). Individuals in the EMDR group will receive ten 90-minute sessions of EMDR fortnightly in addition to TAU. The control group will receive TAU alone. The post-treatment assessments will take place two weeks after the last EMDR session and six months later. The primary outcome will be the change in the intensity of CBP within the last four weeks (numeric rating scale 0–10) from the pre-treatment assessment to the post-treatment assessment two weeks after the completion of treatment. In addition, the patients will undergo a thorough assessment of the change in the experience of pain, disability, trauma-associated distress, mental co-morbidities, resilience, and quality of life to explore distinct treatment effects. To explore the mechanisms of action that are involved, changes in pain perception and pain processing (quantitative sensory testing, conditioned pain modulation) will also be assessed. The statistical analysis of the primary outcome will be performed

  2. Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

    NASA Technical Reports Server (NTRS)

    Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

    2003-01-01

    Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

  3. The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia.

    PubMed

    Yazawa, Ryosuke; Takeuchi, Yutaka; Morita, Tetsuro; Ishida, Masashi; Yoshizaki, Goro

    2013-10-01

    We developed a spermatogonial transplantation technique to produce donor-derived gametes in surrogate fish. Our ultimate aim is to establish surrogate broodstock that can produce bluefin tuna. We previously determined that only type A spermatogonia (ASG) could colonize recipient gonads in salmonids. Therefore, it is necessary to develop a precise molecular marker that can distinguish ASG in order to develop efficient spermatogonial transplantation methods. In this study, the Pacific bluefin tuna (Thunnus orientalis) dead end (BFTdnd) gene was identified as a specific marker for ASG. In situ hybridization and RT-PCR analysis with various types of spermatogenic cell populations captured by laser microdissection revealed that localization of BFTdnd mRNA was restricted to ASG, and not detected in other differentiated spermatogenic cells. In order to determine if BFTdnd can be used as a molecular marker to identify germ cells with high transplantability, transplantation of dissociated testicular cells isolated from juvenile, immature, and mature Pacific bluefin tuna, which have different proportions of dnd-positive ASG, were performed using chub mackerel as the surrogate recipient species. Colonization of transplanted donor germ cells was only successful with testicular cells from immature Pacific Bluefin tuna, which contained higher proportions of dnd-positive ASG than juvenile and mature fish. Thus, BFTdnd is a useful tool for identifying highly transplantable ASG for spermatogonial transplantation.

  4. Immune cell-specific transcriptional profiling highlights distinct molecular pathways controlled by Tob1 upon experimental autoimmune encephalomyelitis

    PubMed Central

    Didonna, Alessandro; Cekanaviciute, Egle; Oksenberg, Jorge R.; Baranzini, Sergio E.

    2016-01-01

    Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by focal lymphocytic infiltration, demyelination and neurodegeneration. Despite the recent advances in understanding MS molecular basis, no reliable biomarkers have been identified yet to monitor disease progression. Our group has previously reported that low levels of TOB1 in CD4+ T cells are strongly associated with a higher risk of MS conversion in individuals experiencing an initial demyelinating event. Consistently, Tob1 ablation in mice exacerbates the clinical phenotype of the MS model experimental autoimmune encephalomyelitis (EAE). To shed light on Tob1 molecular functions in the immune system, we have conducted the first cell-based transcriptomic analysis in Tob1−/− and wildtype mice upon EAE. Next-generation sequencing was employed to characterize the changes in gene expression in T and B cells at pre- and post-symptomatic EAE stages. Remarkably, we found only modest overlap among the different genetic signatures, suggesting that Tob1 may control distinct genetic programs in the different cytotypes. This hypothesis was corroborated by gene ontology and global interactome analyses, which highlighted specific cellular pathways in each cellular subset before and after EAE induction. In summary, our work pinpoints a multifaceted activity of Tob1 in both homeostasis and disease progression. PMID:27546286

  5. Cationic conjugated polyelectrolyte/molecular beacon complex for sensitive, sequence-specific, real-time DNA detection.

    PubMed

    Feng, Xuli; Duan, Xinrui; Liu, Libin; An, Lingling; Feng, Fude; Wang, Shu

    2008-11-01

    A new fluorescence method has been developed for DNA detection at room temperature in a sensitive, selective, economical, and real-time manner that interfaces the superiority of a molecular beacon in mismatch discrimination with the light-harvesting property of water-soluble conjugated polyelectrolytes. The probe solution contains a cationic conjugated polyelectrolyte (PFP-NMe3+), a molecular beacon with a five base pairs double-stranded stem labeled at the 5'-terminus with fluorescein (DNA P-Fl), and ethidium bromide (EB, a specific intercalator of dsDNA). The electrostatic interactions between DNA P-Fl and PFP-NMe3+ keep them in close proximity, facilitating the fluorescence resonance energy transfer (FRET) from PFP-NMe3+ to fluorescein. Upon adding a complementary strand to the probe solution, the conformation of DNA P-Fl transits into dsDNA followed by the intercalation of EB into the grooves. Two-step FRET, from PFP-NMe3+ to DNA P-Fl (FRET-1), followed by FRET from DNA P-Fl to EB (FRET-2) takes place. In view of the observed fluorescein or EB emission changes, DNA can be detected in aqueous solution. Because the base mismatch in target DNA inhibits the transition of DNA P-Fl from the stem-loop to duplex structure, single nucleotide mismatch can be clearly detected.

  6. Competitive fluorescence assay for specific recognition of atrazine by magnetic molecularly imprinted polymer based on Fe3O4-chitosan.

    PubMed

    Liu, Guangyang; Li, Tengfei; Yang, Xin; She, Yongxin; Wang, Miao; Wang, Jing; Zhang, Min; Wang, Shanshan; Jin, Fen; Jin, Maojun; Shao, Hua; Jiang, Zejun; Yu, Hailong

    2016-02-10

    A novel fluorescence sensing strategy for determination of atrazine in tap water involving direct competition between atrazine and 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF), and which exploits magnetic molecularly imprinted polymer (MMIP), has been developed. The MMIP, based on Fe3O4-chitosan nanoparticles, was synthesized to recognize specific binding sites of atrazine. The recognition capability and selectivity of the MMIP for atrazine and other triazine herbicides was investigated. Under optimal conditions, the competitive reaction between 5-DTAF and atrazine was performed to permit quantitation. Fluorescence intensity changes at 515 nm was linearly related to the logarithm of the atrazine concentration for the range 2.32-185.4 μM. The detection limit for atrazine was 0.86μM (S/N=3) and recoveries were 77.6-115% in spiked tap water samples.

  7. DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

    PubMed Central

    Toukdarian, A E; Lidstrom, M E

    1984-01-01

    DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight. Images PMID:6321444

  8. Additive and synergistic effects of a low-molecular-weight, heparin-like molecule and low doses of cyclosporin in preventing arterial graft rejection in rats.

    PubMed

    Plissonnier, D; Amichot, G; Lecagneux, J; Duriez, M; Gentric, D; Michel, J B

    1993-01-01

    Arteriosclerotic intimal proliferation is one of the main long-term complications of organ transplantation. Low-molecular-weight, heparin-like molecules prevent myointimal proliferation in arterial wall injury and limit rejection in skin allografts. Cyclosporin limits rejection but has no major effect on intimal proliferation. Therefore, an experimental protocol was designed to test whether heparin-like molecules interacted with low doses of cyclosporin to prevent arterial wall immune system injury and response in a model of arterial graft rejection in normotensive and hypertensive rats. Aortic allografts were performed in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) normotensive control rats. Four groups of 10 allografted (SHR and WKY) rats were used: one group was treated with placebo, one with low doses of cyclosporin (2 mg/kg body wt per day), one with low-molecular-weight, heparin-like molecule (1 mg/kg body wt per hour), and one with low doses of cyclosporin plus low-molecular-weight, heparin-like molecule. Ten SHRs and 10 WKYs were isografted and served as the control groups. All rats were killed 8 weeks after aortic grafting. Structural parameters of the grafted segment were measured by morphometric analysis on formalin-fixed sections with specific stains. The classical signs of immune system injury and response were present in the untreated allografts in SHRs and WKYs: inflammatory infiltration of the adventitia, medial injury, and intimal proliferative response. Low doses of cyclosporin had a significant beneficial effect on immune medial injury by increasing medial thickness and the number of remaining smooth muscle cells and decreasing the extracellular matrix injury. Cyclosporin had no protective effect on intimal proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

    PubMed

    Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

    2016-09-15

    Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. PMID:27491593

  10. Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation.

    PubMed

    Koide, Takaki; Asada, Shinichi; Takahara, Yoshifumi; Nishikawa, Yoshimi; Nagata, Kazuhiro; Kitagawa, Kouki

    2006-02-10

    The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).

  11. Effect of Ag addition to L1{sub 0} FePt and L1{sub 0} FePd films grown by molecular beam epitaxy

    SciTech Connect

    Tokuoka, Y.; Seto, Y.; Kato, T.; Iwata, S.

    2014-05-07

    L1{sub 0} ordered FePt-Ag (5 nm) and FePd-Ag (5 nm) films were grown on MgO (001) substrate at temperatures of 250–400 °C by using molecular beam epitaxy method, and their crystal and surface structures, perpendicular magnetic anisotropies and Curie temperatures were investigated. In the case of FePt-Ag, Ag addition with the amount of 10–20 at. % was effective to promote L1{sub 0} ordering and granular growth, resulting in the increase of the perpendicular magnetic anisotropy and coercivity of the FePt-Ag films. On the other hand, in the case of FePd-Ag, Ag addition changed the surface morphology from island to continuous film associated with the reductions of its coercivity and perpendicular anisotropy. The variations of lattice constants and Curie temperature with Ag addition were significantly different between FePt-Ag and FePd-Ag. For FePd-Ag, the c and a axes lattice spacings and Curie temperature gradually changed with increasing Ag content, while they unchanged for FePt-Ag. These results suggest the possibility of the formation of FePdAg alloy in FePd-Ag, while Ag segregation in FePt-Ag.

  12. Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition.

    PubMed

    Grob, Carolina; Jardillier, Ludwig; Hartmann, Manuela; Ostrowski, Martin; Zubkov, Mikhail V; Scanlan, David J

    2015-04-01

    To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition. PMID:25345650

  13. Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition.

    PubMed

    Grob, Carolina; Jardillier, Ludwig; Hartmann, Manuela; Ostrowski, Martin; Zubkov, Mikhail V; Scanlan, David J

    2015-04-01

    To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition.

  14. Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways.

    PubMed

    Capra, Emily J; Perchuk, Barrett S; Lubin, Emma A; Ashenberg, Orr; Skerker, Jeffrey M; Laub, Michael T

    2010-11-24

    Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

  15. Molecularly imprinted polymers for the pre-concentration of polar organic micropollutants for compound-specific isotope analysis

    NASA Astrophysics Data System (ADS)

    Bakkour, Rani; Hofstetter, Thomas B.

    2014-05-01

    Compound-specific isotope analysis (CSIA) is a promising tool for assessing transformations of polar organic micropollutants such as pesticides, pharmaceuticals and consumer chemicals in aquatic systems. There are, however, two major challenges: (1) Polar organic micropollutants occur at very low levels and, as a consequence, large amounts of water are required to achieve analyte enrichment with factors of 50'000 and more, inevitably leading to large interferences from the aqueous matrix. (2) The polarity of these micropollutants impedes the use of typical non-polar sorbates for solid-phase enrichment. In view of these challenges, the use of molecularly imprinted polymers (MIP) is a promising approach to produce tailor-made materials for highly selective enrichment of polar organic micropollutants with reduced matrix interferences. In this work, we explore the use of MIP to selectively enrich benzotriazoles, an important class of polar aquatic micropollutants. Polymers were synthesized in the presence of 5,6-dimethyl-1H-benzotriazole as a template, which leaves cavities in the polymer matrix with a very high affinity to the template and closely related structures including our main target analyte, 1H-benzotrizole. After extraction of the template, specific recognition of substituted benzotriazoles is expected by the synthesized MIPs. As the MIP has no specific affinity to the matrix, there is also expected to be negligible enrichment of the matrix. Retention factors of the MIP are compared for different synthetic procedures and to non-imprinted polymers where no specific intermolecular interactions with benzotriazoles are expected. Optimum performance of the MIP is demonstrated in this study in terms of the selectivity of enrichment, recoveries of analytes and the goodness of carbon and nitrogen isotope ratios measured by gas chromatography isotopic ratio mass spectrometry (GC/IRMS). This approach will enable us to enrich large amounts of aqueous samples while

  16. Elucidation of the Molecular Basis of Cholecystokinin Peptide Docking to Its Receptor Using Site-Specific Intrinsic Photoaffinity Labeling and Molecular Modeling†

    PubMed Central

    Dong, Maoqing; Lam, Polo C.-H.; Pino, Delia I.; Abagyan, Ruben; Miller, Laurence J.

    2012-01-01

    G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu199, while the position 31 probe labeled first extracellular loop residue Phe107. Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family. PMID:19441839

  17. Integrative View of α2,3-Sialyltransferases (ST3Gal) Molecular and Functional Evolution in Deuterostomes: Significance of Lineage-Specific Losses

    PubMed Central

    Petit, Daniel; Teppa, Elin; Mir, Anne-Marie; Vicogne, Dorothée; Thisse, Christine; Thisse, Bernard; Filloux, Cyril; Harduin-Lepers, Anne

    2015-01-01

    Sialyltransferases are responsible for the synthesis of a diverse range of sialoglycoconjugates predicted to be pivotal to deuterostomes’ evolution. In this work, we reconstructed the evolutionary history of the metazoan α2,3-sialyltransferases family (ST3Gal), a subset of sialyltransferases encompassing six subfamilies (ST3Gal I–ST3Gal VI) functionally characterized in mammals. Exploration of genomic and expressed sequence tag databases and search of conserved sialylmotifs led to the identification of a large data set of st3gal-related gene sequences. Molecular phylogeny and large scale sequence similarity network analysis identified four new vertebrate subfamilies called ST3Gal III-r, ST3Gal VII, ST3Gal VIII, and ST3Gal IX. To address the issue of the origin and evolutionary relationships of the st3gal-related genes, we performed comparative syntenic mapping of st3gal gene loci combined to ancestral genome reconstruction. The ten vertebrate ST3Gal subfamilies originated from genome duplication events at the base of vertebrates and are organized in three distinct and ancient groups of genes predating the early deuterostomes. Inferring st3gal gene family history identified also several lineage-specific gene losses, the significance of which was explored in a functional context. Toward this aim, spatiotemporal distribution of st3gal genes was analyzed in zebrafish and bovine tissues. In addition, molecular evolutionary analyses using specificity determining position and coevolved amino acid predictions led to the identification of amino acid residues with potential implication in functional divergence of vertebrate ST3Gal. We propose a detailed scenario of the evolutionary relationships of st3gal genes coupled to a conceptual framework of the evolution of ST3Gal functions. PMID:25534026

  18. A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity.

    PubMed Central

    Dennis, C A; Videler, H; Pauptit, R A; Wallis, R; James, R; Moore, G R; Kleanthous, C

    1998-01-01

    We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity. PMID:9639578

  19. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  20. Identification of Barramundi (Lates calcarifer) DC-SCRIPT, a Specific Molecular Marker for Dendritic Cells in Fish

    PubMed Central

    Zoccola, Emmanuelle; Delamare-Deboutteville, Jérôme; Barnes, Andrew C.

    2015-01-01

    Antigen presentation is a critical step bridging innate immune recognition and specific immune memory. In mammals, the process is orchestrated by dendritic cells (DCs) in the lymphatic system, which initiate clonal proliferation of antigen-specific lymphocytes. However, fish lack a classical lymphatic system and there are currently no cellular markers for DCs in fish, thus antigen-presentation in fish is poorly understood. Recently, antigen-presenting cells similar in structure and function to mammalian DCs were identified in various fish, including rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). The present study aimed to identify a potential molecular marker for DCs in fish and therefore targeted DC-SCRIPT, a well-conserved zinc finger protein that is preferentially expressed in all sub-types of human DCs. Putative dendritic cells were obtained in culture by maturation of spleen and pronephros-derived monocytes. DC-SCRIPT was identified in barramundi by homology using RACE PCR and genome walking. Specific expression of DC-SCRIPT was detected in barramundi cells by Stellaris mRNA FISH, in combination with MHCII expression when exposed to bacterial derived peptidoglycan, suggesting the presence of DCs in L. calcarifer. Moreover, morphological identification was achieved by light microscopy of cytospins prepared from these cultures. The cultured cells were morphologically similar to mammalian and trout DCs. Migration assays determined that these cells have the ability to move towards pathogens and pathogen associated molecular patterns, with a preference for peptidoglycans over lipopolysaccharides. The cells were also strongly phagocytic, engulfing bacteria and rapidly breaking them down. Barramundi DCs induced significant proliferation of responder populations of T-lymphocytes, supporting their role as antigen presenting cells. DC-SCRIPT expression in head kidney was higher 6 and 24 h following intraperitoneal challenge with peptidoglycan and

  1. Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays.

    PubMed

    Kumar, Rajesh; Grover, Sunita; Batish, Virender Kumar

    2011-12-01

    In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.

  2. Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana.

    PubMed

    Gangl, Roman; Behmüller, Robert; Tenhaken, Raimund

    2015-01-01

    Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress. PMID:26483807

  3. Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana

    PubMed Central

    Gangl, Roman; Behmüller, Robert; Tenhaken, Raimund

    2015-01-01

    Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress. PMID:26483807

  4. The flexibility of P-glycoprotein for its poly-specific drug binding from molecular dynamics simulations.

    PubMed

    Liu, Ming; Hou, Tingjun; Feng, Zhiwei; Li, Youyong

    2013-01-01

    The multidrug efflux pump P-glycoprotein (P-gp) contributes to multidrug resistance in about half of human cancers. Recently, high resolution X-ray crystal structures of mouse P-gp (inward-facing) were reported, which significantly facilitates the understanding of the function of P-gp and the structure-based design of inhibitors for P-gp. Here we perform 20 ns molecular dynamics simulations of inward-facing P-gp with/without ligand in explicit lipid and water to investigate the flexibility of P-gp for its poly-specific drug binding. By analyzing the interactions between P-gp and QZ59-RRR or QZ59-SSS, we summarize the important residues and the flexibility of different parts of P-gp. Particularly, the flexibility of the side chains of aromatic residues (Phe and Tyr) allows them to form rotamers with different orientations in the binding pocket, which plays a critical role for the poly-specificity of the drug-binding cavity of P-gp. MD simulations reveal that trans-membrane (TM) TM12 and TM6 are flexible and contribute to the poly-specific drug binding, while TM4 and TM5 are rigid and stabilize the whole structure. We also construct outward-facing P-gp based on the MsbA structure and perform 20 ns MD simulations. The comparison between the MD results for outward-facing P-gp and those for inward-facing P-gp shows that the TM parts in outward-facing P-gp undergo significant conformational change to facilitate the export of small molecules. PMID:22888853

  5. Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

    PubMed

    Fregien, N; Dolecki, G J; Mandel, M; Humphreys, T

    1983-06-01

    Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA. PMID:6688291

  6. The molecular basis for the post-translational addition of amino acids by L/F transferase in the N-end rule pathway.

    PubMed

    Fung, Angela Wai S; Fahlman, Richard P

    2015-01-01

    The N-end rule pathway is a conserved targeted proteolytic process observed in organisms ranging from eubacteria to mammals. The N-end rule relates the metabolic stability of a protein to its N-terminal amino acid residue. The identity of the N-terminal amino acid residue is a primary degradation signal, often referred to as an N-degron, which is recognized by the components of the N-end rule when it is a destabilizing N-terminus. N-degrons may be exposed by non-processive proteolytic cleavages or by post-translational modifications. One modification is the post-translational addition of amino acids to the N-termini of proteins, a reaction catalyzed by aminoacyl-tRNA protein transferases. The aminoacyl-tRNA protein transferase in eubacteria like Escherichia coli is L/F transferase. Recent investigations have reported unexpected observations regarding the L/F transferase catalytic mechanism and its mechanisms of substrate recognition. Additionally, recent proteome-wide identification of putative in vivo substrates facilitates hypothesis into the yet elusive biological functions of the prokaryotic N-end rule pathway. Here we summarize the recent findings on the molecular mechanisms of catalysis and substrate recognition by the E. coli L/F transferase in the prokaryotic N-end rule pathway.

  7. Grafting of molecularly imprinted polymers from the surface of silica gel particles via reversible addition-fragmentation chain transfer polymerization: a selective sorbent for theophylline.

    PubMed

    Li, Yong; Zhou, Wen-Hui; Yang, Huang-Hao; Wang, Xiao-Ru

    2009-07-15

    Molecularly imprinted polymers (MIPs) were grafted successfully from the surface of silica gel particles via surface initiated reversible addition-fragmentation chain transfer (RAFT) polymerization using RAFT agent functionalized silica gel as the chain transfer agent. The intrinsic characteristics of the controlled/living polymerization mechanism of RAFT allowed for the effective control of the grafting process. Thus the grafting copolymerization of methacrylic acid and divinyl benzene in the presence of template theophylline led to thin MIP film coating silica gel (MIP-Silica). The thickness of MIP film prepared in this study is about 1.98 nm, which was calculated from the nitrogen sorption analysis results. Measured binding kinetics for theophylline to the MIP-Silica and MIPs prepared by conventional bulk polymerization demonstrated that MIP-Silica had improved mass-transfer properties. In addition, the theophylline-imprinted MIP-Silica was used as the sorbent in solid-phase extraction to determine theophylline in blood serum with satisfactory recovery higher than 90%. Nonspecific adsorption of interfering compounds can be eliminated by a simple elution with acetonitrile, without sacrificing the selective binding of theophylline.

  8. Tissue-Specific Changes in Molecular Clocks During the Transition from Pregnancy to Lactation in Mice1

    PubMed Central

    Casey, Theresa M.; Crodian, Jennifer; Erickson, Emily; Kuropatwinski, Karen K.; Gleiberman, Anatoli S.; Antoch, Marina P.

    2014-01-01

    ABSTRACT Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates. PMID:24759789

  9. Sporadic Early-Onset Colorectal Cancer Is a Specific Sub-Type of Cancer: A Morphological, Molecular and Genetics Study

    PubMed Central

    Kirzin, Sylvain; Marisa, Laetitia; Guimbaud, Rosine; De Reynies, Aurélien; Legrain, Michèle; Laurent-Puig, Pierre; Cordelier, Pierre; Pradère, Bernard; Bonnet, Delphine; Meggetto, Fabienne; Portier, Guillaume; Brousset, Pierre; Selves, Janick

    2014-01-01

    Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45–60 years were excluded to help define “young” and “old” groups. Thirty-nine cases of sporadic EOCRC (patients≤45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways

  10. Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

    PubMed Central

    Friis, Kristina Pagh; Iturrioz, Xavier; Thomsen, Jonas; Alvear-Perez, Rodrigo; Bahrami, Shervin; Llorens-Cortes, Catherine

    2015-01-01

    ABSTRACT We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCE Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry. PMID:26608314

  11. Apportioning sources of organic matter in streambed sediments: an integrated molecular and compound-specific stable isotope approach.

    PubMed

    Cooper, Richard J; Pedentchouk, Nikolai; Hiscock, Kevin M; Disdle, Paul; Krueger, Tobias; Rawlins, Barry G

    2015-07-01

    We present a novel application for quantitatively apportioning sources of organic matter in streambed sediments via a coupled molecular and compound-specific isotope analysis (CSIA) of long-chain leaf wax n-alkane biomarkers using a Bayesian mixing model. Leaf wax extracts of 13 plant species were collected from across two environments (aquatic and terrestrial) and four plant functional types (trees, herbaceous perennials, and C3 and C4 graminoids) from the agricultural River Wensum catchment, UK. Seven isotopic (δ13C27, δ13C29, δ13C31, δ13C27-31, δ2H27, δ2H29, and δ2H27-29) and two n-alkane ratio (average chain length (ACL), carbon preference index (CPI)) fingerprints were derived, which successfully differentiated 93% of individual plant specimens by plant functional type. The δ2H values were the strongest discriminators of plants originating from different functional groups, with trees (δ2H27-29=-208‰ to -164‰) and C3 graminoids (δ2H27-29=-259‰ to -221‰) providing the largest contrasts. The δ13C values provided strong discrimination between C3 (δ13C27-31=-37.5‰ to -33.8‰) and C4 (δ13C27-31=-23.5‰ to -23.1‰) plants, but neither δ13C nor δ2H values could uniquely differentiate aquatic and terrestrial species, emphasizing a stronger plant physiological/biochemical rather than environmental control over isotopic differences. ACL and CPI complemented isotopic discrimination, with significantly longer chain lengths recorded for trees and terrestrial plants compared with herbaceous perennials and aquatic species, respectively. Application of a comprehensive Bayesian mixing model for 18 streambed sediments collected between September 2013 and March 2014 revealed considerable temporal variability in the apportionment of organic matter sources. Median organic matter contributions ranged from 22% to 52% for trees, 29% to 50% for herbaceous perennials, 17% to 34% for C3 graminoids and 3% to 7% for C4 graminoids. The results presented here

  12. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

    PubMed Central

    Wilkins, Christopher E.; Marlow, Laura A.; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O.; Kislinger, Thomas; Copland, John A.; Drake, Richard R.

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  13. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma.

    PubMed

    Neely, Benjamin A; Wilkins, Christopher E; Marlow, Laura A; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O; Kislinger, Thomas; Copland, John A; Drake, Richard R

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  14. Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

    DOE PAGES

    Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; Podar, Mircea; Hurt, Jr., Richard A.; Santillan, Eugenio U.; Soren, Ally; Brandt, Craig C.; Brown, Steven D.; Palumbo, Anthony V.; et al

    2016-07-15

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

  15. Different low-molecular-mass organic acids specifically control leaching of arsenic and lead from contaminated soil.

    PubMed

    Ash, Christopher; Tejnecký, Václav; Borůvka, Luboš; Drábek, Ondřej

    2016-04-01

    Low-molecular-mass organic acids (LMMOA) are of key importance for mobilisation and fate of metals in soil, by functioning as ligands that increase the amount of dissolved metal in solution or by dissociation of metal binding minerals. Column leaching experiments were performed on soil polluted with As and Pb, in order to determine the specificity of LMMOA related release for individual elements, at varying organic acid concentrations. Acetic, citric and oxalic acids were applied in 12h leaching experiments over a concentration range (0.5-25 mM) to soil samples that represent organic and mineral horizons. The leaching of As followed the order: oxalic>citric>acetic acid in both soils. Arsenic leaching was attributed primarily to ligand-enhanced dissolution of mineral oxides followed by As released into solution, as shown by significant correlation between oxalic and citric acids and content of Al and Fe in leaching solutions. Results suggest that subsurface mineral soil layers are more vulnerable to As toxicity. Leaching of Pb from both soils followed the order: citric>oxalic>acetic acid. Mineral soil samples were shown to be more susceptible to leaching of Pb than samples characterised by a high content of organic matter. The leaching efficiency of citric acid was attributed to formation of stable complexes with Pb ions, which other acids are not capable of. Results obtained in the study are evidence that the extent of As and Pb leaching in contaminated surface and subsurface soil depends significantly on the types of carboxylic acid involved. The implications of the type of acid and the specific element that can be mobilised become increasingly significant where LMMOA concentrations are highest, such as in rhizosphere soil. PMID:26849837

  16. Different low-molecular-mass organic acids specifically control leaching of arsenic and lead from contaminated soil

    NASA Astrophysics Data System (ADS)

    Ash, Christopher; Tejnecký, Václav; Borůvka, Luboš; Drábek, Ondřej

    2016-04-01

    Low-molecular-mass organic acids (LMMOA) are of key importance for mobilisation and fate of metals in soil, by functioning as ligands that increase the amount of dissolved metal in solution or by dissociation of metal binding minerals. Column leaching experiments were performed on soil polluted with As and Pb, in order to determine the specificity of LMMOA related release for individual elements, at varying organic acid concentrations. Acetic, citric and oxalic acids were applied in 12 h leaching experiments over a concentration range (0.5-25 mM) to soil samples that represent organic and mineral horizons. The leaching of As followed the order: oxalic > citric > acetic acid in both soils. Arsenic leaching was attributed primarily to ligand-enhanced dissolution of mineral oxides followed by As released into solution, as shown by significant correlation between oxalic and citric acids and content of Al and Fe in leaching solutions. Results suggest that subsurface mineral soil layers are more vulnerable to As toxicity. Leaching of Pb from both soils followed the order: citric > oxalic > acetic acid. Mineral soil samples were shown to be more susceptible to leaching of Pb than samples characterised by a high content of organic matter. The leaching efficiency of citric acid was attributed to formation of stable complexes with Pb ions, which other acids are not capable of. Results obtained in the study are evidence that the extent of As and Pb leaching in contaminated surface and subsurface soil depends significantly on the types of carboxylic acid involved. The implications of the type of acid and the specific element that can be mobilised become increasingly significant where LMMOA concentrations are highest, such as in rhizosphere soil.

  17. Different low-molecular-mass organic acids specifically control leaching of arsenic and lead from contaminated soil.

    PubMed

    Ash, Christopher; Tejnecký, Václav; Borůvka, Luboš; Drábek, Ondřej

    2016-04-01

    Low-molecular-mass organic acids (LMMOA) are of key importance for mobilisation and fate of metals in soil, by functioning as ligands that increase the amount of dissolved metal in solution or by dissociation of metal binding minerals. Column leaching experiments were performed on soil polluted with As and Pb, in order to determine the specificity of LMMOA related release for individual elements, at varying organic acid concentrations. Acetic, citric and oxalic acids were applied in 12h leaching experiments over a concentration range (0.5-25 mM) to soil samples that represent organic and mineral horizons. The leaching of As followed the order: oxalic>citric>acetic acid in both soils. Arsenic leaching was attributed primarily to ligand-enhanced dissolution of mineral oxides followed by As released into solution, as shown by significant correlation between oxalic and citric acids and content of Al and Fe in leaching solutions. Results suggest that subsurface mineral soil layers are more vulnerable to As toxicity. Leaching of Pb from both soils followed the order: citric>oxalic>acetic acid. Mineral soil samples were shown to be more susceptible to leaching of Pb than samples characterised by a high content of organic matter. The leaching efficiency of citric acid was attributed to formation of stable complexes with Pb ions, which other acids are not capable of. Results obtained in the study are evidence that the extent of As and Pb leaching in contaminated surface and subsurface soil depends significantly on the types of carboxylic acid involved. The implications of the type of acid and the specific element that can be mobilised become increasingly significant where LMMOA concentrations are highest, such as in rhizosphere soil.

  18. Molecular and Ecological Evidence for Species Specificity and Coevolution in a Group of Marine Algal-Bacterial Symbioses

    PubMed Central

    Ashen, Jon B.; Goff, Lynda J.

    2000-01-01

    The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the α subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation. PMID:10877801

  19. Tricolour fluorescence detection of sequence-specific DNA with a new molecular beacon and a nucleic acid dye TOTO-3.

    PubMed

    Xiang, Dongshan; Zhang, Cuiling; Chen, Lu; Ji, Xinghu; He, Zhike

    2012-12-21

    We have developed a tricolor fluorescence quantitative method for sequence-specific DNA detection using a new molecular beacon (MB) and a nucleic acid dye TOTO-3. This new MB is designed with two fluorophores of FAM and TAMRA instead of one fluorophore and one quencher of traditional MB, and a nucleotide with guanine base is attached directly to FAM as a quencher. In the absence of target DNA, MBs are in the stem-loop state. The fluorescence of FAM is absorbed by TAMRA, and the fluorescence of TAMRA is quenched by guanine base. Meanwhile, the interaction between TOTO-3 and MBs is very weak. In the presence of target DNA, MBs hybridize with target DNA to form a double-stranded structure. TAMRA is separated from FAM and guanine base, and the fluorescence of FAM and TAMRA recovers simultaneously. At the same time TOTO-3 binds to double-stranded DNA, the fluorescence of TOTO-3 significantly enhances. In this strategy, the false-positive signals of MBs caused by non-specific interactions can be distinguished by the change of the ratio of the total fluorescence intensities of FAM and TAMRA to that of TOTO-3 at different concentrations of target DNA. In the simple sample, the detection of target DNA can be achieved with the total fluorescence intensity of three dyes, which results in a significant improvement of the detection sensitivity. In the complex sample, the detection of target DNA can be achieved with the fluorescence intensity of TOTO-3 which can overcome the false-positive signals of MBs and improve the detection accuracy.

  20. Design, Synthesis, Biological Activity and Molecular Dynamics Studies of Specific Protein Tyrosine Phosphatase 1B Inhibitors over SHP-2

    PubMed Central

    Sun, Su-Xia; Li, Xiao-Bo; Liu, Wen-Bo; Ma, Ying; Wang, Run-Ling; Cheng, Xian-Chao; Wang, Shu-Qing; Liu, Wei

    2013-01-01

    Over expressing in PTPN1 (encoding Protein tyrosine phosphatase 1B, PTP1B), a protein tyrosine phosphatase (PTP) that plays an overall positive role in insulin signaling, is linked to the pathogenesis of diabetes and obesity. The relationship between PTP1B and human diseases exhibits PTP1B as the target to treat these diseases. In this article, small weight molecules of the imidazolidine series were screened from databases and optimized on silicon as the inhibitors of PTP1B based on the steric conformation and electronic configuration of thiazolidinedione (TZD) compounds. The top three candidates were tested using an in vitro biological assay after synthesis. Finally, we report a novel inhibitor, Compound 13, that specifically inhibits PTP1B over the closely related phosphatase Src homology 2 (SH2) domain-containing phosphatase 2 (SHP-2) at 80 μM. Its IC50 values are reported in this paper as well. This compound was further verified by computer analysis for its ability to combine the catalytic domains of PTP1B and SHP-2 by molecular dynamics (MD) simulations. PMID:23774838

  1. Arraying prostate specific antigen PSA and Fab anti-PSA using light-assisted molecular immobilization technology

    PubMed Central

    Parracino, Antonietta; Neves-Petersen, Maria Teresa; di Gennaro, Ane Kold; Pettersson, Kim; Lövgren, Timo; Petersen, Steffen B

    2010-01-01

    We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems. PMID:20665692

  2. Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

    PubMed

    Garg, Rohini; Shankar, Rama; Thakkar, Bijal; Kudapa, Himabindu; Krishnamurthy, Lakshmanan; Mantri, Nitin; Varshney, Rajeev K; Bhatia, Sabhyata; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea. PMID:26759178

  3. Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea

    PubMed Central

    Garg, Rohini; Shankar, Rama; Thakkar, Bijal; Kudapa, Himabindu; Krishnamurthy, Lakshmanan; Mantri, Nitin; Varshney, Rajeev K.; Bhatia, Sabhyata; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea. PMID:26759178

  4. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

    PubMed

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent

  5. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

    PubMed

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent

  6. Specific effects of Ca(2+) ions and molecular structure of β-lactoglobulin interfacial layers that drive macroscopic foam stability.

    PubMed

    Braunschweig, Björn; Schulze-Zachau, Felix; Nagel, Eva; Engelhardt, Kathrin; Stoyanov, Stefan; Gochev, Georgi; Khristov, Khr; Mileva, Elena; Exerowa, Dotchi; Miller, Reinhard; Peukert, Wolfgang

    2016-07-01

    β-Lactoglobulin (BLG) adsorption layers at air-water interfaces were studied in situ with vibrational sum-frequency generation (SFG), tensiometry, surface dilatational rheology and ellipsometry as a function of bulk Ca(2+) concentration. The relation between the interfacial molecular structure of adsorbed BLG and the interactions with the supporting electrolyte is additionally addressed on higher length scales along the foam hierarchy - from the ubiquitous air-water interface through thin foam films to macroscopic foam. For concentrations <1 mM, a strong decrease in SFG intensity from O-H stretching bands and a slight increase in layer thickness and surface pressure are observed. A further increase in Ca(2+) concentrations above 1 mM causes an apparent change in the polarity of aromatic C-H stretching vibrations from interfacial BLG which we associate to a charge reversal at the interface. Foam film measurements show formation of common black films at Ca(2+) concentrations above 1 mM due to considerable decrease of the stabilizing electrostatic disjoining pressure. These observations also correlate with a minimum in macroscopic foam stability. For concentrations >30 mM Ca(2+), micrographs of foam films show clear signatures of aggregates which tend to increase the stability of foam films. Here, the interfacial layers have a higher surface dilatational elasticity. In fact, macroscopic foams formed from BLG dilutions with high Ca(2+) concentrations where aggregates and interfacial layers with higher elasticity are found, showed the highest stability with much smaller bubble sizes.

  7. Improving the mining soil quality for a vegetation cover after addition of sewage sludges: inorganic ions and low-molecular-weight organic acids in the soil solution.

    PubMed

    Peña, Aránzazu; Mingorance, Ma Dolores; Guzmán-Carrizosa, Ignacio; Fernández-Espinosa, Antonio J

    2015-03-01

    We assessed the effects of applying stabilized sewage sludge (SSL) and composted sewage sludge (CLV), at 5 and 10% to an acid mining soil. Limed soil (NCL) amended or not with SSL and CLV was incubated for 47 days. We studied the cations and organic and inorganic anions in the soil solution by means of ion chromatography. Liming led to big increases in Ca(2+) and SO4(2-) and to significant decreases in K(+), Mg(2+), NH4(+) and NO3(-). Addition of both organic amendments increased some cations (NH4(+), K(+), Mg(2+), Na(+)) and anions (Cl(-), NO3(-) only with CLV and PO4(3-) only with SSL) and provided a greater amount of low-molecular-weight organic acids (LMWOAs) (SSL more than CLV). Incubation led to decreases in all cations, particularly remarkable for Ca(2+) and Mg(2+) in SSL-10. A decrease in NH4(+) was associated with variations in NO2(-) and NO3(-) resulting from nitrification reactions. During incubation the LMWOAs content tended to decrease similarly to the cations, especially in SSL-10. Chemometric tools revealed a clear discrimination between SSL, CLV and NCL. Furthermore, treatment effects depended upon dose, mainly in SSL. Amendment nature and dose affect the quality of a mining soil and improve conditions for plant establishment.

  8. Synthesis of magnetic molecularly imprinted polymers by reversible addition fragmentation chain transfer strategy and its application in the Sudan dyes residue analysis.

    PubMed

    Xie, Xiaoyu; Chen, Liang; Pan, Xiaoyan; Wang, Sicen

    2015-07-31

    Magnetic molecularly imprinted polymers (MMIPs) have become a hotspot owing to the dual functions of target recognition and magnetic separation. In this study, the MMIPs were obtained by the surface-initiated reversible addition fragmentation chain transfer (RAFT) polymerization using Sudan I as the template. The resultant MMIPs were characterized by transmission electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometer, and X-ray diffraction. Benefiting from the controlled/living property of the RAFT strategy, the uniform MIP layer was successfully grafted on the surface of RAFT agent-modified Fe3O4@SiO2 nanoparticles, favoring the fast mass transfer and rapid binding kinetics. The developed MMIPs were used as the solid-phase extraction sorbents to selectively extract four Sudan dyes (Sudan I, II, III, and IV) from chili powder samples. The recoveries of the spiked samples in chili powder samples ranged from 74.1 to 93.3% with RSD lower than 6.4% and the relative standard uncertainty lower than 0.029. This work provided a good platform for the extraction and removal of Sudan dyes in complicated matrixes and demonstrated a bright future for the application of the well-constructed MMIPs in the field of solid-phase extraction. PMID:26077971

  9. Quantum mechanics/molecular mechanics modeling of covalent addition between EGFR-cysteine 797 and N-(4-anilinoquinazolin-6-yl) acrylamide.

    PubMed

    Capoferri, Luigi; Lodola, Alessio; Rivara, Silvia; Mor, Marco

    2015-03-23

    Irreversible epidermal growth factor receptor (EGFR) inhibitors can circumvent resistance to first-generation ATP-competitive inhibitors in the treatment of nonsmall-cell lung cancer. They covalently bind a noncatalytic cysteine (Cys797) at the surface of EGFR active site by an acrylamide warhead. Herein, we used a hybrid quantum mechanics/molecular mechanics (QM/MM) potential in combination with umbrella sampling in the path-collective variable space to investigate the mechanism of alkylation of Cys797 by the prototypical covalent inhibitor N-(4-anilinoquinazolin-6-yl) acrylamide. Calculations show that Cys797 reacts with the acrylamide group of the inhibitor through a direct addition mechanism, with Asp800 acting as a general base/general acid in distinct steps of the reaction. The obtained reaction free energy is negative (ΔA = -12 kcal/mol) consistent with the spontaneous and irreversible alkylation of Cys797 by N-(4-anilinoquinazolin-6-yl) acrylamide. Our calculations identify desolvation of Cys797 thiolate anion as a key step of the alkylation process, indicating that changes in the intrinsic reactivity of the acrylamide would have only a minor impact on the inhibitor potency.

  10. Improving the mining soil quality for a vegetation cover after addition of sewage sludges: inorganic ions and low-molecular-weight organic acids in the soil solution.

    PubMed

    Peña, Aránzazu; Mingorance, Ma Dolores; Guzmán-Carrizosa, Ignacio; Fernández-Espinosa, Antonio J

    2015-03-01

    We assessed the effects of applying stabilized sewage sludge (SSL) and composted sewage sludge (CLV), at 5 and 10% to an acid mining soil. Limed soil (NCL) amended or not with SSL and CLV was incubated for 47 days. We studied the cations and organic and inorganic anions in the soil solution by means of ion chromatography. Liming led to big increases in Ca(2+) and SO4(2-) and to significant decreases in K(+), Mg(2+), NH4(+) and NO3(-). Addition of both organic amendments increased some cations (NH4(+), K(+), Mg(2+), Na(+)) and anions (Cl(-), NO3(-) only with CLV and PO4(3-) only with SSL) and provided a greater amount of low-molecular-weight organic acids (LMWOAs) (SSL more than CLV). Incubation led to decreases in all cations, particularly remarkable for Ca(2+) and Mg(2+) in SSL-10. A decrease in NH4(+) was associated with variations in NO2(-) and NO3(-) resulting from nitrification reactions. During incubation the LMWOAs content tended to decrease similarly to the cations, especially in SSL-10. Chemometric tools revealed a clear discrimination between SSL, CLV and NCL. Furthermore, treatment effects depended upon dose, mainly in SSL. Amendment nature and dose affect the quality of a mining soil and improve conditions for plant establishment. PMID:25506677

  11. Synthesis of magnetic molecularly imprinted polymers by reversible addition fragmentation chain transfer strategy and its application in the Sudan dyes residue analysis.

    PubMed

    Xie, Xiaoyu; Chen, Liang; Pan, Xiaoyan; Wang, Sicen

    2015-07-31

    Magnetic molecularly imprinted polymers (MMIPs) have become a hotspot owing to the dual functions of target recognition and magnetic separation. In this study, the MMIPs were obtained by the surface-initiated reversible addition fragmentation chain transfer (RAFT) polymerization using Sudan I as the template. The resultant MMIPs were characterized by transmission electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometer, and X-ray diffraction. Benefiting from the controlled/living property of the RAFT strategy, the uniform MIP layer was successfully grafted on the surface of RAFT agent-modified Fe3O4@SiO2 nanoparticles, favoring the fast mass transfer and rapid binding kinetics. The developed MMIPs were used as the solid-phase extraction sorbents to selectively extract four Sudan dyes (Sudan I, II, III, and IV) from chili powder samples. The recoveries of the spiked samples in chili powder samples ranged from 74.1 to 93.3% with RSD lower than 6.4% and the relative standard uncertainty lower than 0.029. This work provided a good platform for the extraction and removal of Sudan dyes in complicated matrixes and demonstrated a bright future for the application of the well-constructed MMIPs in the field of solid-phase extraction.

  12. Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

    PubMed

    Martin, F N; Coffey, M D; Zeller, K; Hamelin, R C; Tooley, P; Garbelotto, M; Hughes, K J D; Kubisiak, T; Bilodeau, G J; Levy, L; Blomquist, C; Berger, P H

    2009-04-01

    Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately

  13. Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection.

    PubMed

    Stensvold, C Rune; Lebbad, Marianne; Victory, Emma L; Verweij, Jaco J; Tannich, Egbert; Alfellani, Mohammed; Legarraga, Paulette; Clark, C Graham

    2011-07-01

    To expand the representation for phylogenetic analysis, ten additional complete Entamoeba small-subunit rRNA gene sequences were obtained from humans, non-human primates, cattle and a tortoise. For some novel sequences no corresponding morphological data were available, and we suggest that these organisms should be referred to as ribosomal lineages (RL) rather than being assigned species names at present. To investigate genetic diversity and host specificity of selected Entamoeba species, a total of 91 new partial small subunit rRNA gene sequences were obtained, including 49 from Entamoeba coli, 18 from Entamoeba polecki, and 17 from Entamoeba hartmanni. We propose a new nomenclature for significant variants within established Entamoeba species. Based on current data we propose that the uninucleated-cyst-producing Entamoeba infecting humans is called Entamoeba polecki and divided into four subtypes (ST1-ST4) and that Entamoeba coli is divided into two subtypes (ST1-ST2). New hosts for several species were detected and, while host specificity and genetic diversity of several species remain to be clarified, it is clear that previous reliance on cultivated material has given us a misleading and incomplete picture of variation within the genus Entamoeba. PMID:21295520

  14. Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection.

    PubMed

    Stensvold, C Rune; Lebbad, Marianne; Victory, Emma L; Verweij, Jaco J; Tannich, Egbert; Alfellani, Mohammed; Legarraga, Paulette; Clark, C Graham

    2011-07-01

    To expand the representation for phylogenetic analysis, ten additional complete Entamoeba small-subunit rRNA gene sequences were obtained from humans, non-human primates, cattle and a tortoise. For some novel sequences no corresponding morphological data were available, and we suggest that these organisms should be referred to as ribosomal lineages (RL) rather than being assigned species names at present. To investigate genetic diversity and host specificity of selected Entamoeba species, a total of 91 new partial small subunit rRNA gene sequences were obtained, including 49 from Entamoeba coli, 18 from Entamoeba polecki, and 17 from Entamoeba hartmanni. We propose a new nomenclature for significant variants within established Entamoeba species. Based on current data we propose that the uninucleated-cyst-producing Entamoeba infecting humans is called Entamoeba polecki and divided into four subtypes (ST1-ST4) and that Entamoeba coli is divided into two subtypes (ST1-ST2). New hosts for several species were detected and, while host specificity and genetic diversity of several species remain to be clarified, it is clear that previous reliance on cultivated material has given us a misleading and incomplete picture of variation within the genus Entamoeba.

  15. Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of bunching onion (A. fistulosum L.).

    PubMed

    Yaguchi, Shigenori; Hang, Tran Thi Minh; Tsukazaki, Hikaru; Hoa, Vu Quynh; Masuzaki, Shin-ichi; Wako, Tadayuki; Masamura, Noriya; Onodera, Shuichi; Shiomi, Norio; Yamauchi, Naoki; Shigyo, Masayoshi

    2009-02-01

    To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA)--A. fistulosum monosomic addition plants (AA+nF) and 62 shallot--A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs.

  16. Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of bunching onion (A. fistulosum L.).

    PubMed

    Yaguchi, Shigenori; Hang, Tran Thi Minh; Tsukazaki, Hikaru; Hoa, Vu Quynh; Masuzaki, Shin-ichi; Wako, Tadayuki; Masamura, Noriya; Onodera, Shuichi; Shiomi, Norio; Yamauchi, Naoki; Shigyo, Masayoshi

    2009-02-01

    To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA)--A. fistulosum monosomic addition plants (AA+nF) and 62 shallot--A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs. PMID:19420800

  17. Molecular signatures of lineage-specific adaptive evolution in a unique sea basin: the example of an anadromous goby Leucopsarion petersii.

    PubMed

    Kokita, Tomoyuki; Takahashi, Sayaka; Kumada, Hiroki

    2013-03-01

    Climate changes on various time scales often shape genetic novelty and adaptive variation in many biotas. We explored molecular signatures of directional selection in populations of the ice goby Leucopsarion petersii inhabiting a unique sea basin, the Sea of Japan, where a wide variety of environments existed in the Pleistocene in relation to shifts in sea level by repeated glaciations. This species consisted of two historically allopatric lineages, the Japan Sea (JS) and Pacific Ocean (PO) lineages, and these have lived under contrasting marine environments that are expected to have imposed different selection regimes caused by past climatic and current oceanographic factors. We applied a limited genome-scan approach using seven candidate genes for phenotypic differences between two lineages in combination with 100 anonymous microsatellite loci. Neuropeptide Y (NPY) gene, which is an important regulator of food intake and potent orexigenic agent, and three anonymous microsatellites were identified as robust outliers, that is, candidate loci potentially under directional selection, by multiple divergence- and diversity-based outlier tests in comparisons focused on multiple populations of the JS vs. PO lineages. For these outlier loci, populations of the JS lineage had putative signals of selective sweeps. Additionally, real-time quantitative PCR analysis using fish reared in a common environment showed a higher expression level for NPY gene in the JS lineage. Thus, this study succeeded in identifying candidate genomic regions under selection across populations of the JS lineage and provided evidence for lineage-specific adaptive evolution in this unique sea basin.

  18. Multi-objective optimization and design of experiments as tools to tailor molecularly imprinted polymers specific for glucuronic acid.

    PubMed

    Kunath, Stephanie; Marchyk, Nataliya; Haupt, Karsten; Feller, Karl-Heinz

    2013-02-15

    We present a multi-objective optimization of the binding properties of a molecularly imprinted polymer (MIP) which specifically binds glucuronic acid (GA). A design of experiments approach is used to improve four different parameters that describe the binding properties of the polymer. Eleven different methacrylamide-co-ethyleneglycol dimethacrylate polymers imprinted with GA were synthesized according to a full factorial experimental design plan with 3 influencing factors (degree of cross-linking, molar equivalent of monomer to template and initiator concentration). These polymers were characterized by adsorption of the radiolabeled target analyte in methanol:water 9:1. The binding parameters were computed to optimize the polymer composition, taking into account four objective variables: the maximum binding capacity at high (Bmax) and low (B2) analyte concentrations, the equilibrium constant K50, and the imprinting factor (IF, binding to MIP/binding to NIP). With the multi-objective optimization method based on a desirability approach the composition of a twelfth "ideal" polymer could be predicted. This predicted polymer with highest "desirability" was synthesized with a composition of 0.65 mol% of initiator and a 1:4:20 ratio of template:functional monomers:cross-linker (T:M:X) (80% of cross-linking), and found to be the overall best MIP. Improvements over the original starting polymer were a 6 times lower K50, which corresponds to higher affinity, 20% higher capacity at low analyte concentration (B2), 40% higher capacity (Bmax) and 1.3 times increased imprinting factor (IF). Binding assays were also performed in aqueous solvents. Good binding properties were obtained in pure water with an imprinting factor of 3.2. Thus, this polymer is potentially applicable to biological samples like urine where glucuronides occur.

  19. Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

  20. Molecularly imprinted polymers prepared using protein-conjugated cleavable monomers followed by site-specific post-imprinting introduction of fluorescent reporter molecules.

    PubMed

    Suga, Yusuke; Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2013-10-01

    Molecularly imprinted polymers were prepared using a protein-conjugated disulfide cleavable monomer. After removing the protein by disulfide reduction, a thiol-reactive fluorophore was introduced into the thiol residue located only inside the imprinted cavity, resulting in specific transduction of the binding events into fluorescence spectral change.

  1. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.

    PubMed

    Mas, Guillaume; Crublet, Elodie; Hamelin, Olivier; Gans, Pierre; Boisbouvier, Jérôme

    2013-11-01

    The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.

  2. In vitro analysis of the cytotoxic and anti-inflammatory effects of antioxidant compounds used as additives in ultra high-molecular weight polyethylene in total joint replacement components.

    PubMed

    Bladen, C L; Tzu-Yin, L; Fisher, J; Tipper, J L

    2013-04-01

    Ultra high-molecular weight polyethylene (UHMWPE) remains the most commonly used material in modern joint replacement prostheses. However, UHMWPE wear particles, formed as the bearing articulates, are one of the main factors leading to joint replacement failure via the induction of osteolysis and subsequent aseptic loosening. Previous studies have shown that the addition of antioxidants such as vitamin E to UHMWPE can improve wear resistance of the polymer and reduce oxidative fatigue. However, little is known regarding the biological consequences of such antioxidant chemicals. This study investigated the cytotoxic and anti-inflammatory effects of a variety of antioxidant compounds currently being tested experimentally for use in hip and knee prostheses, including nitroxides, hindered phenols, and lanthanides on U937 human histocyte cells and human peripheral blood mononuclear cells (PBMNCs) in vitro. After addition of the compounds, cell viability was determined by dose response cytotoxicity studies. Anti-inflammatory effects were determined by quantitation of TNF-α release in lipopolysaccharide (LPS)-stimulated cells. This study has shown that many of these compounds were cytotoxic to U937 cells and PBMNCs, at relatively low concentrations (micromolar), specifically the hindered phenol 3,5-di-tert-butyl-4-hydroxyhydrocinnamate (HPAO1), and the nitroxide 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO). Lanthanides were only cytotoxic at very high concentrations and were well tolerated by the cells at lower concentrations. Cytotoxic compounds also showed reduced anti-inflammatory effects, particularly in PBMNCs. Careful consideration should therefore be given to the use of any of these compounds as potential additives to UHMWPE.

  3. In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

    PubMed

    Alexandersen, S; Bloom, M E; Wolfinbarger, J; Race, R E

    1987-08-01

    Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.

  4. Comparison of breast specific gamma imaging and molecular breast tomosynthesis in breast cancer detection: Evaluation in phantoms

    PubMed Central

    Gong, Zongyi; Williams, Mark B.

    2015-01-01

    Purpose: Breast specific gamma imaging or molecular breast imaging (BSGI) obtains 2D images of 99mTc sestamibi distribution in the breast. Molecular breast tomosynthesis (MBT) maps the tracer distribution in 3D by acquiring multiple projections over a limited angular range. Here, the authors compare the performance of the two technologies in terms of spatial resolution, lesion contrast, and contrast-to-noise ratio (CNR) in phantom studies under conditions of clinically relevant sestamibi dose and imaging time. Methods: The systems tested were a Dilon 6800 and a MBT prototype developed at the University of Virginia. Both systems comprise a pixelated sodium iodide scintillator, an array of position sensitive photomultipliers, and a parallel hole collimator. The active areas and energy resolution of the systems are similar. System sensitivity, spatial resolution, lesion contrast, and CNR were measured using a Petri dish, a point source phantom, and a breast phantom containing simulated lesions at two depths, respectively. A single BSGI projection was acquired. Five MBT projections were acquired over ±20°. For both modalities, the total scan count density was comparable to that observed for each in typical 10 min human scans following injection of 22 mCi (814 MBq) of 99mTc-sestamibi. To assess the impact of reducing the tracer dose, the pixel counts of projection images were later binomially subsampled by a factor of 2 to give images corresponding to an injected activity of approximately 11 mCi (407 MBq). Both unprocessed (pixelated) BSGI projections and interpolated (smoothed) BSGI images displayed by default on the Dilon 6800 workstation were analyzed. Volumetric images were reconstructed from the MBT projections using a maximum likelihood expectation maximization algorithm and extracted slices were analyzed. Results: Over a depth range of 1.5–7.5 cm, BSGI spatial resolution was 5.6–11.5 mm in unprocessed projections and 5.7–12.0 mm in interpolated images

  5. VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.

    PubMed

    Deng, W; Chen, L; Peng, W T; Liang, X; Sekiguchi, S; Gordon, M P; Comai, L; Nester, E W

    1999-03-01

    Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an

  6. Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of β-alanine.

    PubMed

    Anandhakumar, Chandran; Li, Yue; Kizaki, Seiichiro; Pandian, Ganesh N; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2014-12-15

    The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the β-alanine that links SAHA to PIP, and led in turn to the design of ββ-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of β-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove.

  7. Molecular cloning of a small DNA binding protein with specificity for a tissue-specific negative element within the rps1 promoter.

    PubMed Central

    Zhou, D X; Bisanz-Seyer, C; Mache, R

    1995-01-01

    A cDNA encoding a specific binding activity for the tissue-specific negative cis-element S1F binding site of spinach rps1 was isolated from a spinach cDNA expression library. This cDNA of 0.7 kb encodes an unusual small peptide of only 70 amino acids, with a basic domain which contains a nuclear localization signal and a putative DNA binding helix. This protein, named S1Fa, is highly conserved between dicotyledonous and monocotyledonous plants and may represent a novel class of DNA binding proteins. The corresponding mRNA is accumulated more in roots and in etiolated seedlings than in green leaves. This expression pattern is correlated with the tissue-specific function of the S1F binding site which represses the rps1 promoter preferentially in roots and in etiolated plants. Images PMID:7739894

  8. Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

    PubMed

    Pan, H C; Yang, H Q; Zhao, F X; Qian, X C

    2014-01-01

    The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of β-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

  9. “A draft Musa balbisiana genome sequence for molecular genetics in polyploid, inter- and intra-specific Musa hybrids”

    PubMed Central

    2013-01-01

    Background Modern banana cultivars are primarily interspecific triploid hybrids of two species, Musa acuminata and Musa balbisiana, which respectively contribute the A- and B-genomes. The M. balbisiana genome has been associated with improved vigour and tolerance to biotic and abiotic stresses and is thus a target for Musa breeding programs. However, while a reference M. acuminata genome has recently been released (Nature 488:213–217, 2012), little sequence data is available for the corresponding B-genome. To address these problems we carried out Next Generation gDNA sequencing of the wild diploid M. balbisiana variety ‘Pisang Klutuk Wulung’ (PKW). Our strategy was to align PKW gDNA reads against the published A-genome and to extract the mapped consensus sequences for subsequent rounds of evaluation and gene annotation. Results The resulting B-genome is 79% the size of the A-genome, and contains 36,638 predicted functional gene sequences which is nearly identical to the 36,542 of the A-genome. There is substantial sequence divergence from the A-genome at a frequency of 1 homozygous SNP per 23.1 bp, and a high degree of heterozygosity corresponding to one heterozygous SNP per 55.9 bp. Using expressed small RNA data, a similar number of microRNA sequences were predicted in both A- and B-genomes, but additional novel miRNAs were detected, including some that are unique to each genome. The usefulness of this B-genome sequence was evaluated by mapping RNA-seq data from a set of triploid AAA and AAB hybrids simultaneously to both genomes. Results for the plantains demonstrated the expected 2:1 distribution of reads across the A- and B-genomes, but for the AAA genomes, results show they contain regions of significant homology to the B-genome supporting proposals that there has been a history of interspecific recombination between homeologous A and B chromosomes in Musa hybrids. Conclusions We have generated and annotated a draft reference Musa B-genome and

  10. Pan-cancer subtyping in a 2D-map shows substructures that are driven by specific combinations of molecular characteristics.

    PubMed

    Taskesen, Erdogan; Huisman, Sjoerd M H; Mahfouz, Ahmed; Krijthe, Jesse H; de Ridder, Jeroen; van de Stolpe, Anja; van den Akker, Erik; Verheagh, Wim; Reinders, Marcel J T

    2016-01-01

    The use of genome-wide data in cancer research, for the identification of groups of patients with similar molecular characteristics, has become a standard approach for applications in therapy-response, prognosis-prediction, and drug-development. To progress in these applications, the trend is to move from single genome-wide measurements in a single cancer-type towards measuring several different molecular characteristics across multiple cancer-types. Although current approaches shed light on molecular characteristics of various cancer-types, detailed relationships between patients within cancer clusters are unclear. We propose a novel multi-omic integration approach that exploits the joint behavior of the different molecular characteristics, supports visual exploration of the data by a two-dimensional landscape, and inspection of the contribution of the different genome-wide data-types. We integrated 4,434 samples across 19 cancer-types, derived from TCGA, containing gene expression, DNA-methylation, copy-number variation and microRNA expression data. Cluster analysis revealed 18 clusters, where three clusters showed a complex collection of cancer-types, squamous-cell-carcinoma, colorectal cancers, and a novel grouping of kidney-cancers. Sixty-four samples were identified outside their tissue-of-origin cluster. Known and novel patient subgroups were detected for Acute Myeloid Leukemia's, and breast cancers. Quantification of the contributions of the different molecular types showed that substructures are driven by specific (combinations of) molecular characteristics. PMID:27109935

  11. Pan-cancer subtyping in a 2D-map shows substructures that are driven by specific combinations of molecular characteristics

    PubMed Central

    Taskesen, Erdogan; Huisman, Sjoerd M. H.; Mahfouz, Ahmed; Krijthe, Jesse H.; de Ridder, Jeroen; van de Stolpe, Anja; van den Akker, Erik; Verheagh, Wim; Reinders, Marcel J. T.

    2016-01-01

    The use of genome-wide data in cancer research, for the identification of groups of patients with similar molecular characteristics, has become a standard approach for applications in therapy-response, prognosis-prediction, and drug-development. To progress in these applications, the trend is to move from single genome-wide measurements in a single cancer-type towards measuring several different molecular characteristics across multiple cancer-types. Although current approaches shed light on molecular characteristics of various cancer-types, detailed relationships between patients within cancer clusters are unclear. We propose a novel multi-omic integration approach that exploits the joint behavior of the different molecular characteristics, supports visual exploration of the data by a two-dimensional landscape, and inspection of the contribution of the different genome-wide data-types. We integrated 4,434 samples across 19 cancer-types, derived from TCGA, containing gene expression, DNA-methylation, copy-number variation and microRNA expression data. Cluster analysis revealed 18 clusters, where three clusters showed a complex collection of cancer-types, squamous-cell-carcinoma, colorectal cancers, and a novel grouping of kidney-cancers. Sixty-four samples were identified outside their tissue-of-origin cluster. Known and novel patient subgroups were detected for Acute Myeloid Leukemia’s, and breast cancers. Quantification of the contributions of the different molecular types showed that substructures are driven by specific (combinations of) molecular characteristics. PMID:27109935

  12. Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific "Bacteroidales" Populations

    EPA Science Inventory

    In this study we evaluated specificity, distribution, and sensitivity of Bacteroidales – (PF163 and PigBac1) and methanogen-based (P23-2) assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 220 fecal DNA extracts derived from t...

  13. Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases.

    PubMed

    Bezerra, Gustavo Arruda; Dobrovetsky, Elena; Dong, Aiping; Seitova, Almagul; Crombett, Lissete; Shewchuk, Lisa M; Hassell, Annie M; Sweitzer, Sharon M; Sweitzer, Thomas D; McDevitt, Patrick J; Johanson, Kyung O; Kennedy-Wilson, Karen M; Cossar, Doug; Bochkarev, Alexey; Gruber, Karl; Dhe-Paganon, Sirano

    2012-01-01

    Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.

  14. Specific detection of CD133-positive tumor cells with iron oxide nanoparticles labeling using noninvasive molecular magnetic resonance imaging

    PubMed Central

    Chen, Ya-Wen; Liou, Gunn-Guang; Pan, Huay-Ben; Tseng, Hui-Hwa; Hung, Yu-Ting; Chou, Chen-Pin

    2015-01-01

    Background The use of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to visualize cells has been applied clinically, showing the potential for monitoring cells in vivo with magnetic resonance imaging (MRI). USPIO conjugated with anti-CD133 antibodies (USPIO-CD133 Ab) that recognize the CD133 molecule, a cancer stem cell marker in a variety of cancers, was studied as a novel and potent agent for MRI contrast enhancement of tumor cells. Materials and methods Anti-CD133 antibodies were used to conjugate with USPIO via interaction of streptavidin and biotin for in vivo labeling of CD133-positive cells in xenografted tumors and N-ethyl-N-nitrosourea (ENU)-induced brain tumors. The specific binding of USPIO-CD133 Ab to CD133-positive tumor cells was subsequently detected by Prussian blue staining and MRI with T2-weighted, gradient echo and multiple echo recombined gradient echo images. In addition, the cellular toxicity of USPIO-CD133 Ab was determined by analyzing cell proliferation, apoptosis, and reactive oxygen species production. Results USPIO-CD133 Ab specifically recognizes in vitro and labels CD133-positive cells, as validated using Prussian blue staining and MRI. The assays of cell proliferation, apoptosis, and reactive oxygen species production showed no significant differences in tumor cells with or without labeling of USPIO-CD133 Ab. In vivo imaging of CD133-positive cells was demonstrated by intravenous injection of USPIO-CD133 Ab in mice with HT29 xenografted tumors. The MRI of HT29 xenografts showed several clusters of hypotensive regions that correlated with CD133 expression and Prussian blue staining for iron. In rat, brain tumors induced by transplacental ENU mutagenesis, several clusters of hypointensive zones were observed in CD133-expressing brain tumors by MRI and intravenously administered USPIO-CD133 Ab. Conclusion Combination of USPIO-CD133 Ab and MRI is valuable in recognizing CD133-expressing tumor cells in vitro, extracellularly

  15. Short term feeding of a high fat diet exerts an additive effect on hepatocellular damage and steatosis in liver-specific PTEN knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatospecific deletion of PTEN results in constitutive activation of Akt and increased lipogenesis. In mice, the addition of a high fat diet (HFD) downregulates lipogenesis. The aim of this study was to determine the effects of a HFD on hepatocellular damage induced by deletion of PTEN. Twelve-week...

  16. The molecular structure and conformational characteristics of some specific benzodiazepine receptor ligands: A molecular orbital study of C3-substituted betacarboline derivatives

    SciTech Connect

    Konschin, H.; Tylli, H. ); Gynther, J. ); Rouvinen, J. )

    1989-01-01

    The molecular structures of the benzodiazepine receptor ligands {beta}-carboline-3-carboxylic acid (BCCA), its methyl, ethyl, and propyl esters (BCCM, BCCE, and BCCP, respectively), and 3-CN-{beta}-carboline (BC-3-CN) have been investigated on a minimal basis STO-3G level of accuracy. For BCCM, BCCE, and BCCP semiempirical AM 1 calculations have also been performed. Fully optimized molecular geometries are reported. Comparisons with available experimental structures indicate that minimal basis results may have a useful predictive value. For the mobile ester side chains, a study of chosen points on the conformational surface was made. Both the STO-3G and the AM 1 results give the planar conformers is the most stable structures with small barriers to internal rotation, provided the ester side chain remains extended. The calculated STO-3G rotational barriers are higher than are the corresponding AM 1 barriers. Partial optimization, i.e., of side-chain structure parameters only, seems sufficient to map the conformational characteristics of these compounds. The orientation of the dipole moment vector and its magnitude may have consequences for possible interaction with a receptor. On the basis of the sidechain internal dynamics, the intramolecular flexibility tends to be confined to certain regions of conformational space.

  17. [Study on Extraction Conditions of Water-Soluble Substances—Purity Test (4) for Polyvinylpolypyrrolidone Listed in Japan's Specifications and Standards for Food Additives].

    PubMed

    Yanagi, Tokue; Matsumoto, Makoto; Shimizu, Sachiko; Iwamura, Tetsuro; Ogaki, Mamiko

    2015-01-01

    The food additive polyvinylpolypyrrolidone is approved for use as a filter aid. The water-soluble substances test of polyvinylpolypyrrolidone often shows poor reproducibility. The instruction "boil gently while stirring using a stirrer" was considered critical, and so this issue was examined. The results showed that the use of a combination of both an oil bath and a stirrer provided good reproducibility without decomposition or other problems.

  18. Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the ß-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in ...

  19. Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

    NASA Astrophysics Data System (ADS)

    Takiguchi, Kingo; Negishi, Makiko; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Yoshikawa, Kenichi

    2014-12-01

    Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

  20. Integrated analysis of microRNAs, transcription factors and target genes expression discloses a specific molecular architecture of hyperdiploid multiple myeloma

    PubMed Central

    Caracciolo, Daniele; Agnelli, Luca; Neri, Antonino; Walker, Brian A.; Morgan, Gareth J.; Cannataro, Mario

    2015-01-01

    Multiple Myeloma (MM) is a malignancy characterized by the hyperdiploid (HD-MM) and the non-hyperdiploid (nHD-MM) subtypes. To shed light within the molecular architecture of these subtypes, we used a novel integromics approach. By annotated MM patient mRNA/microRNA (miRNA) datasets, we investigated mRNAs and miRNAs profiles with relation to changes in transcriptional regulators expression. We found that HD-MM displays specific gene and miRNA expression profiles, involving the Signal Transducer and Activator of Transcription (STAT)3 pathway as well as the Transforming Growth Factor–beta (TGFβ) and the transcription regulator Nuclear Protein-1 (NUPR1). Our data define specific molecular features of HD-MM that may translate in the identification of novel relevant druggable targets. PMID:26056083

  1. Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species.

    PubMed

    Kobayashi, Ayako; Kobayashi, Yukihiro; Shiomi, Kazuo

    2016-10-01

    Allergenic characteristics of purified parvalbumins from different fish species have not been thoroughly investigated. We revealed that purified parvalbumins from nine different fish species have identical IgE-reactivities and high cross-reactivities. We also showed that fish allergenicity is associated with the parvalbumin content of the fish species, rather than species-specific differences in the molecular characteristics of the individual parvalbumin proteins. PMID:27251554

  2. Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses V. Isolation of Additional Hybrids Which Differ in Their Simian Virus 40-Specific Biological Properties

    PubMed Central

    Lewis, Andrew M.; Levine, Arthur S.; Crumpacker, Clyde S.; Levin, Myron J.; Samaha, Richard J.; Henry, Patrick H.

    1973-01-01

    Four new nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated. Although these viruses (designated Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5) were clonal derivatives of the same Ad2-SV40 hybrid population, they differ significantly from each other and from the previously isolated nondefective hybrid, Ad2+ND1, in their biological properties or in the amount of SV40-specific RNA induced during lytic infection. Like Ad2+ND1, Ad2+ND2, and Ad2+ND4 pass serially in both human embryonic kidney (HEK) and primary African green monkey kidney cells. In contrast, Ad2+ND3 and Ad2+ND5 pass serially only in HEK cells. Ad2+ND2 is like Ad2+ND1 in that it induces the SV40 U antigen, but not SV40 T antigen; however, in contrast to the perinuclear SV40 antigen induced by Ad2+ND1, the SV40 antigen induced by Ad2+ND2 is located peripherally in the cytoplasm as well as in the perinuclear region of infected cells. Ad2+ND4 induces both the SV40 T and U antigens. Ad2+ND3 and Ad2+ND5 do not induce serologically detectable SV40 antigens and are distinguished from each other on the basis of the relative quantities of SV40-specific RNA which they induce. The induction of different SV40-specific functions suggests the incorporation of different segments of SV40 DNA within the genomes of the respective hybrid viruses. PMID:4350710

  3. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    PubMed

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  4. Utilization of elongation factor Tu gene (tuf) sequencing and species-specific PCR (SS-PCR) for the molecular identification of Acetobacter species complex.

    PubMed

    Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Lina; Chu, Wen-Shen

    2014-02-01

    The aim of this study was to use tuf gene as a molecular target for species discrimination in the Acetobacter genus, as well as to develop species-specific PCR method for direct species identification of Acetobacter aceti. The results showed that most Acetobacter species could be clearly distinguished, and the average sequence similarity for the tuf gene (89.5%) among type strains was significantly lower than that of the 16S rRNA gene sequence (98.0%). A pair of species-specific primers were designed and used to specifically identify A. aceti, but none of the other Acetobacter strains. Our data indicate that the phylogenetic relationships of most strains in the Acetobacter genus can be resolved using tuf gene sequencing, and the novel species-specific primer pair could be used to rapidly and accurately identify the species of A. aceti by the PCR based assay.

  5. [Specific features of gene amplification on the long arm of chromosome 17 in different molecular genetic subtypes of breast cancer].

    PubMed

    Zavalishina, L E; Danilova, N V; Matsionis, A E; Pavlenko, I A

    2014-01-01

    The frequency of gene amplification and coamplification of HER2/neu, TOP2A and the centromeric region of chromosome 17 (CEP17) was examined in 265 breast cancer (BC) cases belonging to different molecular genetic subgroups. Luminal B breast cancer was found to be characterized by the increased probability of coamplifications (CEP17 and HER/neu, HER2/neu, and TOP2A) on chromosome 17. At the same time, the amplification of just three loci on one chromosome is a rare event and encountered in 17% of luminal B breast cancer cases (or 1.1% of all BC cases). That of HER2/neu in conjunction with elevated CEP17 count is statistically significantly more rarely accompanied by deletion of TOP2A rather than its amplification. The findings suggest that there are different amplification mechanisms in different BC molecular genetic subgroups. PMID:25051718

  6. Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures

    PubMed Central

    Zeng, Hongkui; Shen, Elaine H.; Hohmann, John G.; Oh, Wook Seung; Bernard, Amy; Royall, Joshua J.; Glattfelder, Katie J.; Sunkin, Susan M.; Morris, John A.; Guillozet-Bongaarts, Angela L.; Smith, Kimberly A.; Ebbert, Amanda J.; Swanson, Beryl; Kuan, Leonard; Page, Damon T.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hof, Patrick R.; Hyde, Thomas M.; Kleinman, Joel E.; Jones, Allan R.

    2012-01-01

    Summary Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ~1,000 genes important for neural functions, by in situ hybridization with cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene’s expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain. PMID:22500809

  7. [Specific molecular and morphological changes in cardiomyocytes of hibernating ground squirrels in different periods of annual cycle].

    PubMed

    Karaduleva, E V; Santalova, I M; Zakharova, N M

    2014-01-01

    Structural and molecular changes in cardiomyocytes of hibernating ground squirrels in different periods of the annual cycle were analyzed by means of electron microscopy and polymerase chain reaction. Morphological analysis showed an increase in relative area of sarcoplasmic reticulum in cardiac muscle of ground squirrels preparing to torpor compared to active summer animals. The size of sarcoplasmic reticulum in cardiomyocytes of torpid animals was reliably less than in any other condition of ground squirrels in the annual cycle. The results of molecular analysis showed the decrease in sarcoplasmic reticulum Ca(2+)-ATPase gene (SERCA2a) expression .at all stages of hibernation process and also in periods of autumn activity compared to control mRNA level in active summer animals. The revealed season changes in structure of sarcoplasmic reticulum and sarcoplasmic reticulum Ca(2+)-ATPase gene expression are discussed in regard to adaptation of ground squirrels to hibernation.

  8. Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons.

    PubMed

    Svilar, David; Vens, Conchita; Sobol, Robert W

    2012-01-01

    We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

  9. High molecular weight complexes of mutant superoxide dismutase 1: age-dependent and tissue-specific accumulation.

    PubMed

    Wang, Jiou; Xu, Guilian; Borchelt, David R

    2002-03-01

    Mutations in the cytosolic enzyme, superoxide dismutase 1, have been identified as the cause of motor neuron disease in a subset of cases of familial amyotrophic lateral sclerosis. It has been postulated that the injurious property of mutant enzyme resides in its propensity to aggregate or its propensity to catalyze deleterious, copper-mediated, chemistries. Aggregates of SOD1 have been identified, histologically, in neurons and astroglia of the spinal cords of SOD1-linked FALS patients and in transgenic mice that express these mutant proteins. In the present study, we have employed a technique used in detecting and quantifying aggregates of mutant huntingtin (cellulose acetate filtration) to examine the molecular characteristics of mutant SOD1 in three previously characterized transgenic mouse models of FALS. We show that the brains and spinal cords of these mice accumulate mutant SOD1 complexes that can be trapped by cellulose acetate filtration. The relative abundance of these structures increases dramatically with age. Although expressed to the same level in nonnervous tissues, mutant SOD1 was not found in high molecular weight structures. We conclude that some aspect of the biology of neural tissues (in a setting of declining motor neuron function) predisposes to the accumulation of high molecular weight complexes of mutant SOD1.

  10. IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family.

    PubMed

    Wang, Huanchen; DeRose, Eugene F; London, Robert E; Shears, Stephen B

    2014-06-24

    Inositol trisphosphate kinases (IP3Ks) and inositol hexakisphosphate kinases (IP6Ks) each regulate specialized signalling activities by phosphorylating either InsP3 or InsP6 respectively. The molecular basis for these different kinase activities can be illuminated by a structural description of IP6K. Here we describe the crystal structure of an Entamoeba histolytica hybrid IP6K/IP3K, an enzymatic parallel to a 'living fossil'. Through molecular modelling and mutagenesis, we extrapolated our findings to human IP6K2, which retains vestigial IP3K activity. Two structural elements, an α-helical pair and a rare, two-turn 310 helix, together forge a substrate-binding pocket with an open clamshell geometry. InsP6 forms substantial contacts with both structural elements. Relative to InsP6, enzyme-bound InsP3 rotates 55° closer to the α-helices, which provide most of the protein's interactions with InsP3. These data reveal the molecular determinants of IP6K activity, and suggest an unusual evolutionary trajectory for a primordial kinase that could have favored efficient bifunctionality, before propagation of separate IP3Ks and IP6Ks.

  11. Polyimide processing additives

    NASA Technical Reports Server (NTRS)

    Fletcher, James C. (Inventor); Pratt, J. Richard (Inventor); St.clair, Terry L. (Inventor); Stoakley, Diane M. (Inventor); Burks, Harold D. (Inventor)

    1992-01-01

    A process for preparing polyimides having enhanced melt flow properties is described. The process consists of heating a mixture of a high molecular weight poly-(amic acid) or polyimide with a low molecular weight amic acid or imide additive in the range of 0.05 to 15 percent by weight of additive. The polyimide powders so obtained show improved processability, as evidenced by lower melt viscosity by capillary rheometry. Likewise, films prepared from mixtures of polymers with additives show improved processability with earlier onset of stretching by TMA.

  12. Polyimide processing additives

    NASA Technical Reports Server (NTRS)

    Pratt, J. Richard (Inventor); St.clair, Terry L. (Inventor); Stoakley, Diane M. (Inventor); Burks, Harold D. (Inventor)

    1993-01-01

    A process for preparing polyimides having enhanced melt flow properties is described. The process consists of heating a mixture of a high molecular weight poly-(amic acid) or polyimide with a low molecular weight amic acid or imide additive in the range of 0.05 to 15 percent by weight of the additive. The polyimide powders so obtained show improved processability, as evidenced by lower melt viscosity by capillary rheometry. Likewise, films prepared from mixtures of polymers with additives show improved processability with earlier onset of stretching by TMA.

  13. A systematic review of image segmentation methodology, used in the additive manufacture of patient-specific 3D printed models of the cardiovascular system

    PubMed Central

    Byrne, N; Velasco Forte, M; Tandon, A; Valverde, I

    2016-01-01

    Background Shortcomings in existing methods of image segmentation preclude the widespread adoption of patient-specific 3D printing as a routine decision-making tool in the care of those with congenital heart disease. We sought to determine the range of cardiovascular segmentation methods and how long each of these methods takes. Methods A systematic review of literature was undertaken. Medical imaging modality, segmentation methods, segmentation time, segmentation descriptive quality (SDQ) and segmentation software were recorded. Results Totally 136 studies met the inclusion criteria (1 clinical trial; 80 journal articles; 55 conference, technical and case reports). The most frequently used image segmentation methods were brightness thresholding, region growing and manual editing, as supported by the most popular piece of proprietary software: Mimics (Materialise NV, Leuven, Belgium, 1992–2015). The use of bespoke software developed by individual authors was not uncommon. SDQ indicated that reporting of image segmentation methods was generally poor with only one in three accounts providing sufficient detail for their procedure to be reproduced. Conclusions and implication of key findings Predominantly anecdotal and case reporting precluded rigorous assessment of risk of bias and strength of evidence. This review finds a reliance on manual and semi-automated segmentation methods which demand a high level of expertise and a significant time commitment on the part of the operator. In light of the findings, we have made recommendations regarding reporting of 3D printing studies. We anticipate that these findings will encourage the development of advanced image segmentation methods. PMID:27170842

  14. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  15. High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

    PubMed

    Missirian, Victor; Conklin, Phillip A; Culligan, Kevin M; Huefner, Neil D; Britt, Anne B

    2014-01-01

    Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants.

  16. Molecular distributions and compound-specific stable carbon isotopic compositions of lipids in wintertime aerosols from Beijing.

    PubMed

    Ren, Lujie; Fu, Pingqing; He, Yue; Hou, Juzhi; Chen, Jing; Pavuluri, Chandra Mouli; Sun, Yele; Wang, Zifa

    2016-01-01

    Molecular distributions and stable carbon isotopic compositions (δ(13)C) of n-alkanes, fatty acids and n-alcohols were investigated in urban aerosols from Beijing, northern China to better understand the sources and long-range atmospheric transport of terrestrial organic matter during polluted and clear days in winter. n-Alkanes (C19-C36), fatty acids (C8-C32) and n-alcohols (C16-C32) detected in Beijing aerosols are characterized by the predominance of C23, C16 and C28, respectively. Carbon preference index (CPI) values of n-alkanes, the ratios of the sum of odd-numbered n-alkanes to the sum of even-numbered n-alkanes, are close to 1, indicating a heavy influence of fossil fuel combustion. Relatively higher ratios of C(18:0+16:0)/C(18:n+16:1) (fatty acids) on clear days than polluted days indicate that long-distance transport and/or photochemical aging are more significant during clear days. δ(13)C values of n-alkanes and low molecular weight fatty acids (C16:0, C18:0) ranged from -34.1 to -24.7% and -26.9 to -24.6%, respectively, which are generally heavier on polluted days than those on clear days. Such a wide range suggests that atmospheric lipids in Beijing aerosols originate from multiple sources and encounter complicated atmospheric processes during long-range transport in North China. PMID:27270951

  17. Specific binding and inhibition of 6-benzylaminopurine to catalase: multiple spectroscopic methods combined with molecular docking study.

    PubMed

    Xu, Qin; Lu, Yanni; Jing, Longyun; Cai, Lijuan; Zhu, Xinfeng; Xie, Ju; Hu, Xiaoya

    2014-04-01

    6-Benzylaminopurine (6-BA) is a kind of cytokinin which could regulate the activities of the antioxidant defense system of plants. In this work, its interaction with and inhibition of beef liver catalase have been systematically investigated using spectroscopic, isothermal titration calorimetric and molecular docking methods under physiological conditions. The fluorescence quenching of beef liver catalase (BLC) by 6-BA is due to the formation of 6-BA-BLC complex. Hydrogen bonds and van der Waals interactions play major roles in stabilizing the complex. The Stern-Volmer quenching constant, binding constant, the corresponding thermodynamic parameters and binding numbers were measured. The results of UV-vis absorption, three-dimensional fluorescence, synchronous fluorescence and circular dichroism spectroscopic results demonstrate that the binding of 6-BA results in the micro-environment change around tyrosine (Tyr) and tryptophan (Trp) residues of BLC. The BLC-mediated conversion of H2O2 to H2O and O2, in the presence and absence of 6-BA, was also studied. Lineweaver-Burk plot indicates a noncompetitive type of inhibition. Molecular docking study was used to find the binding sites. PMID:24412785

  18. Metal Ion Coordination Essential for Specific Molecular Interactions of Butea monosperma Lectin: ITC and MD Simulation Studies.

    PubMed

    Abhilash, J; Haridas, M

    2015-05-01

    Crystal structure of Butea monosperma seed lectin (BML) was analyzed and the metal ion geometry identified. In order to understand the role of metal ions for the structural stability and ligand binding, studies of demetallized protein were carried out. Binding of different ligands like GalNAc, lactose, and galactose onto native and demetallized protein was studied by isothermal titration calorimetry as well as molecular simulation methods. Molecular dynamics was applied to the structure after removing the coordinates of metal ions, to identify the effect of demetallization in silico. Docking studies of different sugar molecules as well as the fungal α-amylase was carried out and compared the interactions in the native and apo states. It was found that metal ions are important for the ligand binding with increased affinity. However, their absence did not make any alteration to the secondary structure. Though the metal ions were not coordinated to the loops contacting the α-amylase, the absence of metal ions reduced the protein-protein binding strength due to long-range changes in irregular structures of the lectin.

  19. A novel and versatile nanomachine for ultrasensitive and specific detection of microRNAs based on molecular beacon initiated strand displacement amplification coupled with catalytic hairpin assembly with DNAzyme formation.

    PubMed

    Yan, Yurong; Shen, Bo; Wang, Hong; Sun, Xue; Cheng, Wei; Zhao, Hua; Ju, Huangxian; Ding, Shijia

    2015-08-21

    MicroRNAs are small regulatory molecules that can be used as potential biomarkers of clinical diagnosis, and efforts have been directed towards the development of a simple, rapid, and sequence-selective analysis of microRNAs. Here, we report a simple and versatile colorimetric strategy for ultrasensitive and specific determination of microRNAs based on molecular beacon initiated strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) with DNAzyme formation. The presence of target microRNAs triggers strand displacement amplification to release nicking DNA triggers, which initiate CHA to produce large amounts of CHA products. Meanwhile, the numerous CHA products can combine with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, catalyzing a colorimetric reaction. Moreover, the purification of the SDA mixture has been developed for eliminating matrix interference to decrease nonspecific CHA products. Under the optimal conditions and using the promising amplification strategy, the established colorimetric nanomachine (biosensor) shows high sensitivity and selectivity in a dynamic response range from 5 fM to 5 nM with a detection limit as low as 1.7 fM (S/N = 3). In addition, a versatile colorimetric biosensor has been developed for detection of different miRNAs by only changing the miRNA-recognition domain of molecular beacon. Thus, this colorimetric biosensor may become a potential alternative tool for biomedical research and clinical molecular diagnostics.

  20. A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

    PubMed

    Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

    2014-12-15

    A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring.

  1. First principles molecular dynamics simulation of a task-specific ionic liquid based on silver-olefin complex: atomistic insights into a separation process.

    PubMed

    Jiang, De-en; Dai, Sheng

    2008-08-21

    First principles molecular dynamics based on density functional theory is applied to a hypothetical ionic liquid whose cations and anions are silver-ethylene complex [Ag(C2H4)2+] and tetrafluoroborate [BF4-], respectively. This ionic liquid represents a group of task-specific silver complex-based ionic liquids synthesized recently. Molecular dynamics simulations at two temperatures are performed for five picoseconds. Events of association, dissociation, exchange, and recombination of ethylene with silver cation are found. A mechanism of ethylene transfer similar to the Grotthus type of proton transfer in water is identified, where a silver cation accepts one ethylene molecule and donates another to a neighboring silver cation. This mechanism may contribute to fast transport of olefins through ionic liquid membranes based on silver complexes for olefin/paraffin separation.

  2. Specific features of propagation of femtosecond laser pulses through a molecular gaseous medium under conditions of efficient alignment of molecules

    SciTech Connect

    Gulyaev, A V; Tikhonova, O V

    2013-07-31

    The propagation of femtosecond laser pulses in a molecular gaseous medium is studied with the rotational dynamics of molecules under the action of these pulses taken into account. Based on the simultaneous numerical solution of the wave equation describing the laser pulse evolution and the Schroedinger equation that determines the quantum state evolution of the medium molecules, the rotational dynamics of molecules in the laser field and the laser pulse evolution are analysed with their mutual influence taken into account. Efficient dynamic alignment of molecules along the direction of laser radiation polarisation is observed, which causes variation in the spatiotemporal pulse profile, as well as significant broadening and deformation of its spectrum in the course of propagation through the medium. The physical mechanisms of the observed phenomena are analysed by using the approximate analytical solution of the problem, derived for the case of preliminary excitation of the medium by a pump pulse (the pump-probe scheme). (nonlinear optical phenomena)

  3. Molecular distributions and compound-specific stable carbon isotopic compositions of lipids in wintertime aerosols from Beijing

    NASA Astrophysics Data System (ADS)

    Ren, Lujie; Fu, Pingqing; He, Yue; Hou, Juzhi; Chen, Jing; Pavuluri, Chandra Mouli; Sun, Yele; Wang, Zifa

    2016-06-01

    Molecular distributions and stable carbon isotopic compositions (δ13C) of n-alkanes, fatty acids and n-alcohols were investigated in urban aerosols from Beijing, northern China to better understand the sources and long-range atmospheric transport of terrestrial organic matter during polluted and clear days in winter. n-Alkanes (C19–C36), fatty acids (C8–C32) and n-alcohols (C16–C32) detected in Beijing aerosols are characterized by the predominance of C23, C16 and C28, respectively. Carbon preference index (CPI) values of n-alkanes, the ratios of the sum of odd-numbered n-alkanes to the sum of even-numbered n-alkanes, are close to 1, indicating a heavy influence of fossil fuel combustion. Relatively higher ratios of C(18:0+16:0)/C(18:n+16:1) (fatty acids) on clear days than polluted days indicate that long-distance transport and/or photochemical aging are more significant during clear days. δ13C values of n-alkanes and low molecular weight fatty acids (C16:0, C18:0) ranged from –34.1 to ‑24.7% and ‑26.9 to ‑24.6%, respectively, which are generally heavier on polluted days than those on clear days. Such a wide range suggests that atmospheric lipids in Beijing aerosols originate from multiple sources and encounter complicated atmospheric processes during long-range transport in North China.

  4. Molecular basis of the interaction specificity between the human glucocorticoid receptor and its endogenous steroid ligand cortisol.

    PubMed

    von Langen, Johannes; Fritzemeier, Karl-Heinrich; Diekmann, Stephan; Hillisch, Alexander

    2005-06-01

    We analyzed the binding of five steroids to the human glucocorticoid receptor (hGR) experimentally as well as theoretically. In vitro, we measured the binding affinity of aldosterone, cortisol, estradiol, progesterone, and testosterone to hGR in competition with the ligand dexamethasone. The binding affinity relative to the endogenous ligand cortisol (100%) is reduced for progesterone (22%) and aldosterone (20%) and is very weak for testosterone (1.5%) and estradiol (0.2%). In parallel, we constructed a homology model of the hGR ligand-binding domain (LBD) based on the crystal structure of the human progesterone receptor (hPR). After docking the five steroids into the hGR model ligand-binding pocket, we performed five separate 4-ns molecular dynamics (MD) simulations with these complexes in order to study the complex structures. We calculated the binding affinities with two different approaches (MM/PBSA, FlexX) and compared them with the values of the experimentally determined relative binding affinities. Both theoretical methods allowed discrimination between strongly and weakly binding ligands and recognition of cortisol as the endogenous ligand of the hGR in silico. Cortisol binds most strongly due to a nearly perfect steric and electrostatic complementarity with the hGR binding pocket. Chemically similar ligands such as estradiol, testosterone, and progesterone also fit into the hGR binding pocket, but they are unable to form all those contacts with the amino acids of the protein that are necessary to yield a stable, transcriptionally active receptor conformation. Our analysis thus explains the selectivity of the human glucocorticoid receptor for its endogenous ligand cortisol at a molecular level. PMID:15883974

  5. Molecular distributions and compound-specific stable carbon isotopic compositions of lipids in wintertime aerosols from Beijing

    PubMed Central

    Ren, Lujie; Fu, Pingqing; He, Yue; Hou, Juzhi; Chen, Jing; Pavuluri, Chandra Mouli; Sun, Yele; Wang, Zifa

    2016-01-01

    Molecular distributions and stable carbon isotopic compositions (δ13C) of n-alkanes, fatty acids and n-alcohols were investigated in urban aerosols from Beijing, northern China to better understand the sources and long-range atmospheric transport of terrestrial organic matter during polluted and clear days in winter. n-Alkanes (C19–C36), fatty acids (C8–C32) and n-alcohols (C16–C32) detected in Beijing aerosols are characterized by the predominance of C23, C16 and C28, respectively. Carbon preference index (CPI) values of n-alkanes, the ratios of the sum of odd-numbered n-alkanes to the sum of even-numbered n-alkanes, are close to 1, indicating a heavy influence of fossil fuel combustion. Relatively higher ratios of C(18:0+16:0)/C(18:n+16:1) (fatty acids) on clear days than polluted days indicate that long-distance transport and/or photochemical aging are more significant during clear days. δ13C values of n-alkanes and low molecular weight fatty acids (C16:0, C18:0) ranged from –34.1 to −24.7% and −26.9 to −24.6%, respectively, which are generally heavier on polluted days than those on clear days. Such a wide range suggests that atmospheric lipids in Beijing aerosols originate from multiple sources and encounter complicated atmospheric processes during long-range transport in North China. PMID:27270951

  6. Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification.

    PubMed

    Li, Xiaolu; Guo, Jing; Zhai, Qian; Xia, Jing; Yi, Gang

    2016-08-31

    Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo(-)), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring. PMID:27506343

  7. Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification.

    PubMed

    Li, Xiaolu; Guo, Jing; Zhai, Qian; Xia, Jing; Yi, Gang

    2016-08-31

    Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo(-)), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.

  8. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

    PubMed

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-01-01

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

  9. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

    PubMed Central

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-01-01

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

  10. Loss of the Y chromosome PAR2 region and additional rearrangements in two familial cases of satellited Y chromosomes: cytogenetic and molecular analysis.

    PubMed

    Velissariou, V; Sismani, C; Christopoulou, S; Kaminopetros, P; Hatzaki, A; Evangelidou, P; Koumbaris, G; Bartsocas, C S; Stylianidou, G; Skordis, N; Diakoumakos, A; Patsalis, P C

    2007-01-01

    Two cases of rare structural aberrations of the Y chromosome were detected: a del(Y) (q12) chromosome in a child with mild dysmorphic features, obesity and psychomotor delay, and two identical satellited Y chromosomes (Yqs) in a normal twin, which were originally observed during routine prenatal diagnosis. In both cases a Yqs chromosome was detected in the father which had arisen from a reciprocal translocation involving the short arm of chromosome 15 and the heterochromatin of the long arm of the Y chromosome (Yqh). Cytogenetic and molecular studies demonstrated that in the reciprocal product of chromosomes 15 and Y PAR2 could not be detected, showing that PAR2 had been deleted. It is discussed whether the translocation of the short arm of an acrocentric chromosome to the heterochromatin of the long arm of the Y chromosome causes instability of this region which results either in loss of genetic material or interference with the normal mechanism of disjunction.

  11. Molecular characterisation and chromosomal mapping of transcripts having tissue-specific expression in the malaria mosquito Anopheles gambiae: possible involvement in visual or olfactory processes.

    PubMed

    Ricci, Irene; Santolamazza, Federica; Costantini, Carlo; Favia, Guido

    2002-01-01

    We have compared the transcriptional activity of heads, antennae + palps, and carcasses in the mosquito Anopheles gambiae by means of differential display PCR (DD-PCR). Three transcripts specifically or preferentially expressed in the heads and in the antennae + palps have been selected. All are very similar to genes related to visual and olfactory mechanisms of several different organisms. They have been named Ag arrestin, Ag rLDL, and Ag dynamin. The potential of the DD-PCR technique in identifying genes involved in mosquito behaviour and the usefulness of the molecular characterisation of these transcripts are discussed. PMID:11822731

  12. Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy

    NASA Astrophysics Data System (ADS)

    Freudiger, Christian W.; Min, Wei; Holtom, Gary R.; Xu, Bingwei; Dantus, Marcos; Sunney Xie, X.

    2011-02-01

    Label-free microscopy that has chemical contrast and high acquisition speeds up to video rates has recently been made possible using stimulated Raman scattering (SRS) microscopy. SRS imaging offers high sensitivity, but the spectral specificity of the original narrowband implementation is limited, making it difficult to distinguish chemical species with overlapping Raman bands. Here, we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species, based on tailored multiplex excitation of its vibrational spectrum. This is implemented by spectral modulation of a broadband pump beam at a high frequency (>1 MHz), allowing detection of the SRS signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live Caenorhabditis elegans.

  13. Molecular Insights into the Fungus-Specific Serine/Threonine Protein Phosphatase Z1 in Candida albicans

    PubMed Central

    Chen, Emily; Choy, Meng S.; Petrényi, Katalin; Kónya, Zoltán; Erdődi, Ferenc; Dombrádi, Viktor; Peti, Wolfgang

    2016-01-01

    ABSTRACT The opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans. We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae]). Unexpectedly, we also discovered that, despite its 66% sequence identity to PP1α, the catalytic domain of PPZ1 contains novel structural elements that are not present in PP1α. We then used activity and pulldown assays to show that these structural differences block a large subset of PP1/GLC7 regulatory proteins from effectively binding PPZ1, demonstrating that PPZ1 does not compete with GLC7 for its regulatory proteins. Equally important, these unique structural elements provide new pockets suitable for the development of PPZ1-specific inhibitors. Together, these studies not only reveal why PPZ1 does not negatively impact GLC7 activity in vivo but also demonstrate that the family of fungus-specific phosphatases—especially PPZ1 from C. albicans—are highly suitable targets for the development of novel drugs that specifically target C. albicans without cross-reacting with human phosphatases. PMID:27578752

  14. New pathogen-specific immunoPET/MR tracer for molecular imaging of a systemic bacterial infection

    PubMed Central

    Wiehr, Stefan; Warnke, Philipp; Rolle, Anna-Maria; Schütz, Monika; Oberhettinger, Philipp; Kohlhofer, Ursula; Quintanilla-Martinez, Leticia; Maurer, Andreas; Thornton, Christopher; Boschetti, Frederic; Reischl, Gerald; Autenrieth, Ingo B.; Pichler, Bernd J.; Autenrieth, Stella E.

    2016-01-01

    The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions. PMID:26934329

  15. V{delta}1 T cell receptor binds specifically to MHC I chain related A: Molecular and biochemical evidences

    SciTech Connect

    Zhao Jianqing; Huang Jie; Chen Hui; Cui Lianxian; He Wei . E-mail: heweiimu@public.bta.net.cn

    2006-01-06

    Human MHC class I chain-related A (MICA) is a tumor-associated antigen that can be recognized by V{delta}1 subset of tumor-infiltrating {gamma}{delta} T cells. We previously reported that immobilized recombinant MICA protein could induce the proliferation of tumor-infiltrating V{delta}1 {gamma}{delta} T cells in vitro. But there has been no direct evidence showing the engagement of {gamma}{delta} T cell receptor