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Sample records for alanine dipeptide model

  1. Stabilization of helices in glycine and alanine dipeptides in a reaction field model of solvent

    SciTech Connect

    Shang, H.S. Lawrence Berkeley Lab., CA ); Head-Gordon, T. )

    1994-02-23

    We present molecular orbital calculations of the full conformational space of blocked glycine and alanine dipeptide in the presence of a reaction field representation of water. Secondary structures of right- and left-handed helices are found, in contrast to recent gas-phase results, indicating that the origin of helical stabilization in dipeptides is strictly due to environment. Limitations of the reaction field model and the various implications of stabilization due to environment are discussed. 43 refs., 2 figs., 3 tabs.

  2. A theoretical study of alanine dipeptide and analogs

    SciTech Connect

    Head-Gordon, T.; Head-Gordon, M.; Brooks, C. III; Pople, J. ); Frisch, M.J. )

    1989-01-01

    We Present a preliminary report on the conformational and energetic analysis of the molecule (S)-2-acetylamino-N-methylpropanamide (alanine dipeptide) and an analog molecule, (S)-{alpha}-formylaminopropanamide, using high-quality ab initio methods. Alanine dipeptide and its analogs are of interest since they incorporate many of the structural features found in proteins, such as intramolecular hydrogen bonds, conformational flexibility, and a variety of chemical functionality. One purpose of this study is to provide a useful benchmark calculation, MP2/6-31+G{sup **}//HF/6-31+G{sup *}, for a number of conformations of the alanine system. Based on the comparison of these benchmark calculations with lower-level basis sets, HF/3-21G was chosen to generate a fully relaxed {phi}, {psi} dihedral map. These calculations are the first of their kind on systems of this size. Features of the {phi},{psi} alanine dipeptide map that are discussed include the energetically accessible conformations and possible pathways for their interconversion. In addition, we illustrate the importance of fully optimized geometries and the proper evaluation of correlation energies,

  3. Probing the energy landscape of alanine dipeptide and decalanine using temperature as a tunable parameter in molecular dynamics

    NASA Astrophysics Data System (ADS)

    Chatterjee, A.; Bhattacharya, S.

    2016-10-01

    We perform several molecular dynamics (MD) calculations of solvated alanine dipeptide and decalanine in vacuum with temperature as a tunable parameter and in the process, generate Markov state models (MSMs) at each temperature. An interesting observation that the kinetic rates appear to obey the Arrhenius rate law allows us to predict the dynamics of alanine dipeptide at 300 K at the microsecond timescales using the nanoseconds long high temperature calculations without actually performing MD simulations at 300 K. We conclude that the energy landscape of alanine dipeptide contains superbasins deeper than kBT and determine the energy barriers associated with the moves from the Arrhenius rate expression. Similar insights regarding the energy landscape associated with folding/unfolding pathways of a deca-alanine molecule are obtained using kinetic rates calculated at different temperatures.

  4. The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: L-Alanyl-L-alanine

    NASA Astrophysics Data System (ADS)

    Silva, J. G.; Arruda, L. M.; Pinheiro, G. S.; Lima, C. L.; Melo, F. E. A.; Ayala, A. P.; Filho, J. Mendes; Freire, P. T. C.

    2015-09-01

    A single-crystal of peptide L-alanyl-L-alanine (C6H12N2O3) was studied by Raman spectroscopy at low-temperature, and a tentative assignment of the normal modes was given. Evidence of a second order structural phase transition was found through Raman spectroscopy between the temperatures of 80 K and 60 K. Group theory considerations suggest that the transition leads the sample from the tetragonal to a monoclinic structure. Additionally, our study suggests that the mechanism for the structural phase transition is governed by the occupation of non-equivalent C1 local symmetry sites by the CH3 molecular groups. Analysis based on group theory suggests L-alanyl-L-alanine presents C2 symmetry at low temperatures.

  5. The temperature-dependent single-crystal Raman spectroscopy of a model dipeptide: L-Alanyl-L-alanine.

    PubMed

    Silva, J G; Arruda, L M; Pinheiro, G S; Lima, C L; Melo, F E A; Ayala, A P; Filho, J Mendes; Freire, P T C

    2015-09-05

    A single-crystal of peptide L-alanyl-L-alanine (C6H12N2O3) was studied by Raman spectroscopy at low-temperature, and a tentative assignment of the normal modes was given. Evidence of a second order structural phase transition was found through Raman spectroscopy between the temperatures of 80K and 60K. Group theory considerations suggest that the transition leads the sample from the tetragonal to a monoclinic structure. Additionally, our study suggests that the mechanism for the structural phase transition is governed by the occupation of non-equivalent C1 local symmetry sites by the CH3 molecular groups. Analysis based on group theory suggests L-alanyl-L-alanine presents C2 symmetry at low temperatures.

  6. On the fragmentation of biomolecules: Fragmentation of alanine dipeptide along the polypeptide chain

    SciTech Connect

    Solov'yov, I. A. Yakubovich, A. V.; Solov'yov, A. V.; Greiner, W.

    2006-09-15

    The interaction potential between amino acids in alanine dipeptide has been studied for the first time taking into account exact molecular geometry. Ab initio calculation has been performed in the framework of density functional theory taking into account all electrons in the system. The fragmentation of dipeptide along the polypeptide chain, as well as the interaction between alanines, has been considered. The energy of the system has been analyzed as a function of the distance between fragments for all possible dipeptide fragmentation channels. Analysis of the energy barriers makes it possible to estimate the characteristic fragmentation times and to determine the degree of applicability of classical electrodynamics for describing the system energy.

  7. D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity.

    PubMed

    Sato, Masaru; Kirimura, Kohtaro; Kino, Kuniki

    2005-06-01

    D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

  8. Free-energy profile along an isomerization pathway: Conformational isomerization in alanine dipeptide

    NASA Astrophysics Data System (ADS)

    Lee, In-Ho

    2013-02-01

    The free-energy profile for the conformational isomerization process in alanine dipeptide is presented in atomistic detail by using an action-derived molecular dynamics (ADMD) method and replica-exchange molecular dynamics (REMD) method. First, by employing ADMD, a dynamic isomerization pathway model of the alanine dipeptide with two available low-energy conformations, C7 ax and C7 eq , is determined. The pathway model is chosen to be the reaction coordinate, so the isomerization process is characterized by the ADMD step index, which is not an a-priori reaction coordinate as found in conventional studies of molecular conformational changes. Second, by employing the REMD method, the free-energy profile is calculated as a function of temperature. This couple of procedures is a quite natural protocol for conformational isomerization process simulations, irrespective of the arbitrary selection of the reaction coordinate. The alliance between the two simulation methods, ADMD and REMD, is demonstrated to have a great synergy effect on understanding the conformational changes in molecules.

  9. Determinants of Alanine Dipeptide Conformational Equilibria on Graphene and Hydroxylated Derivatives.

    PubMed

    Poblete, Horacio; Miranda-Carvajal, Ingrid; Comer, Jeffrey

    2017-03-24

    Understanding the interaction of carbon nanomaterials with proteins is essential for determining the potential effects of these materials on health and in the design of biotechnology based on them. Here we leverage explicit-solvent molecular simulation and multidimensional free-energy calculations to investigate how adsorption to carbon nanomaterial surfaces affects the conformational equilibrium of alanine dipeptide, a widely used model of protein backbone structure. We find that the two most favorable structures of alanine dipeptide on graphene (or large carbon nanotubes) correspond to the two amide linkages lying in the same plane, flat against the surface, rather than the nonplanar α-helix-like and β-sheet-like conformations that predominate in aqueous solution. On graphenic surfaces, the latter conformations are metastable and most often correspond to amide-π stacking of the N-terminal amide. The calculations highlight the key role of amide-π interactions in determining the conformational equilibrium. Lesser but significant contributions from hydrogen bonding to the high density interfacial water layer or to the hydroxy groups of hydroxylated graphene also define the most favorable conformations. This work should yield insight on the influence of carbon nanotubes, graphene, and their functionalized derivatives on protein structure.

  10. Conformational Changes of the Alanine Dipeptide in Water-Ethanol Binary Mixtures.

    PubMed

    Almeida, Glauco G; Cordeiro, João M M; Martín, M Elena; Aguilar, Manuel A

    2016-04-12

    Experimental work developed in the last years has evidenced the capacity of alcohols and polyalcohols to modify the energy landscape of peptides and proteins. However, the mechanism underlying this effect is not clear. Taking as a model system the alanine dipeptide (AD) we perform a QM/MM study in water, ethanol, and a 40-60% in volume water-ethanol mixture. The AD molecule was described at the MP2/aug-cc-pVDZ level. In polar solution, only αR and PPII conformers contribute in an appreciable way to the conformational equilibrium. The final in solution αR-PPII free energy difference is determined from the interplay between the internal energy of the dipeptide and the solute-solvent interaction free energy. Internal energy favors the formation of PPII, whereas, on the contrary, solute-solvent interaction is favorable to αR, so any factor that decreases the solute-solvent interaction free energy will increase the PPII population. The addition of ethanol increases the stability of the PPII conformer. Our results point to the presence of preferential solvation in this system, the composition of the first solvation shell in the binary mixture being dominated by water molecules. Remarkably, this fact does not affect the differential conformational stability that is controlled by long-range interactions. From the analysis of solvent density maps it is concluded that, in the water-ethanol mixture, ethanol molecules are more likely found around the alanine side chain and the carbonyl group, but while in PPII ethanol molecules interact mainly with the carbonyl group of the N-terminal end, in C5 the interaction is with the carbonyl group of the C-terminal end. In αR, ethanol interacts with both carbonyl groups.

  11. The potential of mean force surface for the alanine dipeptide in aqueous solution: a theoretical approach

    NASA Astrophysics Data System (ADS)

    Montgomery Pettitt, B.; Karplus, Martin

    1985-11-01

    Results of an application of integral equation theory to the determination of the intramolecular potential of mean force for the alanine dipeptide. N-methyl alanine acetamide, in aqueous solution are presented. The calculations are based on Ornstein—Zernike-like equations for polar systems with an intramolecular superposition approximation. The solvated free energy surface for the dipeptide as a function of the dihedral angles φ and ψ (Ramachandran plot) is determined and compared with the vaccum surface calculations. Conformations that are essentially forbidden in vaccum are found to be significant in aqueous solution. The solvent contributions to the free energy surface are decomposed into enthalpic and entropic terms. Possible applications and extensions of the method are outlined.

  12. A molecular dynamics study of the conformation of the alanine dipeptide in aqueous solution using a quantum mechanical potential

    NASA Astrophysics Data System (ADS)

    Buesnel, Robert; Hillier, Ian H.; Masters, Andrew J.

    Molecular dynamics simulations of the aqueous solution of alanine dipeptide have been carried out for seven configurations characteristic of important regions of the Ramachandran plot. A hybrid quantum mechanical-molecular mechanical potential was used that describes the solute using the AM1 Hamiltonian and the solvent using the TIP3P model. The importance of differential solute polarization and the preferential stabilization of the extended structures alphaL, alphaR and beta have been identified. The results are compared with experiment and with the predictions of the ab initio polarizable continuum model of solvation.

  13. Solvent induced conformational fluctuation of alanine dipeptide studied by using vibrational probes

    NASA Astrophysics Data System (ADS)

    Cai, Kaicong; Du, Fenfen; Liu, Jia; Su, Tingting

    2015-02-01

    The solvation effect on the three dimensional structure and the vibrational feature of alanine dipeptide (ALAD) was evaluated by applying the implicit solvents from polarizable continuum solvent model (PCM) through ab initio calculations, by using molecular dynamic (MD) simulations with explicit solvents, and by combining these two approaches. The implicit solvent induced potential energy fluctuations of ALAD in CHCl3, DMSO and H2O are revealed by means of ab initio calculations, and a global view of conformational and solvation environmental dependence of amide I frequencies is achieved. The results from MD simulations with explicit solvents show that ALAD trends to form PPII, αL, αR, and C5 in water, PPII and C5 in DMSO, and C5 in CHCl3, ordered by population, and the demonstration of the solvated structure, the solute-solvent interaction and hydrogen bonding is therefore enhanced. Representative ALAD-solvent clusters were sampled from MD trajectories and undergone ab initio calculations. The explicit solvents reveal the hydrogen bonding between ALAD and solvents, and the correlation between amide I frequencies and the Cdbnd O bond length is built. The implicit solvents applied to the ALAD-solvent clusters further compensate the solvation effect from the bulk, and thus enlarge the degree of structural distortion and the amide I frequency red shift. The combination of explicit solvent in the first hydration shell and implicit solvent in the bulk is helpful for our understanding about the conformational fluctuation of solvated polypeptides through vibrational probes.

  14. Solvent induced conformational fluctuation of alanine dipeptide studied by using vibrational probes.

    PubMed

    Cai, Kaicong; Du, Fenfen; Liu, Jia; Su, Tingting

    2015-02-25

    The solvation effect on the three dimensional structure and the vibrational feature of alanine dipeptide (ALAD) was evaluated by applying the implicit solvents from polarizable continuum solvent model (PCM) through ab initio calculations, by using molecular dynamic (MD) simulations with explicit solvents, and by combining these two approaches. The implicit solvent induced potential energy fluctuations of ALAD in CHCl3, DMSO and H2O are revealed by means of ab initio calculations, and a global view of conformational and solvation environmental dependence of amide I frequencies is achieved. The results from MD simulations with explicit solvents show that ALAD trends to form PPII, αL, αR, and C5 in water, PPII and C5 in DMSO, and C5 in CHCl3, ordered by population, and the demonstration of the solvated structure, the solute-solvent interaction and hydrogen bonding is therefore enhanced. Representative ALAD-solvent clusters were sampled from MD trajectories and undergone ab initio calculations. The explicit solvents reveal the hydrogen bonding between ALAD and solvents, and the correlation between amide I frequencies and the CO bond length is built. The implicit solvents applied to the ALAD-solvent clusters further compensate the solvation effect from the bulk, and thus enlarge the degree of structural distortion and the amide I frequency red shift. The combination of explicit solvent in the first hydration shell and implicit solvent in the bulk is helpful for our understanding about the conformational fluctuation of solvated polypeptides through vibrational probes.

  15. Solvation and hydrogen bonding in alanine- and glycine-containing dipeptides probed using solution- and solid-state NMR spectroscopy.

    PubMed

    Bhate, Manasi P; Woodard, Jaie C; Mehta, Manish A

    2009-07-15

    The NMR chemical shift is a sensitive reporter of peptide secondary structure and its solvation environment, and it is potentially rich with information about both backbone dihedral angles and hydrogen bonding. We report results from solution- and solid-state (13)C and (15)N NMR studies of four zwitterionic model dipeptides, L-alanyl-L-alanine, L-alanyl-glycine, glycyl-L-alanine, and glycyl-glycine, in which we attempt to isolate structural and environmental contributions to the chemical shift. We have mapped hydrogen-bonding patterns in the crystalline states of these dipeptides using the published crystal structures and correlated them with (13)C and (15)N magic angle spinning chemical shift data. To aid in the interpretation of the solvated chemical shifts, we performed ab initio quantum chemical calculations to determine the low-energy conformers and their chemical shifts. Assuming low energy barriers to interconversion between thermally accessible conformers, we compare the Boltzmann-averaged chemical shifts with the experimentally determined solvated-state shifts. The results allow us to correlate the observed differences in chemical shifts between the crystalline and solvated states to changes in conformation and hydrogen bonding that occur upon solvation.

  16. Conformational Study of the Alanine Dipeptide at the MP2 and DFT Levels

    SciTech Connect

    Vargas, Rubicelia; Garza, Jorge; Hay, Benjamin P.; Dixon, David A.

    2002-04-04

    Conformations of the important model system, the alanine dipeptide, have been calculated by using high level, ab initio electronic structure theory. A Ramachandran plot, with the angle f in the range [-180o,90o] and the angle y in the range [-60o,180o], was generated by using density functional theory with the generalized-gradient BLYP functional and a polarized triple-z basis set (TZVP+). Six conformers, C7eq, C5, C7ax, b2, aL, and a', have been identified in this region of the Ramachandran plot. A second derivative (frequency) analysis showed that all conformers are stable at this level of theory. These structures were used as starting points for geometry optimizations at the MP2/aug-cc-pVDZ level. Single point energies were calculated at the MP2/aug-cc-pVTZ and MP2/aug-cc-pVQZ levels at the final MP2/aug-cc-pVDZ structures and together with the MP2/aug-cc-pVDZ results were used in extrapolations to the complete basis set limit. The N-HoooO, N-HoooN, and C-HoooO hydrogen bond interactions that are key to the energetics are discussed. In general, the results obtained at the BLYP/TZVP+, MP2/aug-cc-pVDZ, MP2/aug-cc-pVTZ//aug-cc-pVDZ, and MP2/aug-cc-pVQZ//aug-cc-pVDZ levels are in reasonable agreement with each other, except for the b2 conformation where there are significant differences in the structures. Although the same stability order is obtained at all levels of theory that were used, there are significant differences in the magnitude of the relative conformational energies.

  17. Water-mediated conformations of the alanine dipeptide as revealed by distributed umbrella sampling simulations, quantum mechanics based calculations, and experimental data.

    PubMed

    Cruz, Víctor; Ramos, Javier; Martínez-Salazar, Javier

    2011-04-28

    An exhaustive umbrella sampling simulation of the alanine dipeptide in solution is reported. The presence of stable Y conformations in solution is assessed by both quantum calculations and experimental data from X-ray and NMR protein-solved structures available in the protein coil library. The agreement between experimental and simulation Ramachandran plots of the dipeptide in solution is excellent. A suitable explanation of the stabilization of the Y conformation mediated by water for the alanine dipeptide is discussed on the basis of Car-Parrinello MD calculations of the dipeptide in water.

  18. Ramachandran Plot for Alanine Dipeptide as Determined from Raman Optical Activity.

    PubMed

    Parchaňský, Václav; Kapitán, Josef; Kaminský, Jakub; Šebestík, Jaroslav; Bouř, Petr

    2013-08-15

    Accessible values of the φ and ψ torsional angles determining peptide main chain conformation are traditionally displayed in the form of Ramachandran plots. The number of experimental methods making it possible to determine such conformational distribution is limited. In the present study, Raman optical activity (ROA) spectra of Ac-Ala-NHMe were measured and fit by theoretical curves. This revealed the most favored conformers and a large part of the potential energy surface (PES) of this model dipeptide. Such experimental PES compares well to quantum chemical computations, whereas molecular dynamics (MD) modeling reproduces it less faithfully. The surface shape is consistent with the temperature dependence of the spectra, as observed experimentally and predicted by MD. Despite errors associated with spectral modeling and the measurement, the results are likely to facilitate future applications of ROA spectroscopy.

  19. Dipeptide Nanotubes Containing Unnatural Fluorine-Substituted β(2,3)-Diarylamino Acid and L-Alanine as Candidates for Biomedical Applications.

    PubMed

    Bonetti, Andrea; Pellegrino, Sara; Das, Priyadip; Yuran, Sivan; Bucci, Raffaella; Ferri, Nicola; Meneghetti, Fiorella; Castellano, Carlo; Reches, Meital; Gelmi, Maria Luisa

    2015-09-18

    The synthesis and the structural characterization of dipeptides composed of unnatural fluorine-substituted β(2,3)-diarylamino acid and L-alanine are reported. Depending on the stereochemistry of the β amino acid, these dipeptides are able to self-assemble into proteolytic stable nanotubes. These architectures were able to enter the cell and locate in the cytoplasmic/perinuclear region and represent interesting candidates for biomedical applications.

  20. ON THE FORMATION OF DIPEPTIDES IN INTERSTELLAR MODEL ICES

    SciTech Connect

    Kaiser, R. I.; Kim, Y. S.; Stockton, A. M.; Jensen, E. C.; Mathies, R. A.

    2013-03-10

    The hypothesis of an exogenous origin and delivery of biologically important molecules to early Earth presents an alternative route to their terrestrial in situ formation. Dipeptides like Gly-Gly detected in the Murchison meteorite are considered as key molecules in prebiotic chemistry because biofunctional dipeptides present the vital link in the evolutionary transition from prebiotic amino acids to early proteins. However, the processes that could lead to the exogenous abiotic synthesis of dipeptides are unknown. Here, we report the identification of two proteinogenic dipeptides-Gly-Gly and Leu-Ala-formed via electron-irradiation of interstellar model ices followed by annealing the irradiated samples to 300 K. Our results indicate that the radiation-induced, non-enzymatic formation of proteinogenic dipeptides in interstellar ice analogs is facile. Once synthesized and incorporated into the ''building material'' of solar systems, biomolecules at least as complex as dipeptides could have been delivered to habitable planets such as early Earth by meteorites and comets, thus seeding the beginning of life as we know it.

  1. The amide III vibrational circular dichroism band as a probe to detect conformational preferences of alanine dipeptide in water.

    PubMed

    Mirtič, Andreja; Merzel, Franci; Grdadolnik, Jože

    2014-07-01

    The conformational preferences of blocked alanine dipeptide (ADP), Ac-Ala-NHMe, in aqueous solution were studied using vibrational circular dichroism (VCD) together with density functional theory (DFT) calculations. DFT calculations of three most representative conformations of ADP surrounded by six explicit water molecules immersed in a dielectric continuum have proven high sensitivity of amide III VCD band shape that is characteristic for each conformation of the peptide backbone. The polyproline II (PII ) and αR conformation of ADP are associated with a positive VCD band while β conformation has a negative VCD band in amide III region. Knowing this spectral characteristic of each conformation allows us to assign the experimental amide III VCD spectrum of ADP. Moreover, the amide III region of the VCD spectrum was used to determine the relative populations of conformations of ADP in water. Based on the interpretation of the amide III region of VCD spectrum we have shown that dominant conformation of ADP in water is PII which is stabilized by hydrogen bonded water molecules between CO and NH groups on the peptide backbone.

  2. Instantaneous normal mode analysis of the vibrational relaxation of the amide I mode of alanine dipeptide in water.

    PubMed

    Farag, Marwa H; Zúñiga, José; Requena, Alberto; Bastida, Adolfo

    2013-05-28

    Nonequilibrium Molecular Dynamics (MD) simulations coupled to instantaneous normal modes (INMs) analysis are used to study the vibrational relaxation of the acetyl and amino-end amide I modes of the alanine dipeptide (AlaD) molecule dissolved in water (D2O). The INMs are assigned in terms of the equilibrium normal modes using the Effective Atomic Min-Cost algorithm as adapted to make use of the outputs of standard MD packages, a method which is well suited for the description of flexible molecules. The relaxation energy curves of both amide I modes show multiexponential decays, in good agreement with the experimental findings. It is found that ~85%-90% of the energy relaxes through intramolecular vibrational redistribution. The main relaxation pathways are also identified. The rate at which energy is transferred into the solvent is similar for the acetyl-end and amino-end amide I modes. The conformational changes occurring during relaxation are investigated, showing that the populations of the alpha and beta region conformers are altered by energy transfer in such a way that it takes 15 ps for the equilibrium conformational populations to be recovered after the initial excitation of the AlaD molecule.

  3. Metadynamics combined with auxiliary density functional and density functional tight-binding methods: alanine dipeptide as a case study.

    PubMed

    Cuny, Jerome; Korchagina, Kseniia; Menakbi, Chemseddine; Mineva, Tzonka

    2017-03-01

    Application of ab initio molecular dynamics to study free energy surfaces (FES) is still not commonly performed because of the extensive sampling required. Indeed, it generally necessitates computationally costly simulations of more than several hundreds of picoseconds. To achieve such studies, efficient density functional theory (DFT) formalisms, based on various levels of approximate computational schemes, have been developed, and provide a good alternative to commonly used DFT implementations. We report benchmark results on the conformational change FES of alanine dipeptide obtained with auxiliary density functional theory (ADFT) and second- and third-order density functional tight-binding (DFTB) methods coupled to metadynamics simulations. The influence of an explicit water solvent is also studied with DFTB, which was made possible by its lower computational cost compared to ADFT. Simulations lengths of 2.1 and 15 ns were achieved with ADFT and DFTB, respectively, in a reasonably short computational time. ADFT leads to a free energy difference (ΔF eq-ax) of ∼ -3 kcal mol(-1) between the two low energy conformers, C7eq and C7ax, which is lower by only 1.5 kcal mol(-1) than the ΔF eq-ax computed with DFTB. The two minima in ADFT FES are separated by an energy barrier of 9 kcal mol(-1), which is higher than the DFTB barriers by 2-4 kcal mol(-1). Despite these small quantitative differences, the DFTB method reveals FES shapes, confor-mation geometries and energies of the stationary points in good agreement with these found with ADFT. This validates the promising applicability of DFTB to FES of reactions occurring in larger-size systems placed in complex environments.

  4. Role of Intramolecular Aromatic π-π Interactions in the Self-Assembly of Di-l-Phenylalanine Dipeptide Driven by Intermolecular Interactions: Effect of Alanine Substitution.

    PubMed

    Reddy, Samala Murali Mohan; Shanmugam, Ganesh

    2016-09-19

    Although the role of intermolecular aromatic π-π interactions in the self-assembly of di-l-phenylalanine (l-Phe-l-Phe, FF), a peptide that is known for hierarchical structure, is well established, the influence of intramolecular π-π interactions on the morphology of the self-assembled structure of FF has not been studied. Herein, the role of intramolecular aromatic π-π interactions is investigated for FF and analogous alanine (Ala)-containing dipeptides, namely, l-Phe-l-Ala (FA) and l-Ala-l-Phe (AF). The results reveal that these dipeptides not only form self-assemblies, but also exhibit remarkable differences in structural morphology. The morphological differences between FF and the analogues indicate the importance of intramolecular π-π interactions, and the structural difference between FA and AF demonstrates the crucial role of the nature of intramolecular side-chain interactions (aromatic-aliphatic or aliphatic-aromatic), in addition to intermolecular interactions, in deciding the final morphology of the self-assembled structure. The current results emphasise that intramolecular aromatic π-π interaction may not be essential to induce self-assembly in smaller peptides, and π (aromatic)-alkyl or alkyl-π (aromatic) interactions may be sufficient. This work also illustrates the versatility of aromatic and a combination of aromatic and aliphatic residues in dipeptides in the formation of structurally diverse self-assembled structures.

  5. Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

    PubMed

    Raizel, Raquel; Leite, Jaqueline Santos Moreira; Hypólito, Thaís Menezes; Coqueiro, Audrey Yule; Newsholme, Philip; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation.

  6. Study on the conformational equilibrium of the alanine dipeptide in water solution by using the averaged solvent electrostatic potential from molecular dynamics methodology.

    PubMed

    García-Prieto, Francisco F; Fdez Galván, Ignacio; Aguilar, Manuel A; Martín, M Elena

    2011-11-21

    The ASEP/MD method has been employed for studying the solvent effect on the conformational equilibrium of the alanine dipeptide in water solution. MP2 and density functional theory (DFT) levels of theory were used and results were compared. While in gas phase cyclic structures showing intramolecular hydrogen bonds were found to be the most stable, the stability order is reversed in water solution. Intermolecular interaction with the solvent causes the predominance of extended structures as the stabilizing contacts dipeptide-water are favoured. Free-energy differences in solution were calculated and PPII, α(R), and C5 conformers were identified as the most stable at MP2 level. Experimental data from Raman and IR techniques show discrepancies about the relative abundance of α(R) y C5, our results support the Raman data. The DFT level of theory agrees with MP2 in the location and stability of PPII and α(R) forms but fails in the location of C5. MP2 results suggest the possibility of finding traces of C7eq conformer in water solution, in agreement with recent experiments.

  7. Dipeptide interactions with Zn(II)-cyclen artificial model for molecular recognition.

    PubMed

    Rostášová, I; Vilková, M; Vargová, Z; Walko, M; Almáši, M; Imrich, J; Hermann, P; Lukeš, I

    2015-04-01

    The Zn(II)-cyclen-dipeptide ternary systems (where cyclen is abbreviated as L and dipeptide is glycylglycine (HL(1)) or glycyl-(S)-alanine (HL(2))) were investigated by potentiometry applying both "out-of-cell" and direct titrations and by (1) H NMR spectroscopy. Especially, the (1)H NMR study was found to be very efficient to estimate speciation in the systems. The results obtained under full equilibria indicated two main species, [Zn(L)(HL(1,2))](2+) and [Zn(L)(L(1,2))](+), in both the systems. In the [Zn(L)(HL(1,2))](2+) complex, presence of carbonyl-carboxylate chelate was confirmed, and in the [Zn(L)(L(1,2))](+) species, the peptide coordination is re-organized to carbonyl-amine chelate or only terminal amino group is coordinated. Equilibrium constants describing [Zn(L)](2+)-dipeptide interaction are relatively low, log K = 3.4 for Gly-Gly and 4.1 for Gly-(S)-Ala, respectively. Nevertheless, the values are slightly higher than stability constants for interaction of Zn(II) with the dipeptides (i.e. [Zn(L(1,2))](+) species) where a chelate formation is expected. It indicates that interaction between Zn(II) ion in [Zn(L)](2+) and the dipeptides should be supported by some additional interactions. Potentiometry carried out under non-equilibrum condition showed different species where these additional stabilizing forces play more important role.

  8. A line integral reaction path approximation for large systems via nonlinear constrained optimization: application to alanine dipeptide and the beta hairpin of protein G.

    PubMed

    Khavrutskii, Ilja V; Byrd, Richard H; Brooks, Charles L

    2006-05-21

    A variation of the line integral method of Elber with self-avoiding walk has been implemented using a state of the art nonlinear constrained optimization procedure. The new implementation appears to be robust in finding approximate reaction paths for small and large systems. Exact transition states and intermediates for the resulting paths can easily be pinpointed with subsequent application of the conjugate peak refinement method [S. Fischer and M. Karplus, Chem. Phys. Lett. 194, 252 (1992)] and unconstrained minimization, respectively. Unlike previous implementations utilizing a penalty function approach, the present implementation generates an exact solution of the underlying problem. Most importantly, this formulation does not require an initial guess for the path, which makes it particularly useful for studying complex molecular rearrangements. The method has been applied to conformational rearrangements of the alanine dipeptide in the gas phase and in water, and folding of the beta hairpin of protein G in water. In the latter case a procedure was developed to systematically sample the potential energy surface underlying folding and reconstruct folding pathways within the nearest-neighbor hopping approximation.

  9. Polarizable Simulations with Second order Interaction Model (POSSIM) force field: Developing parameters for alanine peptides and protein backbone

    PubMed Central

    Ponomarev, Sergei Y.; Kaminski, George A.

    2011-01-01

    A previously introduced POSSIM (POlarizable Simulations with Second order Interaction Model) force field has been extended to include parameters for alanine peptides and protein backbones. New features were introduced into the fitting protocol, as compared to the previous generation of the polarizable force field for proteins. A reduced amount of quantum mechanical data was employed in fitting the electrostatic parameters. Transferability of the electrostatics between our recently developed NMA model and the protein backbone was confirmed. Binding energy and geometry for complexes of alanine dipeptide with a water molecule were estimated and found in a good agreement with high-level quantum mechanical results (for example, the intermolecular distances agreeing within ca. 0.06Å). Following the previously devised procedure, we calculated average errors in alanine di- and tetra-peptide conformational energies and backbone angles and found the agreement to be adequate (for example, the alanine tetrapeptide extended-globular conformational energy gap was calculated to be 3.09 kcal/mol quantim mechanically and 3.14 kcal/mol with the POSSIM force field). However, we have now also included simulation of a simple alpha-helix in both gas-phase and water as the ultimate test of the backbone conformational behavior. The resulting alanine and protein backbone force field is currently being employed in further development of the POSSIM fast polarizable force field for proteins. PMID:21743799

  10. Free Energy Landscapes of Alanine Oligopeptides in Rigid-Body and Hybrid Water Models.

    PubMed

    Nayar, Divya; Chakravarty, Charusita

    2015-08-27

    Replica exchange molecular dynamics is used to study the effect of different rigid-body (mTIP3P, TIP4P, SPC/E) and hybrid (H1.56, H3.00) water models on the conformational free energy landscape of the alanine oligopeptides (acAnme and acA5nme), in conjunction with the CHARMM22 force field. The free energy landscape is mapped out as a function of the Ramachandran angles. In addition, various secondary structure metrics, solvation shell properties, and the number of peptide-solvent hydrogen bonds are monitored. Alanine dipeptide is found to have similar free energy landscapes in different solvent models, an insensitivity which may be due to the absence of possibilities for forming i-(i + 4) or i-(i + 3) intrapeptide hydrogen bonds. The pentapeptide, acA5nme, where there are three intrapeptide backbone hydrogen bonds, shows a conformational free energy landscape with a much greater degree of sensitivity to the choice of solvent model, though the three rigid-body water models differ only quantitatively. The pentapeptide prefers nonhelical, non-native PPII and β-sheet populations as the solvent is changed from SPC/E to the less tetrahedral liquid (H1.56) to an LJ-like liquid (H3.00). The pentapeptide conformational order metrics indicate a preference for open, solvent-exposed, non-native structures in hybrid solvent models at all temperatures of study. The possible correlations between the properties of solvent models and secondary structure preferences of alanine oligopeptides are discussed, and the competition between intrapeptide, peptide-solvent, and solvent-solvent hydrogen bonding is shown to be crucial in the relative free energies of different conformers.

  11. Formation of pyrazines in Maillard model systems of lysine-containing dipeptides.

    PubMed

    Van Lancker, Fien; Adams, An; De Kimpe, Norbert

    2010-02-24

    Whereas most studies concerning the Maillard reaction have focused on free amino acids, little information is available on the impact of peptides and proteins on this important reaction in food chemistry. Therefore, the formation of flavor compounds from the model reactions of glucose, methylglyoxal, or glyoxal with eight dipeptides with lysine at the N-terminus was studied in comparison with the corresponding free amino acids by means of stir bar sorptive extraction (SBSE) followed by GC-MS analysis. The reaction mixtures of the dipeptides containing glucose, methylglyoxal, and glyoxal produced 27, 18, and 2 different pyrazines, respectively. Generally, the pyrazines were produced more in the case of dipeptides as compared to free amino acids. For reactions with glucose and methylglyoxal, this difference was mainly caused by the large amounts of 2,5(6)-dimethylpyrazine and trimethylpyrazine produced from the reactions with dipeptides. For reactions with glyoxal, the difference in pyrazine production was rather small and mostly unsubstituted pyrazine was formed. A reaction mechanism for pyrazine formation from dipeptides was proposed and evaluated. This study clearly illustrates the capability of peptides to produce flavor compounds that can differ from those obtained from the corresponding reactions with free amino acids.

  12. The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties.

    PubMed

    Chang, Yu-Jen; Jeng, U-Ser; Chiang, Ya-Ling; Hwang, Ing-Shouh; Chen, Yun-Ru

    2016-03-04

    Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)15 DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)15 with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-β sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)15 DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.

  13. Assessment of CCSD(T), MP2, DFT-D, CBS-QB3, and G4(MP2) methods for conformational study of alanine and proline dipeptides

    NASA Astrophysics Data System (ADS)

    Kang, Young Kee; Park, Hae Sook

    2014-04-01

    The CCSD(T), MP2, dispersion-corrected DFT, CBS-QB3, and G4(MP2) levels of theory with various basis sets are assessed for their ability to describe the conformational preferences of the Ala and Pro dipeptides. The ωB97X-D/6-311++G(d,p) level of theory provided the rotational constants of the most stable conformer of the Ala dipeptide consistent with the values from microwave experiments. The double-hybrid DSD-PBEP86-D3BJ DFT method provided the best performance for relative energies of both dipeptides consistent with CCSD(T)/CBS limit values. The DSD-PBEP86-D3BJ/cc-pVTZ//M06-2X/6-31+G(d), B2PLYPD-D3BJ/cc-pVTZ//M06-2X/6-31+G(d), and M06-2X/cc-pVTZ//M06-2X/6-31+G(d) levels of theory may be an alternative to the CCSD(T)/CBS limit//ωB97X-D/6-311++G(d,p) level of theory with marginal deviations for conformational study of peptides.

  14. Solvation effects on alanine dipeptide: A MP2/cc-pVTZ//MP2/6-31G** study of (Phi, Psi) energy maps and conformers in the gas phase, ether, and water.

    PubMed

    Wang, Zhi-Xiang; Duan, Yong

    2004-11-15

    The effects of solvation on the conformations and energies of alanine dipeptide (AD) have been studied by ab initio calculations up to MP2/cc-pVTZ//MP2/6-31G**, utilizing the polarizable continuum model (PCM) to mimic solvation effects. The energy surfaces in the gas phase, ether, and water bear similar topological features carved by the steric hindrance, but the details differ significantly due to the solvent effects. The gas-phase energy map is qualitatively consistent with the Ramachandran plot showing seven energy minima. With respect to the gas-phase map, the significant changes of the aqueous map include (1) the expanded low-energy regions, (2) the emergence of an energy barrier between C5-beta and alpha(R)-beta(2) regions, (3) a clearly pronounced alpha(R) minimum, a new beta-conformer, and the disappearance of the gas-phase global minimum, and (4) the shift of the dominant region in LEII from the gas-phase C7(ax) region to the alpha(L) region. These changes bring the map in water to be much closer to the Ramachandran plot than the gas-phase map. The solvent effects on the geometries include the elongation of the exposed N-H and C=O bonds, the shortening of the buried HN--CO peptide bonds, and the enhanced planarity of the peptide bonds. The energy surface in ether has features similar to those both in the gas phase and in water. The free energy order computed in the gas phase and in ether is in good agreement with experimental studies that concluded that C5 and C7(eq) are the dominant species in both the gas phase and nonpolar solvents. The free energy order in water is consistent with the experimental observation that the dominant C7(eq) in the nonpolar solvent was largely replaced by P(II)-like (i.e., beta) and alpha(R) in the strong polar solvents. Based on calculations on AD + 4H(2)O and other AD-water clusters, we suggest that explicit water-AD interactions may distort C5 and beta (or alpha(R) and beta) to an intermediate conformation. Our analysis

  15. Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

    PubMed Central

    Lukk, Tiit; Sakai, Ayano; Kalyanaraman, Chakrapani; Brown, Shoshana D.; Imker, Heidi J.; Song, Ling; Fedorov, Alexander A.; Fedorov, Elena V.; Toro, Rafael; Hillerich, Brandan; Seidel, Ronald; Patskovsky, Yury; Vetting, Matthew W.; Nair, Satish K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.; Jacobson, Matthew P.

    2012-01-01

    The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion. PMID:22392983

  16. Solvation model induced structural changes in peptides. A quantum chemical study on Ramachandran surfaces and conformers of alanine diamide using the polarizable continuum model.

    PubMed

    Hudáky, Ilona; Hudáky, Péter; Perczel, András

    2004-09-01

    Potential energy surfaces of the model peptide HCO-L-Ala-NH2 were calculated using polarizable continuum model (PCM) for the description of aqueous solution at RHF/3-21G, RHF/6-31+G(d), and B3LYP/6-31+G(d) levels of theory. Energy minima were optimized at all three levels as well as at B3LYP/PCM/6-311++G(d,p) level of theory. Results were correlated to experimental data of protein structures retrieved from PDB SELECT. It is concluded that alanine residues of proteins are modeled better by PCM results than by gas-phase calculations on the alanine diamide model (frequently called alanine dipeptide model). The currently available version of the PCM model implemented in Gaussian 03 provides a reasonable alternative to anticipate solvation effects without the computational costs of introducing explicit solvent molecules into the model system. Frequencies calculated at RHF/PCM/6-31+G(d) and B3LYP/PCM/6-31+G(d) levels of theory show high correlation; thus, RHF results have their own merit.

  17. Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury

    PubMed Central

    2014-01-01

    Introduction Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined. Methods Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily. Results Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Plasma glutamate remained unchanged and cerebral glutamate was decreased without any signs of cerebral impairment. Urea and ammonia were significantly increased within normal limits without signs of organ dysfunction (urea: from 2.7 ± 1.6 to 5.5 ± 1.5 mmol/ l; ammonia: from 12 ± 6.3 to 26 ± 8.3 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Conclusions High dose L-alanine-L-glutamine infusion (0

  18. A silaproline-containing dipeptide.

    PubMed

    Vivet, B; Cavelier, F; Martinez, J; Didierjean, C; Marraud, M; Aubry, A

    2000-12-01

    The silaproline-containing dipeptide N-(3, 3-dimethyl-1-pivaloyl-1-aza-3-sila-5-cyclopentylcarbonyl)-L- alanine isopropylamide, C(17)H(33)N(3)O(3)Si, has two independent molecules in the asymmetric unit and each adopts a beta-II folded conformation, where the amide on the terminal C interacts intramolecularly with the pivaloyl O atom. The five-membered silaproline ring is C(beta)-puckered, an infrequent conformation for the homologous proline ring.

  19. A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine.

    PubMed

    Fonseca, Andrea S C; Gonçalves, M Sameiro T; Costa, Susana P G

    2012-12-01

    A novel fluorescent amino acid, L-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.

  20. Function of the D-alanine:D-alanine ligase lid loop: a molecular modeling and bioactivity study.

    PubMed

    Hrast, Martina; Vehar, Blaž; Turk, Samo; Konc, Janez; Gobec, Stanislav; Janežič, Dušanka

    2012-08-09

    D-Alanine:D-alanine ligase (Ddl) is an essential ATP-dependent bacterial enzyme involved in peptidoglycan biosynthesis. Discovery of Ddl inhibitors not competitive with ATP has proven to be difficult because the Ddl bimolecular d-alanine binding pocket is very restricted, as is accessibility to the active site for larger molecules in the catalytically active closed conformation of Ddl. A molecular dynamics study of the opening and closing of the Ddl lid loop informs future structure-based design efforts that allow for the flexibility of Ddl. A virtual screen on generated enzyme conformations yielded some hit inhibitors whose bioactivity was determined.

  1. Formation of Peptide Bound Pyrraline in the Maillard Model Systems with Different Lys-Containing Dipeptides and Tripeptides.

    PubMed

    Liang, Zhili; Li, Lin; Qi, Haiping; Wan, Liting; Cai, Panfu; Xu, Zhenbo; Li, Bing

    2016-04-07

    Peptide-bound advanced glycation end-products (peptide-bound AGEs) can be formed when peptides are heated with reducing saccharides. Pyrraline is the one of most commonly studied AGEs in foods, but the relative importance of the precursor peptide structure is uncertain. In the present study, model systems were prepared by heating peptides with glucose from 60 °C to 220 °C for up to 65 min, and the amounts of peptide-bound pyrraline formed were monitored to evaluate the effect of the neighboring amino acids on the peptide-bound pyrraline formation. The physico-chemical properties were introduced to explore the quantitative structure-reactivity relationships between physicochemical properties and peptide bound formation. 3-DG content in dipeptide-glucose model system was higher than that in the corresponding tripeptide-glucose model systems. Dipeptides produced higher amounts of peptide-bound pyrraline than the corresponding tripeptides. The peptide-bound pyrraline and 3-DG production were influenced by the physico-chemical properties of the side chain of amino acids adjacent to Lys in the following order: Lys-Leu/glucose > Lys-Ile/glucose > Lys-Val/ glucose > Lys-Thr/glucose > Lys-Ser/glucose > Lys-Ala/ glucose > Lys-Gly/glucose; Lys-Leu-Gly/glucose > Lys-Ile-Gly/glucose > Lys-Val-Gly/glucose > Lys-Thr-Gly/glucose > Lys-Ser-Gly/glucose > Lys-Ala-Gly/glucose > Lys-Gly-Gly/glucose. For the side chain of amino acids adjacent to Lys in dipeptides, residue volume, polarizability, molecular volume and localized electrical effect were positively related to the yield of peptide bound pyrraline, while hydrophobicity and pKb were negatively related to the yield of peptide bound pyrraline. In terms of side chain of amino acid adjacent to Lys in tripeptides, a similar result was observed, except hydrophobicity was positively related to the yield of peptide bound pyrraline.

  2. Effect of the chirality of residues and γ-turns on the electronic excitation spectra, excited-state reaction paths and conical intersections of capped phenylalanine-alanine dipeptides.

    PubMed

    Shemesh, Dorit; Domcke, Wolfgang

    2011-07-11

    The capped dipeptides Ac-L-Phe-Xxx-NH(2) , Xxx=L-Ala, D-Ala, Aib, where Aib (aminoisobutyric acid) is a non-chiral amino acid, have been investigated by means of UV/IR double-resonance spectroscopy in supersonic jets and density functional theory calculations by Gloaguen et al. [Phys. Chem. Chem. Phys. 2007, 9, 4491]. The UV and IR spectra of five different species were observed and their structures assigned by comparison with calculated vibrational frequencies in the NH-stretching region. The peptides with two chiral residues can form homochiral or heterochiral species. In addition, γ-turns exist as two helical forms (γ(D), γ(L)) of opposite handedness. Herein, we explore the excited-state potential-energy surfaces of these dipeptides with ab initio calculations. Vertical and adiabatic excitation energies, excited-state reaction paths and conical intersections are characterized with the ADC(2) propagator method. It is shown that electron/proton transfer along the hydrogen bond of the γ-turn gives rise to efficient radiationless deactivation of the (1)ππ* state of the chromophore via several conical intersections. While the homo/hetero chirality of the residues appears to have a negligible effect on the photophysical dynamics, we found evidence that the γ(L) conformers may have shorter excited-state lifetimes (and thus higher photostability) than the γ(D) conformers.

  3. Amide I vibrational circular dichroism of dipeptide: Conformation dependence and fragment analysis

    NASA Astrophysics Data System (ADS)

    Choi, Jun-Ho; Cho, Minhaeng

    2004-03-01

    The amide I vibrational circular dichroic response of alanine dipeptide analog (ADA) was theoretically investigated and the density functional theory calculation and fragment analysis results are presented. A variety of vibrational spectroscopic properties, local and normal mode frequencies, coupling constant, dipole, and rotational strengths, are calculated by varying two dihedral angles determining the three-dimensional ADA conformation. Considering two monopeptide fragments separately, we show that the amide I vibrational circular dichroism of the ADA can be quantitatively predicted. For several representative conformations of the model ADA, vibrational circular dichroism spectra are calculated by using both the density functional theory calculation and fragment analysis methods.

  4. Glutamyl cysteine dipeptide suppresses ferritin expression and alleviates liver injury in iron-overload rat model.

    PubMed

    Salama, Samir A; Al-Harbi, Mohammad S; Abdel-Bakky, Mohamed S; Omar, Hany A

    2015-08-01

    Despite its biological importance, iron is a pro-oxidant element and its accumulation results in tissue injury. Iron overload diseases such as thalassemia and hereditary hemochromatosis are commonly associated with liver tissue injury. Glutamyl cysteine (GC) is a dipeptide with antioxidant properties owing to its cysteine residue. The aim of the current work was to investigate the hepatoprotective effect of GC against iron overload-induced liver injury. Rats were distributed into five groups; normal control, GC control, iron-treated (150 mg/kg ip injection) and both iron and GC-treated (total iron: 150 mg/kg ip and GC: 50 mg or 100 mg/kg/day ip for 30 days). Our results showed that treatment with GC at the two-dose levels attenuated iron-induced liver tissue injury as evidenced by significant reduction in serum activity of liver enzymes ALT and AST, amelioration of iron-induced histopathological alteration, suppression of iron-induced oxidative stress as demonstrated by significant reduction of malondialdehyde and protein carbonyl content beside elevation of total antioxidant capacity, reduced glutathione and the antioxidant enzymes GPx and SOD in liver tissue. In addition, GC significantly reduced levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β and activity of the apoptotic marker caspase-3 in liver tissues. To our surprise, GC reduced liver iron content and ferritin expression, denoting the possible iron chelation competency. Collectively our results highlight evidence for the hepatoprotective effect of GC against iron overload-induced liver injury that is potentially mediated through suppression of oxidative tissue injury, attenuation of inflammatory response, amelioration of hepatocellular apoptosis and possibly through iron chelation.

  5. Morphological Versatility in the Self-Assembly of Val-Ala and Ala-Val Dipeptides.

    PubMed

    Erdogan, Hakan; Babur, Esra; Yilmaz, Mehmet; Candas, Elif; Gordesel, Merve; Dede, Yavuz; Oren, Ersin Emre; Demirel, Gokcen Birlik; Ozturk, Mustafa Kemal; Yavuz, Mustafa Selman; Demirel, Gokhan

    2015-07-07

    Since the discovery of dipeptide self-assembly, diphenylalanine (Phe-Phe)-based dipeptides have been widely investigated in a variety of fields. Although various supramolecular Phe-Phe-based structures including tubes, vesicles, fibrils, sheets, necklaces, flakes, ribbons, and wires have been demonstrated by manipulating the external physical or chemical conditions applied, studies of the morphological diversity of dipeptides other than Phe-Phe are still required to understand both how these small molecules respond to external conditions such as the type of solvent and how the peptide sequence affects self-assembly and the corresponding molecular structures. In this work, we investigated the self-assembly of valine-alanine (Val-Ala) and alanine-valine (Ala-Val) dipeptides by varying the solvent medium. It was observed that Val-Ala dipeptide molecules may generate unique self-assembly-based morphologies in response to the solvent medium used. Interestingly, when Ala-Val dipeptides were utilized as a peptide source instead of Val-Ala, we observed distinct differences in the final dipeptide structures. We believe that such manipulation may not only provide us with a better understanding of the fundamentals of the dipeptide self-assembly process but also may enable us to generate novel peptide-based materials for various applications.

  6. Many-body Green's function GW and Bethe-Salpeter study of the optical excitations in a paradigmatic model dipeptide.

    PubMed

    Faber, C; Boulanger, P; Duchemin, I; Attaccalite, C; Blase, X

    2013-11-21

    We study within the many-body Green's function GW and Bethe-Salpeter formalisms the excitation energies of a paradigmatic model dipeptide, focusing on the four lowest-lying local and charge-transfer excitations. Our GW calculations are performed at the self-consistent level, updating first the quasiparticle energies, and further the single-particle wavefunctions within the static Coulomb-hole plus screened-exchange approximation to the GW self-energy operator. Important level crossings, as compared to the starting Kohn-Sham LDA spectrum, are identified. Our final Bethe-Salpeter singlet excitation energies are found to agree, within 0.07 eV, with CASPT2 reference data, except for one charge-transfer state where the discrepancy can be as large as 0.5 eV. Our results agree best with LC-BLYP and CAM-B3LYP calculations with enhanced long-range exchange, with a 0.1 eV mean absolute error. This has been achieved employing a parameter-free formalism applicable to metallic or insulating extended or finite systems.

  7. Crystal structure of the Apo form of D-Alanine:D-Alanine ligase (DDl) from Streptococcus mutans.

    PubMed

    Lu, Yongzhi; Xu, Hongyan; Zhao, Xiaojun

    2010-08-01

    D-Alanine:D-Alanine ligase (DDl) catalyzes the formation of D-Alanine:D-Alanine dipeptide and is an essential enzyme in bacterial cell wall biosynthesis.. This enzyme does not have a human ortholog, making it an attractive target for developing new antibiotic drugs. We determined the crystal structure at 2.23 A resolution of DDl from Streptococcus mutans (SmDDl), the principal aetiological agent of human dental caries. This structure reveals that SmDDl is a dimer and has a disordered omega-loop region.

  8. A simple model for polyproline II structure in unfolded states of alanine-based peptides

    PubMed Central

    Pappu, Rohit V.; Rose, George D.

    2002-01-01

    The striking similarity between observed circular dichroism spectra of nonprolyl homopolymers and that of regular left-handed polyproline II (PII) helices prompted Tiffany and Krimm to propose in 1968 that unordered peptides and unfolded proteins are built of PII segments linked by sharp bends. A large body of experimental evidence, accumulated over the past three decades, provides compelling evidence in support of the original hypothesis of Tiffany and Krimm. Of particular interest are the recent experiments of Shi et al. who find significant PII structure in a short unfolded alanine-based peptide. What is the physical basis for PII helices in peptide and protein unfolded states? The widely accepted view is that favorable chain-solvent hydrogen bonds lead to a preference for dynamical fluctuations about noncooperative PII helices in water. Is this preference simply a consequence of hydrogen bonding or is it a manifestation of a more general trend for unfolded states which are appropriately viewed as chains in a good solvent? The prevalence of closely packed interiors in folded proteins suggests that under conditions that favor folding, water—which is a better solvent for itself than for any polypeptide chain—expels the chain from its midst, thereby maximizing chain packing. Implicit in this view is a complementary idea: under conditions that favor unfolding, chain-solvent interactions are preferred and in a so-called good solvent, chain packing density is minimized. In this work we show that minimization of chain packing density leads to preferred fluctuations for short polyalanyl chains around canonical, noncooperative PII-like conformations. Minimization of chain packing is modeled using a purely repulsive soft-core potential between polypeptide atoms. Details of chain-solvent interactions are ignored. Remarkably, the simple model captures the essential physics behind the preference of short unfolded alanine-based peptides for PII helices. Our results are

  9. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    PubMed

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  10. Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity

    PubMed Central

    Krans, Amy; Sawaya, Michael R.; Paulson, Henry L.; Todd, Peter K.; Barmada, Sami J.; Ivanova, Magdalena I.

    2016-01-01

    Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity. PMID:27776165

  11. Discovery of a Dipeptide Epimerase Enzymatic Function Guided by Homology Modeling and Virtual Screening

    SciTech Connect

    Kalyanaraman, C.; Imker, H; Fedorov, A; Fedorov, E; Glasner, M; Babbitt, P; Almo, S; Gerlt, J; Jacobson, M

    2008-01-01

    We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse enolase superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function. This prediction was experimentally confirmed, resulting in the assignment of epimerase activity for L-Ala-D/L-Phe, L-Ala-D/L-Tyr, and L-Ala-D/L-His, whereas the enzyme is annotated incorrectly in GenBank as muconate cycloisomerase. Subsequently, crystal structures of the enzyme were determined in complex with three substrates, showing close agreement with the computational models and revealing the structural basis for the observed substrate selectivity.

  12. [Effect of the novel nootropic and neuroprotective dipeptide noopept on the streptozotocin-induced model of sporadic Alzheimer disease in rats].

    PubMed

    Ostrovskaia, R U; Tsaplina, A P; Vakhitova, Iu V; Salimgareeva, M Kh; Iamidanov, R S

    2010-01-01

    Streptozotocin-intracerebroventricularly treated rats are proposed as an experimental model of sporadic Alzheimer disease (AD). Diabetogenic toxin streptozotocin (STZ) administered in both cerebral ventricles in a dose of 3 mg/kg decreases the expression of NGF and BDNF mainly in the hippocampus and increases the content of malonic dialdehyde (MDA)--a product of lipid peroxidation--in the brain tissues. These metabolic changes are accompanied by a pronounced cognitive deficiency, which is manifested by long-term memory deterioration in the passive avoidance test. These manifestations of pathology are not accompanied by hyperglycemia in the case of intraventricular STZ administration, in contrast to the systemic (in particular, intraperitoneal) route of introduction that causes a pronounced increase in the blood glucose level. These results are consistent with the existing notions that (i) STZ administered intraventricularly provokes a complex of changes imitating the sporadic AD and (ii) this disease can be considered as a manifestation of type-III diabetes. The new original cognition enhancing and neuroprotective dipeptide noopept decreases the aforementioned metabolic changes and the accompanying long-term deterioration of the memory. Previously, this systemically active dipeptide was shown to be capable of increasing expression of NGF and BDNF in the hippocampus, stimulating the antibody production to beta-amyloid, inhibiting the lipid peroxidation, activating the endogenous antioxidant systems, and decreasing the rate of glutamate release (cholinopositive effect). Taken together, these data indicate that noopept can be considered as a multipotent substance acting upon several important pathogenic chainsof the sporadic AD.

  13. Hydrolysis of protein and model dipeptide substrated by attached and nonattached marine Pseudomonas sp. strain NCIMB 2021

    SciTech Connect

    Griffith, P.C.; Fletcher, M. )

    1991-08-01

    Rates of substrate hydrolysis by nonattached bacteria and by bacteria attached to particles derived from marine diatom frustules were estimated by using two substrates, a dipeptide analog and a protein. Adsorption of the two substrates onto the particles was also evaluated. Methyl-coumarinyl-amide-leucine (MCA-leucine) was used to estimate hydrolysis of dipeptides by measuring an increase in fluorescence as MCA-leucine was hydrolyzed to leucine and the fluorochrome methylcoumarin. To examine hydrolysis of a larger molecule, was prepared a radiolabeled protein by {sup 14}C-methylation of bovine serum albumin. The rate of protein hydrolysis in samples of particle-attached or nonattached bacteria was estimated by precipitating all nonhydrolyzed protein with cold trichloroacetic acid and then determining the trichloroacetic acid-soluble radiolabeled material, which represented methyl-{sup 14}C-peptides and -amino acids. About 25% of the MCA-leucine adsorbed to the particles. MCA-leucine was hydrolyzed faster by nonattached than attached bacteria, which was probably related to its tendency to remain dissolved in the liquid phase. In contrast, almost 100% of the labeled protein adsorbed to the particles. Accordingly, protein was much less available to nonattached bacteria but was rapidly hydrolyzed by attached bacteria.

  14. Addition of amino acids and dipeptides to fullerene C{sub 60} giving rise to monoadducts

    SciTech Connect

    Romanova, V.S.; Tsyryapkin, V.A.; Vol`pin, M.E.

    1994-12-01

    The authors have developed a general method for the direct addition of amino acids and dipeptides of various structures to fullerene C{sub 60}. In all cases the addition involves the amino group. The reaction proceeds when the solutions of fullerene and an amino acid (or dipeptide) are mixed at 50-100 {degrees}C. The fullerene derivatives of the following amino acids and dipeptides have been obtained: glycine, p-aminobenzoic acid, {omega}-aminocaproic acid, L-proline, L-alanine, L-alanyl-Lalanine, D,L-alanyl-D,L-alanine, glycyl-L-valine. The adduct of methyl L-ananinate with C{sub 60} was also prepared.

  15. Inducible l-Alanine Exporter Encoded by the Novel Gene ygaW (alaE) in Escherichia coli ▿

    PubMed Central

    Hori, Hatsuhiro; Yoneyama, Hiroshi; Tobe, Ryuta; Ando, Tasuke; Isogai, Emiko; Katsumata, Ryoichi

    2011-01-01

    We previously isolated a mutant hypersensitive to l-alanyl-l-alanine from a non-l-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular l-alanine and a reduction in the l-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the l-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of l-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular l-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular l-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for l-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export. PMID:21531828

  16. Vibrational Spectroscopy and Gas-Phase Thermochemistry of the Model Dipeptide N-Acetyl Glycine Methyl Amide

    NASA Astrophysics Data System (ADS)

    Leavitt, Christopher; Raston, Paul; Moody, Grant; Shirley, Caitlyne; Douberly, Gary

    2014-06-01

    The structure-function relationship in proteins is widely recognized, motivating numerous investigations of isolated neutral and ionic polypeptides that generally employ conformation specific, multidimensional UV and IR spectroscopies. This data taken in conjunction with computed harmonic frequencies has provided a snapshot of the underlying molecular physics at play in many polypeptides, but few experiments have been able to probe the energetics of these systems. In this study, we use vibrational spectroscopy to measure the gas-phase enthalpy change for isomerization between two conformations of the dipeptide N-acetyl glycine methyl amide (NAGMA). A two-stage oven source is implemented producing a gas-phase equilibrium distribution of NAGMA molecules that is flash frozen upon pickup by He nanodroplets. Using polarization spectroscopy, the IR spectrum is assigned to a mixture of two conformers having intramolecular hydrogen bonds made up of either five- or seven-membered rings, C5 and C7, respectively. The interconversion enthalpy, obtained from the van't Hoff relation, is 4.52{±}0.12 kJ/mol for isomerization from the C7 to the C5-conformer. This experimental measurement is compared to computations employing a broad range of theoretical methods.

  17. New triorganotin (IV) derivatives of dipeptides as models for metal-protein interactions: Synthesis, structural characterization and biological studies

    NASA Astrophysics Data System (ADS)

    Nath, Mala; Pokharia, Sandeep; Eng, George; Song, Xueqing; Kumar, Ashok

    2006-01-01

    New non-electrolytic triorganotin(IV) derivatives of dipeptides with general formulae R3Sn(HL), where R = Ph and HL = monoanion of glycylisoleucine (H2L-1), valylvaline (H2L-2), alanylvaline (H2L-3), leucylalanine (H2L-4), leucylleucine (H2L-5); R = n-Bu and HL = monoanion of glycylisoleucine (H2L-1) and leucylalanine (H2L-4); and R = Me and HL = monoanion of leucylalanine (H2L-4) have been synthesized and characterized on the basis of infrared, multinuclear 1H, 13C and 119Sn NMR and 119Sn Mössbauer spectroscopic studies. These investigations suggest that all the ligands in R3Sn(HL) act as monoanionic bidentates coordinating through the COO- and NH2 groups. The 119Sn Mössbauer studies, together with the NMR data, indicate that, for these polymeric derivatives, the polyhedron around tin in R3Sn(HL) is a trigonal-bipyramid with the three organic groups in the equatorial positions, while the axial positions are occupied by a carboxylic oxygen and the amino nitrogen atom from the adjacent molecule. The anti-inflammatory and cardiovascular activities and toxicity of all these compounds have been determined. Four of the complexes have also been screened against some of the chosen bacterial and fungal strains. The Ph3Sn(IV) compounds exhibit better anti-inflammatory and cardiovascular activities in comparison to the Me3Sn(IV) and n-Bu3Sn(IV) analogues. n-Bu3Sn(Gly-Ile) and Ph3Sn(Ala-Val) exhibit good antibacterial activity against all the chosen strains.

  18. Cysteine containing dipeptides show a metal specificity that matches the composition of seawater.

    PubMed

    Belmonte, Luca; Rossetto, Daniele; Forlin, Michele; Scintilla, Simone; Bonfio, Claudia; Mansy, Sheref S

    2016-07-27

    Model prebiotic dipeptide sequences were identified by bioinformatics and DFT and molecular dynamics calculations. The peptides were then synthesized and evaluated for metal affinity and specificity. Cysteine containing dipeptides were not associated with metal affinities that followed the Irving-Williams series but did follow the concentration trends found in seawater.

  19. Probing micro-solvation in "numbers": the case of neutral dipeptides in water.

    PubMed

    Takis, Panteleimon G; Papavasileiou, Konstantinos D; Peristeras, Loukas D; Melissas, Vasilios S; Troganis, Anastassios N

    2013-05-21

    How many solvent molecules and in what way do they interact directly with biomolecules? This is one of the most challenging questions regarding a deep understanding of biomolecular functionalism and solvation. We herein present a novel NMR spectroscopic study, achieving for the first time the quantification of the directly interacting water molecules with several neutral dipeptides. Our proposed method is supported by both molecular dynamics simulations and density functional theory calculations, advanced analysis of which allowed the identification of the direct interactions between solute-solvent molecules in the zwitterionic L-alanyl-L-alanine dipeptide-water system. Beyond the quantification of dipeptide-water molecule direct interactions, this NMR technique could be useful for the determination and elucidation of small to moderate bio-organic molecular groups' direct interactions with various polar solvent molecules, shedding light on the biomolecular micro-solvation processes and behaviour in various solvents.

  20. The role of a dipeptide outer-coordination sphere on H2-production catalysts: influence on catalytic rates and electron transfer.

    PubMed

    Reback, Matthew L; Ginovska-Pangovska, Bojana; Ho, Ming-Hsun; Jain, Avijita; Squier, Thomas C; Raugei, Simone; Roberts, John A S; Shaw, Wendy J

    2013-02-04

    The outer-coordination sphere of enzymes acts to fine-tune the active site reactivity and control catalytic rates, suggesting that incorporation of analogous structural elements into molecular catalysts may be necessary to achieve rates comparable to those observed in enzyme systems at low overpotentials. In this work, we evaluate the effect of an amino acid and dipeptide outer-coordination sphere on [Ni(P(Ph)(2)N(Ph-R)(2))(2)](2+) hydrogen production catalysts. A series of 12 new complexes containing non-natural amino acids or dipeptides was prepared to test the effects of positioning, size, polarity and aromaticity on catalytic activity. The non-natural amino acid was either 3-(meta- or para-aminophenyl)propionic acid terminated as an acid, an ester or an amide. Dipeptides consisted of one of the non-natural amino acids coupled to one of four amino acid esters: alanine, serine, phenylalanine or tyrosine. All of the catalysts are active for hydrogen production, with rates averaging ∼1000 s(-1), 40 % faster than the unmodified catalyst. Structure and polarity of the aliphatic or aromatic side chains of the C-terminal peptide do not strongly influence rates. However, the presence of an amide bond increases rates, suggesting a role for the amide in assisting catalysis. Overpotentials were lower with substituents at the N-phenyl meta position. This is consistent with slower electron transfer in the less compact, para-substituted complexes, as shown in digital simulations of catalyst cyclic voltammograms and computational modeling of the complexes. Combining the current results with insights from previous results, we propose a mechanism for the role of the amino acid and dipeptide based outer-coordination sphere in molecular hydrogen production catalysts.

  1. The Role of a Dipeptide Outer-Coordination Sphere on H2 -Production Catalysts: Influence on Catalytic Rates and Electron Transfer

    SciTech Connect

    Reback, Matthew L.; Ginovska-Pangovska, Bojana; Ho, Ming-Hsun; Jain, Avijita; Squier, Thomas C.; Raugei, Simone; Roberts, John A.; Shaw, Wendy J.

    2013-02-04

    The outer-coordination sphere of enzymes acts to fine-tune the active site reactivity and control catalytic rates, suggesting that incorporation of analogous structural elements into molecular catalysts may be necessary to achieve rates comparable to those observed in enzyme systems at low overpotentials. In this work, we evaluate the effect of an amino acid and dipeptide outer-coordination sphere on [Ni(PPh2NPh-R2)2]2+ hydrogen production catalysts. A series of 12 new complexes containing non-natural amino acids or dipeptides were prepared to test the effects of positioning, size, polarity and aromaticity on catalytic activity. The non-natural amino acid was either 3-(meta- or para-aminophenyl)propionic acid terminated as an acid, an ester or an amide. Dipeptides consisted of one of the non-natural amino acids coupled to one of four amino acid esters: alanine, serine, phenylalanine or tyrosine. All of the catalysts are active for hydrogen production, with rates averaging ~1000 s-1, 40% faster than the unmodified catalyst. Structure and polarity of the aliphatic or aromatic side chains of the C-terminal peptide do not strongly influence rates. However, the presence of an amide bond increases rates, suggesting a role for the amide in assisting catalysis. Overpotentials were lower with substituents at the N-phenyl meta position. This is consistent with slower electron transfer in the less compact, para-substituted complexes, as shown in digital simulations of catalyst cyclic voltammograms and computational modeling of the complexes. Combining the current results with insights from previous results, we propose a mechanism for the role of the amino acid and dipeptide based outer-coordination sphere in molecular hydrogen production catalysts.

  2. Energetics of Cyclic Dipeptide Crystal Packing and Solvation

    PubMed Central

    Brady, G. Patrick; Sharp, Kim A.

    1997-01-01

    Calculations of the thermodynamics of transfer of the cyclic alanine-alanine (cAA) and glycine-glycine (cGG) dipeptides between the gas, water, and crystal phases were carried out using a combination of molecular mechanics, normal mode analysis, and continuum electrostatics. The experimental gas-to-water solvation free energy and the enthalpy of gas-to-crystal transfer of cGG are accurately reproduced by the calculations. The enthalpies of cGG and cAA crystal-to-water transfer are also close to the experimental values. A combination of experimental data and normal mode analysis of cGG provides an accurate estimate of the association entropy penalty (loss of rotational and translational entropy and gain in vibrational entropy) for “binding” in the crystalline phase of -14.1 cal/mol/K. This is a smaller number than most previous theoretical estimates, but it is similar to previous experimental estimates. Calculated entropies of the crystal phase under-estimate the experimental entropy by about 15 cal/mol/K because of neglect of longe-range lattice motions. Comparison of the intermolecular interactions in the crystals of cGG and cAA provides a possible explanation of the puzzling decrease in enthalpy, with increasing hydrophobicity seen previously for both cyclic dipeptide dissolution and protein unfolding. This decrease arises from a favorable long-range electrostatic interaction between dipeptide molecules in the crystals, which is attenuated by the more hydrophobic side chains. PMID:9017216

  3. Acute β-N-Methylamino-L-alanine Toxicity in a Mouse Model

    PubMed Central

    Al-Sammak, Maitham Ahmed; Rogers, Douglas G.; Hoagland, Kyle D.

    2015-01-01

    The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) is considered to be an “excitotoxin,” and its suggested mechanism of action is killing neurons. Long-term exposure to L-BMAA is believed to lead to neurodegenerative diseases including Parkinson's and Alzheimer's diseases and amyotrophic lateral sclerosis (Lou Gehrig's disease). Objectives of this study were to determine the presumptive median lethal dose (LD50), the Lowest-Observed-Adverse-Effect Level (LOAEL), and histopathologic lesions caused by the naturally occurring BMAA isomer, L-BMAA, in mice. Seventy NIH Swiss Outbred mice (35 male and 35 female) were used. Treatment group mice were injected intraperitoneally with 0.03, 0.3, 1, 2, and 3 mg/g body weight L-BMAA, respectively, and control mice were sham-injected. The presumptive LD50 of L-BMAA was 3 mg/g BW and the LOAEL was 2 mg/g BW. There were no histopathologic lesions in brain, liver, heart, kidney, lung, or spleen in any of the mice during the 14-day study. L-BMAA was detected in brains and livers in all of treated mice but not in control mice. Males injected with 0.03 mg/g BW, 0.3 mg/g BW, and 3.0 mg/g BW L-BMAA showed consistently higher concentrations (P < 0.01) in brain and liver samples as compared to females in those respective groups. PMID:26604922

  4. Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1.

    PubMed

    Oswald, Matthew C W; West, Ryan J H; Lloyd-Evans, Emyr; Sweeney, Sean T

    2015-12-15

    Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1, or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominant-negative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1, consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER-Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1.

  5. Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1

    PubMed Central

    Oswald, Matthew C. W.; West, Ryan J. H.; Lloyd-Evans, Emyr; Sweeney, Sean T.

    2015-01-01

    Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1, or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominant-negative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1, consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER–Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1. PMID:26395456

  6. [Effects of ß-alanine supplementation on athletic performance].

    PubMed

    Domínguez, Raúl; Hernández Lougedo, Juan; Maté-Muñoz, José Luis; Garnacho-Castaño, Manuel Vicente

    2014-10-06

    Carnosine, dipeptide formed by amino acids ß-alanine and L-histidine, has important physiological functions among which its antioxidant and related memory and learning. However, in connection with the exercise, the most important functions would be associated with muscle contractility, improving calcium sensitivity in muscle fibers, and the regulatory function of pH. Thus, it is proposed that carnosine is the major intracellular buffer, but could contribute to 7-10% in buffer or buffer capacity. Since carnosine synthesis seems to be limited by the availability of ß-alanine supplementation with this compound has been gaining increasing popularity among the athlete population. Therefore, the objective of this study literature review was to examine all those research works have shown the effect of ß-alanine supplementation on athletic performance. Moreover, it also has attempted to establish a specific dosage that maximizing the potential benefits, minimize paresthesia, the main side effect presented in response to supplementation.

  7. Alanine water complexes.

    PubMed

    Vaquero, Vanesa; Sanz, M Eugenia; Peña, Isabel; Mata, Santiago; Cabezas, Carlos; López, Juan C; Alonso, José L

    2014-04-10

    Two complexes of alanine with water, alanine-(H2O)n (n = 1,2), have been generated by laser ablation of the amino acid in a supersonic jet containing water vapor and characterized using Fourier transform microwave spectroscopy. In the observed complexes, water molecules bind to the carboxylic group of alanine acting as both proton donors and acceptors. In alanine-H2O, the water molecule establishes two intermolecular hydrogen bonds forming a six-membered cycle, while in alanine-(H2O)2 the two water molecules establish three hydrogen bonds forming an eight-membered ring. In both complexes, the amino acid moiety is in its neutral form and shows the conformation observed to be the most stable for the bare molecule. The microsolvation study of alanine-(H2O)n (n = 1,2) can be taken as a first step toward understanding bulk properties at a microscopic level.

  8. Terahertz spectra and normal mode analysis of the crystalline VA class dipeptide nanotubes.

    PubMed

    Zhang, Hailiang; Siegrist, Karen; Plusquellic, David F; Gregurick, Susan K

    2008-12-31

    Terahertz (THz) vibrational modes are characterized by nonlocal, collective molecular motions which are relevant to conformational changes and molecular functions in biological systems. We have investigated the THz spectra of a set of small bionanotubes which can serve as very simple models of membrane pores, and have examined the character of the THz modes which can impact transport processes. In this work, THz spectra of the crystalline VA class dipeptide nanotubes were calculated at both the harmonic and vibrational self-consistent field (VSCF) level using the CHARMM22 force field with periodic boundary conditions. Comparison of the calculated THz spectra against the experimental spectra revealed that the VSCF corrections generally improved the predictions in the low-frequency region. The improvements were especially manifested in the overall blue-shifts of the VSCF frequencies relative to the harmonic values, and blue shifts were attributed to the overall positive coupling strengths in all systems. Closer examination of the motions in the most significantly coupled normal mode pairs leads us to propose that, when two similar side-chain squeezing modes are coupled, the rapidly increased van der Waals interactions can lead to a stiffening of the effective potential, which in turn leads to the observed blue-shifts. However, we also noted that when the side-chain atoms become unphysically proximate and the van der Waals repulsion becomes too large, the VSCF calculations tend to deviate in the high frequency region and for the system of l-isoleucyl-l-valine. In addition, normal-mode analysis revealed a series of channel-breathing motions in all systems except l-valyl-l-alanine. We show that the inner products of the backbone vibrations between these channel-breathing motions divided the remaining VA class dipeptide systems into two subgroups. It is suggested that these modes may facilitate a pathway for the guest molecule absorption, substitution and removal in the

  9. Di-peptide Based Models of Nickel Superoxide Dismutase: Solvent Effects Highlight a Critical Role to Ni-S Bonding and Active Site Stabilization

    PubMed Central

    Gale, Eric M.; Cowart, Darin M.; Scott, Robert A.

    2013-01-01

    Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of the superoxide radical to O2 and H2O2 utilizing the Ni(III/II) redox couple. The Ni center in Ni-SOD resides in an unusual coordination environment that is distinct from other SODs. In the reduced state (Ni-SODred), the Ni(II) center is ligated to a primary amine-N from His1, anionic carboxamido-N/thiolato-S from Cys2, and a second thiolato-S from Cys6 to complete a NiN2S2 square-planar coordination motif. Utilizing the dipeptide N2S2− ligand, H2N-Gly-L-Cys-OMe (GC-OMeH2) that accurately models the structural and electronic contributions provided by His1 and Cys2 in Ni-SODred, we have constructed the dinuclear sulfur-bridged metallosynthon, [Ni2(GC-OMe)2] (1). From 1 we have prepared the following monomeric Ni(II)-N2S2 complexes: K[Ni(GC-OMe)(SC6H4-p-Cl)] (2), K[Ni(GC-OMe)(StBu)] (3), K[Ni(GC-OMe)(SC6H4-p-OMe)] (4), and K[Ni(GC-OMe)(S-NAc)] (5). The design strategy in utilizing GC-OMe2− is analogous to one which we have reported before (see Inorg. Chem. 2009, 48, 5620 and Inorg. Chem. 2010, 49, 7080) wherein Ni-SODred active site mimics can be constructed at will with electronically variant RS− ligands. Discussed herein is the first account pertaining to the aqueous behavior of isolable, small molecule Ni-SOD model complexes (non-maquette based). Spectroscopic (FTIR, UV-vis, XAS) and electrochemical (CV) measurements suggest that 2–5 successfully simulate many of the electronic features of the Ni-SODred active site but also reveal, in conjunction with 1H NMR and ESI-MS studies, that these models are dynamic species with regards to RS− lability and bridging interactions in aqueous media suggesting a stabilizing role brought about by the protein architecture. PMID:21932766

  10. Mass Spectrometry Identification of N-Chlorinated Dipeptides in Drinking Water.

    PubMed

    Huang, Guang; Jiang, Ping; Li, Xing-Fang

    2017-03-14

    We report the identification of N-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N,N-di-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, (35)Cl/(37)Cl isotopic patterns, and MS/MS spectra. These identified N-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except N,N-di-Cl-Phe-Gly. To enable sensitive detection of N-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with multiple reaction monitoring (MRM) mode. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the N-chlorinated dipeptides were at background levels. These results suggest that the N-chlorinated dipeptides are produced by chlorination. This study has identified N-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water.

  11. Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2017-01-01

    The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

  12. N-( p-Ethynylbenzoyl) derivatives of amino acid and dipeptide methyl esters - Synthesis and structural study

    NASA Astrophysics Data System (ADS)

    Eißmann, Frank; Weber, Edwin

    2011-11-01

    A series of N-( p-ethynylbenzoyl) derivatives ( 1-4) of the amino acids glycine and L-alanine as well as the dipeptides glycylglycine and L-alanylglycine has been synthesized via a two-step reaction sequence including the reaction of an appropriate N-( p-bromobenzoyl) precursor with trimethylsilylacetylene followed by deprotection of the trimethylsilyl protecting group, respectively. X-ray crystal structures of the amino acid and dipeptide methyl esters 1-4 are reported. The amide and peptide bonds within each molecular structure are planar and adopt the trans-configuration. The packing structures are governed by N sbnd H⋯O interactions leading to the formation of characteristic strand motifs. Further stabilization results from weaker C sbnd H⋯O and C sbnd H⋯π contacts.

  13. Microscale Synthesis and Analysis of a Dipeptide.

    ERIC Educational Resources Information Center

    Blatchly, Richard A.; And Others

    1989-01-01

    Described is a microscale chemistry laboratory in which a dipeptide is synthesized from its component amino acids and analyzed using chiral-phase thin-layer chromatography. Experimental procedures, and materials are discussed. Twelve references are listed. (CW)

  14. The nootropic and neuroprotective proline-containing dipeptide noopept restores spatial memory and increases immunoreactivity to amyloid in an Alzheimer's disease model.

    PubMed

    Ostrovskaya, Rita U; Gruden, Marina A; Bobkova, Natalya A; Sewell, Robert D E; Gudasheva, Tatyana A; Samokhin, Alexander N; Seredinin, Sergey B; Noppe, Wim; Sherstnev, Vladimir V; Morozova-Roche, Ludmilla A

    2007-08-01

    The effects of the novel proline-containing nootropic and neuroprotective dipeptide, noopept (GVS-111, N-phenylacetyl-L-prolylglycine ethyl ester) were investigated in NMRI mice following olfactory bulbectomy. We have shown previously that these animals developed Alzheimer's disease (AD)-like behaviour, morphology and biochemistry including impairment of spatial memory, regional neuronal degeneration and elevated Abeta peptide brain levels. In the current investigation, spatial memory was assessed using the Morris water maze and serum antibodies to in vitro morphologically characterized amyloid structures of both Abeta((25-35)) peptide and equine lysozyme, as well as to neurotrophic glial factor S100b, were analyzed by enzyme-linked immunosorbent assay (ELISA). Noopept (administered at a dose of 0.01 mg/kg for a period of 21 days and during a further 5 days training) restored spatial memory and increased serum antibody levels to oligomers of Abeta((25-35)) peptide but not to equine lysozyme amyloid or S100b protein in bulbectomized animals. The positive immunotropic effect of noopept to Abeta((25-35)) peptide prefibrillar aggregates was more marked in sham-operated compared to the bulbectomized subjects which were characterized by an overall suppression of immunoreactivity. Enhancement of the immune response to Abeta((25-35)) peptide prefibrils caused by noopept may attenuate the neurotoxic consequences of amyloid fibrillization and also be associated with an improvement in spatial memory in bulbectomized mice. These actions of noopept, combined with its previously reported neuroprotective and cholinomimetic properties, suggests that this dipeptide may well be useful for improving cognitive deficits induced by neurodegenerative diseases.

  15. DNA Oxidative Cleavage Induced by the Novel Peptide Derivatives of 3-(quinoxalin-6-yl)alanine in Combination with Cu(II) or Fe(II) Ions

    PubMed Central

    Szczepanik, Wojciech; Kucharczyk-Klamińska, Marzena; Stefanowicz, Piotr; Staszewska, Anna; Szewczuk, Zbigniew; Skała, Jacek; Mysiak, Andrzej; Jeżowska-Bojczuk, Małgorzata

    2009-01-01

    Three model dipeptides containing 3-(2,3-di(pyridin-2-yl)quinoxalin-6-yl)alanine, 3-(dipyrido[3,2-a:2,3-c]phenazin-11-yl)alanine, and 3-(2,3-diphenylquinoxalin-6-yl)alanine were studied with respect to their ability to bind selected transition metal ions, such as Cu(II), Fe(II), Ni(II), Co(II), Mn(II), and Cr(III). It was found that only Cu(II) and Fe(II) ions could form stable complex species with the studied compounds. The ability to form the complexes correlated well with DNA damage experiments. Only the ferrous and cupric complexes are capable of generating both single- and double-strand scissions. However, double-strand breakages appear to be dominating lesions in the presence of hydrogen peroxide, especially for copper(II) containing systems. The quantity of breakage products in the presence of N-(3-(dipyrido[3,2-a:2,3-c]phenazine-11-yl)alanyl)glycine complexes was the highest as compared to the complexes of the remaining compounds. Moreover, this ligand was the only one that cleaved DNA in the absence of either Cu(II) or Fe(II) ions. PMID:20224817

  16. Programming the supramolecular helical polymerization of dendritic dipeptides via the stereochemical information of the dipeptide.

    PubMed

    Rosen, Brad M; Peterca, Mihai; Morimitsu, Kentaro; Dulcey, Andrés E; Leowanawat, Pawaret; Resmerita, Ana-Maria; Imam, Mohammad R; Percec, Virgil

    2011-04-06

    Many natural biomacromolecules are homochiral and are built from constituents possessing identical handedness. The construction of synthetic molecules, macromolecules, and supramolecular structures with tailored stereochemical sequences can detail the relationship between chirality and function and provide insight into the process that leads to the selection of handedness and amplification of chirality. Dendritic dipeptides, previously reported from our laboratory, self-assemble into helical porous columns and serve as fundamental mimics of natural porous helix-forming proteins and supramolecular polymers. Herein, the synthesis of all stereochemical permutations of a self-assembling dendritic dipeptide including homochiral, heterochiral, and differentially racemized variants is reported. A combination of CD/UV-vis spectroscopy in solution and in film, X-ray diffraction, and differential scanning calorimetry studies in solid state established the role of the stereochemistry of the dipeptide on the thermodynamics and mechanism of self-assembly. It was found that the highest degree of stereochemical purity, enantiopure homochiral dendritic dipeptides, exhibits the most thermodynamically favorable self-assembly process in solution corresponding to the greatest degree of helical order and intracolumnar crystallization in solid state. Reducing the stereochemical purity of the dendritic dipeptide through heterochirality or by partially or fully racemizing the dendritic dipeptide destructively interferes with the self-assembly process. All dendritic dipeptides were shown to coassemble into single columns regardless of their stereochemistry. Because these columns exhibit no deracemization, the thermodynamic advantage of enantiopurity and homochirality suggests a mechanism for stereochemical selection and chiral amplification.

  17. Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

    PubMed

    Maini, Rumit; Dedkova, Larisa M; Paul, Rakesh; Madathil, Manikandadas M; Chowdhury, Sandipan Roy; Chen, Shengxi; Hecht, Sidney M

    2015-09-09

    Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

  18. Arginine dipeptides affect insulin aggregation in a pH- and ionic strength-dependent manner.

    PubMed

    Nuhu, Mariam M; Curtis, Robin

    2015-03-01

    Solutions containing arginine or mixtures of arginine and other amino acids are commonly used for protein liquid formulations to overcome problems such as high viscosities, aggregation, and phase separation. The aim of this work is to examine whether the stabilizing properties of arginine can be improved by incorporating the amino acid into a dipeptide. A series of arginine-containing dipeptides have been tested for their ability to suppress insulin aggregation over a range of pH and ionic strength. The aggregation is monitored at room temperature using a combination of turbidimetry and light scattering for solutions at pH 5.5 or 3.7, whereas thermal-induced aggregation is measured at pH 7.5. In addition, intrinsic fluorescence has been used to quantify additive binding to insulin. The dipeptide diArg is the most effective additive in solutions at pH 5.5 and 3.7, whereas the dipeptide Arg-Phe almost completely eliminates thermally-induced aggregation of insulin at pH 7.5 up to temperature of 90°C. Insulin has been chosen as a model system because the molecular forces controlling its aggregation are well known. From this understanding, we are able to provide a molecular basis for how the various dipeptides affect insulin aggregation.

  19. gamma-Glutamyl dipeptides in Petiveria alliacea.

    PubMed

    Kubec, Roman; Musah, Rabi A

    2005-10-01

    Three gamma-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S(C2)R(C7))-gamma-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S(C2)R(C7)R(S))- and (S(C2)R(C7)R(S))-gamma-glutamyl-S-benzylcysteine S-oxides (gamma-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.

  20. Molecular Mechanisms of Several Novel Dipeptides with Angiotensin-I-converting Enzyme Inhibitory Activity from In-silico Screening of Silkworm Pupae Protein.

    PubMed

    Wang, Wei; Zhang, Yu; Wang, Nan; Zhu, Zuoyi

    2014-01-01

    An in silico screening method for novel dipeptides from a dipeptide training set was performed. A 3D pharmacophore model with three H-bond acceptor projections (Acc2), and one H-bond donor projection (Don2) was obtained. To validate the pharmacophore model, a test set containing 10 reported dipeptides was screened and all were found to be bioactive. Eleven novel dipeptides (IF, GD, DA, TE, TA, ES, SS, ST, SD, QD, QE) from the silkworm pupae protein peptide database were predicted to have ACE inhibitory bioactivity. The interaction mechanisms of the dipeptides with the ACE active pocket were elucidated by molecule docking, and besides involving interaction bonds, the interaction force equipoise of the peptides was also an important factor in their bioactivity.

  1. Effects of glycyl-glutamine dipeptide supplementation on myocardial damage and cardiac function in rats after severe burn injury.

    PubMed

    Zhang, Yong; Yan, Hong; Lv, Shang-Gun; Wang, Lin; Liang, Guang-Ping; Wan, Qian-Xue; Peng, Xi

    2013-01-01

    Glutamine decreases myocardial damage in ischemia/reperfusion injury. However, the cardioprotective effect of glutamine after burn injury remains unclear. Present study was to explore the protective effect of glycyl-glutamine dipeptide on myocardial damage in severe burn rats. Seventy-two Wistar rats were randomly divided into three groups: normal control (C), burned control (B) and glycyl-glutamine dipeptide-treated (GG) groups. B and GG groups were inflicted with 30% total body surface area of full thickness burn. The GG group was given 1.5 g/kg glycyl-glutamine dipeptide per day and the B group was given the same dose of alanine via intraperitoneal injection for 3 days. The serum CK, LDH, AST, and, blood lactic acid levels, as well as the myocardium ATP and GSH contents, were measured. The indices of cardiac contractile function and histopathological change were analyzed at 12, 24, 48, and 72 post-burn hours (PBH). The serum CK, LDH, AST and blood lactic acid levels increased, and the myocardium ATP and GSH content decreased in both burned groups. Compared with B group, the CK, LDH, AST and blood lactic acid levels reduced, myocardium ATP and GSH content increased in GG group. Moreover, the inhibition of cardiac contractile function and myocardial histopathological damage were reduced significantly in GG group. We conclude that myocardial histological structure and function were damaged significantly after burn injury, glycyl-glutamine dipeptide supplementation is beneficial to myocardial preservation by improving cardiocyte energy metabolism, increasing ATP and glutathione synthesis.

  2. β-N-methylamino-L-alanine (BMAA) uptake by the animal model, Daphnia magna and subsequent oxidative stress.

    PubMed

    Esterhuizen-Londt, Maranda; Wiegand, Claudia; Downing, Tim G

    2015-06-15

    β-N-methylamino-l-alanine (BMAA), produced by cyanobacteria, is a neurotoxin implicated in Amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). BMAA concentrations in cyanobacteria are lower than those thought to be necessary to result in neurological damage thus bioaccumulation or biomagnification is required to achieve concentrations able to cause neurodegeneration. Many cyanobacteria produce BMAA and uptake routes into the food web require examination. In this study we investigate the uptake of BMAA by adult phytoplanktivorus Daphnia magna via exposure to dissolved pure BMAA and BMAA containing cyanobacteria, as well as the subsequent oxidative stress response in the daphnia. Free BMAA and protein-associated BMAA were quantified by LC-MS/MS. Dissolved BMAA was taken up and was found as free BMAA in D. magna. No protein-associated BMAA was detected in D. magna after a 24-h exposure period. No BMAA was detectable in D. magna after exposure to BMAA containing cyanobacteria. BMAA inhibited the oxidative stress defence and biotransformation enzymes within 24-h exposure in the tested Daphnia and could therefore impair the oxidant status and the capability of detoxifying other substances in D. magna.

  3. Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

    SciTech Connect

    Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia; Barletta, Raúl G.; Sacchettini, James C.

    2011-09-28

    D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

  4. Theoretical UV circular dichroism of aliphatic cyclic dipeptides.

    PubMed

    Carlson, Kristine L; Lowe, Stephen L; Hoffmann, Mark R; Thomasson, Kathryn A

    2005-06-23

    Four cyclic dipeptides (piperazine-2,5-diones), cyclo(L-Pro-Gly), cyclo(L-Pro-L-Leu), cyclo(L-Ala-L-Ala), and cyclo(L-Pro-L-Ala), were modeled from crystal structure data. Conformations resulting from energy minimization using molecular mechanics were compared with traditional ab initio and density functional theory geometric optimizations for each dipeptide. In all computational cases, the gas phase was assumed. The pi-pi transition feature of the UV circular dichroic (CD) spectra was predicted for each peptide structure via the classical dipole interaction model. The dipole interaction model predicted CD spectra that qualitatively agreed with experiment when MP2 or DFT geometries were used. By coupling MP2 or DFT geometric optimizations with the classical physics method of the dipole interaction model, significantly better CD spectra were calculated than those using geometries obtained by molecular mechanics. Thus, one can couple quantum mechanical geometries with a classical physics model for calculation of circular dichroism.

  5. Self-assembled dipeptide-gold nanoparticle hybrid spheres for highly sensitive amperometric hydrogen peroxide biosensors.

    PubMed

    Gong, Yufei; Chen, Xu; Lu, Yanluo; Yang, Wensheng

    2015-04-15

    Novel self-assembled dipeptide-gold nanoparticle (DP-AuNP) hybrid microspheres with a hollow structure have been prepared in aqueous solution by a simple one-step method. Diphenylalanine (FF) dipeptide was used as a precursor to form simultaneously peptide spheres and a reducing agent to reduce gold ions to gold nanoparticles in water at 60°C. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that formed AuNPs were localized both inside and on the surface of the dipeptide spheres. Horseradish peroxidase (HRP) as a model enzyme was further immobilized on the dipeptide-AuNP hybrid spheres to construct a mediate H2O2 amperometric biosensor. UV-vis spectroscopy showed that the immobilized HRP retained its original structure. Cyclic voltammetry characterization demonstrated that the HRP/dipeptide-AuNP hybrid spheres modified glassy carbon electrode showed high electrocatalytic activity to H2O2. The proposed biosensor exhibited a wide linear response in the range from 5.0×10(-7) to 9.7×10(-4)M with a high sensitivity of 28.3µAmM(-1). A low detection limit of 1.0×10(-7)M was estimated at S/N=3. In addition, the biosensor possessed satisfactory reproducibility and long-term stability. These results indicated that the dipeptide-AuNP hybrid sphere is a promising matrix for application in the fabrication of electrochemical biosensors due to its excellent biocompatibility and good charge-transfer ability.

  6. Biomedical applications of dipeptides and tripeptides.

    PubMed

    Santos, Sara; Torcato, Inês; Castanho, Miguel A R B

    2012-01-01

    Peptides regulate many physiological processes, acting at some sites as endocrine or paracrine signals and at others as neurotransmitters or growth factors, for instance. These molecules represent a major evolution in medical and industrial fields, as it is becoming mandatory to design and exploit molecules that do not necessarily fit the description of classical drug classes. The list of peptides with potential biomedical applications is huge and is growing each year. These biomedical applications range from uses as drugs to flavor-active peptides as ingredients in natural health products, nutraceuticals and functional foods. Among the peptide family, dipeptides and tripeptides are very appealing for drug discovery and development because of their cost-effectiveness, possibility of oral administration, and simplicity to perform molecular structural and quantitative structure-activity studies. Our objective is to review different actual and future uses of dipeptides and tripeptides as well as the major advances and obstacles in this growing area.

  7. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    PubMed

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  8. Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin.

    PubMed

    Monti, Patrizia; Taddei, Paola; Freddi, Giuliano; Ohgo, Kosuke; Asakura, Tetsuo

    2003-01-01

    This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.

  9. Repeated Supramaximal Exercise-Induced Oxidative Stress: Effect of β-Alanine Plus Creatine Supplementation

    PubMed Central

    Belviranli, Muaz; Okudan, Nilsel; Revan, Serkan; Balci, Serdar; Gokbel, Hakki

    2016-01-01

    Background: Carnosine is a dipeptide formed from the β-alanine and histidine amino acids and found in mainly in the brain and muscle, especially fast twitch muscle. Carnosine and creatine has an antioxidant effect and carnosine accounts for about 10% of the muscle's ability to buffer the H+ ions produced by exercise. Objectives: The aim of the study was to investigate the effects of beta alanine and/or creatine supplementation on oxidant and antioxidant status during repeated Wingate tests (WTs). Patients and Methods: Forty four sedentary males participated in the study. Participants performed three 30s WTs with 2 minutes rest between exercise bouts. After the first exercise session, the subjects were assigned to one of four groups: Placebo, Creatine, Beta-alanine and Beta-alanine plus creatine. Participants ingested twice per day for 22 consecutive days, then four times per day for the following 6 days. After the supplementation period the second exercise session was applied. Blood samples were taken before and immediately after the each exercise session for the analysis of oxidative stress and antioxidant markers. Results: Malondialdehyde levels and superoxide dismutase activities were affected by neither supplementation nor exercise. During the pre-supplementation session, protein carbonyl reduced and oxidized glutathione (GSH and GSSG) levels increased immediately after the exercise. However, during the post-supplementation session GSH and GSSG levels increased in beta-alanine and beta-alanine plus creatine groups immediately after the exercise compared to pre-exercise. In addition, during the post-supplementation session total antioxidant capacity increased in beta-alanine group immediately after the exercise. Conclusions: Beta-alanine supplementation has limited antioxidant effect during the repeated WTs. PMID:27217925

  10. Salt Taste Enhancing l-Arginyl Dipeptides from Casein and Lysozyme Released by Peptidases of Basidiomycota.

    PubMed

    Harth, Lisa; Krah, Ulrike; Linke, Diana; Dunkel, Andreas; Hofmann, Thomas; Berger, Ralf G

    2016-08-24

    Some l-arginyl dipeptides were recently identified as salt taste enhancers, thus opening the possibility to reduce dietary sodium uptake without compromising palatability. A screening of 15 basidiomycete fungi resulted in the identification of 5 species secreting a high peptidolytic activity (>3 kAU/mL; azocasein assay). PFP-LC-MS/MS and HILIC-MS/MS confirmed that l-arginyl dipeptides were liberated when casein or lysozyme served as substrate. Much higher yields of dipeptides (42-75 μmol/g substrate) were released from lysozyme than from casein. The lysozyme hydrolysate generated by the complex set of peptidases of Trametes versicolor showed the highest l-arginyl dipeptide yields and a significant salt taste enhancing effect in a model cheese matrix and in a curd cheese. With a broad spectrum of novel specific and nonspecific peptidases active in the slightly acidic pH range, T. versicolor might be a suitable enzyme source for low-salt dairy products.

  11. Insight into the intermolecular recognition mechanism between Keap1 and IKKβ combining homology modelling, protein-protein docking, molecular dynamics simulations and virtual alanine mutation.

    PubMed

    Jiang, Zheng-Yu; Chu, Hong-Xi; Xi, Mei-Yang; Yang, Ting-Ting; Jia, Jian-Min; Huang, Jing-Jie; Guo, Xiao-Ke; Zhang, Xiao-Jin; You, Qi-Dong; Sun, Hao-Peng

    2013-01-01

    Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

  12. Structure of dipeptides having N-terminal selenocysteine residues: a DFT study in gas and aqueous phase.

    PubMed

    Mandal, Shilpi; Das, Gunajyoti

    2013-06-01

    Over the last few decades, dipeptides as well as their analogues have served as important model systems for the computational studies concerning the structure of protein and energetics of protein folding. Here, we present a density functional structural study on a set of seven dipeptides having N-terminal selenocysteine residues (the component in the C-terminus is varied with seven different combinations viz. Ala, Phe, Glu, Thr, Asn, Arg and Sec) in gas and simulated aqueous phase using a polarizable continuum model (PCM). The molecular geometries of the dipeptides are fully optimized at B3LYP/6-311++G(d,p) level and subsequent frequency calculations confirm them as true minima. The effects of solvation and identity of the varying C-terminal residue on the energetics, structural features of the peptide planes, values of the ψ and ф dihedrals, geometry around the α-carbon atoms and theoretically predicted vibrational spectra of the dipeptides are investigated. Two types of intramolecular H-bonds, namely N…H-N and O…H-C, are found to play important roles in influencing the planarity of the peptide planes and geometry around the α-carbon atoms of the dipeptides. The identity of the varying C-terminal residue influences the values of ф, planarity of the peptide planes and geometry around the C₇ α-carbon atoms while the solvation effects are evident on the values of bond lengths and bond angles of the amide planes.

  13. Bestatin Induces Specific Changes in Trypanosoma cruzi Dipeptide Pool

    PubMed Central

    Creek, Darren J.; Faral-Tello, Paula; Barrett, Michael P.

    2015-01-01

    Proteases and peptidases in Trypanosoma cruzi are considered potential targets for antichagasic chemotherapy. We monitored changes in low-mass metabolites in T. cruzi epimastigotes treated with bestatin, a dipeptide metalloaminopeptidase inhibitor. After treatment, multiple dipeptides were shown to be increased, confirming in situ inhibition of the leucine aminopeptidase of T. cruzi (LAPTc) and probably other peptidases. PMID:25712359

  14. Probing gelation ability for a library of dipeptide gelators.

    PubMed

    Awhida, Salmah; Draper, Emily R; McDonald, Tom O; Adams, Dave J

    2015-10-01

    Functionalised dipeptides are a class of interesting and useful low molecular weight hydrogelators. Here, we report a significantly expanded library of materials, including dipeptides conjugated to carbazole, phenanthracene, anthracene, pyrene and substituted naphthalenes. We assess the effect of using two different gelation methods; a pH-switch and a solvent switch on the gelation behaviour and properties of the dipeptides. Importantly, we investigate the relationship between the structure of these dipeptides and their ability to form gels. From an analysis of the gelation ability of all these dipeptides, it is clear that those containing a phenylalanine as either of the constituent amino acids are much more likely to lead to a gelator being formed as opposed to using non-aromatic amino acids only.

  15. Investigations of dipeptide structures containing pyrrolysine as N-terminal residues: a DFT study in gas and aqueous phase.

    PubMed

    Das, Gunajyoti

    2013-04-01

    A set of six dipeptides containing pyrrolysine invariably at their N-terminal positions is studied in gas and aqueous phase using a polarizable continuum model (PCM). The molecular geometries of the dipeptides are fully optimized at B3LYP/6-31++G(d,p) level of theory and a second derivative (frequency) analysis confirms that all the optimized geometries are true minima. The effects of solvation and identity of the varying C-terminal residue on the energetics, structural features of the peptide planes, values of the ψ and ϕ dihedrals, geometry around the α-carbon atoms and theoretically predicted vibrational spectra of the dipeptides are thoroughly analyzed. Solvation effects are found to modify the gas phase conformation of the dipeptides around ψ dihedrals while the identity of the varying C-terminal residue affect the values of ϕ, planarity of the peptide planes and geometry around the α-carbon atoms. The presence or absence of three types of intramolecular H-bonds, namely O...H-N, N...H-N and O...H-C that leave noticeable signatures in the IR spectra, play crucial roles in influencing the geometry of the peptide planes and in determining the energetics of the dipeptides.

  16. β-N-methylamino-l-alanine causes neurological and pathological phenotypes mimicking Amyotrophic Lateral Sclerosis (ALS): the first step towards an experimental model for sporadic ALS.

    PubMed

    de Munck, Estefanía; Muñoz-Sáez, Emma; Miguel, Begoña G; Solas, M Teresa; Ojeda, Irene; Martínez, Ana; Gil, Carmen; Arahuetes, Rosa Ma

    2013-09-01

    β-N-methylamino-l-alanine (L-BMAA) is a neurotoxic amino acid that has been related to various neurodegenerative diseases. The aim of this work was to analyze the biotoxicity produced by L-BMAA in vivo in rats, trying to elucidate its physiopathological mechanisms and to search for analogies between the found effects and pathologies like Amyotrophic Lateral Sclerosis (ALS). Our data demonstrated that the neurotoxic effects in vivo were dosage-dependent. For evaluating the state of the animals, a neurological evaluation scale was developed as well as a set of functional tests. Ultrastructural cell analysis of spinal motoneurons has revealed alterations both in endoplasmic reticulum and mitochondria. Since GSK3β could play a role in some neuropathological processes, we analyzed the alterations occurring in GSK3β levels in L-BMAA treated rats, we have observed an increase in the active form of GSK3β levels in lumbar spinal cord and motor cerebral cortex. On the other hand, (TAR)-DNA-binding protein 43 (TDP-43) increased in L-BMAA treated animals. Our results indicated that N-acetylaspartate (NAA) declined in animals treated with L-BMAA, and the ratio of N-acetylaspartate/choline (NAA/Cho), N-acetylaspartate/creatine (NAA/Cr) and N-acetylaspartate/choline+creatine (NAA/Cho+Cr) tended to decrease in lumbar spinal cord and motor cortex. This project offers some encouraging results that could help establishing the progress in the development of an animal model of sporadic ALS and L-BMAA could be a useful tool for this purpose.

  17. L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus.

    PubMed

    Shiau, Chia-Yang; Liu, Yu-Tien

    2002-04-12

    L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

  18. Density functional theory calculations on dipeptide gallic acid interaction

    NASA Astrophysics Data System (ADS)

    Madhan, B.; Parthasarathi, R.; Subramanian, V.; Raghava Rao, J.; Nair, Balachandran Unni; Ramasami, T.

    2003-02-01

    In the present investigation, an attempt has been made to study the interaction of dipeptides with gallic acid, using Becke3 parameter Lee Yang Parr (B3LYP) method employing 3-21G*, 6-31G* and 6-31+G* basis sets. The interaction energies of the dipeptide-gallic acid complexes are in the range of -5 to -18 kcal/mol depending on the mode of intermolecular complexation. Calculated molecular electrostatic potential (MESP) for the various intermolecular complexes revealed the electrostatic nature of the interaction. Qualitative estimations based on chemical hardness and chemical potential demonstrated fractional electron transfer from dipeptide to gallic acid.

  19. Β-alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply.

    PubMed

    Usui, Takuya; Kubo, Yoshiyuki; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

    2013-08-01

    The supply of L-carnosine, a bioactive dipeptide of β-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [(3)H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [(3)H]β-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]β-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]β-alanine uptake, suggesting that a carrier-mediated process was involved in β-alanine transport across the inner BRB. [(3)H]β-Alanine uptake was inhibited by taurine and β-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of β-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [(3)H]l-histidine was identified, and the in vitro [(3)H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of β-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.

  20. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    SciTech Connect

    Ikebe, T.; Iribe, H.; Hirata, M.; Yanaga, F.; Koga, T. )

    1990-12-01

    Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.

  1. Detoxification of aldehydes by histidine-containing dipeptides: from chemistry to clinical implications

    PubMed Central

    Xie, Zhengzhi; Baba, Shahid P.; Sweeney, Brooke R.; Barski, Oleg A.

    2015-01-01

    Aldehydes are generated by oxidized lipids and carbohydrates at increased levels under conditions of metabolic imbalance and oxidative stress during atherosclerosis, myocardial and cerebral ischemia, diabetes, neurodegenerative diseases and trauma. In most tissues, aldehydes are detoxified by oxidoreductases that catalyze the oxidation or the reduction of aldehydes or enzymatic and nonenzymatic conjugation with low molecular weight thiols and amines, such as glutathione and histidine dipeptides. Histidine dipeptides are present in micromolar to millimolar range in the tissues of vertebrates, where they are involved in a variety of physiological functions such as pH buffering, metal chelation, oxidant and aldehyde scavenging. Histidine dipeptides such as carnosine form Michael adducts with lipid-derived unsaturated aldehydes, and react with carbohydrate-derived oxo- and hydroxy- aldehydes forming products of unknown structure. Although these peptides react with electrophilic molecules at lower rate than glutathione, they can protect glutathione from modification by oxidant and they may be important for aldehyde quenching in glutathione-depleted cells or extracellular space where glutathione is scarce. Consistent with in vitro findings, treatment with carnosine has been shown to diminish ischemic injury, improve glucose control, ameliorate the development of complications in animal models of diabetes and obesity, promote wound healing and decrease atherosclerosis. The protective effects of carnosine have been linked to its anti-oxidant properties, it ability to promote glycolysis, detoxify reactive aldehydes and enhance histamine levels. Thus, treatment with carnosine and related histidine dipeptides may be a promising strategy for the prevention and treatment of diseases associated with high carbonyl load. PMID:23313711

  2. The polyproline II conformation in short alanine peptides is noncooperative.

    PubMed

    Chen, Kang; Liu, Zhigang; Kallenbach, Neville R

    2004-10-26

    The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure (Phi=-75 degrees, Psi=+145 degrees ) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGGAnGGNH2 (n=1-3) over a temperature range from -10 degrees C to +80 degrees C. Use of 15N-labeled alanine substitutions makes it possible to measure 3JalphaN coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter sigma of PII nucleation is evaluated from the coupling constant data. The finding that sigma is close to unity (1 +/- 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine Psi angle, we estimate that, at 40 degrees C, the time-averaged alanine conformation (Phi=-80 degrees, Psi=+170 degrees ) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended beta structure basin, but not a coil.

  3. Functional architectures based on self-assembly of bio-inspired dipeptides: Structure modulation and its photoelectronic applications.

    PubMed

    Chen, Chengjun; Liu, Kai; Li, Junbai; Yan, Xuehai

    2015-11-01

    Getting inspiration from nature and further developing functional architectures provides an effective way to design innovative materials and systems. Among bio-inspired materials, dipeptides and its self-assembled architectures with functionalities have recently been the subject of intensive studies. However, there is still a great challenge to explore its applications likely due to the lack of effective adaptation of their self-assembled structures as well as a lack of understanding of the self-assembly mechanisms. In this context, taking diphenylalanine (FF, a core recognition motif for molecular self-assembly of the Alzheimer's β-amyloid polypeptides) as a model of bio-inspired dipeptides, recent strategies on modulation of dipeptide-based architectures were introduced with regard to both covalent (architectures modulation by coupling functional groups) and non-covalent ways (controlled architectures by different assembly pathways). Then, applications are highlighted in some newly emerging fields of innovative photoelectronic devices and materials, such as artificial photosynthetic systems for renewable solar energy storage and renewable optical waveguiding materials for optoelectronic devices. At last, the challenges and future perspectives of these bio-inspired dipeptides are also addressed.

  4. Pseudopeptidic cages as receptors for N-protected dipeptides.

    PubMed

    Faggi, Enrico; Moure, Alejandra; Bolte, Michael; Vicent, Cristian; Luis, Santiago V; Alfonso, Ignacio

    2014-05-16

    The molecular recognition of short peptides is a challenge in supramolecular chemistry, and the use of peptide-like cage receptors represents a promising approach. Here we report the synthesis and characterization of a diverse family of pseudopeptidic macrobicycles, as well as their binding abilities toward N-protected dipeptides using a combination of different techniques (NMR, ESI-MS, and fluorescence spectroscopy). The cage hosts were assayed for dipeptide binding using competition ESI-MS experiments as high-throughput screening to obtain general trends for the recognition phenomena. Selected hosts were additionally studied by NMR spectroscopy ((1)H NMR titration and diffusion-ordered spectroscopy experiments) in different solvents. The results unambiguously demonstrated the formation of the [cage·dipeptide] supramolecular complexes and rendered quantitative information about the strength of the interaction (K(ass)). The structural variables within the pseudopeptidic cage framework that produced a stronger and more selective recognition were thus identified. The cages showed a remarkable selectivity for N-protected dipeptides with an aromatic amino acid at the carboxylic terminus, which prompted us to propose a mode of binding based on polar and nonpolar noncovalent interactions. Accordingly, we faced the molecular recognition of a target dipeptide (Ac-EY-OH) mimicking a biologically relevant sequence by NMR and fluorescence spectroscopy in highly competitive media.

  5. Synthesis and conformational analysis of hybrid α/β-dipeptides incorporating S-glycosyl-β(2,2)-amino acids.

    PubMed

    García-González, Iván; Mata, Lara; Corzana, Francisco; Jiménez-Osés, Gonzalo; Avenoza, Alberto; Busto, Jesús H; Peregrina, Jesús M

    2015-01-12

    We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α-amino acid attached to a quaternary glyco-β-amino acid. In particular, we combined a S-glycosylated β(2,2)-amino acid and two different types of α-amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β-dipeptides. The key step in the synthesis involved the ring-opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur-containing nucleophile by using 1-thio-β-D-glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time-averaged restraints (MD-tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β-amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α-amino acids due to the presence of CH-π interactions between the phenyl or indole ring and the methyl groups of the β-amino acid unit.

  6. Synthesis of a Gln-Phe hydroxy-ethylene dipeptide isostere.

    PubMed

    Haug, Bengt Erik; Rich, Daniel H

    2004-12-09

    [structure: see text] The protected Gln-Phe hydroxyethylene dipeptide isostere 1 was synthesized as a precursor for preparation of potential inhibitors of Botulinum neurotoxin B metalloprotease. The method allows for the synthesis of additional hydroxyethylene dipeptide isosteres such as 2 with functionalized P1 side chains. The isosteres prepared were coupled with a dipeptide to produce protected pseudotetrapeptide derivatives.

  7. Inhibitors of alanine racemase enzyme: a review.

    PubMed

    Azam, Mohammed Afzal; Jayaram, Unni

    2016-08-01

    Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alaphosphin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.

  8. [Raman scattering study of DL-alanine].

    PubMed

    Gong, Yan; Wang, Wen-qing

    2006-01-01

    Studies of Raman vibration spectra are useful to obtaining information on biomolecular crystals. The cell dimensions of the L- and DL-alanine crystals are nearly identical, and both structures belong to the orthorhombic system, but the space group is P2(1) 2(1) 2(1) for the L-isomer, and Pna2(1) for the racemate crystal. The Raman spectrum of L-alanine has been measured by many authors. The present work is focusing on the Raman scattering study of DL-alanine powder. Based on the analysis of the differences between DL-alanine and L-alanine Raman spectra, the authors obtained indispensable information on hydrogen bond and the motion of the molecular conformation in alanine crystals.

  9. Synthesis and alkylation of aza-glycinyl dipeptide building blocks.

    PubMed

    Garcia-Ramos, Yesica; Lubell, William D

    2013-12-01

    Aza-glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza-glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by-products. Employing N,N'-disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza-glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza-glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza-propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross-couplings, dipolar cycloadditions and intramolecular exo-dig cycloadditions to furnish a variety of azapeptide building blocks.

  10. Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

    NASA Astrophysics Data System (ADS)

    Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

    2000-02-01

    Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

  11. A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases

    SciTech Connect

    Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Joachimiak, Andrzej; Mucha, Artur

    2016-07-01

    N0 -substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki ¼ 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2- diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region.

  12. Vibrational dynamics of crystalline L-alanine

    SciTech Connect

    Bordallo, H.N.; Eckert, J.; Barthes, M.

    1997-11-01

    The authors report a new, complete vibrational analysis of L-alanine and L-alanine-d{sub 4} which utilizes IINS intensities in addition to frequency information. The use of both isotopomers resulted in a self-consistent force field for and assignment of the molecular vibrations in L-alanine. Some details of the calculation as well as a comparison of calculated and observed IINS spectra are presented. The study clarifies a number of important issues on the vibrational dynamics of this molecule and presents a self-consistent force field for the molecular vibrations in crystalline L-alanine.

  13. Catalytic Stereoinversion of L-Alanine to Deuterated D-Alanine.

    PubMed

    Moozeh, Kimia; So, Soon Mog; Chin, Jik

    2015-08-03

    A combination of an achiral pyridoxal analogue and a chiral base has been developed for catalytic deuteration of L-alanine with inversion of stereochemistry to give deuterated D-alanine under mild conditions (neutral pD and 25 °C) without the use of any protecting groups. This system can also be used for catalytic deuteration of D-alanine with retention of stereochemistry to give deuterated D-alanine. Thus a racemic mixture of alanine can be catalytically deuterated to give an enantiomeric excess of deuterated D-alanine. While catalytic deracemization of alanine is forbidden by the second law of thermodynamics, this system can be used for catalytic deracemization of alanine with deuteration. Such green and biomimetic approach to catalytic stereocontrol provides insights into efficient amino acid transformations.

  14. Insertion of beta-alanine in model peptides for copper binding to His96 and His111 of the human prion protein.

    PubMed

    Rivillas-Acevedo, Lina; Maciel-Barón, Luis; García, Javier E; Juaristi, Eusebio; Quintanar, Liliana

    2013-09-01

    The prion protein coordinates copper with high affinity in the regions encompassing residues 92-99 (GGGTHSQW) and 106-115 (KTNMKHMAGA). Cu(II) binding to these sites involves the coordination of the His96/His111 imidazole ring and backbone deprotonated amides that precede the His residue. Such a coordination arrangement involves the formation of hexa- and penta-membered cycles that provide further stabilization of the metal-peptide complex. The purpose of the present study is to introduce a methylene group in the peptide backbone, to evaluate the impact of increasing the size of these cycles in Cu(II) binding. Thus, a β-alanine residue was inserted at different positions preceding the His residue in these prion fragments, and their Cu(II) coordination properties were assessed by UV-Visible absorption, circular dichroism, and electron paramagnetic resonance. Spectroscopic data show that the insertion of a methylene group leads to a completely different Cu(II) coordination that involves the His96/His111 imidazole ring and nitrogen or oxygen atoms provided by the peptide backbone towards the C-terminal. This study clearly shows that two main factors determine the nature of Cu(II)-peptide complexes involving an anchoring His residue and deprotonated amides from the backbone chain: i) the stabilization of Cu(II)-peptide complexes due to the formation of cyclic structures (i.e. chelate effect) and ii) the nature of the residues associated to the deprotonated amide groups that participate in metal ion coordination.

  15. The Role of Dipeptide Repeats in C9ORF72-Related ALS-FTD

    PubMed Central

    Freibaum, Brian D.; Taylor, J. Paul

    2017-01-01

    Expansion of a hexanucleotide (GGGGCC) repeat in the gene chromosome 9 open reading frame 72 (C9ORF72) is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Three non-exclusive mechanisms have been proposed to contribute to the pathology initiated by this genetic insult. First, it was suggested that decreased expression of the C9orf72 protein product may contribute to disease. Second, the recognition that C9ORF72-related disease is associated with accumulation of GGGGCC repeat-containing RNA in nuclear foci led to the suggestion that toxic gain of RNA function, perhaps related to sequestration of RNA-binding proteins, might be an important driver of disease. Third, it was subsequently appreciated that GGGGCC repeat-containing RNA undergoes unconventional translation to produce unnatural dipeptide repeat (DPR) proteins that accumulate in patient brain early in disease. DPRs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DPRs: glycine–alanine (GA), glycine–arginine (GR), proline–alanine (PA), proline–arginine (PR) and glycine–proline (GP; GP is generated from both the sense and antisense reading frames). However, the relative contribution of each DPR to disease pathogenesis remains unclear. Here, we review evidence for the contribution of each specific DPR to pathogenesis and examine the probable mechanisms through which these DPRs induce neurodegeneration. We also consider the association of the toxic DPRs with impaired RNA metabolism and alterations to the liquid-like state of non-membrane-bound organelles. PMID:28243191

  16. Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry

    PubMed Central

    2015-01-01

    An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited Ki values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models. PMID:26985300

  17. Specific inhibition of endopeptidase 24.16 by dipeptides.

    PubMed

    Dauch, P; Vincent, J P; Checler, F

    1991-12-05

    The inhibitory effect of various dipeptides on the neurotensin-degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10-Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro-Xaa dipeptides, the most potent inhibitory effect was elicited by Pro-Ile (Ki approximately 90 microM) with Pro-Ile greater than Pro-Met greater than Pro-Phe. All the Xaa-Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro-Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro-Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin-converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro-Ile was carboxypeptidase A, although it was with a 50-fold lower potency (Ki approximately 5 mM) than for endopeptidase 24.16. By means of fluorimetric substrates with a series of hydrolysing activities, we demonstrate that Pro-Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.

  18. The Dipeptide Ala-Gly in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Bermúdez, Celina; Varela, Marcelino; Cabezas, Carlos; Peña, Isabel; Alonso, José L.

    2014-06-01

    The dipeptide Ala-Gly has been examined in gas phase by laser ablation molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopy in the frequency region 3-12 GHz. Three rotamers have been detected in the supersonic expansion. The quadrupole hyperfine structure of two 14N (I=1) nuclei has been totally resolved allowing the conclusive identification of one conformer.

  19. Functional characterization of dipeptide transport system in human jejunum.

    PubMed

    Adibi, S A; Soleimanpour, M R

    1974-05-01

    The present studies were performed to determine whether dipeptide absorption in human jejunum exhibits the characteristics of carrier-mediated transport. 15-cm jejunal segments from human volunteers were perfused with test solutions containing varying amounts of either glycylglycine, glycylleucine, glycine, leucine, glycylglycine with leucine or glycine, glycylglycine with glycylleucine, or glycylleucine with an equimolar mixture of free glycine and leucine. Jejunal absorption rates of both glycylglycine and glycylleucine followed the kinetics of a saturable process. The K(m) value in millimoles/liter of glycylglycine was significantly greater than the K(m) value of glycylleucine (43.3+/-2.6 vs. 26.8+/-5.9, P < 0.05); and the K(m) value of glycine was also significantly greater than the K(m) value of leucine (42.7+/-7.5 vs. 20.4+/-5.4, P < 0.05). While overlapping occurred among the K(m) values of free amino acids and dipeptides, the transport kinetics of dipeptides were characterized by higher V(max) values (in micromoles per minute per 15 centimeters) than those of free amino acids. For example, the V(max) values for glycylglycine and glycine were 837+/-62 and 590+/-56, respectively (P < 0.02). While jejunal absorption rates of glycylglycine were not significantly affected by free leucine or free glycine, they were competitively inhibited by glycylleucine. The jejunal absorption rate of glycylleucine was not significantly altered by an equimolar mixture of free glycine and leucine. The selective absorption of dipeptides was investigated by infusing three equimolar mixtures, each containing two different dipeptides. Among the three dipeptides examined, glycylglycine was the least absorbed. There was no significant difference between the absorption of glycylleucine and leucylglycine. The above studies suggest that absorption of both glycylglycine and glycylleucine is mediated by a carrier which is not shared with free neutral amino acids; and that both COOH- and NH

  20. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.

    PubMed

    Jovičić, Ana; Mertens, Jerome; Boeynaems, Steven; Bogaert, Elke; Chai, Noori; Yamada, Shizuka B; Paul, Joseph W; Sun, Shuying; Herdy, Joseph R; Bieri, Gregor; Kramer, Nicholas J; Gage, Fred H; Van Den Bosch, Ludo; Robberecht, Wim; Gitler, Aaron D

    2015-09-01

    C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.

  1. l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

    PubMed

    Leite, Jaqueline Santos Moreira; Raizel, Raquel; Hypólito, Thaís Menezes; Rosa, Thiago Dos Santos; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

  2. Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, D-alanine-D-alanine ligase A, from Xanthomonas oryzae pv. oryzae.

    PubMed

    Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Kim, Hyesoon; Ahn, Yeh-Jin; Kim, Jeong-Gu; Lee, Byoung-Moo; Kang, Lin-Woo

    2008-12-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. D-Alanine-D-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in D-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of D-alanyl-D-alanine from two D-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 A resolution and belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 83.0, c = 97.6 A. There is one molecule in the asymmetric unit, with a corresponding V(M) of 1.88 A(3) Da(-1) and a solvent content of 34.6%. The initial structure was determined by molecular replacement using D-alanine-D-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.

  3. Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

    PubMed

    Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Wang-Chuan; Wang, Yeng-Tseng; Lee, Yu-Ching; Lu, Chung-Kuang; Don, Ming-Jaw; Chang, Chang-Yu; Lee, Ching-Hsiao; Lin, Hui-Hsiung; Hsu, Hung-Ju; Hsiao, Nai-Wan

    2015-07-15

    Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

  4. Alanine increases blood pressure during hypotension

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Maher, T. J.; Wurtman, R. J.

    1990-01-01

    The effect of L-alanine administration on blood pressure (BP) during haemorrhagic shock was investigated using anesthetized rats whose left carotid arteries were cannulated for BP measurement, blood removal, and drug administration. It was found that L-alanine, in doses of 10, 25, 50, 100, and 200 mg/kg, increased the systolic BP of hypotensive rats by 38 to 80 percent (while 100 mg/kg pyruvate increased BP by only 9.4 mmhg, not significantly different from saline). The results suggest that L-alanine might influence cardiovascular function.

  5. How similar is the electronic structures of β-lactam and alanine?

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhojyoti; Ahmed, Marawan; Wang, Feng

    2016-02-01

    The C1s spectra of β-lactam i.e. 2-azetidinone (C3H5NO), a drug and L-alanine (C3H7NO2), an amino acid, exhibit striking similarities, which may be responsible for the competition between 2-azetidinone and the alanyl-alanine moiety in biochemistry. The present study is to reveal the degree of similarities and differences between their electronic structures of the two model molecular pairs. It is found that the similarities in C1s and inner valence binding energy spectra are due to their bonding connections but other properties such as ring structure (in 2-azetidinone) and chiral carbon (alanine) can be very different. Further, the inner valence region of ionization potential greater than 18 eV for 2-azetidinone and alanine is also significantly similar. Finally the strained lactam ring exhibits more chemical reactivity measured at all non-hydrogen atoms by Fukui functions with respect to alanine.

  6. Marked enhancement in vivo of adjuvant activity of muramyl dipeptide to protein antigens and to synthetic weak immunogens with monoclonal anti-muramyl dipeptide antibodies.

    PubMed Central

    Bahr, G M; Tello, D S; Chedid, L A

    1985-01-01

    Priming of mice with complexes of antigen coupled to muramyl dipeptide and monoclonal anti-muramyl dipeptide antibodies enhanced the adjuvant activity of muramyl dipeptide on the humoral response to the antigen. The enhancement did not occur with free (uncoupled) muramyl dipeptide and required the presence of an adjuvant-active hapten within the complex as well as the Fc fragment of the monoclonal antibody. This system proved highly effective in eliciting antibodies to synthetic weak immunogens whereas muramyl dipeptide, on its own, exerts very little or no adjuvant activity. The effect was not due to a general polyclonal activation and was restricted to the antigen coupled to the synthetic adjuvant. Possible pathways involved in this phenomenon are discussed. PMID:4018873

  7. Muramyl dipeptide-Lys stimulates the function of human dendritic cells.

    PubMed

    Todate, A; Suda, T; Kuwata, H; Chida, K; Nakamura, H

    2001-11-01

    Muramyl dipeptide (MDP)-Lys (L18), a synthetic MDP analogue derived from bacterial cell walls, has been reported to be a potent immunoadjuvant that enhances protective immunity against pathogens and tumors by stimulating immune-competent cells, such as monocytes and macrophages. However, it is not known whether MDP-Lys modulates the function of dendritic cells (DCs), which are the most potent antigen-presenting cells and play a crucial role in initiating T cell-mediated immunity. Therefore, we examined the effects of MDP-Lys on the expression of surface molecules, cytokine production, and antigen-presenting function of human DCs generated from peripheral blood cells in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. We found that MDP-Lys markedly up-regulated the expression of CD80, CD83, CD86, and CD40, but not human leukocyte antigen-DR, and stimulated the production of tumor necrosis factor-alpha, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs in a dose-dependent manner. Furthermore, MDP-Lys-treated DCs showed enhanced antigen-presenting function compared with untreated DCs, as assessed by an allogeneic mixed lymphocyte reaction. These results suggested that the immunoadjuvant activity of MDP-Lys in vivo is mediated, in part, by its stimulation of DC function.

  8. Production of Alanine by Fusarium moniliforme

    PubMed Central

    Carito, Sebastian L.; Pisano, Michael A.

    1966-01-01

    Fusarium moniliforme grown in a chemically defined medium in submerged culture accumulated amino acids extracellularly. Alanine and glutamic acid were present in greatest amounts, with traces of glycine, lysine, threonine, and valine detectable. Increasing the glucose and urea concentrations of the medium increased yields of alanine. Further increases in alanine production occurred with elevated levels of mineral salts in the medium, whereas the addition of a vitamin mixture proved to be inhibitory. Chemical changes resulting from the growth of F. moniliforme in the final fermentation medium disclosed maximal alanine production, mycelial weight, and glucose consumption after 72 hr of incubation at 28.5 C. Total soluble nitrogen, by contrast, was minimal at the same time period. The pH remained in the alkaline range throughout the fermentation. PMID:5914495

  9. The unresolved puzzle why alanine extensions cause disease.

    PubMed

    Winter, Reno; Liebold, Jens; Schwarz, Elisabeth

    2013-08-01

    The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

  10. Solved? The reductive radiation chemistry of alanine.

    PubMed

    Pauwels, Ewald; De Cooman, Hendrik; Waroquier, Michel; Hole, Eli O; Sagstuen, Einar

    2014-02-14

    The structural changes throughout the entire reductive radiation-induced pathway of l-α-alanine are solved on an atomistic level with the aid of periodic DFT and nudged elastic band (NEB) simulations. This yields unprecedented information on the conformational changes taking place, including the protonation state of the carboxyl group in the "unstable" and "stable" alanine radicals and the internal transformation converting these two radical variants at temperatures above 220 K. The structures of all stable radicals were verified by calculating EPR properties and comparing those with experimental data. The variation of the energy throughout the full radiochemical process provides crucial insight into the reason why these structural changes and rearrangements occur. Starting from electron capture, the excess electron quickly localizes on the carbon of a carboxyl group, which pyramidalizes and receives a proton from the amino group of a neighboring alanine molecule, forming a first stable radical species (up to 150 K). In the temperature interval 150-220 K, this radical deaminates and deprotonates at the carboxyl group, the detached amino group undergoes inversion and its methyl group sustains an internal rotation. This yields the so-called "unstable alanine radical". Above 220 K, triggered by the attachment of an additional proton on the detached amino group, the radical then undergoes an internal rotation in the reverse direction, giving rise to the "stable alanine radical", which is the final stage in the reductive radiation-induced decay of alanine.

  11. Facile access to unnatural dipeptide-alcohols based on cis-2,5-disubstituted pyrrolidines.

    PubMed

    Jia, Yan-Yan; Li, Xiao-Ye; Wang, Ping-An; Wen, Ai-Dong

    2015-02-11

    Well-defined unnatural dipeptide-alcohols based on a cis-2,5-disubstitued pyrrolidine backbone were synthesized from commercially available starting materials meso-diethyl-2,5-dibromoadipate, (S)-(-)-1-phenylethylamine, and phenylalaninol. The structures of these unnatural dipeptide-alcohols are supported by HRMS, 1H- and 13C-NMR spectroscopy. These unnatural dipeptide-alcohols can act as building blocks for peptidomimetics.

  12. Evaluation of toxicity on epithelial and tumor cells of biaryl dipeptide tyrosines.

    PubMed

    Vasconcelos, Stanley N S; Drewes, Carine C; de Vinci Kanda Kupa, Leonard; Farsky, Sandra H P; Stefani, Hélio A

    2016-05-23

    We report a method to obtain biaryl dipeptide tyrosine via Suzuki-Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells.

  13. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.

  14. Synthesis and characterization of novel dipeptide ester prodrugs of acyclovir

    NASA Astrophysics Data System (ADS)

    Nashed, Yasser E.; Mitra, Ashim K.

    2003-07-01

    Four dipeptide (Gly-Gly, Gly-Val, Val-Val, Val-Gly) ester prodrugs of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV) were synthesized. LC/MS was used to characterize the new prodrugs. Both 1H NMR and 13C NMR spectra of the four prodrugs of ACV were measured and assigned based on spectral comparison with compounds of similar structures.

  15. Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli.

    PubMed

    Hayashi, Mikiro; Tabata, Kazuhiko; Yagasaki, Makoto; Yonetani, Yoshiyuki

    2010-03-01

    L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.

  16. Mimosine Dipeptide Enantiomsers: Improved Inhibitors against Melanogenesis and Cyclooxygenase.

    PubMed

    Nguyen, Binh Cao Quan; Tawata, Shinkichi

    2015-08-06

    Melanogenesis plays an important role in the protection of skin against UV through production of melanin pigments, but abnormal accumulation of this pigment causes unaesthetic hyperpigmentation. Much effort is being made to develop effective depigmenting agents. Here, we show for the first time that a small library of mimosine dipeptide enantiomers (Mi-L/D-amino acid) inhibit the melanogenesis in B16F10 melanoma cells by down-regulating the cellular tyrosinase with little effect on their growth or viability. Two of them, Mi-D-Trp and Mi-D-Val, turned out to be the most potent inhibitors on melanin content and cellular tyrosinase in B16F10 melanoma cells. In addition, most of the mimosine dipeptides were more potent than mimosine for inhibiting cyclooxygenase 1 (COX-1) with IC50 of 18-26 μM. Among them, Mi-L-Val and Mi-L-Trp inhibited cyclooxygenase 2 (COX-2) more potently than indomethacin, with IC50 values of 22 and 19 μM, respectively. Taken together, our results suggest the possibility that mimosine dipeptides could be better candidates (than mimosine) for anti-melanogenic (skin hyperpigmentation treatment) and cyclooxygenase (COX) inhibition.

  17. Failure of synthetic muramyl dipeptide to increase antibacterial resistance.

    PubMed Central

    Finger, H; Wirsing von König, C H

    1980-01-01

    Synthetic muranyl dipeptide, which potentiates antibody production and cellular immune responses at a dosage of 100 to 500 micrograms, did not enhance resistance to intravenous infection with a sublethal dose of 2 X 10(3) to 4 X 10(3) viable Listeria monocytogenes cells in mice when intraperitoneally injected either 20 min or 5 days before infection. Similarly, blockade of the mononuclear phagocyte system by dextran sulfate 500 could not be overcome by pretreatment with muramyl dipeptide. In contrast, dextran sulfate 500-induced loss of antibacterial resistance was found to be completely abolished by intraperitoneal injection of 3 X 10(9) killed Bordetella pertussis organisms when given 4 days before injection of dextran sulfate 500, i.e., 5 days before infection. B. pertussis were also effective in enhancing antibacterial resistance when administered 5 days before infection. The different behavior of the two adjuvants tested is assumed to be due to their different nonspecific proliferative capacities. Thus, B. pertussis are assumed to act by direct stimulation of the mononuclear phagocyte system whereas muramyl dipeptide does not. PMID:6155329

  18. Lipophilic derivative of muramyl dipeptide is more active than muramyl dipeptide in priming macrophages to release superoxide anion.

    PubMed Central

    Pabst, M J; Cummings, N P; Shiba, T; Kusumoto, S; Kotani, S

    1980-01-01

    Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement. PMID:6260655

  19. Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria.

    PubMed

    Reichmann, Nathalie T; Cassona, Carolina Picarra; Gründling, Angelika

    2013-09-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with D-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA-D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers D-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for D-alanine incorporation through a process that has been proposed to proceed via a D-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of D-alanine, indicating that LTA has a role, either direct or indirect, in the efficient D-alanine incorporation into WTA in living cells.

  20. Modeling solvation contributions to conformational free energy changes of biomolecules using a potential of mean force expansion

    SciTech Connect

    Pellegrini, M.; Doniach, S.

    1995-08-15

    The standard free energy perturbation (FEP) techniques for the calculation of conformational free energy changes of a solvated biomolecule involve long molecular dynamics (MD) simulations. We have developed a method for performing the same calculations many orders of magnitude faster. We model the average solvent density around a solute as the product of the relevant solute--solvent correlation functions (CF), following the work of Garcia, Hummer, and Soumpasis. We calculate the CF`s by running Monte Carlo simulations of a single solute atom in a box of explicit water molecules and also angular dependent CF`s for selected pairs of solute atoms. We then build the water shell around a larger solute (e.g., alanine dipeptide) by taking the product of the appropriate CF`s. Using FEP techniques we are able to calculate free energy changes as we rotate the dihedral angles of the alanine dipeptide and we find they are in close agreement with the MD results. We also compute the potential of mean force as a function of distance between two solvated methanes and calculate the contribution of the solvent to the free energy change that results from rotating {ital n}-butane about its dihedral angle. {copyright} {ital 1995} {ital American} {ital Institute} {ital of} {ital Physics}.

  1. Effects of Monovalent Cations on the Sodium-Alanine Interaction in Rabbit Ileum

    PubMed Central

    Frizzell, Raymond A.; Schultz, Stanley G.

    1970-01-01

    H, K, Rb, and Li inhibit Na-dependent alanine influx across the brush border of rabbit ileum. Kinetic analysis indicates that H and K behave as competitive inhibitors of influx so that increasing the concentration of H or K in the mucosal solution is kinetically indistinguishable from decreasing the Na concentration. In addition the coupling between alanine and Na influxes is markedly reduced at pH 2.5. With the exception of H and Li, none of these monovalent cations significantly affects carrier-mediated alanine influx in the absence of Na indicating that their inhibitory effects are largely restricted to the Na-dependent fraction of influx. Increasing H concentration from 0.03 to 3 mM does not affect influx in the absence of Na but markedly inhibits influx in the presence of Na. Li significantly enhances alanine influx in the absence of Na. Ag, UO2, and La also inhibit the Na-dependent fraction of alanine influx. These findings suggest that anionic groups having a pKa of approximately 4 are involved in the interaction between Na and the alanine-carrier complex; present evidence implicates carboxylate groups however, phosphoryl residues cannot be ruled out. The previously proposed kinetic model for the Na-alanine interaction has been extended to accommodate these effects of H and other monovalent cations. The mechanistic and physiological implications of these findings are discussed. PMID:5507092

  2. Characterization of alanine catabolism in Pseudomonas aeruginosa and its importance for proliferation in vivo.

    PubMed

    Boulette, Megan L; Baynham, Patricia J; Jorth, Peter A; Kukavica-Ibrulj, Irena; Longoria, Aissa; Barrera, Karla; Levesque, Roger C; Whiteley, Marvin

    2009-10-01

    The opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised individuals, including individuals with the heritable disease cystic fibrosis. Like the carbon sources metabolized by many disease-causing bacteria, the carbon sources metabolized by P. aeruginosa at the host infection site are unknown. We recently reported that l-alanine is a preferred carbon source for P. aeruginosa and that two genes potentially involved in alanine catabolism (dadA and dadX) are induced during in vivo growth in the rat peritoneum and during in vitro growth in sputum (mucus) collected from the lungs of individuals with cystic fibrosis. The goals of this study were to characterize factors required for alanine catabolism in P. aeruginosa and to assess the importance of these factors for in vivo growth. Our results reveal that dadA and dadX are arranged in an operon and are required for catabolism of l-alanine. The dad operon is inducible by l-alanine, d-alanine, and l-valine, and induction is dependent on the transcriptional regulator Lrp. Finally, we show that a mutant unable to catabolize dl-alanine displays decreased competitiveness in a rat lung model of infection.

  3. Influence of ionization on the conformational preferences of peptide models. Ramachandran surfaces of N-formyl-glycine amide and N-formyl-alanine amide radical cations.

    PubMed

    Gil, Adrià; Sodupe, Mariona; Bertran, Juan

    2009-09-01

    Ramachandran maps of neutral and ionized HCO-Gly-NH2 and HCO-Ala-NH2 peptide models have been built at the B3LYP/6-31++G(d,p) level of calculation. Direct optimizations using B3LYP and the recently developed MPWB1K functional have also been carried out, as well as single-point calculations at the CCSD(T) level of theory with the 6-311++G(2df,2p) basis set. Results indicate that for both peptide models ionization can cause drastic changes in the shape of the PES in such a way that highly disallowed regions in neutral PES become low-energy regions in the radical cation surface. The structures localized in such regions, epsilonL+* and epsilonD+* are highly stabilized due to the formation of 2-centre-3-electron interactions between the two carbonyl oxygens. Inclusion of solvent effects by the conductor-like polarizable continuum model (CPCM) shows that the solute-solvent interaction energy plays an important role in determining the stability order.

  4. Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

    PubMed

    Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

    2001-08-15

    This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

  5. Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin.

    PubMed

    Taddei, Paola; Asakura, Tetsuo; Yao, Juming; Monti, Patrizia

    2004-11-01

    For a deeper insight into the structure of Bombyx mori silk fibroin, some model peptides containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid-phase method and analyzed by Raman spectroscopy in order to clarify their conformation and to evaluate the formation and/or disruption of the ordered structure typical of B. mori silk fibroin upon incorporation of Y, V, and S residues into the basic (AG)n sequence. The Raman results indicated that the silk I structure remains stable only when the Y residue is positioned near the chain terminus; otherwise, a silk I --> silk II conformational transition occurs. The peptides AGVGAGYGAGVGAGYGAGVGAGYG(AG)3 and (AG)3YG(AG)2VGYG(AG)3YG(AG)3 treated with LiBr revealed a prevalent silk II conformation; moreover, the former contained a higher amount of random coil than the latter. This result was explained in relation to the different degrees of interruption of the (AG)n sequence. The Raman analysis of the AGSGAG-containing samples confirmed that the AGSGAG hexapeptide is a good model for the silk II crystalline domain. As the number of AGSGAG repeating units decreased, the random coil content increased. The study of the Y domain (I850/I830 intensity ratio) allowed us to hypothesize that in the packing characteristic of Silk I and Silk II conformations the Y residues experience different environments and hydrogen-bonding arrangements; the packing typical of silk I structure traps the tyrosyl side chains in environments more unfavorable to phenoxyl hydrogen-bonding interactions.

  6. A tandem mass spectrometric investigation of N-(3-ferrocenyl-2-naphthoyl) dipeptide ethyl esters and N-(6-ferrocenyl-2-naphthoyl) dipeptide ethyl esters.

    PubMed

    Rai, Dilip K; Mooney, Áine; Kenny, Peter T M

    2011-10-15

    N-(3-Ferrocenyl-2-naphthoyl) dipeptide ethyl esters 1-4 and N-(6-ferrocenyl-2-naphthoyl) dipeptide ethyl esters 5-8 were prepared by coupling either 3-ferrocenylnaphthalene-2-carboxylic acid or 6-ferrocenylnaphthalene-2-carboxylic acid to the dipeptide ethyl esters GlyGly(OEt) (1, 5), AlaGly(OEt) (2, 6), GlyPhe(OEt) (3, 7) and GlyLeu(OEt) (4, 8), using the standard N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole protocol. Electrospray ionization mass spectrometry (ESI-MS) and laser desorption ionization mass spectrometry (LDI-MS) were employed in conjunction with tandem mass spectrometry in the analysis of N-(3-ferrocenyl-2-naphthoyl) dipeptide ethyl esters 1-4 and N-(6-ferrocenyl-2-naphthoyl) dipeptide ethyl esters 5-8. Radical cations, [M](+•) and [M + H](+) species were both observed in the mass spectra. Intense sodium [M + Na](+) and potassium [M + K](+) adducts were also present. An important diagnostic ion at m/z [M-65](+) was observed in both the MS and MS/MS spectra of the N-(3-ferrocenyl-2-naphthoyl) dipeptide derivatives. Sequence-specific ions were generally not observed in the MS/MS spectra of the N-(3-ferrocenyl-2-naphthoyl) series due to formation of the diagnostic [M-65](+) ion. Sequence-specific ions were observed in the MS/MS spectra of the N-(6-ferrocenyl-2-naphthoyl) dipeptide esters with charge retention on the derivatized N-terminal of the dipeptide. Both series of compounds could be successfully analyzed by MALDI without the use of a matrix (LDI).

  7. The Prototype Dipeptide Gly-Gly a Rotational Study

    NASA Astrophysics Data System (ADS)

    Varela, M.; Cabezas, C.; Mata, S.; Alonso, J. L.

    2013-06-01

    The simplest dipeptide Gly-Gly has been examined for the first time in the gas phase by laser ablation molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopy. The nuclear quadrupole hyperfine structure of two ^{14}N nuclei has been totally resolved allowing the conclusive identification of three conformers in the supersonic expansion. Intramolecular hydrogen bonding interactions have been analyzed on the bases of the structure of the observed conformers. Present results indicate that it is possible to face the study larger peptides using LA-MB-FTMW spectroscopy.

  8. Principles of self-assembly of helical pores from dendritic dipeptides.

    PubMed

    Percec, Virgil; Dulcey, Andrés E; Peterca, Mihai; Ilies, Monica; Nummelin, Sami; Sienkowska, Monika J; Heiney, Paul A

    2006-02-21

    The self-assembly of the dendritic dipeptides (4-3,4-3,5)nG2-CH2-Boc-L-Tyr-L-Ala-OMe and their achiral dendritic alcohol (4-3,4-3,5)nG2-CH2OH precursors, both with n = 1-16, where n represents the number of methylenic units in the alkyl groups of the dendron, are reported. All chiral dendritic dipeptides and achiral dendritic alcohols self-assemble into helical porous columns that are stable in both solution and solid state. The pore diameter (D(pore)) of the columns self-assembled from dendritic dipeptides is approximately 10 A larger than that of structures assembled from dendritic alcohols. The increase of the D(pore) at the transition from dendritic alcohol to dendritic dipeptide is accompanied by a decreased solid angle of the building block. This trend is in agreement with previous pore size-solid angle dependences observed with different protective groups of the dipeptide and primary structures of the dendron. However, within the series of dendritic alcohols and dendritic dipeptides with various n, the D(pore) increases when the solid angle increases. The results of these investigations together with those of previous studies on the role of dipeptide stereochemistry and protective groups on this self-assembly process provide the molecular principles required to program the construction of supramolecular helical pores with diameter controlled at the A level from a single dendritic dipeptide architecture. These principles are expected to be valid for libraries of dendritic dipeptides based on dendrons and dipeptides with various primary structures.

  9. Blood-brain barrier (BBB) toxicity and permeability assessment after L-(4-¹⁰Boronophenyl)alanine, a conventional B-containing drug for boron neutron capture therapy, using an in vitro BBB model.

    PubMed

    Roda, E; Nion, S; Bernocchi, G; Coccini, T

    2014-10-02

    Since brain tumours are the primary candidates for treatment by Boron Neutron Capture Therapy, one major challenge in the selective drug delivery to CNS is the crossing of the blood-brain barrier (BBB). The present pilot study investigated (i) the transport of a conventional B-containing product (i.e., L-(4-(10)Boronophenyl)alanine, L-(10)BPA), already used in medicine but still not fully characterized regarding its CNS interactions, as well as (ii) the effects of the L-(10)BPA on the BBB integrity using an in vitro model, consisting of brain capillary endothelial cells co-cultured with glial cells, closely mimicking the in vivo conditions. The multi-step experimental strategy (i.e. Integrity test, Filter study, Transport assay) checked L-(10)BPA toxicity at 80 µg Boron equivalent/ml, and its ability to cross the BBB, additionally by characterizing the cytoskeletal and TJ's proteins by immunocytochemistry and immunoblotting. In conclusion, a lack of toxic effects of L-(10)BPA was demonstrated, nevertheless accompanied by cellular stress phenomena (e.g. vimentin expression modification), paralleled by a low permeability coefficient (0.39 ± 0.01 × 10(-3)cm min(-1)), corroborating the scarce probability that L-(10)BPA would reach therapeutically effective cerebral concentration. These findings emphasized the need for novel strategies aimed at optimizing boron delivery to brain tumours, trying to ameliorate the compound uptake or developing new targeted products suitable to safely and effectively treat head cancer. Thus, the use of in vitro BBB model for screening studies may provide a useful early safety assessment for new effective compounds.

  10. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  11. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  12. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  13. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  14. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  15. Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2.

    PubMed

    Lauro, Mackenzie L; D'Ambrosio, Elizabeth A; Bahnson, Brian J; Grimes, Catherine Leimkuhler

    2017-04-14

    Genetic mutations in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (Nod2) have demonstrated increased susceptibility to Crohn's disease, an inflammatory bowel disease that is hypothesized to be accompanied by changes in the gut microbiota. Nod2 responds to the presence of bacteria, specifically a fragment of the bacterial cell wall, muramyl dipeptide (MDP). The proposed site of this interaction is the leucine-rich repeat (LRR) domain. Surface plasmon resonance and molecular modeling were used to investigate the interaction of the LRR domain with MDP. A functional and pure LRR domain was obtained from Escherichia coli expression in high yield. The LRR domain binds to MDP with high affinity, with a KD of 212 ± 24 nM. Critical portions of the receptor were determined by mutagenesis of putative binding residues. Fragment analysis of MDP revealed that both the peptide and carbohydrate portion contribute to the binding interaction.

  16. Structure-Functional Study of Tyrosine and Methionine Dipeptides: An Approach to Antioxidant Activity Prediction

    PubMed Central

    Torkova, Anna; Koroleva, Olga; Khrameeva, Ekaterina; Fedorova, Tatyana; Tsentalovich, Mikhail

    2015-01-01

    Quantum chemical methods allow screening and prediction of peptide antioxidant activity on the basis of known experimental data. It can be used to design the selective proteolysis of protein sources in order to obtain products with antioxidant activity. Molecular geometry and electronic descriptors of redox-active amino acids, as well as tyrosine and methionine-containing dipeptides, were studied by Density Functional Theory method. The calculated data was used to reveal several descriptors responsible for the antioxidant capacities of the model compounds based on their experimentally obtained antioxidant capacities against ABTS (2,2′-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonate)) and peroxyl radical. A formula to predict antioxidant activity of peptides was proposed. PMID:26512651

  17. Angiogenic Effects of Dimeric Dipeptide Mimetic of Loop 4 of Nerve Growth Factor.

    PubMed

    Kryzhanovskii, S A; Antipova, T A; Tsorin, I B; Pekeldina, E S; Stolyaruk, V N; Nikolaev, S V; Sorokina, A V; Gudasheva, T A; Seredenin, S B

    2016-08-01

    Angiogenic action of compound GK-2, a dimeric dipeptide mimetic of loop 4 of nerve growth factor (NGF), was studied in in vitro and in vivo experiments. Experiments on human endothelial cell culture HUVEC showed that compound GK-2 significantly (p<0.05) stimulated the initial stage of angiogenesis, and its angiogenic activity was not inferior to the reference neurotrophin NGF. In experiments with hindlimb ischemia modeled in rats, GK-2 (1 mg/kg intraperitoneally for 14 days) significantly increased the total length of capillary vessels (p<0.003) and the number of vessels per 1 mm2 ischemic tissue (p<0.001) in comparison with the control. Our findings indicate that under experimental conditions compound GK-2 exhibits not only angiogenic, but also anti-ischemic activity.

  18. Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei.

    PubMed

    Sinz, Quirin; Schwab, Wilfried

    2012-04-01

    The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively. Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles. It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.

  19. Cyclo-Gly-Pro, a cyclic dipeptide, attenuates nociceptive behaviour and inflammatory response in mice.

    PubMed

    Ferro, Jamylle Nunes de Souza; de Aquino, Fernanda Lima Torres; de Brito, Renan Guedes; dos Santos, Priscila Laíse; Quintans, Jullyana de Souza Siqueira; de Souza, Lucas Costa; de Araújo, Almair Ferreira; Diaz, Bruno Lourenço; Lucca-Júnior, Waldecy; Quintans-Júnior, Lucindo José; Barreto, Emiliano

    2015-12-01

    The present study aimed to investigate the antinociceptive and anti-inflammatory effects of the cyclic dipeptide cyclo-Gly-Pro (CGP) in mice. Antinociceptive activity was assessed by employing different pain models, such as formalin test, acetic acid-induced writhing, hot plate test, and carrageenan-induced hyperalgesia, in mice. The number of c-Fos-immunoreactive cells in the periaqueductal gray (PAG) was evaluated in CGP-treated mice. Anti-inflammatory activity was evaluated using paw oedema induced by carrageenan, compound 48/80, serotonin, and prostaglandin E2 (PGE2) and analyzed by plethysmometry. Quantitation of myeloperoxidase (MPO) in the paw was carried out to analyze the presence of neutrophils in the tissue. Intraperitoneal injection of CGP produced a significant inhibition in both neurogenic and inflammatory phases of formalin-induced pain. The antinociceptive effect of CGP, evaluated in the acetic acid-induced writhing test, was detected for up to 6 h after treatment. Further, in the hot plate test, antinociceptive behaviour was evoked by CGP, and this response was inhibited by naloxone. Animals treated with CGP did not present changes in motor performance. In CGP-treated mice there was an increase in the number of c-Fos-positive neurons in the periaqueductal gray. In another set of experiments, CGP attenuated the hyperalgesic response induced by carrageenan. Furthermore, CGP also reduced the carrageenan-increased MPO activity in paws. In addition, CGP also reduced the paw oedema evoked by compound 48/80, serotonin, and PGE2 . Taken together, these results may support a possible therapeutic application of the cyclic dipeptide cyclo-Gly-Pro toward alleviating nociception and damage caused by inflammation conditions.

  20. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  1. On the existence of ‘L-alanine cadmium bromide'

    NASA Astrophysics Data System (ADS)

    Srinivasan, Bikshandarkoil R.

    2013-12-01

    It is argued that the recently reported nonlinear optical crystal L-alanine cadmium bromide, grown by slow solvent evaporation method at room temperature [P. Ilayabarathi, J. Chandrasekaran, Spectrochim. Acta 96A (2012) 684-689] is the well-known L-alanine crystal. The isolation of L-alanine crystal is explained due to fractional crystallization.

  2. On the existence of 'L-alanine cadmium bromide'.

    PubMed

    Srinivasan, Bikshandarkoil R

    2013-12-01

    It is argued that the recently reported nonlinear optical crystal L-alanine cadmium bromide, grown by slow solvent evaporation method at room temperature [P. Ilayabarathi, J. Chandrasekaran, Spectrochim. Acta 96A (2012) 684-689] is the well-known L-alanine crystal. The isolation of L-alanine crystal is explained due to fractional crystallization.

  3. Convergent Synthesis of a Deuterium Labeled Serine Dipeptide Lipid for Analysis of Biological Samples.

    PubMed

    Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

    2017-03-08

    Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-L-serine, (3S)-L-serine] isolated from Porphyromonas gingivalis,(1) in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria.

  4. A preliminary optimization of alanine blends for ESR dosimetry in a mixed n-γ field: Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Hoseininaveh, M.; Ranjbar, A. H.

    2016-04-01

    In this study, a preliminary work on the enhancement of ESR response of several arrangements of alanine and boron compounds, exposed to a thermal neutron beam, is presented using FLUKA code. A multi-layer dosimeter consist of consecutive layers of alanine and boron compounds showed that the amount of energy deposited in the alanine layers is maximized when their thickness is 5 μm and the thickness of boron compound layers are between 2 and 3 μm. Furthermore, the optimum number of 10B layers in the dosimeter was found to be 35 layers. Moreover, the alanine samples consisting of small spherical grains of boron compounds, arranged regularly in the middle plane of the dosimeters, exposed to a thermal neutron beam, were modeled. The dependence of energy deposition in the alanine material on the size of grains, and on their composition were also studied, as well.

  5. Mycobacterium abscessus ᴅ-alanyl-ᴅ-alanine dipeptidase induces the maturation of dendritic cells and promotes Th1-biased immunity

    PubMed Central

    Lee, Seung Jun; Jang, Jong-Hwa; Yoon, Gun Young; Kang, Da Rae; Park, Hee Jo; Shin, Sung Jae; Han, Hee Dong; Kang, Tae Heung; Park, Won Sun; Yoon, Young Kyung; Soh, Byoung Yul; Jung, In Duk; Park, Yeong-Min

    2016-01-01

    Mycobacterium abscessus, a member of the group of non-tuberculous mycobacteria, has been identified as an emerging pulmonary pathogen in humans. However, little is known about the protective immune response of antigen-presenting cells, such as dendritic cells (DCs), which guard against M. abscessus infection. The M. abscessus gene MAB1843 encodes ᴅ-alanyl-ᴅ-alanine dipeptidase, which catalyzes the hydrolysis of ᴅ-alanyl-ᴅ-alanine dipeptide. We investigated whether MAB1843 is able to interact with DCs to enhance the effectiveness of the host’s immune response. MAB1843 was found to induce DC maturation via toll-like receptor 4 and its downstream signaling pathways, such as the mitogen-activated protein kinase and nuclear factor kappa B pathways. In addition, MAB1843-treated DCs stimulated the proliferation of T cells and promoted Th1 polarization. Our results indicate that MAB1843 could potentially regulate the immune response to M. abscessus, making it important in the development of an effective vaccine against this mycobacterium. [BMB Reports 2016; 49(10): 554-559] PMID:27439605

  6. Differential laser-induced perturbation Raman spectroscopy: a comparison with Raman spectroscopy for analysis and classification of amino acids and dipeptides.

    PubMed

    Oztekin, Erman K; Smith, Sarah E; Hahn, David W

    2015-04-01

    Differential-laser induced perturbation spectroscopy (DLIPS) is a new spectral analysis technique for classification and identification, with key potential applications for analysis of complex biomolecular systems. DLIPS takes advantage of the complex ultraviolet (UV) laser–material interactions based on difference spectroscopy by coupling low intensity UV laser perturbation with a traditional spectroscopy probe. Here, we quantify the DLIPS performance using a Raman scattering probe in classification of basic constituents of collagenous tissues, namely, the amino acids glycine, L-proline, and L-alanine, and the dipeptides glycine–glycine, glycine–alanine and glycine–proline and compare the performance to a traditional Raman spectroscopy probe via several multivariate analyses. We find that the DLIPS approach yields an ~40% improvement in discrimination among these tissue building blocks. The effects of the 193-nm perturbation laser are further examined by assessing the photodestruction of targeted material molecular bonds. The DLIPS method with a Raman probe holds promise for future tissue diagnosis, either as a stand-alone technique or as part of an orthogonal biosensing scheme.

  7. Stimulation of chemiluminescence by synthetic muramyl dipeptide and analogs.

    PubMed

    Masihi, K N; Azuma, I; Brehmer, W; Lange, W

    1983-04-01

    The effect on respiratory burst of murine splenic cells after in vitro exposure to synthetic muramyl dipeptide (MDP) and 6-O-acyl and quinonyl derivatives was studied at an early phase of interaction by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. The MDP molecule enhanced CL, but the degree of CL response varied with the kinds of fatty acids introduced in the chemical structure of synthetic glycopeptide analogs. A 6-O-acyl derivative possessing an alpha-branched fatty acid chain, B30-MDP, stimulated maximum levels of CL activity. High CL responses were obtained with L8-MDP having a short chain of linear fatty acids and with QS-10-MDP-66 containing a ubiquinone compound. CL was also stimulated by MDP and its analogs in the spleen cells of nude mice lacking mature T lymphocytes, but the extent of stimulation was decreased compared with that of normal spleen cells.

  8. Toward very potent, non-covalent organophosphonate inhibitors of cathepsin C and related enzymes by 2-amino-1-hydroxy-alkanephosphonates dipeptides.

    PubMed

    Drąg, Marcin; Wieczerzak, Ewa; Pawełczak, Małgorzata; Berlicki, Łukasz; Grzonka, Zbigniew; Kafarski, Paweł

    2013-08-01

    Cathepsins play an important role in several human disorders and therefore the design and synthesis of their inhibitors attracts considerable interest in current medicinal chemistry approaches. Due to the presence of a strong sulphydryl nucleophile in the active center of the cysteine type cathepsins, most strategies to date have yielded covalent inhibitors. Here we present a series of non-covalent β-amino-α-hydroxyalkanephosphonate dipeptidic inhibitors of cathepsin C, ranking amongst the best low-molecular weight inhibitors of this enzyme. Their binding modes determined by molecular modelling indicate that the hydroxymethyl fragment of the molecule, not the phosphonate moiety, acts as a transition state analogue of peptide bond hydrolysis. These dipeptide mimetics appear also to be potent inhibitors of other cysteine proteases such as papain, cathepsin B and cathepsin K, thus providing new leading structures for these medicinally important enzymes.

  9. Effects of muramyl dipeptide treatment on resistance to infection with Toxoplasma gondii in mice.

    PubMed Central

    Krahenbuhl, J L; Sharma, S D; Ferraresi, R W; Remington, J S

    1981-01-01

    Studies were carried out to determine whether treatment of mice with the synthetic adjuvant muramyl dipeptide afforded any resistance to infection with the obligate intracellular protozoan Toxoplasma gondii. Marked resistance to lethal challenge infection was observed in CBA but not C57BL/6 mice pretreated with muramyl dipeptide. In CBA mice, a single muramyl dipeptide treatment administered 14, 7, or 4 days before Toxoplasma challenge did not afford protection, whereas mice treated at -1 day were highly resistant. Additional studies carried out to investigate the mechanisms underlying the enhanced resistance to Toxoplasma in muramyl dipeptide-treated mice failed to reveal either enhanced cytolytic antibodies to the parasite or evidence that peritoneal macrophages from treated mice were activated as determined in vitro by their microbicidal capacity for Toxoplasma or cytotoxic capacity for tumor target cells. PMID:7216470

  10. Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase

    PubMed Central

    Lee, Hyun-e; Kim, Eun-Hyun; Choi, Hye-Ryung; Sohn, Uy Dong; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan

    2012-01-01

    This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents. PMID:22915995

  11. Dipeptide-derived nitriles containing additional electrophilic sites: potentially irreversible inhibitors of cysteine proteases.

    PubMed

    Löser, Reik; Gütschow, Michael

    2009-12-01

    Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.

  12. Utilization of tyrosine- and histidine-containing dipeptides to enhance productivity and culture viability.

    PubMed

    Kang, Sohye; Mullen, Johanna; Miranda, Les P; Deshpande, Rohini

    2012-09-01

    Adequate supply of nutrients, especially providing a sufficient level of specific amino acids, is essential for cell survival and production. Complex raw materials such as soy hydrolysates or yeast extracts are the source for both free amino acids and peptides. However, typical chemically defined (CD) media provide amino acids only in free form. While most amino acids are highly soluble in media and can be provided at fairly high concentrations, certain amino acids such as tyrosine have poor solubility and thus, only a limited amount can be added as a media component. The limited solubility of amino acids in media can raise the risk of media precipitation and instability, and could contribute to suboptimal culture performance due to insufficient nutrient levels to meet cellular demands. In this study, we examine the use of chemically synthesized dipeptides as an alternative method for delivering amino acids to various monoclonal antibody producing cell lines. In particular, we focus on tyrosine-containing dipeptides. Due to their substantially higher solubility (up to 250-fold as compared with free tyrosine), tyrosine-containing dipeptides can efficiently provide large amounts of tyrosine to cultured cells. When tested in fed-batch processes, these supplemental dipeptides exerted positive effects, including enhanced culture viability and titer. Moreover, dipeptide-supplemented cultures displayed improved metabolic profiles including lower lactate and NH 4(+) production, and better pH maintenance. In bioreactor studies using two-sided pH control, a lactate spike occurring on Day 10 and the concomitant high levels of base addition could be prevented with dipeptide supplementation. These beneficial effects could be obtained by one-time addition of dipeptides during inoculation, and did not require further feeds during the entire 11-15-day process. Non-tyrosine-containing dipeptides, such as His-Gly, also showed improved productivity and viability over control cultures.

  13. Induction of gamma-turn-like structure in ferrocene bearing dipeptide chains via conformational control.

    PubMed

    Moriuchi, Toshiyuki; Nagai, Takayoshi; Hirao, Toshikazu

    2006-01-05

    [structure: see text] A combination of the ferrocene scaffold as a central reverse-turn unit with the dipeptide chains (-L-Pro-L-Ala-NHPy) was demonstrated to induce both inverse gamma-turn-like and antiparallel beta-sheet-like structures. Only the antiparallel beta-sheet-like structure was formed in the ferrocene bearing the heterochiral dipeptide chains (-L-Pro-D-Ala-NHPy), in which highly organized self-assembly was achieved through a network of intermolecular hydrogen bonds.

  14. Interaction of a Model Peptide with a Water--Bilayer System

    NASA Technical Reports Server (NTRS)

    Pohorille, A.; Wilson, M. A.

    1994-01-01

    We discuss a molecular dynamics study of the alanine dipeptide at the interface between water and a glycerol-1-monooleate (GMO) bilayer. The dipeptide is interfacially active and incorporates into the bilayer without disrupting its structure. The interfacial region that is readily penetrated by the dipeptide spans the entire head group portion of the bilayer. The polar groups of the alanine dipeptide mostly remain well solvated by water and the oxygen atoms of GMO, and conformations of the dipeptide are characterized by (phi, psi) angles typical of alpha-helix and beta-sheet structures. When the molecule is deeper in the bilayer, the C(sub 7eq) state also becomes stable. The barrier to the isomerization reaction at the interface is lower than in bulk phases. After 7 ns of trajectories, the system is not fully equilibrated, due to slow collective motions involving GMO head groups. These result in decreased mobility and lower rates of isomerization of the dipeptide at the interface.

  15. Thermodynamics of Deca-alanine Folding in Water.

    PubMed

    Hazel, Anthony; Chipot, Christophe; Gumbart, James C

    2014-07-08

    The determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems. Numerous computational models and methods have been developed for studying structure formation at the atomic level. Due to its small size and simple structure, deca-alanine is used as a model system in molecular dynamics (MD) simulations. The free energy of unfolding in vacuum has been studied extensively using the end-to-end distance of the peptide as the reaction coordinate. However, few studies have been conducted in the presence of explicit solvent. Previous results show a significant decrease in the free energy of extended conformations in water, but the α-helical state is still notably favored over the extended state. Although sufficient in vacuum, we show that end-to-end distance is incapable of capturing the full complexity of deca-alanine folding in water. Using α-helical content as a second reaction coordinate, we deduce a more descriptive free-energy landscape, one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the α-helical state. Equilibrium simulations demonstrate the relative stability of the extended and α-helical states in water as well as the transition between the two states. This work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process.

  16. NQR in Alanine and Lysine Iodates

    NASA Astrophysics Data System (ADS)

    Petrosyan, A. M.; Burbelo, V. M.; Tamazyan, R. A.; Karapetyan, H. A.; Sukiasyan, R. P.

    2000-02-01

    The structure o f iodates of α- and β-alanine ( Ala) (2(β-Ala • HIO3) • H2O , β-Ala-2HIO3 , D L-Ala• HIO3 • 2H2O, L-Ala • HIO3) and L-lysine (L-Lys) (L-Lys • HIO3, L-Lys • 2HIO3,L-Lys • 3HIO3, L-Lys • 6HIO3) have been investigated by means of iodine-127 NQR, IR spectroscopy and X-ray diffraction

  17. Crystal structure of N-(tert-but-oxy-carbon-yl)phenyl-alanylde-hydro-alanine isopropyl ester (Boc-Phe-ΔAla-OiPr).

    PubMed

    Lenartowicz, Paweł; Makowski, Maciej; Zarychta, Bartosz; Ejsmont, Krzysztof

    2014-12-01

    In the title compound, the de-hydro-dipeptide (Boc-Phe-ΔAla-OiPr, C20H28N2O5), the mol-ecule has a trans conformation of the N-methyl-amide group. The geometry of the de-hydro-alanine moiety is to some extent different from those usually found in simple peptides, indicating conjugation between the H2C=C group and the peptide bond. The bond angles around de-hydro-alanine have unusually high values due to the steric hindrance, the same inter-action influencing the slight distortion from planarity of the de-hydro-alanine. The mol-ecule is stabilized by intra-molecular inter-actions between the isopropyl group and the N atoms of the peptide main chain. In the crystal, an N-H⋯O hydrogen bond links the mol-ecules into ribbons, giving a herringbone head-to-head packing arrangement extending along the [100] direction. In the stacks, the mol-ecules are linked by weak C-H⋯O hydrogen-bonding associations.

  18. Discrimination of Enantiomers of Dipeptide Derivatives with Two Chiral Centers by Tetraaza Macrocyclic Chiral Solvating Agents Using (1)H NMR Spectroscopy.

    PubMed

    Fang, Lixia; Lv, Caixia; Wang, Guo; Feng, Lei; Stavropoulos, Pericles; Gao, Guangpeng; Ai, Lin; Zhang, Jiaxin

    2016-12-01

    (1)H NMR spectroscopy is often used to discriminate enantiomers of chiral analytes and determine their enantiomeric excess (ee) by various chiral auxiliaries. In reported research, these studies were mainly focused on chiral discriminantion of chiral analytes with only one chiral center. However, many chiral compounds possessing two or more chiral centers are often found in natural products, chiral drugs, products of asymmetric synthesis and biological systems. Therefore, it is necessary to investigate their chiral discrimination by effective chiral auxiliaries using (1)H NMR spectroscopy. In this paper, a new class of tetraaza macrocyclic chiral solvating agents (TAMCSAs) with two amide (CONH), two amino (NH) and two phenolic hydroxyl (PhOH) groups has been designed and synthsized for chiral discrimination towards dipeptide derivatives with two chiral centers. These dipeptide derivatives are important chiral species because some of them are used as clinical drugs and special dietary supplements for treatment of human diseases, such as L-alanyl-L-glutamine and aspartame. The results show that these TAMCSAs have excellent chiral discriminating properties and offer multiple detection possibilities pertaining to (1)H NMR signals of diagnostic split protons. The nonequivalent chemical shifts (up to 0.486 ppm) of various types of protons of these dipeptide derivatives were evaluated with the assistance of well-resolved (1)H NMR signals in most cases. In addition, enantiomeric excesses (ee) of the dipeptide derivatives with different optical compositions have been calculated based on integration of well-separeted proton signals. At the same time, the possible chiral discriminating behaviors have been discussed by means of Job plots, ESI mass spectra and a proposed theoretical model of (±)-G1 with TAMCSA 1c. Additionally, the association constants of enantiomers of (±)-G5 with TAMCSA 1a were calculated by employing the nonlinear curve-fitting method.

  19. [Search for chaperon-like anticataract drugs, the antiaggregants of lens crystallins. Communication. 1. Chaperon-like activity of N-acetyl carnosine dipeptide: in vitro study on a model of ultraviolet-induced aggregation of betaL-crystallin].

    PubMed

    Muranov, K O; Dizhevskaia, A K; Boldyrev, A A; Karpova, O E; Sheremet, N L; Polunin, G S; Avetisov, S E; Ostrovskiĭ, M A

    2008-01-01

    Aggregation ofcrystallins, the lens proteins, is one of the basic stages of cataract formation. Among the protein aggregation models used to study the molecular mechanisms of the initial stages of lenticular opacity, UV-induced aggregation of betaL-crystallin is most close to the in vivo conditions. The carnosine derivative N-acetyl carnosine has been shown to be effective in inhibiting the UV-induced aggregation of betaL-crystallin. Examination of the accumulation kinetics of carbonyl groups in betaL-crystallin under UV irradiation has indicated that neither carnosine nor N-acetyl carnosine fails to affect this parameter--an indicator of oxidative protein damage. By taking into account also the fact that N-acetyl carnosine is not an antioxidant, it can be believed that the molecular mechanism of action of this compound on UV-induced aggregation of betaL is unassociated with its antioxidative properties. The authors hypothesize that the molecular chaperon-like properties similar to those of alpha-crystallin underlie the mechanism of action of the acetyl derivative carnosine. The prospects for searching anticataract agents of a new chaperon-like class are discussed.

  20. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  1. Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes

    PubMed Central

    Blättner, Sebastian; Das, Sudip; Paprotka, Kerstin; Eilers, Ursula; Krischke, Markus; Kretschmer, Dorothee; Remmele, Christian W.; Dittrich, Marcus; Müller, Tobias; Schuelein-Voelk, Christina; Hertlein, Tobias; Mueller, Martin J.; Huettel, Bruno; Reinhardt, Richard; Ohlsen, Knut; Rudel, Thomas

    2016-01-01

    Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. PMID:27632173

  2. Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

    PubMed

    Blättner, Sebastian; Das, Sudip; Paprotka, Kerstin; Eilers, Ursula; Krischke, Markus; Kretschmer, Dorothee; Remmele, Christian W; Dittrich, Marcus; Müller, Tobias; Schuelein-Voelk, Christina; Hertlein, Tobias; Mueller, Martin J; Huettel, Bruno; Reinhardt, Richard; Ohlsen, Knut; Rudel, Thomas; Fraunholz, Martin J

    2016-09-01

    Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

  3. Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells.

    PubMed

    Bauer, Peter O

    2016-01-26

    A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), together referred to as c9FTD/ALS. It has been suggested that a loss of C9orf72 protein expression, the formation of toxic RNA foci and dipeptide-repeat proteins contribute to C9orf72-related diseases. Interestingly, it has been shown that trimethylation of histones and methylation of CpG islands near the repeat expansion may play a role in the pathogenesis c9FTD/ALS. Recently, methylation of expanded repeat itself has been reported. To further elucidate the mechanisms underlying these diseases, the influence of epigenetic modification in the repeat expansion on its pathogenic effect was assessed. Here, a reduced formation of toxic RNA foci and dipeptide-repeat proteins upon methylation of the GGGGCC repeat in a cellular model of c9FTD/ALS is shown. Additionally, a novel methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels is presented, potentially usable for methylation analysis in patients carrying C9orf72 repeat expansion carriers as a diagnostic tool. Presented results suggest that increased level of pathogenic GGGGCC expansion methylation may be sufficient to alleviate the molecular pathology of the C9orf72-related diseases.

  4. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

  5. Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

    SciTech Connect

    Faraci, W.S.; Walsh, C.T.

    1988-05-03

    Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.

  6. Surface enhanced Raman spectroscopy of L-alanyl-L-tryptophan dipeptide adsorbed on Si substrate decorated with triangular silver nanoplates

    NASA Astrophysics Data System (ADS)

    Ramanauskaite, Lina; Snitka, Valentinas

    2015-03-01

    Raman and surface enhanced Raman spectroscopies have been used to investigate interaction of L-alanyl-L-tryptophan (Ala-Trp) dipeptide with nanostructured silver surface. A highly sensitive surface enhanced Raman scattering (SERS)-based biosensor has been employed for label-free dipeptide detection and its orientation on the nanostructured surface. The synthesis of SERS substrate was based on direct silver ions reduction on hydrofluoric acid etched silicon wafer. Raman and SERS spectra of Ala-Trp were recorded in liquid to keep native environment for dipeptide. To the best of our knowledge, this work is a first attempt to analyze the structure of Ala-Trp dipeptide by Raman spectroscopy.

  7. Absorbed dose determination in kilovoltage X-ray synchrotron radiation using alanine dosimeters.

    PubMed

    Butler, D J; Lye, J E; Wright, T E; Crossley, D; Sharpe, P H G; Stevenson, A W; Livingstone, J; Crosbie, J C

    2016-12-01

    Alanine dosimeters from the National Physical Laboratory (NPL) in the UK were irradiated using kilovoltage synchrotron radiation at the imaging and medical beam line (IMBL) at the Australian Synchrotron. A 20 × 20 mm(2) area was irradiated by scanning the phantom containing the alanine through the 1 mm × 20 mm beam at a constant velocity. The polychromatic beam had an average energy of 95 keV and nominal absorbed dose to water rate of 250 Gy/s. The absorbed dose to water in the solid water phantom was first determined using a PTW Model 31014 PinPoint ionization chamber traceable to a graphite calorimeter. The alanine was read out at NPL using correction factors determined for (60)Co, traceable to NPL standards, and a published energy correction was applied to correct for the effect of the synchrotron beam quality. The ratio of the doses determined by alanine at NPL and those determined at the synchrotron was 0.975 (standard uncertainty 0.042) when alanine energy correction factors published by Waldeland et al. (Waldeland E, Hole E O, Sagstuen E and Malinen E, Med. Phys. 2010, 37, 3569) were used, and 0.996 (standard uncertainty 0.031) when factors by Anton et al. (Anton M, Büermann L., Phys Med Biol. 2015 60 6113-29) were used. The results provide additional verification of the IMBL dosimetry.

  8. Structural and Biochemical Analyses of Mycobacterium tuberculosis N-Acetylmuramyl-l-alanine Amidase Rv3717 Point to a Role in Peptidoglycan Fragment Recycling*

    PubMed Central

    Prigozhin, Daniil M.; Mavrici, Daniela; Huizar, John P.; Vansell, Hilary J.; Alber, Tom

    2013-01-01

    Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-l-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn2+ binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn2+ is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling. PMID:24019530

  9. On the roles of the alanine and serine in the β-sheet structure of fibroin.

    PubMed

    Carrascoza Mayen, Juan Francisco; Lupan, Alexandru; Cosar, Ciprian; Kun, Attila-Zsolt; Silaghi-Dumitrescu, Radu

    2015-02-01

    In its silk II form, fibroin is almost exclusively formed from layers of β-sheets, rich in glycine, alanine and serine. Reported here are computational results on fibroin models at semi-empirical, DFT levels of theory and molecular dynamics (MD) for (Gly)10, (Gly-Ala)5 and (Gly-Ser)5 decapeptides. While alanine and serine introduce steric repulsions, the alanine side-chain adds to the rigidity of the sheet, allowing it to maintain a properly pleated structure even in a single β-sheet, and thus avoiding two alternative conformations which would interfere with the formation of the multi-layer pleated-sheet structure. The role of the serine is proposed to involve modulation of the hydrophobicity in order to construct the supramolecular assembly as opposed to random precipitation due to hydrophobicity.

  10. International society of sports nutrition position stand: Beta-Alanine.

    PubMed

    Trexler, Eric T; Smith-Ryan, Abbie E; Stout, Jeffrey R; Hoffman, Jay R; Wilborn, Colin D; Sale, Craig; Kreider, Richard B; Jäger, Ralf; Earnest, Conrad P; Bannock, Laurent; Campbell, Bill; Kalman, Douglas; Ziegenfuss, Tim N; Antonio, Jose

    2015-01-01

    The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of beta-alanine supplementation. Based on the current available literature, the conclusions of the ISSN are as follows: 1) Four weeks of beta-alanine supplementation (4-6 g daily) significantly augments muscle carnosine concentrations, thereby acting as an intracellular pH buffer; 2) Beta-alanine supplementation currently appears to be safe in healthy populations at recommended doses; 3) The only reported side effect is paraesthesia (tingling), but studies indicate this can be attenuated by using divided lower doses (1.6 g) or using a sustained-release formula; 4) Daily supplementation with 4 to 6 g of beta-alanine for at least 2 to 4 weeks has been shown to improve exercise performance, with more pronounced effects in open end-point tasks/time trials lasting 1 to 4 min in duration; 5) Beta-alanine attenuates neuromuscular fatigue, particularly in older subjects, and preliminary evidence indicates that beta-alanine may improve tactical performance; 6) Combining beta-alanine with other single or multi-ingredient supplements may be advantageous when supplementation of beta-alanine is high enough (4-6 g daily) and long enough (minimum 4 weeks); 7) More research is needed to determine the effects of beta-alanine on strength, endurance performance beyond 25 min in duration, and other health-related benefits associated with carnosine.

  11. Electrospray ionization tandem mass spectrometric study of protonated and alkali- cationized α/ε-hybrid peptides: differentiation of a pair of dipeptide positional isomers.

    PubMed

    Ramesh Babu, A; Raju, G; Purna Chander, C; Shoban Babu, B; Srinivas, R; Sharma, G V M

    2016-01-01

    A new class of Boc-N-protected hybrid peptides derived from L- Ala and ε(6)-Caa (L-Ala = L-Alanine, Caa = C-linked carboamino acid derived from D-xylose) have been studied by positive ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS(n) spectra of protonated and alkali-cationized hybrid peptides produce characteristic fragmentation involving the peptide backbone, the tert-butyloxycarbonyl (Boc) group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated and alkali-cationized peptides. The CID of [M + H](+) ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1) shows a prominent [M + H - C4H8](+) ion, which is totally absent for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6), which instead shows significant loss of t-butanol. The formation of the [M + Cat - C4H8](+) ion is totally absent and [M + Cat - Boc + H](+) is prominent in the CID of the [M + Cat](+) ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1), whereas the former is highly abundant and the latter is of low abundance for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6). It is observed that 'b' ions are abundant when oxazolone structures are formed through a five-membered cyclic transition state in tetra-, penta-, and hexapeptides and the cyclization process for larger 'b' ions led to an insignificant abundance. However, the significant 'b' ion is formed in ε,α-dipeptide, which may have a seven-membered substituted 2-oxoazepanium ion structure. The MS(n) spectra of [M + Cat - Boc + H](+) ions of these peptides are found to be significantly different to those of [M + H - Boc + H](+) ions. The CID spectra of [M + Cat - Boc + H](+) ions of peptide acids containing L-Ala at the C-terminus show an abundant N-terminal rearrangement ion, [b(n) + 17 + Cat](+), which is absent for the peptide acids containing ε-Caa at the C-terminus. Thus, the results of these hybrid peptides

  12. Alanine aminotransferase controls seed dormancy in barley

    PubMed Central

    Sato, Kazuhiro; Yamane, Miki; Yamaji, Nami; Kanamori, Hiroyuki; Tagiri, Akemi; Schwerdt, Julian G.; Fincher, Geoffrey B.; Matsumoto, Takashi; Takeda, Kazuyoshi; Komatsuda, Takao

    2016-01-01

    Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. PMID:27188711

  13. Predicting cancerlectins by the optimal g-gap dipeptides

    NASA Astrophysics Data System (ADS)

    Lin, Hao; Liu, Wei-Xin; He, Jiao; Liu, Xin-Hui; Ding, Hui; Chen, Wei

    2015-12-01

    The cancerlectin plays a key role in the process of tumor cell differentiation. Thus, to fully understand the function of cancerlectin is significant because it sheds light on the future direction for the cancer therapy. However, the traditional wet-experimental methods were money- and time-consuming. It is highly desirable to develop an effective and efficient computational tool to identify cancerlectins. In this study, we developed a sequence-based method to discriminate between cancerlectins and non-cancerlectins. The analysis of variance (ANOVA) was used to choose the optimal feature set derived from the g-gap dipeptide composition. The jackknife cross-validated results showed that the proposed method achieved the accuracy of 75.19%, which is superior to other published methods. For the convenience of other researchers, an online web-server CaLecPred was established and can be freely accessed from the website http://lin.uestc.edu.cn/server/CalecPred. We believe that the CaLecPred is a powerful tool to study cancerlectins and to guide the related experimental validations.

  14. Rational and combinatorial tailoring of bioactive cyclic dipeptides.

    PubMed

    Giessen, Tobias W; Marahiel, Mohamed A

    2015-01-01

    Modified cyclic dipeptides represent a diverse family of microbial secondary metabolites. They display a broad variety of biological and pharmacological activities and have long been recognized as privileged structures with the ability to bind to a wide range of receptors. This is due to their conformationally constrained 2, 5-diketopiperazine (DKP) scaffold and the diverse set of DKP tailoring enzymes present in nature. After initial DKP assembly through different biosynthetic systems modifying enzymes are responsible for installing functional groups crucial for the biological activities of the resulting modified DKPs. They represent a vast and largely untapped enzyme repository very useful for synthetic biology approaches aiming at introducing structural variations into DKP scaffolds. In this review we focus on these DKP modification enzymes found in various microbial secondary metabolite gene clusters. We will give a brief overview of their distribution and highlight a select number of characterized DKP tailoring enzymes before turning to their application potential in combinatorial biosynthesis with the aim of producing molecules with improved or entirely new biological and medicinally relevant properties.

  15. Identifying Antioxidant Proteins by Using Optimal Dipeptide Compositions.

    PubMed

    Feng, Pengmian; Chen, Wei; Lin, Hao

    2016-06-01

    Antioxidant proteins are a kind of molecules that can terminate cellular and DNA damages caused by free radical intermediates. The use of antioxidant proteins for prevention of diseases has been intensively studied in recent years. Thus, accurate identification of antioxidant proteins is essential for understanding their roles in pharmacology. In this study, a support vector machine-based predictor called AodPred was developed for identifying antioxidant proteins. In this predictor, the sequence was formulated by using the optimal 3-gap dipeptides obtained by using feature selection method. It was observed by jackknife cross-validation test that AodPred can achieve an overall accuracy of 74.79 % in identifying antioxidant proteins. As a user-friendly tool, AodPred is freely accessible at http://lin.uestc.edu.cn/server/AntioxiPred . To maximize the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web server to obtain the desired results.

  16. Rational and combinatorial tailoring of bioactive cyclic dipeptides

    PubMed Central

    Giessen, Tobias W.; Marahiel, Mohamed A.

    2015-01-01

    Modified cyclic dipeptides represent a diverse family of microbial secondary metabolites. They display a broad variety of biological and pharmacological activities and have long been recognized as privileged structures with the ability to bind to a wide range of receptors. This is due to their conformationally constrained 2, 5-diketopiperazine (DKP) scaffold and the diverse set of DKP tailoring enzymes present in nature. After initial DKP assembly through different biosynthetic systems modifying enzymes are responsible for installing functional groups crucial for the biological activities of the resulting modified DKPs. They represent a vast and largely untapped enzyme repository very useful for synthetic biology approaches aiming at introducing structural variations into DKP scaffolds. In this review we focus on these DKP modification enzymes found in various microbial secondary metabolite gene clusters. We will give a brief overview of their distribution and highlight a select number of characterized DKP tailoring enzymes before turning to their application potential in combinatorial biosynthesis with the aim of producing molecules with improved or entirely new biological and medicinally relevant properties. PMID:26284060

  17. Sterically allowed configuration space for amino acid dipeptides

    NASA Astrophysics Data System (ADS)

    Caballero, Diego; Maatta, Jukka; Sammalkorpi, Maria; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite recent improvements in computational methods for protein design, we still lack a quantitative, predictive understanding of the intrinsic propensities for amino acids to be in particular backbone or side-chain conformations. This question has remained unsettled for years because of the discrepancies between different experimental approaches. To address it, I performed all-atom hard-sphere simulations of hydrophobic residues with stereo-chemical constraints and non-attractive steric interactions between non-bonded atoms for ALA, ILE, LEU and VAL dipeptide mimetics. For these hard-sphere MD simulations, I show that transitions between α-helix and β-sheet structures only occur when the bond angle τ(N -Cα - C) >110° , and the probability distribution of bond angles for structures in the `bridge' region of ϕ- ψ space is shifted to larger angles compared to that in other regions. In contrast, the relevant bond-angle distributions obtained from most molecular dynamics packages are broader and shifter to larger values. I encounter similar correlations between bond angles and side-chain dihedral angles. The success of these studies is an argument for re-incorporating local stereochemical constraints into computational protein design methodology.

  18. Pharmacological Potential of the Endogenous Dipeptide Kyotorphin and Selected Derivatives

    PubMed Central

    Perazzo, Juliana; Castanho, Miguel A. R. B.; Sá Santos, Sónia

    2017-01-01

    The endogenous peptide kyotorphin (KTP) has been extensively studied since it was discovered in 1979. The dipeptide is distributed unevenly over the brain but the majority is concentrated in the cerebral cortex. The putative KTP receptor has not been identified yet. As many other neuropeptides, KTP clearance is mediated by extracellular peptidases and peptide transporters. From the wide spectrum of biological activity of KTP, analgesia was by far the most studied. The mechanism of action is still unclear, but researchers agree that KTP induces Met-enkephalins release. More recently, KTP was proposed as biomarker of Alzheimer disease. Despite all that, KTP limited pharmacological value prompted researchers to develop derivatives more lipophilic and therefore more prone to cross the blood–brain barrier (BBB), and also more resistant to enzymatic degradation. Conjugation of KTP with functional molecules, such as ibuprofen, generated a new class of compounds with additional biological properties. Moreover, the safety profile of these derivatives compared to opioids and their efficacy as neuroprotective agents greatly increases their pharmacological value. PMID:28127286

  19. Predicting cancerlectins by the optimal g-gap dipeptides.

    PubMed

    Lin, Hao; Liu, Wei-Xin; He, Jiao; Liu, Xin-Hui; Ding, Hui; Chen, Wei

    2015-12-09

    The cancerlectin plays a key role in the process of tumor cell differentiation. Thus, to fully understand the function of cancerlectin is significant because it sheds light on the future direction for the cancer therapy. However, the traditional wet-experimental methods were money- and time-consuming. It is highly desirable to develop an effective and efficient computational tool to identify cancerlectins. In this study, we developed a sequence-based method to discriminate between cancerlectins and non-cancerlectins. The analysis of variance (ANOVA) was used to choose the optimal feature set derived from the g-gap dipeptide composition. The jackknife cross-validated results showed that the proposed method achieved the accuracy of 75.19%, which is superior to other published methods. For the convenience of other researchers, an online web-server CaLecPred was established and can be freely accessed from the website http://lin.uestc.edu.cn/server/CalecPred. We believe that the CaLecPred is a powerful tool to study cancerlectins and to guide the related experimental validations.

  20. Energetics of oxo- and thio-dipeptide formation via amino acid condensation: a systematic computational analysis.

    PubMed

    Torsello, Mauro; Orian, Laura; De Zotti, Marta; Saini, Roberta; Formaggio, Fernando; Polimeno, Antonino

    2014-09-07

    Oxo-dipeptides and thio-dipeptides are built via condensation between couples of amino acids and amino thioacids, the latter with the carbonyl oxygen replaced by an sp(2) sulfur. We explored via in silico methods (PBE0/6-31G(d,p) and PBE0/6-311G(d,p)) all the possible combinations and built 800 dipeptides, whose structures were fully optimized. Maps of condensation energies are presented to highlight optimal partners leading to stable dipeptides and critical situations for which lower stability or instability is predicted in terms of Gibbs reaction free energies. To validate the feasibility of our computational investigation, we synthesized and compared the stabilities of two thionated dimers, namely -Gly[Ψ(CSNH)]Gly- and -Phe[Ψ(CSNH)]Phe-, characterized by diverging physico-chemical properties. To the best of our knowledge, this is the first systematic analysis reported for dipeptides built from natural amino acids as well as for their corresponding thio-analogs.

  1. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  2. Synthesis of 1,5-benzothiazepine dipeptide mimetics via two CuI-catalyzed cross coupling reactions.

    PubMed

    Gan, Jiangang; Ma, Dawei

    2009-07-02

    CuI-catalyzed coupling of 4-methylphenyl bromide with amino acids gives N-aryl amino acids, which are converted into linear dipeptides via iodination and condensation with L-cysteine derived acyl chloride. Cyclization is achieved via a CuI/N,N-dimethylglycine catalyzed intramolecular coupling of aryl iodides with the liberated thiol to afford 1,5-benzothiazepine dipeptide mimetics.

  3. The Synthesis of a Dipeptide from its Component Amino Acids: Protecting Groups in the Elementary Organic Laboratory.

    ERIC Educational Resources Information Center

    Young, Paul E.; Campbell, Andrew

    1982-01-01

    A simple, three-step procedure for synthesizing a dipeptide from its component amino acids is described. The dipeptide synthesized uses inexpensive amino acids having hydrocarbon side-chains and can be observed in E/Z forms by nuclear magnetic resonance spectroscopy. Each step in the synthesis produces white crystalline products using standard…

  4. [Behavioral and electrophysiological analysis of the choline-positive effect of nootropic dipeptide acylproline (GVS-111)].

    PubMed

    Ostrovskaia, R U; Mirzoev, T Kh; Firova, F A; Trofimof, S S; Gudasheva, T A; Grechenko, T N; Gutyrchik, E F; Barkova, E B

    2001-01-01

    The behavioral experiments using a passive avoidance learning model showed that the new cognition-enhancing acyl-prolyn containing dipeptide GVS-111 promotes recovery of the test performance in animals with a long-term memory deficit caused by the M-cholinolytic scopolamine (1 mg/kg/day scopolamine for 20 days, followed by 0.5 mg/kg/day GVS-111 for 10 days). At the same time, GVS-111 increased the duration of tremor induced by the M-cholinomimetic arecoline. The results of electrophysicological experiments showed that GVS-111 in a concentration range from 10(-11) to 10(-9) M increased amplitude of the neural response to acetylcholine (Ach) microappications in 75% of the isolated neurons of Helix Pomatum and produced a predominantly facilitating effect upon the endoneuronal pacemaker activity. The effect of GVS-111 upon the Ach response in a part of neurons was attenuated or even blocked by scopolamine, and in the other neurons--by the N-cholinolytic d-tubocurarine. This fact indicates that both muscarinic and nicotinic receptors are involved in the mechanism of the cholino-sensitizing action of GVS-111 upon the neuronal activity.

  5. Dipeptide-based Polyphosphazene and Polyester Blends for Bone Tissue Engineering

    PubMed Central

    Deng, Meng; Nair, Lakshmi S.; Nukavarapu, Syam P.; Jiang, Tao; Kanner, William A.; Li, Xudong; Kumbar, Sangamesh G.; Weikel, Arlin L.; Krogman, Nicholas R.; Allcock, Harry R.; Laurencin, Cato T.

    2010-01-01

    Polyphosphazene-polyester blends are attractive materials for bone tissue engineering applications due to their controllable degradation pattern with non-toxic and neutral pH degradation products. In our ongoing quest for an ideal completely miscible polyphosphazene-polyester blend system, we report synthesis and characterization of a mixed-substituent biodegradable polyphosphazene poly[(glycine ethyl glycinato)1(phenyl phenoxy)1phosphazene] (PNGEG/PhPh) and its blends with a polyester. Two dipeptide-based blends namely 25:75 (Matrix1) and 50:50 (Matrix2) were produced at two different weight ratios of PNGEG/PhPh to poly(lactic acid-glycolic acid) (PLAGA). Blend miscibility was confirmed by differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Both blends resulted in higher tensile modulus and strength than the polyester. The blends showed a degradation rate in the order of Matrix2 < Matrix1 < PLAGA in phosphate buffered saline at 37°C over 12 weeks. Significantly higher pH values of degradation media were observed for blends compared to PLAGA confirming the neutralization of PLAGA acidic degradation by polyphosphazene hydrolysis products. The blend components PLAGA and polyphosphazene exhibited a similar degradation pattern as characterized by the molecular weight loss. Furthermore, blends demonstrated significantly higher osteoblast growth rates compared to PLAGA while maintaining osteoblast phenotype over a 21-day culture. Both blends demonstrated improved biocompatibility in a rat subcutaneous implantation model compared to PLAGA over 12 weeks. PMID:20334909

  6. The discovery and potential of N-sulfonylated dipeptide VLA-4 antagonists.

    PubMed

    Hagmann, William K

    2004-01-01

    Through a directed screening of a combinatorial library containing carboxylic acids, N-sulfonylated dipeptides were identified as leads in the Merck Research Laboratories VLA-4 antagonist program. Further optimization quickly identified subnanomolar compounds with varying degrees of specificity over the related integrin alpha4beta7. Various metabolic liabilities were identified and addressed. However, the pharmacokinetic properties of nearly all compounds in this class were unacceptable. Other leads were identified with apparent good oral bioavailability, but these were generally associated with very high plasma protein binding and a loss of potency. The mechanism of high plasma clearance was identified in the rat as the organic acid transporter, mrp-2. Compounds were identified that were not substrates of mrp-2, but they still suffered from poor oral bioavailability. Finally, a shift in strategy to identifying VLA-4 antagonists that would be suitable as candidates for inhalation therapy resulted in the preparation of compounds with exception tight binding properties. These compounds were superior to BIO-1211 in the ovalbumin-sensitized mouse model of eosinophil trafficking to the lung. One particular compound had an exceptionally long off-rate with a KD < or = 2 pM. The evolution of the structure activity relationships in our laboratories and strategies for improving potencies and pharmacokinetic profiles are the subject of this review.

  7. Carbon nanotube as a carrier in drug delivery system for carnosine dipeptide: A computer simulation study.

    PubMed

    Ketabi, Sepideh; Rahmani, Leila

    2017-04-01

    Biological application of carbon nanotube in drug delivery is our main concern in this investigation. For this purpose interaction of carnosine and carbon nanotube was studied in both gas phase and separately in aqueous media. Three possible interactions of carnosine dipeptide with (5,5) carbon nanotube in physiological media were considered. At first step each species were modeled using quantum mechanical calculations, in the next step, their properties in aqueous solution were studied by applying Monte Carlo simulations. The results of density functional calculations in gas phase showed that interaction of zwitterion of carnosine with carbon nanotube via NH3(+) had relatively higher interaction energy than the other complexes. Computation of solvation free energies in water showed functionalization with carnosine enhanced the solubility of carbon nanotube significantly that improve the medicinal applications of these materials. Calculation of complexation free energies indicated that zwitterion of carnosine with carbon nanotube via NH3(+) produced the most stable complex in aqueous solution. This tendency could be observed in gas and liquid phase similarly.

  8. Micelle-catalyzed reaction between ninhydrin and nickel dipeptide complex [Ni(II)-Gly-Tyr]+.

    PubMed

    Akram, Mohd; Kumar, Dileep; Kabir-ud-Din

    2012-06-01

    The interaction of nickel dipeptide complex [Ni(II)-Gly-Tyr](+) with ninhydrin has been investigated in the absence and presence of cationic cetyltrimethylammonium bromide (CTAB) and gemini (16-s-16, s=4, 5, 6) surfactants spectrophotometrically at 80°C and pH 5.0. The product formed was the same and the reaction followed first- and fractional-order kinetics with respect to [Ni(II)-Gly-Tyr](+) and [ninhydrin], respectively, in both aqueous as well as micellar media. In the presence of CTAB, rate increased and reached up to a maximum, then decreased. Also, whereas typical rate constant (k(Ψ)) increase and leveling-off regions, just like CTAB, were observed with geminis, the latter produced a third region of increasing k(Ψ) at higher concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The micellar catalysis is explained in terms of pseudo-phase model. The binding constants and the values of activation parameters such as activation energy (E(a)), enthalpy of activation (ΔH(#)) and entropy of activation (ΔS(#)) have been evaluated.

  9. A comparison of weighted ensemble and Markov state model methodologies

    NASA Astrophysics Data System (ADS)

    Feng, Haoyun; Costaouec, Ronan; Darve, Eric; Izaguirre, Jesús A.

    2015-06-01

    Computation of reaction rates and elucidation of reaction mechanisms are two of the main goals of molecular dynamics (MD) and related simulation methods. Since it is time consuming to study reaction mechanisms over long time scales using brute force MD simulations, two ensemble methods, Markov State Models (MSMs) and Weighted Ensemble (WE), have been proposed to accelerate the procedure. Both approaches require clustering of microscopic configurations into networks of "macro-states" for different purposes. MSMs model a discretization of the original dynamics on the macro-states. Accuracy of the model significantly relies on the boundaries of macro-states. On the other hand, WE uses macro-states to formulate a resampling procedure that kills and splits MD simulations for achieving better efficiency of sampling. Comparing to MSMs, accuracy of WE rate predictions is less sensitive to the definition of macro-states. Rigorous numerical experiments using alanine dipeptide and penta-alanine support our analyses. It is shown that MSMs introduce significant biases in the computation of reaction rates, which depend on the boundaries of macro-states, and Accelerated Weighted Ensemble (AWE), a formulation of weighted ensemble that uses the notion of colors to compute fluxes, has reliable flux estimation on varying definitions of macro-states. Our results suggest that whereas MSMs provide a good idea of the metastable sets and visualization of overall dynamics, AWE provides reliable rate estimations requiring less efforts on defining macro-states on the high dimensional conformational space.

  10. Production of D-Alanine by Corynebacterium fascians

    PubMed Central

    Yamada, Shigeki; Maeshima, Haruko; Wada, Mitsuru; Chibata, Ichiro

    1973-01-01

    A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH4)2HPO4, and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent. PMID:4699220

  11. The structure of alanine racemase from Acinetobacter baumannii.

    PubMed

    Davis, Emily; Scaletti-Hutchinson, Emma; Opel-Reading, Helen; Nakatani, Yoshio; Krause, Kurt L

    2014-09-01

    Acinetobacter baumannii is an opportunistic Gram-negative bacterium which is a common cause of hospital-acquired infections. Numerous antibiotic-resistant strains exist, emphasizing the need for the development of new antimicrobials. Alanine racemase (Alr) is a pyridoxal 5'-phosphate dependent enzyme that is responsible for racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target. The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic-resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrates a conserved overall fold. The substrate entryway and active site of the enzymes were shown to be highly conserved. The structure of AlrAba will provide the template required for future structure-based drug-design studies.

  12. Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain.

    PubMed

    Ichinose, Takashi; Moriyasu, Kazuki; Nakahata, Akane; Tanaka, Mitsuru; Matsui, Toshiro; Furuya, Shigeki

    2015-01-01

    In this study, we examined the effect of orally administrated dipeptides containing Tyr (Y) on the metabolism of catecholamines in mouse brains. We found that among eight synthetic dipeptides whose sequences are present frequently in soy proteins, Ser-Tyr (SY), Ile-Tyr, and Tyr-Pro had the highest apparent permeability coefficients in monolayers of human intestinal epithelial Caco-2 cells. When administrated orally, SY markedly increased tyrosine content in the cerebral cortex compared to the vehicle control, Ile-Tyr, Tyr-Pro, and Y alone. The oral administration of SY more effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol, the principal metabolite of noradrenaline, in the cerebral cortex and hippocampus than did Ile-Tyr, Tyr-Pro, or Y alone. Central noradrenergic turnover was also markedly stimulated by SY administration. These in vivo observations strongly suggest that SY is more potent in boosting central catecholamine transmission, particularly the noradrenergic system, than Y alone or other dipeptides that include Y.

  13. Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor.

    PubMed

    Lan, Vu Thi Tuyet; Ito, Keisuke; Ohno, Masumi; Motoyama, Takayasu; Ito, Sohei; Kawarasaki, Yasuaki

    2015-05-15

    Human dipeptidyl peptidase IV (hDPPIV) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Because certain peptides are known to act as hDPPIV inhibitors, a dataset of possible peptides with their inhibition intensities will facilitate the development of functional food for type 2 diabetes. In this study, we examined a total of 337 dipeptides with respect to their hDPPIV inhibitory effects. Amino acid residues at N-termini dominated their inhibition intensities. Particularly highly inhibitory dipeptides discovered included the following novel dipeptides: Thr-His, Asn-His, Val-Leu, Met-Leu, and Met-Met. Using our dataset, prime candidates contributing to the hDPPIV inhibitory effect of soy protein hydrolyzates were successfully identified. Possible dietary proteins potentially able to produce particularly highly hDPPIV inhibitory peptides are also discussed on the basis of the dataset.

  14. Discrete dynamic system oriented on the formation of prebiotic dipeptides from Rode's experiment.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Buhse, Thomas; Castañón González, Jorge Alberto

    2014-01-01

    This work attempts to rationalize the possible prebiotic profile of the first dipeptides of about 4 billion years ago based on a computational discrete dynamic system that uses the final yields of the dipeptides obtained in Rode's experiments of salt-induced peptide formation (Rode et al., 1999, Peptides 20: 773-786). The system built a prebiotic scenario that allowed us to observe that (i) the primordial peptide generation was strongly affected by the abundances of the amino acid monomers, (ii) small variations in the concentration of the monomers have almost no effect on the final distribution pattern of the dipeptides and (iii) the most plausible chemical reaction of prebiotic peptide bond formation can be linked to Rode's hypothesis of a salt-induced scenario. The results of our computational simulations were related to former simulations of the Miller, and Fox & Harada experiments on amino acid monomer and oligomer generation, respectively, offering additional information to our approach.

  15. Highly selectively monitoring heavy and transition metal ions by a fluorescent sensor based on dipeptide.

    PubMed

    Neupane, Lok Nath; Thirupathi, Ponnaboina; Jang, Sujung; Jang, Min Jung; Kim, Jung Hwa; Lee, Keun-Hyeung

    2011-09-15

    Fluorescent sensor (DMH) based on dipeptide was efficiently synthesized in solid phase synthesis. The dipeptide sensor shows sensitive response to Ag(I), Hg(II), and Cu(II) among 14 metal ions in 100% aqueous solution. The fluorescent sensor differentiates three heavy metal ions by response type; turn on response to Ag(I), ratiometric response to Hg(II), and turn off detection of Cu(II). The detection limits of the sensor for Ag(I) and Cu(II) were much lower than the EPA's drinking water maximum contaminant levels (MCL). Specially, DMH penetrated live cells and detected intracellular Ag(+) by turn on response. We described the fluorescent change, binding affinity, detection limit for the metal ions. The study of a heavy metal-responsive sensor based on dipeptide demonstrates its potential utility in the environment field.

  16. Comparison of EPR response of alanine and Gd₂O₃-alanine dosimeters exposed to TRIGA Mainz reactor.

    PubMed

    Marrale, M; Schmitz, T; Gallo, S; Hampel, G; Longo, A; Panzeca, S; Tranchina, L

    2015-12-01

    In this work we report some preliminary results regarding the analysis of electron paramagnetic resonance (EPR) response of alanine pellets and alanine pellets added with gadolinium used for dosimetry at the TRIGA research reactor in Mainz, Germany. Two set-ups were evaluated: irradiation inside PMMA phantom and irradiation inside boric acid phantom. We observed that the presence of Gd2O3 inside alanine pellets increases the EPR signal by a factor of 3.45 and 1.24 in case of PMMA and boric acid phantoms, respectively. We can conclude that in the case of neutron beam with a predominant thermal neutron component the addition of gadolinium oxide can significantly improve neutron sensitivity of alanine pellets. Monte Carlo (MC) simulations of both response of alanine and Gd-added alanine pellets with FLUKA code were performed and a good agreement was achieved for pure alanine dosimeters. For Gd2O3-alanine deviations between MC simulations and experimental data were observed and discussed.

  17. In vitro transport and satiety of a beta-lactoglobulin dipeptide and beta-casomorphin-7 and its metabolites.

    PubMed

    Osborne, Simone; Chen, Wei; Addepalli, Rama; Colgrave, Michelle; Singh, Tanoj; Tran, Cuong; Day, Li

    2014-11-01

    Understanding the digestive behaviour and biological activities of dairy proteins may help to develop model dairy products with targeted health outcomes including increased satiety and healthy weight maintenance. Caseins and whey proteins constitute over 95% of milk proteins with consumption of these proteins associated with increased satiety and a decreased prevalence of metabolic disorders. To investigate the in vitro digestive behaviour and satiety of dairy proteins at the intestinal epithelium, the in vitro transport and hydrolysis of 500-2000 μM β-casomorphin-7 (YPFPGPI or β-CM7) and a β-lactoglobulin (β-Lg) dipeptide (YL) was measured using Caco-2 cell monolayers grown on transwells as a model of the intestinal epithelium. Transport of YL was concentration dependent and ranged from 0.37-5.26 × 10(-6) cm s(-1), whereas transport of β-CM7 was only detected at 2000 μM and was significantly lower at 0.13 × 10(-6) cm s(-1). Rapid hydrolysis of β-CM7 in the apical chamber by the Caco-2 cells produced three peptide metabolites: YP, GPI and FPGPI. All of these metabolites were detected in the basolateral chamber after 30 min with both the YP and GPI peptides transporting at a higher rate than intact β-CM7. In vitro satiety was indicated by the secretion of cholecystokinin [26-33] (CCK-8) and glucagon-like peptide 1 (GLP-17-36NH2) in the STC-1 enteroendocrine cell model. CCK-8 secretion was highest in response to β-CM7 followed by the β-CM7 metabolite FPGPI. CCK-8 secretion however was not significantly stimulated by the tri- or dipeptides. Secretion of GLP-1 was not significantly stimulated by β-CM7 or YL. These in vitro results suggest that dairy peptide size enhances CCK-8 secretion, whilst limiting transport across Caco-2 monolayers.

  18. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides.

    PubMed

    Mitsuhashi, Satoshi

    2014-04-01

    Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine).

  19. Chirality organization of ferrocenes bearing podand dipeptide chains: synthesis and structural characterization.

    PubMed

    Moriuchi, T; Nomoto, A; Yoshida, K; Ogawa, A; Hirao, T

    2001-01-10

    A variety of ferrocenes bearing podand dipeptide chains have been synthesized to form an ordered structure in both solid and solution states and have been investigated by 1H NMR, FT-IR, CD, and X-ray crystallographic analyses. Conformational enantiomerization through chirality organization was achieved by the intramolecular hydrogen bondings between the podand dipeptide chains. The single-crystal X-ray structure determination of the ferrocene 2 bearing the podand dipeptide chains (-D-Ala-D-Pro-OEt) revealed two C2-symmetric intramolecular hydrogen bondings between CO (Ala) and NH (another Ala) of each podand dipeptide chain to induce the chirality-organized structure. The molecular structures of the ferrocene 1 composed of the podand L-dipeptide chains (-L-Ala-L-Pro-OEt) and 2 are in a good mirror image relationship, indicating that they are conformational enantiomers. An opposite helically ordered molecular arrangement was formed in the crystal packing of 2 as compared with 1. The ferrocene 2 exhibited induced circular dichroism (CD), which appeared at the absorbance of the ferrocene moiety. The mirror image of the CD signals between 1 and 2 was observed, suggesting that the chirality-organized structure via intramolecular hydrogen bondings is present even in solution. The ferrocene 4 bearing the podand dipeptide chains (-Gly-L-Leu-OEt) also showed an ordered structure in the crystal based on two intramolecular hydrogen bondings between CO (Gly) and NH (another Gly) of each podand dipeptide chain, together with intermolecular hydrogen bondings between CO adjacent to the ferrocene unit and NH (neighboring Leu) to create the highly organized self-assembly. A different self-assembly was observed in the crystal of the ferrocene 5 composed of the podand dipeptide chains (-Gly-L-Phe-OEt), wherein each molecule is bonded to two neighboring molecules through two pairs of symmetrical intermolecular hydrogen bonds to form a 14-membered intermolecularly hydrogen-bonded ring

  20. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  1. Characterization of Nα-Fmoc-protected dipeptide isomers by electrospray ionization tandem mass spectrometry (ESI-MS(n)): effect of protecting group on fragmentation of dipeptides.

    PubMed

    Ramesh, M; Raju, B; Srinivas, R; Sureshbabu, V V; Vishwanatha, T M; Hemantha, H P

    2011-07-30

    A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx=Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx=Leu, Phe) (Fmoc=[(9-fluorenylmethyl)oxy]carbonyl) and Y=CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS(n)). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y(1)(+) and/or a(1)(+) ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b(1)(+) ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b(1)(+) ions. The [M+H](+) ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO(2) to form [M+H-Fmoc+H](+). The MS(3) collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS(3) CID has also been found to be useful for differentiating these isomeric peptide acids. The MS(3) of [M-H-Fmoc+H](-) of isomeric peptide acids produce c(1)(-), z(1)(-) and y(1)(-) ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers.

  2. Contribution of the empirical dispersion correction on the conformation of short alanine peptides obtained by gas-phase QM calculations.

    PubMed

    Fadda, Elisa; Woods, Robert J

    2013-09-01

    In this work we analyze the effect of the inclusion of an empirical dispersion term to standard DFT (DFT-D) in the prediction of the conformational energy of the alanine dipeptide (Ala2) and in assessing the relative stabilities of short polyala-nine peptides in helical conformations, i.e., α and 310 helices, from Ala4 to Ala16. The Ala2 conformational energies obtained with the dispersion-corrected GGA functional B97-D are compared to previously published high level MP2 data. Meanwhile, the B97-D performance on larger polyalanine peptides is compared to MP2, B3LYP and RHF calculations obtained at a lower level of theory. Our results show that electron correlation affects the conformational energies of short peptides with a weight that increases with the peptide length. Indeed, while the contribution of vdW forces is significant for larger peptides, in the case of Ala2 it is negligible when compared to solvent effects. Even for short peptides, the inclusion of an empirical dispersion term greatly improves accuracy of DFT methods, providing results that correlate very well with the MP2 reference at no additional computational cost.

  3. Formation of Diastereoisomeric Piperazine-2,5-dione from dl-Alanine in the Presence of Olivine and Water

    NASA Astrophysics Data System (ADS)

    Fuchida, Shigeshi; Naraoka, Hiroshi; Masuda, Harue

    2017-03-01

    dl-Alanine (Ala) was heated with/without powdered olivine and water at 120 °C for 8 days to investigate the formation of the diastereoisomers of piperazine-2,5-dione (diketopiperazine, DKP). When only dl-Ala was heated with a small amount of water, 3.0 % of dl-Ala changed to cis- and trans-DKP after 8 days. DKPs were not detected after heating when no water was added. The presence of a small amount of water is important factor controlling peptide production rates under thermal conditions. When DL-Ala was heated with olivine powder for 8 days, the yields of cis- and trans-DKP were 6.8 and 4.9 %, respectively. The high yield of cis-DKP compared with trans-DKP was attributed to greater thermal stability of cis-DKP. After heating for 8 days, the diastereoisomeric excess of cis-DKP without olivine was 7.3 %, whereas a much higher value of 16.3 % was obtained in the presence of olivine. Taken together, these results show that olivine is not only an efficient catalyst for the formation of DKPs but that it also play a significant role in determining the diastereoisomer selectivity of these cyclic dipeptides.

  4. Photolytic control and infrared probing of amide I mode in the dipeptide backbone-caged with the 4,5-dimethoxy-2-nitrobenzyl group.

    PubMed

    Rhee, Hanju; Lee, Jang-Soo; Lee, Jihae; Joo, Cheonik; Han, Hogyu; Cho, Minhaeng

    2008-02-21

    Alanine dipeptide analog 1 backbone-caged with a photolabile linker, 4,5-dimethoxy-2-nitrobenzyl (DmNb), was synthesized. UV-pulse-induced photochemical reaction of 1 was monitored by Fourier transform IR absorption spectroscopy under a steady-state condition or in a fast-scan mode. Upon photolysis of 1, the amide I band is changed from a doublet to a singlet with concomitant line shape changes of several IR bands. The change of the amide I band is directly associated with the photocleavage of the covalent N-C bond connecting the backbone amide of 2 to DmNb. Therefore, IR spectroscopy is useful for directly probing the photocleavage of backbone-caged peptide 1 and the concurrent release of native peptide 2. In contrast, UV-vis spectroscopy probing the irradiation-induced structural change of the 2-nitrobenzyl moiety itself may not provide a clue directly relevant to the photocleavage of such N-C bond. Time-resolved IR spectra recorded in a fast-scan mode after pulsed UV irradiation of 1 reveal that such photocleavage occurs at least faster than a few seconds of our instrumental time resolution.

  5. Comparison of Intramolecular Forces in Dipeptides with Two Aromatic Rings: does Dispersion Dominate?

    NASA Astrophysics Data System (ADS)

    Thomas, Jessica A.

    2015-06-01

    IR/UV double resonance spectroscopy has shown that the structure of the capped dipeptide Ac-Trp-Tyr-NH2 is dominated by a hydrophobic interaction between the aromatic rings on the side chains. Using the same method, a similar molecule, Ac-Phe-Phe-NH2, had three experimentally observed conformers including one similar to that of Ac-Trp-Tyr-NH2. In this work, calculations were performed on additional dipeptides containing two aromatic rings to determine if the dispersion-dominated structure was among the lowest energy structures in all such cases. The B3LYP-DCP method developed by DiLabio and Torres was used to calculate the conformations of each dipeptide. Appending dispersion correction potentials (DCP) to B3LYP input files improves results for systems containing dispersion interactions without significantly increasing the calculation time. This method was used first on Ac-Trp-Tyr-NH2 and Ac-Phe-Phe-NH2 to confirm that it successfully identified the experimentally observed structures among the lowest energy results and was then applied to other capped dipeptides containing two aromatic rings including Ac-Phe-Tyr-NH2 and Ac-Tyr-Phe-NH2.

  6. Enantiomeric interactions between liquid crystals and organized monolayers of tyrosine-containing dipeptides.

    PubMed

    Bai, Yiqun; Abbott, Nicholas L

    2012-01-11

    We have examined the orientational ordering of nematic liquid crystals (LCs) supported on organized monolayers of dipeptides with the goal of understanding how peptide-based interfaces encode intermolecular interactions that are amplified into supramolecular ordering. By characterizing the orientations of nematic LCs (4-cyano-4'-pentylbiphenyl and TL205 (a mixture of mesogens containing cyclohexane-fluorinated biphenyls and fluorinated terphenyls)) on monolayers of l-cysteine-l-tyrosine, l-cysteine-l-phenylalanine, or l-cysteine-l-phosphotyrosine formed on crystallographically textured films of gold, we conclude that patterns of hydrogen bonds generated by the organized monolayers of dipeptides are transduced via macroscopic orientational ordering of the LCs. This conclusion is supported by the observation that the ordering exhibited by the achiral LCs is specific to the enantiomers used to form the dipeptide-based monolayers. The dominant role of the -OH group of tyrosine in dictating the patterns of hydrogen bonds that orient the LCs was also evidenced by the effects of phosphorylation of the tyrosine on the ordering of the LCs. Overall, these results reveal that crystallographic texturing of gold films can direct the formation of monolayers of dipeptides with long-range order, thus unmasking the influence of hydrogen bonding, chirality, and phosphorylation on the macroscopic orientational ordering of LCs supported on these surfaces. These results suggest new approaches based on supramolecular assembly for reporting the chemical functionality and stereochemistry of synthetic and biological peptide-based molecules displayed at surfaces.

  7. Profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reactive carbonyl species (RCS), oxidation products of polyunsaturated fatty acids, protein & sugars, play a role in the etiology of certain chronic diseases. Our previous studies revealed that histidine-dipeptides such as carnosine and anserine detoxify cytotoxic carbonyls such as 4-hydroxy-trans-...

  8. Analysis of the biosynthesis of antibacterial cyclic dipeptides in Nocardiopsis alba.

    PubMed

    Li, Yongli; Lai, Ying-Mi; Lu, Yi; Yang, Yu-Liang; Chen, Shawn

    2014-11-01

    Nocardiopsis alba is frequently isolated from environment and has recently been suggested as a casual symbiotic actinobacterium of diverse invertebrates. Using activity-guided fractionation, we purified two antibacterial cyclic dipeptides, cyclo(ΔPhe-ΔLeu) (albonoursin) and cyclo(ΔmTyr-ΔLeu), from a culture of Nocardiopsis alba ATCC BAA-2165. Analysis of N. alba genome revealed genetic information similar to albonoursin biosynthetic gene cluster, albABC. An albABC gene deletion mutant of N. alba was generated. Liquid chromatography-mass spectrometry analysis showed that the mutant could not produce the cyclic dipeptides. Cyclic dipeptide production in the mutant was restored by genetic complementation with the albABC cloned in a native plasmid of Nocardiopsis. β-Glucuronidase reporter assays with a second mutant construct, in which albABC promoter is transcriptionally fused to the reporting gene gusA, indicated that albABC gene expression was subject to osmoregulation. The system presented will be used to study the metabolic and genetic control of cyclic dipeptide biosynthesis in Nocardiopsis.

  9. Influence of the synthesis conditions of gold nanoparticles on the structure and architectonics of dipeptide composites

    NASA Astrophysics Data System (ADS)

    Loskutov, Alexander I.; Guskova, Olga A.; Grigoriev, Sergey N.; Oshurko, Vadim B.; Tarasiuk, Aleksei V.; Uryupina, Olga Ya.

    2016-08-01

    A wide variety of peptides and their natural ability to self-assemble makes them very promising candidates for the fabrication of solid-state devices based on nano- and mesocrystals. In this work, we demonstrate an approach to form peptide composite layers with gold nanoparticles through in situ reduction of chloroauric acid trihydrate by dipeptide and/or dipeptide/formaldehyde mixture in the presence of potassium carbonate at different ratios of components. Appropriate composition of components for the synthesis of highly stable gold colloidal dispersion with particle size of 34-36 nm in dipeptide/formaldehyde solution is formulated. Infrared spectroscopy results indicate that dipeptide participates in the reduction process, conjugation with gold nanoparticles and the self-assembly in 2D, which accompanied by changing peptide chain conformations. The structure and morphology of the peptide composite solid layers with gold nanoparticles on gold, mica and silica surfaces are characterized by atomic force microscopy. In these experiments, the flat particles, dendrites, chains, mesocrystals and Janus particles are observed depending on the solution composition and the substrate/interface used. The latter aspect is studied on the molecular level using computer simulations of individual peptide chains on gold, mica and silica surfaces.

  10. Highly cytotoxic bioconjugated gold(I) complexes with cysteine-containing dipeptides.

    PubMed

    Gutiérrez, Alejandro; Marzo, Isabel; Cativiela, Carlos; Laguna, Antonio; Gimeno, M Concepción

    2015-07-27

    Several gold(I) complexes with cysteine-containing dipeptides have been prepared starting from cystine by coupling different amino acids and using several orthogonal protections. The first step is the reaction of cystine, where the sulfur centre is protected as disulfide, with Boc2 O in order to protect the amino group, followed by coupling of an amino acid ester; finally the disulfide bridge is broken with mercaptoethanol to afford the dipeptide derivative. Further reaction with [AuCl(PPh3 )] gives the gold-dipeptide-phosphine species. Starting from these formally gold(I) thiolate-dipeptide phosphine complexes with the general formula [Au(SR)(PR3 )] different structural modifications, such as change in the type of the amino protecting group, the type of phosphine, the number of gold(I) atoms per molecule, or the use of a non-proteinogenic conformationally restricted amino acid ester, were introduced in order to evaluate their influence in the biological activity of the final complexes. The cytotoxic activity, in vitro, of these complexes was evaluated against different tumour human cell lines (A549, MiaPaca2 and Jurkat). The complexes show an outstanding cytotoxic activity with IC50 values in the very low micromolar range. Structure-activity relationship studies from the complexes open the possibility of designing more potent and promising gold(I) anticancer agents.

  11. Side chain dependence of intensity and wavenumber position of amide I' in IR and visible Raman spectra of XA and AX dipeptides.

    PubMed

    Measey, Thomas; Hagarman, Andrew; Eker, Fatma; Griebenow, Kai; Schweitzer-Stenner, Reinhard

    2005-04-28

    A series of AX and XA dipeptides in D2O have been investigated by FTIR, isotropic, and anisotropic Raman spectroscopy at acidic, neutral, and alkaline pD, to probe the influence of amino acid side chains on the amide I' band. We obtained a set of spectral parameters for each peptide, including intensities, wavenumbers, half-widths, and dipole moments, and found that these amide I' parameters are indeed dependent on the side chain. Side chains with similar characteristic properties were found to have similar effects on the amide I'. For example, dipeptides with aliphatic side chains were found to exhibit a downshift of the amide I' wavenumber, while those containing polar side chains experienced an increase in wavenumber. The N-terminal charge causes a substantial upshift of amide I', whereas the C-terminal charge causes a moderate decrease of the transition dipole moment. Density functional theory (DFT) calculations on the investigated dipeptides in vacuo yielded different correlations between theoretically and experimentally obtained wavenumbers for aliphatic/aromatic and polar/charged side chains, respectively. This might be indicative of a role of the hydration shell in transferring side chain-backbone interactions. For Raman bands, we found a correlation between amide I' depolarization ratio and wavenumber which reflects that some side chains (valine, histidine) have a significant influence on the Raman tensor. Altogether, the obtained data are of utmost importance for utilizing amide I as a tool for secondary structure analysis of polypeptides and proteins and providing an experimental basis for theoretical modeling of this important backbone mode. This is demonstrated by a rather accurate modeling for the amide I' band profiles of the IR, isotropic Raman, and anisotropic Raman spectra of the beta-amyloid fragment Abeta(1-82).

  12. Influence of substituents on conformational preferences of helix foldamers of γ-dipeptides.

    PubMed

    Kee Kang, Young; Yoo, In Kee

    2014-11-01

    Conformational preferences of 9- and 14-helix foldamers have been studied for γ-dipeptides of 2-aminocyclohexylacetic acid (γAc6 a) residues such as Ac-(γAc6 a)2 -NHMe (1), Ac-(C(α) -Et-γAc6 a)2 -NHMe (2), Ac-(γAc6 a)2 -NHBn (3), and Ac-(C(α) -Et-γAc6 a)2 -NHBn (4) at the M06-2X/cc-pVTZ//M06-2X/6-31 + G(d) level of theory to explore the influence of substituents on their conformational preferences. In the gas phase, the 9-helix foldamer H9 and 14-helix foldamer H14-z are found to be most preferred for dipeptides 2 and 4, respectively, as for dipeptides 1 and 3, which indicates no remarkable influence of the C(α) -ethyl substitution on conformational preferences. The benzyl substitution at the C-terminal end lead H14-z to be the most preferred conformer for dipeptides 3 and 4, whereas it is H9 for dipeptides 1 and 2, which can be ascribed to the favored CH···π interactions between the cyclohexyl group of the first residue and the C-terminal benzyl group. There are only marginal changes in backbone structures and the distances and angles of H-bonds for all local minima by C(α) -ethyl and/or benzyl substitutions. Although vibrational frequencies and intensities of the dipeptide 4 calculated at both M06-2X/6-31 + G(d) and M05-2X/6-31 + G(d) levels of theory are consistent with observed results in the gas phase, H14-z is predicted to be most preferred by ΔG only at the former level of theory. Hydration did not bring the significant changes in backbone structures of helix foldamers for both dipeptide 1 and 4. It is expected that the different substitutions at the C-terminal end lead to the different helix foldamers, which may increase the resistance of helical structures to proteolysis and provide the more surface to the helical structures suitable for molecular recognition.

  13. Granuloma formation by muramyl dipeptide associated with branched fatty acids, a structure probably essential for tubercle formation by Mycobacterium tuberculosis.

    PubMed Central

    Emori, K; Nagao, S; Shigematsu, N; Kotani, S; Tsujimoto, M; Shiba, T; Kusumoto, S; Tanaka, A

    1985-01-01

    Muramyl dipeptide, which does not induce epithelioid granuloma when injected alone dissolved in phosphate-buffered saline, could induce extensive granulomas in guinea pigs when chemically conjugated with branched, but not linear, fatty acids. Peptidoglycan fragments of Staphylococcus epidermidis could evoke epithelioid granulomas when incorporated in a water-in-oil emulsion. These findings suggest the importance of a lipid bound to muramyl dipeptide for granuloma formation. In view of the fact that mycobacteria uniquely contain large amounts of branched fatty acids, it was proposed that the complex of muramyl dipeptide and branched fatty acids, mostly mycolic acids, is a structure in tubercle bacilli responsible for tubercle formation. Images PMID:3924828

  14. Dipeptide analysis of p53 mutations and evolution of p53 family proteins.

    PubMed

    Huang, Qiang; Yu, Long; Levine, Arnold J; Nussinov, Ruth; Ma, Buyong

    2014-01-01

    p53 gain-of-function mutations are similar to driver mutations in cancer genes, with both promoting tumorigenesis. Most previous studies focused on residues lost by mutations, providing information related to a dominantly-negative effect. However, to understand gain-of-function mutations, it is also important to investigate what are the distributions of residues gained by mutations. We compile available p53/p63/p73 protein sequences and construct a non-redundant dataset. We analyze the amino acid and dipeptide composition of p53/p63/p73 proteins across evolution and compare them with the gain/loss of amino acids and dipeptides in human p53 following cancer-related somatic mutations. We find that the ratios of amino acids gained via somatic mutations during evolution to those lost through p53 cancer mutations correlate with the ratios found in single nucleotide polymorphisms in the human proteome. The dipeptide mutational gain/loss ratios are inversely correlated with those observed over p53 evolution but tend to follow the increasing p63/p73-like dipeptide propensities. We successfully simulated the p53 cancer mutation spectrum using the dipeptide composition across the p53 family accounting for the likelihood of mutations in p53 codons. The results revealed that the p53 mutation spectrum is dominated not only by p53 evolution but also by reversal of evolution to a certain degree. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.

  15. Effects of dipeptides administered to a perfused area of the skin in Angora goats.

    PubMed

    Pierzynowski, S G; Puchala, R; Sahlu, T

    1997-11-01

    The effect of dipeptide infusion on mohair growth of Angora goats was investigated using a skin perfusion technique. Six Angora wethers (average BW 32 +/- 2 kg) were implanted bilaterally with silicon catheters into the superficial branches of the deep circumflex iliac artery and to the deep circumflex iliac vein. For the first 14 d of the experiment, animals received infusions into the deep circumflex iliac arteries of either a mixture of Met-Leu and Lys-Leu (one side) or saline (other side). Infusion rates of amino acids were .72 mg/h Met-Leu and .72 mg/h Lys-Leu. The area of skin supplied by the deep circumflex iliac artery was approximately 300 cm2. An area of 150 cm2 within the perfused region was used to determine mohair growth. Two weeks after the cessation of infusions, perfused areas were shorn, and greasy and clean mohair production, staple length, and diameter were determined. Greasy and clean mohair production from the perfused region were increased by dipeptide infusion compared to the side infused with saline (1.91 vs 1.66 g, P < .05 and 1.56 vs 1.31 g, P < .04, respectively). No significant changes were observed in mohair diameter; however, staple length tended to increase as a result of dipeptide infusion (18.0 vs 16.1, P < .1). Decreased concentrations of Met, Cys, Lys, Phe, Val, Ileu, Leu, and Arg were observed in the venous blood taken from the deep circumflex iliac vein on the side infused with the amino acid mixture compared with blood taken from the saline side (P < .05). There were no treatment differences in triiodothyronine, thyroxine, or insulin concentrations in venous blood taken from the deep circumflex iliac vein. Direct skin infusion with dipeptide may have resulted in mobilization of amino acids for increased protein synthesis, or the infused dipeptides may have acted as growth promoters stimulating skin amino acid uptake and protein synthesis.

  16. Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

    PubMed Central

    van der Aart, Lizah T.; Lemmens, Nicole; van Wamel, Willem J.

    2016-01-01

    The increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to the d-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variant d-Ala–d-Lac, which is produced by VanA. Here we show that exogenous d-Ala competes with d-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of the d-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations of d-Ala led to the production of a significant amount of wild-type cell wall precursors, while vanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organism Streptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates of Enterococcus faecium (VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity of S. coelicolor cells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance. PMID:27270282

  17. Noncovalent and covalent functionalization of a (5, 0) single-walled carbon nanotube with alanine and alanine radicals.

    PubMed

    Rajarajeswari, Muthusivarajan; Iyakutti, Kombiah; Kawazoe, Yoshiyuki

    2012-02-01

    We have systematically investigated the noncovalent and covalent adsorption of alanine and alanine radicals, respectively, onto a (5, 0) single-walled carbon nanotube using first-principles calculation. It was found that XH···π (X = N, O, C) interactions play a crucial role in the non-ovalent adsorption and that the functional group close to the carbon nanotube exhibits a significant influence on the binding strength. Noncovalent functionalization of the carbon nanotube with alanine enhances the conductivity of the metallic (5, 0) nanotube. In the covalent adsorption of each alanine radical onto a carbon nanotube, the binding energy depends on the adsorption site on CNT and the electronegative atom that binds with the CNT. The strongest complex is formed when the alanine radical interacts with a (5, 0) carbon nanotube through the amine group. In some cases, the covalent interaction of the alanine radical introduces a half-filled band at the Fermi level due to the local sp (3) hybridization, which modifies the conductivity of the tube.

  18. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

  19. Dipeptide species regulate p38MAPK–Smad3 signalling to maintain chronic myelogenous leukaemia stem cells

    PubMed Central

    Naka, Kazuhito; Jomen, Yoshie; Ishihara, Kaori; Kim, Junil; Ishimoto, Takahiro; Bae, Eun-Jin; Mohney, Robert P.; Stirdivant, Steven M.; Oshima, Hiroko; Oshima, Masanobu; Kim, Dong-Wook; Nakauchi, Hiromitsu; Takihara, Yoshihiro; Kato, Yukio; Ooshima, Akira; Kim, Seong-Jin

    2015-01-01

    Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK–Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment. PMID:26289811

  20. Dipeptide species regulate p38MAPK-Smad3 signalling to maintain chronic myelogenous leukaemia stem cells.

    PubMed

    Naka, Kazuhito; Jomen, Yoshie; Ishihara, Kaori; Kim, Junil; Ishimoto, Takahiro; Bae, Eun-Jin; Mohney, Robert P; Stirdivant, Steven M; Oshima, Hiroko; Oshima, Masanobu; Kim, Dong-Wook; Nakauchi, Hiromitsu; Takihara, Yoshihiro; Kato, Yukio; Ooshima, Akira; Kim, Seong-Jin

    2015-08-20

    Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK-Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment.

  1. EPR/alanine dosimetry for two therapeutic proton beams

    NASA Astrophysics Data System (ADS)

    Marrale, Maurizio; Carlino, Antonio; Gallo, Salvatore; Longo, Anna; Panzeca, Salvatore; Bolsi, Alessandra; Hrbacek, Jan; Lomax, Tony

    2016-02-01

    In this work the analysis of the electron paramagnetic resonance (EPR) response of alanine pellets exposed to two different clinical proton beams employed for radiotherapy is performed. One beam is characterized by a passive delivery technique and is dedicated to the eyes treatment (OPTIS2 beam line). Alanine pellets were irradiated with a 70 MeV proton beam corresponding to 35 mm range in eye tissue. We investigated how collimators with different sizes and shape used to conform the dose to the planned target volume influence the delivered dose. For this purpose we performed measurements with varying the collimator size (Output Factor) and the results were compared with those obtained with other dosimetric techniques (such as Markus chamber and diode detector). This analysis showed that the dosimeter response is independent of collimator diameter if this is larger than or equal to 10 mm. The other beam is characterized by an active spot-scanning technique, the Gantry1 beam line (maximum energy 230 MeV), and is used to treat deep-seated tumors. The dose linearity of alanine response in the clinical dose range was tested and the alanine dose response at selected locations in depth was measured and compared with the TPS planned dose in a quasi-clinical scenario. The alanine response was found to be linear in the dose in the clinical explored range (from 10 to 70 Gy). Furthermore, a depth dose profile in a quasi-clinical scenario was measured and compared to the dose computed by the Treatment Planning System PSIPLAN. The comparison of calibrated proton alanine measurements and TPS dose shows a difference under 1% in the SOBP and a "quenching" effect up to 4% in the distal part of SOBP. The positive dosimetric characteristics of the alanine pellets confirm the feasibility to use these detectors for "in vivo" dosimetry in clinical proton beams.

  2. Alanine synthesis from glyceraldehyde and ammonium ion in aqueous solution

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1985-01-01

    The formation of alanine (ala) form C(14)-glyceraldehyde and ammonium phosphate in the presence or absence of a thiol is reported. At ambient temperature, ala synthesis was six times more rapid in the presence of 3-mercaptopropionic acid than in its absence (0.6 and 0.1 percent, respectively, after 60 days). Similarly, the presence of another thiol, N-acetylcysteinate, increased the production of ala, as well as of lactate. The reaction pathway of thiol-catalyzed synthesis of ala, with the lactic acid formed in a bypath, is suggested. In this, dehydration of glyceraldehyde is followed by the formation of hemithioacetal. In the presence of ammonia, an imine is formed, which eventually yields ala. This pathway is consistent with the observation that the rate ratio of ala/lactate remains constant throughout the process. The fact that the reaction takes place under anaerobic conditions in the presence of H2O and with the low concentrations of simple substrates and catalysts makes it an attractive model prebiotic reaction in the process of molecular evolution.

  3. [The ratio between the initial stages of assimilation of intact and hydrolyzed dipeptides in rat small intestine].

    PubMed

    Metel'skiĭ, S T

    2013-04-01

    Absorption of amino acids and dipeptides in the intestine may be coupled with sodium or proton, respectively. In both cases, when these nutrients are added to the mucosal solution (solution bathing the mucosa) through the epithelial layer should flow additional electric current. which is the most convenient to register by the short-circuit current method (SCC). It was shown that at pH 8.5, we can observe more efficient dipeptide transport (SCC response amplitudes on dipeptides have greater amplitude then SCC responses to equimolar mixture of the corresponding amino acid). In contrast, at pH 5.5 we observe the reverse situation - the SCC responses to mixture of amino acids are more then SCC responses to dipeptides. Increased with pH sodium dependent component of the stimulating effect of easily hydrolysable dipeptides is due to the membrane digestion. Sodium independent effect of dipeptides assimilation decreasing with pH caused by, apparently, functioning of proton-dependent transporter PepT1 for oligopeptides in the apical membrane of enterocytes. We believe that sequential "conditioning" of the stomach contents in the ever-increasing pH while its moving along the gastrointestinal tract (from stomach to colon) enables optimal use of both the mechanism of absorption of oligodipeptides, which is accompanied by a more complete digestion of oligodipeptides.

  4. [Kinetics and equilibrium of reactions between nucleotides and methylol derivatives of beta-alanine].

    PubMed

    Khulordava, K G; Kosaganov, Iu N; Lazurkin, Iu S

    1978-01-01

    The rate constants of forward and reverse reactions between methylol derivatives of beta-alanine and deoxycytidine 5'-phosphate, deoxyadenosine 5'phosphate and deoxyguanosine 5'phosphate and the equilibrium constants of these reactions were determined by the spectrophotometric method at 39,5 degrees C and pH 6,95. Besides, the equilibrium constant of the reaction between beta-alanine and formaldehyde was determined. Unlike deoxycytidine and deoxyadenosine 5'-phosphates, interaction of deoxyguanosine 5'phosphate with methylol derivatives is more complicated. A model proposed for the interaction of deoxyguanosine 5'phosphate with methylol derivatives explains the behavior of this nucleotide in the reaction. The kinetic and equilibrium constants of the interaction of methylol derivatives with nucleotides investigated exceed by two or three orders of magnitude the corresponding constants of the interaction of formaldehyde with these nucleotides.

  5. Adsorption of di-l-alanine on Cu(110) investigated with scanning tunneling microscopy [rapid communication

    NASA Astrophysics Data System (ADS)

    Stensgaard, I.

    2003-11-01

    Sub-monolayer growth of a small chiral peptide, di- L-alanine, on Cu(1 1 0) was investigated by variable temperature scanning tunneling microscopy (STM). At low coverage and for temperatures above ≈-220 K the molecules nucleate along the [ 3¯ 3 2] direction to form short, mainly one-dimensional islands. An increase in coverage leads to the formation of [ 3¯ 3 2]-directed, elongated islands. Images with sub-molecular resolution reveal that the orientation of the molecules within one particular island depends on the deposition temperature. At higher coverage, up to one monolayer, the islands coalesce, giving rise to phase boundaries between domains of opposite orientation. An atomic-scale model for di- L-alanine on Cu(1 1 0) is presented.

  6. Chiral effects on helicity studied via the energy landscape of short (D, L)-alanine peptides.

    PubMed

    Neelamraju, Sridhar; Oakley, Mark T; Johnston, Roy L

    2015-10-28

    The homochirality of natural amino acids facilitates the formation of regular secondary structures such as α-helices and β-sheets. Here, we study the relationship between chirality and backbone structure for the example of hexa-alanine. The most stable stereoisomers are identified through global optimisation. Further, the energy landscape, a database of connected low-energy local minima and transition points, is constructed for various neutral and zwitterionic stereoisomers of hexa-alanine. Three order parameters for partial helicity are applied and metric disconnectivity graphs are presented with partial helicity as a metric. We also apply the Zimm-Bragg model to derive average partial helicities for Ace-(L-Ala)6-NHMe, Ace-(D-Ala-L-Ala)3-NHMe, and Ace-(L-Ala)3-(D-Ala)3-NHMe from the database of local minima and compare with previous studies.

  7. Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents

    PubMed Central

    Wang, Limin; Zha, Zhihao; Qu, Wenchao; Qiao, Hongwen; Lieberman, Brian P.; Plössl, Karl; Kung, Hank F.

    2012-01-01

    Introduction This paper reports the synthesis and labeling of 18F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. Methods Three new 18F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labelling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[18F]fluoromethyl)-L-alanine (L[18F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). Results New 18F alanine derivatives were prepared with 7–34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[18F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than those observed for other two alanine derivatives and [18F]FDG in first 1 h. Inhibition of cell uptake studies suggested that L-[18F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[18F]FMA remained stable and was not incorporated into protein within 2 h. In vivo biodistribution studies demonstrated that L-[18F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[18F]FMA in both 9L rat and transgenic mouse. Conclusion L-[18F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor

  8. On the existence of ``l-threonine formate'', ``l-alanine lithium chloride'' and ``bis l-alanine lithium chloride'' crystals

    NASA Astrophysics Data System (ADS)

    Petrosyan, A. M.; Ghazaryan, V. V.; Fleck, M.

    2013-03-01

    We argue that the recently reported crystals "L-threonine formate" as well as "L-alanine lithium chloride" and "bis L-alanine lithium chloride" actually are the well-known crystals L-threonine and L-alanine, respectively.

  9. First-principles studies of pure and fluorine substituted alanines

    NASA Astrophysics Data System (ADS)

    Ahmad, Sardar; Vaizie, Hamide; Rahnamaye Aliabad, H. A.; Ahmad, Rashid; Khan, Imad; Ali, Zahid; Jalali-Asadabadi, S.; Ahmad, Iftikhar; Khan, Amir Abdullah

    2016-05-01

    This paper communicates the structural, electronic and optical properties of L-alanine, monofluoro and difluoro substituted alanines using density functional calculations. These compounds exist in orthorhombic crystal structure and the calculated structural parameters such as lattice constants, bond angles and bond lengths are in agreement with the experimental results. L-alanine is an indirect band gap insulator, while its fluorine substituted compounds (monofluoroalanine and difluoroalanine) are direct band gap insulators. The substitution causes reduction in the band gap and hence these optically tailored direct wide band gap materials have enhanced optical properties in the ultraviolet (UV) region of electromagnetic spectrum. Therefore, optical properties like dielectric function, refractive index, reflectivity and energy loss function are also investigated. These compounds have almost isotropic nature in the lower frequency range while at higher energies, they have a significant anisotropic nature.

  10. Surface Chirality of Gly-Pro Dipeptide Adsorbed on a Cu(110) Surface.

    PubMed

    Cruguel, Hervé; Méthivier, Christophe; Pradier, Claire-Marie; Humblot, Vincent

    2015-07-01

    The adsorption of chiral Gly-Pro dipeptide on Cu(110) has been characterized by combining in situ polarization modulation infrared reflection absorption spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS). The chemical state of the dipeptide, and its anchoring points and adsorption geometry, were determined at various coverage values. Gly-Pro molecules are present on Cu(110) in their anionic form (NH2 /COO(-)) and adsorb under a 3-point binding via both oxygen atoms of the carboxylate group and via the nitrogen atom of the amine group. Low-energy electron diffraction (LEED) and scanning tunneling microscopy (STM) have shown the presence of an extended 2D chiral array, sustained via intermolecular H-bonds interactions. Furthermore, due to the particular shape of the molecule, only one homochiral domain is formed, creating thus a truly chiral surface.

  11. Analogues of arginine vasopressin modified in position 2 and 3 with conformationally constrained dipeptide fragments.

    PubMed

    Łempicka, Elzbieta; Derdowska, Izabela; Kowalczyk, Wioleta; Dawidowska, Olga; Prahl, Adam; Janecki, Marcin; Jasiński, Tomasz; Trzeciak, Henryk I; Lammek, Bernard

    2005-02-01

    This study describes the synthesis and some pharmacological properties of ten new analogues of arginine vasopressin (AVP) containing a conformationally constrained dipeptide fragment in the N-terminal part of their molecules. Amino acid residues in positions 2 and 3 of AVP and some of its agonistic analogues were replaced with -Phe-Phe and D-Phe-D-Phe, dipeptides having a -CH2-CH2- link bridging two nitrogens. All the new peptides were tested for vasopressor and antidiuretic activities. Four peptides with pA2 values ranging from 5.96 to 7.21 turned out to be weak or moderately potent V1a antagonists. The results supplied new information about the structure-activity relationship of AVP analogues. As some of these were unexpected, they point to the need for caution when extrapolating previously known effects of modifications to analogues having conformationally constrained fragments in their molecules.

  12. 157 nm photodissociation of dipeptide ions containing N-terminal arginine.

    PubMed

    Webber, Nathaniel; He, Yi; Reilly, James P

    2014-02-01

    Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

  13. Prediction of the disulfide-bonding state of cysteines in proteins based on dipeptide composition.

    PubMed

    Song, Jiang-Ning; Wang, Ming-Lei; Li, Wei-Jiang; Xu, Wen-Bo

    2004-05-21

    In this paper, a novel approach has been introduced to predict the disulfide-bonding state of cysteines in proteins by means of a linear discriminator based on their dipeptide composition. The prediction is performed with a newly enlarged dataset with 8114 cysteine-containing segments extracted from 1856 non-homologous proteins of well-resolved three-dimensional structures. The oxidation of cysteines exhibits obvious cooperativity: almost all cysteines in disulfide-bond-containing proteins are in the oxidized form. This cooperativity can be well described by protein's dipeptide composition, based on which the prediction accuracy of the oxidation form of cysteines scores as high as 89.1% and 85.2%, when measured on cysteine and protein basis using the rigorous jack-knife procedure, respectively. The result demonstrates the applicability of this new relatively simple method and provides superior prediction performance compared with existing methods for the prediction of the oxidation states of cysteines in proteins.

  14. Linking micellar structures to hydrogelation for salt-triggered dipeptide gelators.

    PubMed

    Cardoso, Andre Zamith; Mears, Laura L E; Cattoz, Beatrice N; Griffiths, Peter C; Schweins, Ralf; Adams, Dave J

    2016-04-21

    Some functionalised dipeptides can form hydrogels when salts are added to solutions at high pH. We have used surface tension, conductivity, rheology, optical, confocal and scanning electron microscopy, (1)H NMR and UV-Vis spectroscopy measurements to characterise fully the phase behaviour of solutions of one specific gelator, 2NapFF, at 25 °C at pH 10.5. We show that this specific naphthalene-dipeptide undergoes structural transformations as the concentration is increased, initially forming spherical micelles, then worm-like micelles, followed by association of these worm-like micelles. On addition of a calcium salt, gels are generally formed as long as worm-like micelles are initially present in solution, although there are structural re-organisations that occur at lower concentrations, allowing gelation at lower than expected concentration. Using IR and SANS, we show the differences between the structures present in the solution and hydrogel phases.

  15. Light-driven topochemical polymerization under organogel conditions of a symmetrical dipeptide-diacetylene system.

    PubMed

    Mazzier, Daniela; Mosconi, Dario; Marafon, Giulia; Reheman, Aikebaier; Toniolo, Claudio; Moretto, Alessandro

    2017-02-01

    A symmetrical dipeptide-based diacetylene system (DAs) was found to be able to self-assemble in dichloromethane and to form a compact fiber network which resulted in a stable organogel. As a consequence of the organogel formation, we explored the possibility to run a light-induced topochemical polymerization. This is a typical reaction of ordered diacetylene moieties taking advantage from their organized packing mode resulting from fiber formation. Evidence for the generation of peptide-based polydiacetylenes is provided by Raman, UV-Vis, and CD spectroscopies and a set of microscopic techniques. Finally, we succeeded in processing a polymeric composite by use of the electrospinning technique, starting from a mixture of a dipeptide-based diacetylene and polymethyl methacrylate. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  16. Enhancement of the neonate's nonspecific immunity to Klebsiella infection by muramyl dipeptide, a synthetic immunoadjuvant.

    PubMed Central

    Parant, M; Parant, F; Chedid, L

    1978-01-01

    N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) and certain derivatives that are structural analogs of part of the bacterial peptidoglycan monomer have been shown to be adjuvant active and to enhance the nonspecific immunity of adult mice infected by Klebsiella pneumoniae. In the present study muramyl dipeptide and two other synthetic analogs were found to be active in newborn mice. This activity could be demonstrated after administration by subcutaneous or even by oral route. In contrast to what was observed after treatment by lipopolysaccharide, 8-day-old mice were definitively protected against bacterial challenge by these glycopeptides. Therefore such molecules could have a great value in view of studying and correcting the neonate's unresponsiveness. PMID:356054

  17. A multi-stimuli responive, self-assembling, boronic acid dipeptide

    DOE PAGES

    Jones, Brad Howard; Martinez, Alina Marissa; Wheeler, Jill S.; ...

    2015-08-11

    Modification of the dipeptide of phenylalanine, FF, with a boronic acid (BA) functionality imparts unique aqueous self-assembly behavior that responds to multiple stimuli. Changes in pH and ionic strength are used to trigger hydrogelation via the formation of nanoribbon networks. Thus, we show for the first time that the binding of polyols to the BA functionality can modulate a peptide between its assembled and disassembled states.

  18. Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.

    PubMed

    Wen, Xinmei; Tan, Wenzhi; Westergard, Thomas; Krishnamurthy, Karthik; Markandaiah, Shashirekha S; Shi, Yingxiao; Lin, Shaoyu; Shneider, Neil A; Monaghan, John; Pandey, Udai B; Pasinelli, Piera; Ichida, Justin K; Trotti, Davide

    2014-12-17

    Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.

  19. Antisense Proline-Arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death

    PubMed Central

    Wen, Xinmei; Tan, Wenzhi; Westergard, Thomas; Krishnamurthy, Karthik; ShamamandriMarkandaiah, Shashirekha; Shi, Yingxiao; Lin, Shaoyu; Shneider, Neil A.; Monaghan, John; Pandey, Udai B.; Pasinelli, Piera; Ichida, Justin K.; Trotti, Davide

    2015-01-01

    SUMMARY Expanded GGGGCC nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granules formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human-induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms. PMID:25521377

  20. Atomic Layer Deposition of L-Alanine Polypeptide

    SciTech Connect

    Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; Dunphy, Darren R.; Tsai, Andy; Tam, Siu-Yue; Fan, Hongyou Y.; Zhang, Hongxia; Rogers, David; Rempe, Susan; Atanassov, Plamen; Cecchi, Joseph L.; Brinker, C. Jeffrey

    2014-10-30

    L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

  1. Solvation Free Energies of Alanine Peptides: The Effect of Flexibility

    SciTech Connect

    Kokubo, Hironori; Harris, Robert C.; Asthagiri, Dilip; Pettitt, Bernard M.

    2013-12-03

    The electrostatic (?Gel), cavity-formation (?Gvdw), and total (?G) solvation free energies for 10 alanine peptides ranging in length (n) from 1 to 10 monomers were calculated. The free energies were computed both with xed, extended conformations of the peptides and again for some of the peptides without constraints. The solvation free energies, ?Gel, ?Gvdw, and ?G, were found to be linear in n, with the slopes of the best-fit lines being gamma_el, gamma_vdw, and gamma, respectively. Both gamma_el and gamma were negative for fixed and flexible peptides, and gamma_vdw was negative for fixed peptides. That gamma_vdw was negative was surprising, as experimental data on alkanes, theoretical models, and MD computations on small molecules and model systems generally suggest that gamma_vdw should be positive. A negative gamma_vdw seemingly contradicts the notion that ?Gvdw drives the initial collapse of the protein when it folds by favoring conformations with small surface areas, but when we computed ?Gvdw for the flexible peptides, thereby allowing the peptides to assume natural ensembles of more compact conformations, gamma-vdw was positive. Because most proteins do not assume extended conformations, a ?Gvdw that increases with increasing surface area may be typical for globular proteins. An alternative hypothesis is that the collapse is driven by intramolecular interactions. We show that the intramolecular van der Waal's interaction energy is more favorable for the flexible than for the extended peptides, seemingly favoring this hypothesis, but the large fluctuations in this energy may make attributing the collapse of the peptide to this intramolecular energy difficult.

  2. 2, 5-diketopiperazines (cyclic dipeptides) in beef: identification, synthesis, and sensory evaluation.

    PubMed

    Chen, M Z; Dewis, M L; Kraut, K; Merritt, D; Reiber, L; Trinnaman, L; Da Costa, N C

    2009-03-01

    Stewed beef and grilled dry aged beef were analyzed as part of an in-depth analytical program, with the aim of creating new flavors incorporating only compounds identified in the target foods and identifying new synthesis targets. In-house GC-MS analyses of several types of cooked beef have identified over 1000 volatile and semivolatile components; many for the 1st time. Among the semivolatiles detected were ten 2, 5-diketopiperazines (cyclic dipeptides) previously unreported in beef. These cyclic dipeptides are cis-cyclo(L-Ile-L-Pro), cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Pro-L-Pro), cis-cyclo(L-Pro-L-Val), cis-cyclo(L-Ala-L-Pro), cyclo(Gly-L-Pro), cyclo(Gly-L-Leu), cis-cyclo(L-Met-L-Pro), cis-cyclo(L-Phe-L-Pro), and cis-cyclo(L-Phe-L-Val). All 10 cyclic dipeptides were synthesized and evaluated organoleptically. Among them cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Met-L-Pro), and cis-cyclo(L-Phe-L-Pro) were found to be of particular organoleptic interest.

  3. Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors.

    PubMed

    Schmitz, Janina; Furtmann, Norbert; Ponert, Moritz; Frizler, Maxim; Löser, Reik; Bartz, Ulrike; Bajorath, Jürgen; Gütschow, Michael

    2015-08-01

    Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors.

  4. Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations

    SciTech Connect

    Island, M.D.; Naider, F.; Becker, J.M.

    1987-05-01

    Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides. The authors examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 ..mu..M (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L(/sup 3/H)leucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts form tryptophan-induced cultures. The authors concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptidetransport that can respond to very low concentrations of amino acids.

  5. Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

    PubMed

    Vistoli, Giulio; De Maddis, Danilo; Straniero, Valentina; Pedretti, Alessandro; Pallavicini, Marco; Valoti, Ermanno; Carini, Marina; Testa, Bernard; Aldini, Giancarlo

    2013-08-01

    The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.

  6. Design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor 1 antagonist.

    PubMed

    Hwang, Tsong-Long; Hung, Chih-Hao; Hsu, Ching-Yun; Huang, Yin-Ting; Tsai, Yu-Chi; Hsieh, Pei-Wen

    2013-06-14

    Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selectively inhibited neutrophil elastase release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-containing dipeptides were synthesized and their pharmacological activities were investigated in human neutrophils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on FMLP-induced superoxide anion (O2˙(-)) generation and neutrophil elastase release in neutrophils with IC50 values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of structure-activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most suitable as a core structure for interaction with FPR1, and may be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized compounds exhibited separable conformational isomers, and showed diverse bioactivities, the conformation analysis of these compounds is also discussed herein.

  7. Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir.

    PubMed

    Talluri, Ravi S; Samanta, Swapan K; Gaudana, Ripal; Mitra, Ashim K

    2008-09-01

    The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine-l-valine-acyclovir (LLACV), l-valine-d-valine-acyclovir (LDACV), d-valine-l-valine-acyclovir (DLACV) and d-valine-d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC-MS/MS and NMR spectroscopy. Uptake and transport of [(3)H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV>LLACV>DDACV>DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB).

  8. Structure-activity relationships for dipeptide prodrugs of acyclovir: implications for prodrug design.

    PubMed

    Santos, Cledir R; Capela, Rita; Pereira, Cláudia S G P; Valente, Emília; Gouveia, Luís; Pannecouque, Christophe; De Clercq, Erik; Moreira, Rui; Gomes, Paula

    2009-06-01

    A series of water-soluble dipeptide ester prodrugs of the antiviral acyclovir (ACV) were evaluated for their chemical stability, cytotoxicity, and antiviral activity against several strains of Herpes Simplex-1 and -2, vaccinia, vesicular stomatitis, cytomegalovirus and varicella zoster viruses. ACV dipeptide esters were very active against herpetic viruses, independently of the rate at which they liberate the parent drug. Their minimum cytotoxic concentrations were above 100 microM and the resulting MCC/EC(50) values were lower than those of ACV. When comparing the reactivity of Phe-Gly esters and amides (ACV, zidovudine, paracetamol, captopril and primaquine) in pH 7.4 buffer it was found that the rate of drug release increases with drug's leaving group ability. Release of the parent drug from Phe-Gly in human plasma is markedly faster than in pH 7.4 buffer, thus suggesting that the dipeptide-based prodrug approach can be successfully applied to bioactive agents containing thiol, phenol and amine functional groups.

  9. Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kempkes, Lisanne J. M.; Martens, Jonathan; Grzetic, Josipa; Berden, Giel; Oomens, Jos

    2016-11-01

    Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the resulting product ion structures using infrared ion spectroscopy with the free electron laser FELIX. The influence of the side chain length (Asn versus Gln) and of the amino acid sequence on the deamidation reaction has been examined. Molecular structures for the product ions are determined by comparison of experimental IR spectra with spectra predicted by density functional theory (DFT). The reaction mechanisms identified for the four dipeptides AlaAsn, AsnAla, AlaGln, and GlnAla are not the same. For all four dipeptides, primary deamidation takes place from the amide side chain (and not from the N-terminus) and, in most cases, resembles the mechanisms previously identified for the protonated amino acids asparagine and glutamine. Secondary fragmentation reactions of the deamidation products have also been characterized and provide further insight in - and confirmation of - the identified mechanisms. Overall, this study provides a comprehensive molecular structure map of the deamidation chemistry of this series of dipeptides.

  10. Dipeptides and Diketopiperazines in the Yamato-791198 and Murchison Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Shimoyama, Akira; Ogasawara, Ryo

    2002-04-01

    The Yamato-791198 and Murchison carbonaceous chondrites were analyzed for dipeptides and diketopiperazines as well as amino acids and hydantoins by gas chromatography combined with mass spectrometry. Glycylglycine (gly-gly) and cyclo(gly-gly) were detected at the concentrations of 11 and 18 pmol g^-1, respectively, in Yamato-791198, and 4 and 23 pmol g^-1, respectively, in Murchison. No other dipeptide and diketopiperazine were detected. Five hydantoins were detected at 8 to 65 pmol g^-1 in Yamato-791198 and seven in Murchison at 6 to 104 pmol g^-1. Total concentration of the glycine (gly) dimers is approximately four orders of magnitude less than the concentration of free gly in Yamato-791198, and three orders of magnitude less than that in Murchison. The absence of L- and LL-stereoisomers of dipeptides consisting of protein amino acids indicates that gly-gly and cyclo(gly-gly) detected are native to the chondrites and not from terrestrial contaminants. A possibility was discussed that the gly dimers might have been formed by condensation of gly monomers but not formed through N-carboxyanhydrides of gly.

  11. A new cyclic dipeptide penicimutide: the activated production of cyclic dipeptides by introduction of neomycin-resistance in the marine-derived fungus Penicillium purpurogenum G59.

    PubMed

    Wang, Nan; Cui, Cheng-Bin; Li, Chang-Wei

    2016-06-01

    A novel cyclic dipeptide, named penicimutide (1), and four known cyclic dipeptides, cyclo(L-Val-L-Pro) (2), cyclo(L-Ile-L-Pro) (3), cyclo(L-Leu-L-Pro) (4) and cyclo(L-Phe-L-Pro) (5), were isolated from a neomycin-resistant mutant of the marine-derived fungus Penicillium purpurogenum G59. The structure of 1, including the absolute configuration, was determined by spectroscopic and chemical methods, especially NMR and Marfey's analysis. An unusual amino acid in 1, 4,5-didehydro-L-leucine, was found for the first time occurring in nature. HPLC-ESI-MS analysis evidenced that 1-3 were produced only in the mutant strain, but 4 and 5 were produced in both the mutant and parental strains, indicating that the introduction of neomycin-resistance in the mutant activated pathways of 1-3 biosynthesis that were silent in the parental strain. Compound 1 selectively inhibited HeLa cells (among five tested human cancer cell lines) with an inhibition rate (IR %) of 39.4 % at 100 µg/mL, a similar inhibition intensity to that of the positive control 5-fluorouracil (IR % of 41.4 % at 100 µg/mL against HeLa cells). The present work exemplifies the effectiveness of our previous DMSO-mediated method for introducing drug-resistance in fungi to activate silent biosynthetic pathways to obtain new bioactive compounds.

  12. Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

    NASA Technical Reports Server (NTRS)

    Profy, A. T.; Usher, D. A.

    1984-01-01

    The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

  13. Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry.

    PubMed

    Takahashi, Kei; Tokuoka, Masafumi; Kohno, Hiromi; Sawamura, Nobuko; Myoken, Yuka; Mizuno, Akihiro

    2012-06-15

    Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 μM in sake. The analysis was validated by dipeptide recovery of 64.0-107.2% (2.5 μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

  14. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria.

    PubMed

    Peng, Bo; Su, Yu-Bin; Li, Hui; Han, Yi; Guo, Chang; Tian, Yao-Mei; Peng, Xuan-Xian

    2015-02-03

    Multidrug-resistant bacteria are an increasingly serious threat to human and animal health. However, novel drugs that can manage infections by multidrug-resistant bacteria have proved elusive. Here we show that glucose and alanine abundances are greatly suppressed in kanamycin-resistant Edwardsiella tarda by GC-MS-based metabolomics. Exogenous alanine or glucose restores susceptibility of multidrug-resistant E. tarda to killing by kanamycin, demonstrating an approach to killing multidrug-resistant bacteria. The mechanism underlying this approach is that exogenous glucose or alanine promotes the TCA cycle by substrate activation, which in turn increases production of NADH and proton motive force and stimulates uptake of antibiotic. Similar results are obtained with other Gram-negative bacteria (Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacterium (Staphylococcus aureus), and the results are also reproduced in a mouse model for urinary tract infection. This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria.

  15. Surface chemistry of alanine on Cu{111}: Adsorption geometry and temperature dependence

    NASA Astrophysics Data System (ADS)

    Baldanza, Silvia; Cornish, Alix; Nicklin, Richard E. J.; Zheleva, Zhasmina V.; Held, Georg

    2014-11-01

    Adsorption of L-alanine on the Cu{111} single crystal surface was investigated as a model system for interactions between small chiral modifier molecules and close-packed metal surfaces. Synchrotron-based X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy are used to determine the chemical state, bond coordination and out-of-plane orientation of the molecule on the surface. Alanine adsorbs in its anionic form at room temperature, whilst at low temperature the overlayer consists of anionic and zwitterionic molecules. NEXAFS spectra exhibit a strong angular dependence of the π* resonance associated with the carboxylate group, which allows determining the tilt angle of this group with respect to the surface plane (48° ± 2°) at room temperature. Low-energy electron diffraction (LEED) shows a p(2√{ 13} × 2√{ 13}) R 13 ° superstructure with only one domain, which breaks the mirror symmetry of the substrate and, thus, induces global chirality to the surface. Temperature-programmed XPS (TP-XPS) and temperature-programmed desorption (TPD) experiments indicate that the zwitterionic form converts into the anionic species (alaninate) at 293 K. The latter desorbs/decomposes between 435 K and 445 K.

  16. [Regulation of key enzymes of L-alanine biosynthesis by Brevibacterium flavum producer strains].

    PubMed

    Melkonian, L O; Avetisova, G E; Ambartsumian, A A; Chakhalian, A Kh; Sagian, A S

    2013-01-01

    The mechanisms of L-alanine overproduction by Brevibacterium flavum producer strains were studied. It was shown that beta-CI-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine, including alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL1 8 producer strains, which are resistant to the inhibitory effect of beta-Cl-L-alanine, were obtained using a parental B. flavum AA5 producer strain, characterized by a reduced activity of alanine racemase (>or=98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the producer strains developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

  17. Cyclic cis-locked phospho-dipeptides reduce entry of AβPP into amyloidogenic processing pathway

    PubMed Central

    Fisher, Carolyn L.; Resnick, Ross J.; De, Soumya; Acevedo, Lucila A.; Lu, Kun Ping; Schroeder, Frank C.; Nicholson, Linda K.

    2016-01-01

    The cis/trans isomerization of X-Pro peptide bonds in proteins in some instances acts as a molecular switch in biological pathways. Our prior work suggests that the cis isomer of the phospho-Thr668-Pro669 motif, located in the cytoplasmic domain of the amyloid-β precursor protein (AβPP), is correlated with an increase in amyloidogenic processing of AβPP and production of amyloid beta (Aβ), the neurotoxic peptide fragment in Alzheimer’s disease (AD). We designed a 100% cis-locked cyclic dipeptide composed of cyclized phospho-Thr-Pro (pCDP) as a mimic for this putative pathological conformation, and three phosphate-blocked derivatives (pCDP-diBzl, pCDP-Bzl, and pCDP-diPOM). Two H4 neuroglioma cell lines were established as AD cell models for use in testing these compounds: H4-AβPP695 for stable overexpression of wild-type AβPP695, and H4-BACE1 for stable overexpression of β-site AβPP Cleaving Enzyme-1 (BACE1). The level of the secreted AβPP fragment resulting from BACE1 activity, sAβPPβ, served as a key proxy for amyloidogenic processing, since cleavage of AβPP by BACE1 is a requisite first step in Aβ production. Of the compounds tested, pCDP-diBzl decreased sAβPPβ levels in both cell lines, while pCDP-diPOM decreased sAβPPβ levels in only H4-BACE1 cells, all with similar dose-dependences and patterns of proteolytic AβPP fragments. Enzymatic assays showed that none of the pCDP derivatives directly inhibit BACE1 catalytic activity. These results suggest a model in which pCDP-diBzl and pCDP-diPOM act at a common point to inhibit entry of AβPP into the amyloidogenic AβPP processing pathway but through different targets, and provide important insights for the development of novel AD therapeutics. Keywords: Amyloid beta-Protein Precursor, Alzheimer’s Disease, cyclic dipeptides, diketopiperazine, phosphorylated Thr668. PMID:27662285

  18. Formation of {gamma}-alumina nanorods in presence of alanine

    SciTech Connect

    Dabbagh, Hossein A.; Rasti, Elham; Yalfani, Mohammad S.; Medina, Francesc

    2011-02-15

    Graphical abstract: Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. Research highlights: {yields} Research highlights {yields} Boehmite was prepared using a green sol-gel process in the presence of alanine. {yields} Nanorod aluminas with a high surface area were obtained. {yields} Addition of alanine would shape the size of the holes and crevices. {yields} The morphologies of the nanorods were revealed by transmission electron microscope. -- Abstract: Boehmite and alumina nanostructures were prepared using a simple green sol-gel process in the presence of alanine in water medium at room temperature. The uncalcined (dried at 200 {sup o}C) and the calcined materials (at 500, 600 and 700 {sup o}C for 4 h) were characterized using XRD, TEM, SEM, N{sub 2} physisorption and TGA. Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. The surface area was enhanced and crystallization was retarded as the alanine content increased. The morphologies of the nanoparticles and nanorods were revealed by a transmission electron microscope (TEM).

  19. Spectrophotometric readout for an alanine dosimeter for food irradiation applications

    NASA Astrophysics Data System (ADS)

    Ebraheem, S.; Beshir, W. B.; Eid, S.; Sobhy, R.; Kovács, A.

    2003-06-01

    The alanine-electron spin resonance (EPR) readout system is well known as a reference and transfer dosimetry system for the evaluation of high doses in radiation processing. The high cost of an EPR/alanine dosimetry system is a serious handicap for large-scale routine application in irradiation facilities. In this study, the use of a complex produced by dissolving irradiated L-alanine in 1,4-phenyl diammonium dichloride solution was investigated for dosimetry purposes. This complex—having a purple colour—has an increasing absorbance with increasing dose in the range of 1-20 kGy. The applicability of spectrophotometric evaluation was studied by measuring the absorbance intensity of this complex at 360 and 505 nm, respectively. Fluorimetric evaluation was also investigated by measuring the emission of the complex at 435 nm as a function of dose. The present method is easy for routine application. The effect of the dye concentration as well as the suitable amount of irradiated alanine has been studied. With respect to routine application, the stability of the product complex after its formation was also investigated.

  20. Computational alanine scanning with linear scaling semiempirical quantum mechanical methods.

    PubMed

    Diller, David J; Humblet, Christine; Zhang, Xiaohua; Westerhoff, Lance M

    2010-08-01

    Alanine scanning is a powerful experimental tool for understanding the key interactions in protein-protein interfaces. Linear scaling semiempirical quantum mechanical calculations are now sufficiently fast and robust to allow meaningful calculations on large systems such as proteins, RNA and DNA. In particular, they have proven useful in understanding protein-ligand interactions. Here we ask the question: can these linear scaling quantum mechanical methods developed for protein-ligand scoring be useful for computational alanine scanning? To answer this question, we assembled 15 protein-protein complexes with available crystal structures and sufficient alanine scanning data. In all, the data set contains Delta Delta Gs for 400 single point alanine mutations of these 15 complexes. We show that with only one adjusted parameter the quantum mechanics-based methods outperform both buried accessible surface area and a potential of mean force and compare favorably to a variety of published empirical methods. Finally, we closely examined the outliers in the data set and discuss some of the challenges that arise from this examination.

  1. Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

    PubMed

    Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Ngo, Ho-Phuong-Thuy; Tran, Huyen-Thi; Cha, Sun-Shin; Min Chung, Kyung; Huynh, Kim-Hung; Ahn, Yeh-Jin; Kang, Lin-Woo

    2014-03-01

    D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

  2. Trends for isolated amino acids and dipeptides: Conformation, divalent ion binding, and remarkable similarity of binding to calcium and lead

    NASA Astrophysics Data System (ADS)

    Ropo, M.; Blum, V.; Baldauf, C.

    2016-11-01

    We derive structural and binding energy trends for twenty amino acids, their dipeptides, and their interactions with the divalent cations Ca2+, Ba2+, Sr2+, Cd2+, Pb2+, and Hg2+. The underlying data set consists of more than 45,000 first-principles predicted conformers with relative energies up to ~4 eV (~400 kJ/mol). We show that only very few distinct backbone structures of isolated amino acids and their dipeptides emerge as lowest-energy conformers. The isolated amino acids predominantly adopt structures that involve an acidic proton shared between the carboxy and amino function. Dipeptides adopt one of two intramolecular-hydrogen bonded conformations C5 or . Upon complexation with a divalent cation, the accessible conformational space shrinks and intramolecular hydrogen bonding is prevented due to strong electrostatic interaction of backbone and side chain functional groups with cations. Clear correlations emerge from the binding energies of the six divalent ions with amino acids and dipeptides. Cd2+ and Hg2+ show the largest binding energies–a potential correlation with their known high acute toxicities. Ca2+ and Pb2+ reveal almost identical binding energies across the entire series of amino acids and dipeptides. This observation validates past indications that ion-mimicry of calcium and lead should play an important role in a toxicological context.

  3. Trends for isolated amino acids and dipeptides: Conformation, divalent ion binding, and remarkable similarity of binding to calcium and lead

    PubMed Central

    Ropo, M.; Blum, V.; Baldauf, C.

    2016-01-01

    We derive structural and binding energy trends for twenty amino acids, their dipeptides, and their interactions with the divalent cations Ca2+, Ba2+, Sr2+, Cd2+, Pb2+, and Hg2+. The underlying data set consists of more than 45,000 first-principles predicted conformers with relative energies up to ~4 eV (~400 kJ/mol). We show that only very few distinct backbone structures of isolated amino acids and their dipeptides emerge as lowest-energy conformers. The isolated amino acids predominantly adopt structures that involve an acidic proton shared between the carboxy and amino function. Dipeptides adopt one of two intramolecular-hydrogen bonded conformations C5 or . Upon complexation with a divalent cation, the accessible conformational space shrinks and intramolecular hydrogen bonding is prevented due to strong electrostatic interaction of backbone and side chain functional groups with cations. Clear correlations emerge from the binding energies of the six divalent ions with amino acids and dipeptides. Cd2+ and Hg2+ show the largest binding energies–a potential correlation with their known high acute toxicities. Ca2+ and Pb2+ reveal almost identical binding energies across the entire series of amino acids and dipeptides. This observation validates past indications that ion-mimicry of calcium and lead should play an important role in a toxicological context. PMID:27808109

  4. Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

    PubMed Central

    Shi, Kevin Y.; Mori, Eiichiro; Nizami, Zehra F.; Lin, Yi; Kato, Masato; Xiang, Siheng; Wu, Leeju C.; Ding, Ming; Yu, Yonghao; Gall, Joseph G.; McKnight, Steven L.

    2017-01-01

    The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS. PMID:28069952

  5. Formation of simple biomolecules from alanine in ocean by impacts

    NASA Astrophysics Data System (ADS)

    Umeda, Y.; Sekine, T.; Furukawa, Y.; Kakegawa, T.; Kobayashi, T.

    2013-12-01

    The biomolecules on the Earth are thought either to have originated from the extraterrestrial parts carried with flying meteorites or to have been formed from the inorganic materials on the Earth through given energy. From the standpoint to address the importance of impact energy, it is required to simulate experimentally the chemical reactions during impacts, because violent impacts may have occurred 3.8-4.0 Gyr ago to create biomolecules initially. It has been demonstrated that shock reactions among ocean (H2O), atmospheric nitrogen, and meteoritic constitution (Fe) can induce locally reduction environment to form simple bioorganic molecules such as ammonia and amino acid (Nakazawa et al., 2005; Furukawa et al., 2009). We need to know possible processes for alanine how chemical reactions proceed during repeated impacts and how complicated biomolecules are formed. Alanine can be formed from glycine (Umeda et al., in preparation). In this study, we carried out shock recovery experiments at pressures of 4.4-5.7 GPa to investigate the chemical reactions of alanine. Experiments were carried out with a propellant gun. Stainless steel containers (30 mm in diameter, 30 mm long) with 13C-labeled alanine aqueous solution immersed in olivine or hematite powders were used as targets. Air gap was present in the sample room (18 mm in diameter, 2 mm thick) behind the sample. The powder, solution, and air represent meteorite, ocean, and atmosphere on early Earth, respectively. Two powders of olivine and hematite help to keep the oxygen fugacity low and high during experiments, respectively in order to investigate the effect of oxygen fugacity on chemical processes of alanine. The recovered containers, after cleaned completely, were immersed into liquid nitrogen to freeze sample solution and then we drilled on the impact surface to extract water-soluble run products using pure water. Thus obtained products were analyzed by LC/MS for four amino acids (glycine, alanine, valine, and

  6. Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine.

    PubMed

    Prosser, Gareth A; de Carvalho, Luiz Pedro S

    2013-02-01

    D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

  7. A theoretical study of the XP and NEXAFS spectra of alanine: gas phase molecule, crystal, and adsorbate at the ZnO(10 ̅10) surface.

    PubMed

    Gao, You Kun; Traeger, Franziska; Kotsis, Konstantinos; Staemmler, Volker

    2011-06-14

    The adsorption of alanine on the mixed-terminated ZnO(10 ̅10) surface is studied by means of quantum-chemical ab initio calculations. Using a finite cluster model and the adsorption geometry as obtained both by periodic CPMD and embedded cluster calculations, the C1s, N1s and O1s X-ray photoelectron spectra (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectra are calculated for single alanine molecules on ZnO(10 ̅10). These spectra are compared with the spectra calculated for alanine in the gas phase and in its crystalline form and with experimental XPS and NEXAFS data for the isolated alanine molecule and for alanine adsorbed on ZnO(10 ̅10) at multilayer and monolayer coverage. The excellent agreement between the experimental and calculated XP and NEXAFS spectra confirms the calculated adsorption geometry: A single alanine molecule is bound to ZnO(10 ̅10) in a dissociated bidentate form with the two O atoms of the acid group bound to two Zn atoms of the surface and the proton transferred to one O atom of the surface. Other possible structures, such as adsorption of alanine in one of its neutral or zwitterionic forms in which the proton of the -COOH group remains at this group or is transferred to the amino group, can be excluded since they would give rise to quite different XP spectra. In the multilayer coverage regime, on the other hand, alanine is in its crystalline form as is also shown by the analysis of the XP spectra.

  8. The Dipeptides Ile-Tyr and Ser-Tyr Exert Distinct Effects on Catecholamine Metabolism in the Mouse Brainstem

    PubMed Central

    Moriyasu, Kazuki; Ichinose, Takashi; Nakahata, Akane; Tanaka, Mitsuru; Matsui, Toshiro; Furuya, Shigeki

    2016-01-01

    Catecholamine synthesis and transmission in the brain are influenced by the availability of Tyr in the body. In this study, we compared the effects of oral administration of Tyr-containing dipeptides Ile-Tyr, Ser-Tyr, and Tyr-Pro with Tyr alone on catecholamine metabolism in the mouse brainstem. Among these dipeptides, Ile-Tyr administration led to increases in dopamine, the dopamine metabolites homovanillic acid, and 3,4-dihydroxyphenylacetic acid, compared to administration of Ser-Tyr, Tyr-Pro, or Tyr alone. In comparison, administration of Ser-Tyr induced significantly increasing noradrenaline turnover, while Tyr-Pro administration suppressed dopamine turnover. Therefore, oral administration of Ile-Tyr, Ser-Tyr, and Tyr-Pro differentially affected metabolism of dopamine and noradrenaline. These observations strongly suggest that Tyr-containing dipeptides exert distinct effects on catecholamine metabolism in the brainstem when ingested orally. PMID:26981137

  9. Chemical synthesis and crystallization of the dipeptide AcPheIleNH(2) in TTAB/heptane/octanol reversed micelles.

    PubMed

    Dias, A I; Feliciano, A S; Cabral, J M S; Prazeres, D M F

    2007-01-01

    The chemical synthesis of the dipeptide acetyl phenyl iso-leucin-amide (AcPheIleNH(2)) in tetradecyl trimethyl ammonium bromide (TTAB)/ heptane/octanol/carbonate buffer reversed micelles is described. The co-existence of the surfactant bounded minute water pools within the bulk organic solvent enables the simultaneous solubilization of the polar (IleNH(2)) and apolar (AcPheOEt) substrates, thus enabling the synthesis to take place at the micellar interface. The synthesis was favored by increasing the micellar interface via an increase in water content and surfactant concentration. The best dipeptide yield (87%) was obtained at 32 degrees C, with the largest concentrations of TTAB (200 mM) and water (1100 mM) tested. The low solubility of the dipeptide in the micellar media further led to the formation and growth of needle-like crystals during synthesis, thus enabling the removal of product from solution.

  10. Two novel ferrocenyl dipeptide-like compounds generated via the Ugi four-component reaction.

    PubMed

    Shao, Guang-Kui; Zhao, Mei; Wei, Zheng; Ma, Jian-Ping; Guo, Dian-Shun

    2015-08-01

    The Ugi four-component reaction, a powerful method for the synthesis of diverse dipeptide-like derivatives in combinatorial chemistry, was used to synthesize (S)-1'-{N-[1-(anthracen-9-yl)-2-(tert-butylamino)-2-oxoethyl]-N-(4-methoxyphenyl)carbamoyl}ferrocene-1-carboxylic acid dichloromethane disolvate, [Fe(C6H5O2)(C33H31N2O3)]·2CH2Cl2, (I), and (S)-2-(anthracen-9-yl)-N-tert-butyl-2-[N-(4-methylphenyl)ferrocenylformamido]acetamide, [Fe(C5H5)(C33H31N2O2)], (II). They adopt broadly similar molecular conformations, with near-eclipsed cyclopentadienyl rings and near-perpendicular amide planes in their dipeptide-like chains, one of which is almost coplanar with its attached cyclopentadienyl ring but perpendicular to the aromatic ring bound to the N atom. In the supramolecular structure of (I), a two-dimensional network is constructed based on molecular dimers and a combination of intermolecular O-H···O, N-H···O and C-H···O hydrogen bonds, forming R2(2)(11), R2(2)(16), R2(2)(22) and C(9) motifs. These two-dimensional networks are connected by C-H···O and C-H···Cl contacts to create a three-dimensional framework, where one dichloromethane solvent molecule acts as a bridge between two neighbouring networks. In the packing of (II), classical hydrogen bonds are absent and an infinite one-dimensional chain is generated via a combination of C-H···O hydrogen bonds and C-H···π interactions, producing a C(7) motif. This work describes a simple synthesis and the supramolecuar structures of ferrocenyl dipeptide-like compounds and is significant in the development of redox-active receptors.

  11. Potential impact of sitagliptin on collagen-derived dipeptides in diabetic osteoporosis.

    PubMed

    Baerts, L; Glorie, L; Maho, W; Eelen, A; Verhulst, A; D'Haese, P; Covaci, A; De Meester, I

    2015-10-01

    It is known that diabetes coincides with an increased risk of osteoporosis. While a disturbed collagen metabolism is proposed as a possible cause, much remains unknown about the enzymes involved and changes in the collagen-derived dipeptides and amino acids. Therefore, we sought to study this intricate pathway and the effect of dipeptidyl peptidase 4 (DPP4) inhibitors. Control and streptozotocin-nicotinamide-induced diabetic rats were treated for 12 weeks with vehicle or sitagliptin, a DPP4 inhibitor (Con/VH, Con/SG, DM/VH and DM/SG). The activities of four key enzymes involved in collagen breakdown were determined in serum (DPP4, matrix metalloproteinase 2 and 9 and prolidase). Dipeptide (Ala-Pro, Gly-Pro, Pro-Pro and Pro-Hyp) and amino acid (Pro and Hyp) concentrations were measured by liquid chromatography coupled to mass spectrometry. We found three-fold higher MMP9 activities in DM/VH than in controls, while in DM/SG this rise was attenuated. MMP2 and prolidase did not differ in the investigated groups. Furthermore, we are the first to report on two-fold higher Ala-Pro and Pro-Pro levels in diabetes compared to controls. In contrast, Pro-Hyp concentrations were lower in diabetes (DM/VH and DM/SG). DPP4 inhibition does not seem to have a direct influence on the collagen metabolism in streptozotocin-nicotinamide-induced diabetic rats. Instead, it probably acts through its effect on osteoprotective substrates. In diabetes, increased MMP9 activities seem to favour the production of Ala-Pro and Pro-Pro containing collagen fragments. The high Pro-Hyp levels in untreated controls might have a bone-stimulating effect. Nevertheless, the biological significance of these dipeptides is not yet clear and should be further investigated.

  12. Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides.

    PubMed

    Hayward, Steven; Leader, David P; Al-Shubailly, Fawzia; Milner-White, E James

    2014-02-01

    Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of "nests" and "catgrips"; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β-sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β-pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P-loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II' β-turns, or twisted, as in multiple type I and type I' β-turns. Hexa- and octa-peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β-turns, and are the same as the "β-bend ribbons" hitherto identified only in short peptides. One is seen in the GTPase-activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β-bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity.

  13. SCMMTP: identifying and characterizing membrane transport proteins using propensity scores of dipeptides

    PubMed Central

    2015-01-01

    Background Identifying putative membrane transport proteins (MTPs) and understanding the transport mechanisms involved remain important challenges for the advancement of structural and functional genomics. However, the transporter characters are mainly acquired from MTP crystal structures which are hard to crystalize. Therefore, it is desirable to develop bioinformatics tools for the effective large-scale analysis of available sequences to identify novel transporters and characterize such transporters. Results This work proposes a novel method (SCMMTP) based on the scoring card method (SCM) using dipeptide composition to identify and characterize MTPs from an existing dataset containing 900 MTPs and 660 non-MTPs which are separated into a training dataset consisting 1,380 proteins and an independent dataset consisting 180 proteins. The SCMMTP produced estimating propensity scores for amino acids and dipeptides as MTPs. The SCMMTP training and test accuracy levels respectively reached 83.81% and 76.11%. The test accuracy of support vector machine (SVM) using a complicated classification method with a low possibility for biological interpretation and position-specific substitution matrix (PSSM) as a protein feature is 80.56%, thus SCMMTP is comparable to SVM-PSSM. To identify MTPs, SCMMTP is applied to three datasets including: 1) human transmembrane proteins, 2) a photosynthetic protein dataset, and 3) a human protein database. MTPs showing α-helix rich structure is agreed with previous studies. The MTPs used residues with low hydration energy. It is hypothesized that, after filtering substrates, the hydrated water molecules need to be released from the pore regions. Conclusions SCMMTP yields estimating propensity scores for amino acids and dipeptides as MTPs, which can be used to identify novel MTPs and characterize transport mechanisms for use in further experiments. Availability http://iclab.life.nctu.edu.tw/iclab_webtools/SCMMTP/ PMID:26677931

  14. Degradation of glycine and alanine on irradiated quartz.

    PubMed

    Pawlikowski, Maciej; Benko, Aleksandra; Wróbel, Tomasz P

    2013-04-01

    Recent researches suggest participation of minerals in the formation of life under primordial conditions. Among all of the minerals, quartz seems to be one of the most probable to take part in such processes. However, an external source of energy is needed, e.g. electric discharge. A device simulating the proposed conditions was designed and was used to simulate prebiotic conditions. Investigation of processes occurring during the stimulation of quartz with electric discharge was studied by means of Ultraviolet-visible (UV-VIS) spectroscopy, in order to monitor the generation kinetics of free radicals. Additionally, infrared spectroscopy was applied to identify chemical reaction products created in a solution of alanine or glycine, in the presence of quartz treated with electric discharge. Formation of increased amounts of free radicals, compared to experiments performed without quartz and/or amino acid, is reported, along with identification of possible degradation products of alanine. No synthetic reactions were observed.

  15. Clinical applications of alanine/electron spin resonance dosimetry.

    PubMed

    Baffa, Oswaldo; Kinoshita, Angela

    2014-05-01

    This paper discusses the clinical applications of electron spin resonance (ESR) dosimetry focusing on the ESR/alanine system. A review of few past studies in this area is presented offering a critical overview of the challenges and opportunities for extending this system into clinical applications. Alanine/ESR dosimetry fulfills many of the required properties for several clinical applications such as water-equivalent composition, independence of the sensitivity for the energy range used in therapy and high precision. Improvements in sensitivity and the development of minidosimeters coupled with the use of a spectrometer of higher microwave frequency expanded the possibilities for clinical applications to the new modalities of radiotherapy (intensity-modulated radiation therapy and radiosurgery) and to the detection of low doses such as those present in some radiological image procedures.

  16. The Quasi-Static Self Quenching of Trp-X and X-Trp Dipeptides in Water: Ultrafast Fluorescence Decay

    PubMed Central

    Xu, Jianhua; Knutson, Jay R.

    2012-01-01

    Time-resolved fluorescence decay profiles of N-acetyl-L-tryptophan-amide (NATA) and tryptophan (Trp) dipeptides of the form Trp-X and X-Trp, where X is another aminoacyl residue, have been investigated using an ultraviolet upconversion spectrophoto fluorometer with time resolution better than 350 fs, together with a time correlated single photon counting apparatus on the 100ps to 20ns time scale. We analyzed the set of fluorescence decay profiles at multiple wavelengths using the global analysis technique. Nanosecond fluorescence transients for Trp dipeptides all show multiexponential decay, while NATA exhibits a monoexponential decay near 3 ns independent of pH. In the first 100 ps, a time constant for the water “bulk relaxation” around Trp, NATA and Trp dipeptides is seen near 1-2 ps, with an associated preexponential amplitude that is positive or negative depending on emission wavelength, as expected for a population-conserving spectral shift. The initial brightness (sub-ps) we measure for all these dipeptides is less than that of NATA, implying even faster (<200fs) intra-molecular (quasi) static quenching occurs within them. A new, third, ultrafast decay, bearing an exponential time constant of 20-30 ps with positive amplitude, has been found in many of these dipeptides. We believe it verifies our previous predictions of dipeptide QSSQ (“quasi static self quenching”) –the loss of quantum yield to sub-100ps decay process (Chen et al., Biochemistry, 1991, 30, 5184). Most important, this term is found in proteins as well (J.A.C.S., 2006, 128, 1214; Biophysical Journal 2008, 94, 546; 2009, 96, 46a), suggesting an ultrafast quenching mechanism must be common to both. PMID:19708715

  17. Crystal Structures of Aedes Aegypt Alanine Glyoxylate Aminotransferase

    SciTech Connect

    Han,Q.; Robinson, H.; Gao, Y.; Vogelaar, N.; Wilson, S.; Rizzi, M.; Li, J.

    2006-01-01

    Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75{angstrom} high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1{angstrom} resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

  18. Physiological hypercortisolemia increases proteolysis, glutamine, and alanine production

    SciTech Connect

    Darmaun, D.; Matthews, D.E.; Bier, D.M. Cornell Univ. Medical College, New York, NY )

    1988-09-01

    Physiological elevations of plasma cortisol levels, as are encountered in stress and severe trauma, were produced in six normal subjects by infusing them with hydrocortisone for 64 h. Amino acid kinetics were measured in the postabsorptive state using three 4-h infusions of L-(1-{sup 13}C)leucine, L-phenyl({sup 2}H{sub 5})phenylalanine, L-(2-{sup 15}N)glutamine, and L-(1-{sup 13}C)alanine tracers (1) before, (2) at 12 h, and (3) at 60 h of cortisol infusion. Before and throughout the study, the subjects ate a normal diet of adequate protein and energy intake. The cortisol infusion raised plasma cortisol levels significantly from 10 {plus minus} 1 to 32 {plus minus} 4 {mu}g/dl, leucine flux from 83 {plus minus} 3 to 97 {plus minus} 3 {mu}mol{center dot}kg{sup {minus}1}{center dot}h{sup {minus}1}, and phenylalanine flux from 34 {plus minus} 1 to 39 {plus minus} 1 (SE) {mu}mol{center dot}kg{sup {minus}1}{center dot}h{sup {minus}1} after 12 h of cortisol infusion. These increases were maintained until the cortisol infusion was terminated. These nearly identical 15% increases in two different essential amino acid appearance rates are reflective of increased whole body protein breakdown. Glutamine flux rose by 12 h of cortisol infusion and remained elevated at the same level at 64 h. The increase in flux was primarily due to a 55% increase in glutamine de novo synthesis. Alanine flux increased with acute hypercortisolemia and increased further at 60 h of cortisol infusion, a result primarily of increased alanine de novo synthesis. Insulin, alanine, and lactate plasma levels responded similarly with significant rises between the acute and chronic periods of cortisol infusion. Thus hypercortisolemia increases both protein breakdown and the turnover of important nonessential amino acids for periods of up to 64 h.

  19. Crystal structures of Aedes aegypti alanine glyoxylate aminotransferase.

    PubMed

    Han, Qian; Robinson, Howard; Gao, Yi Gui; Vogelaar, Nancy; Wilson, Scott R; Rizzi, Menico; Li, Jianyong

    2006-12-01

    Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75A high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1A resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

  20. The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

    PubMed

    Alligood, K J; Charifson, P S; Crosby, R; Consler, T G; Feldman, P L; Gampe, R T; Gilmer, T M; Jordan, S R; Milstead, M W; Mohr, C; Peel, M R; Rocque, W; Rodriguez, M; Rusnak, D W; Shewchuk, L M; Sternbach, D D

    1998-05-19

    The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

  1. Dipeptide preparation Noopept prevents scopolamine-induced deficit of spatial memory in BALB/c mice.

    PubMed

    Belnik, A P; Ostrovskaya, R U; Poletaeva, I I

    2007-04-01

    The effect of original nootropic preparation Noopept on learning and long-term memory was studied with BALB/c mice. Scopolamine (1 mg/kg) impaired long-term memory trace, while Noopept (0.5 mg/kg) had no significant effect. Noopept completely prevented the development of cognitive disorders induced by scopolamine (blockade of muscarinic cholinergic receptors). Our results confirmed the presence of choline-positive effect in dipeptide piracetam analogue Noopept on retrieval of learned skill of finding a submerged platform (spatial memory). We conclude that the effectiveness of this drug should be evaluated in patients with Alzheimer's disease.

  2. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  3. Novel nootropic dipeptide Noopept increases inhibitory synaptic transmission in CA1 pyramidal cells.

    PubMed

    Kondratenko, Rodion V; Derevyagin, Vladimir I; Skrebitsky, Vladimir G

    2010-05-31

    Effects of newly synthesized nootropic and anxiolytic dipeptide Noopept on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) significantly increased the frequency of spike-dependant spontaneous IPSCs whereas spike-independent mIPSCs remained unchanged. It was suggested that Noopept mediates its effect due to the activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion.

  4. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

    PubMed

    Sousa, Cristovão M; Biancur, Douglas E; Wang, Xiaoxu; Halbrook, Christopher J; Sherman, Mara H; Zhang, Li; Kremer, Daniel; Hwang, Rosa F; Witkiewicz, Agnes K; Ying, Haoqiang; Asara, John M; Evans, Ronald M; Cantley, Lewis C; Lyssiotis, Costas A; Kimmelman, Alec C

    2016-08-25

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.

  5. Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

    PubMed

    Okubo, Y; Yokoigawa, K; Esaki, N; Soda, K; Kawai, H

    1999-03-16

    A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

  6. Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate

    PubMed Central

    Barb, A.W.; Hekmatyar, S.K.; Glushka, J.N.; Prestegard, J.H.

    2013-01-01

    Hyperpolarized metabolites offer a tremendous sensitivity advantage (>104 fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, 13C-enriched analog of pyruvic acid, 13C3D3-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct 13C observation and indirect observation through methyl protons introduced by ALT-catalyzed H–D exchange. Exchange on injecting hyperpolarized 13C3D3-pyruvate into ALT dissolved in buffered 1H2O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5 s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized 13C3-pyruvate and products in imaging applications are discussed. PMID:23357427

  7. Pressure-induced phase transitions in L-alanine, revisited.

    PubMed

    Tumanov, N A; Boldyreva, E V; Kolesov, B A; Kurnosov, A V; Quesada Cabrera, R

    2010-08-01

    The effect of pressure on L-alanine has been studied by X-ray powder diffraction (up to 12.3 GPa), single-crystal X-ray diffraction, Raman spectroscopy and optical microscopy (up to approximately 6 GPa). No structural phase transitions have been observed. At approximately 2 GPa the cell parameters a and b become accidentally equal to each other, but without a change in space-group symmetry. Neither of two transitions reported by others (to a tetragonal phase at approximately 2 GPa and to a monoclinic phase at approximately 9 GPa) was observed. The changes in cell parameters were continuous up to the highest measured pressures and the cells remained orthorhombic. Some important changes in the intermolecular interactions occur, which also manifest themselves in the Raman spectra. Two new orthorhombic phases could be crystallized from a MeOH/EtOH/H(2)O pressure-transmitting mixture in the pressure range 0.8-4.7 GPa, but only if the sample was kept at these pressures for at least 1-2 d. The new phases converted back to L-alanine on decompression. Judging from the Raman spectra and cell parameters, the new phases are most probably not L-alanine but its solvates.

  8. Synthesis of modular dipeptide mimetics on the basis of diazabicycloalkanes and derivatives thereof with sulphur containing side chains.

    PubMed

    Grohs, D C; Maison, W

    2005-08-01

    We present the synthesis of new modular dipeptide mimetics based on diazabicycloalkane backbones. These diazabicycloalkanes are ligands for the prostate specific membrane antigen (PSMA), a well known tumor marker. Our previously described synthetic route to enantiomerically pure diazabicycloalkanes is extended to yield polyfunctional diazabicycloalkanes with a modular character using a new ring closing methodology. This, finally, allows us to attach linker moieties to different positions of the diazabicycloalkane scaffold providing conjugation sites to other functional molecules such as markers or cytostatic compounds. Furthermore, successful synthesis of sulphur-containing dipeptide analogues as for example CysX(AA)- or HCysX(AA)-mimetics on the basis of diazabicycloalkanes is described.

  9. Quality by design development of brivanib alaninate tablets: degradant and moisture control strategy.

    PubMed

    Badawy, Sherif I F; Lin, Judy; Gokhale, Madhushree; Desai, Sachin; Nesarikar, Vishwas V; LaMarche, Keirnan R; Subramanian, Ganeshkumar A; Narang, Ajit S

    2014-07-20

    A quality by design approach was applied to the development of brivanib alaninate tablets. Brivanib alaninate, an ester pro-drug, undergoes hydrolysis to its parent compound, BMS-540215. The shelf-life of the tablets is determined by the rate of the hydrolysis reaction. Hydrolysis kinetics in the tablets was studied to understand its dependence on temperature and humidity. The BMS-540215 amount versus time profile was simulated using a kinetic model for the formation of BMS-540215 as function of relative humidity in the environment and a sorption-desorptiom moisture transfer model for the relative humidity inside the package. The combined model was used to study the effect of initial tablet water content on the rate of degradation and to identify a limit for initial tablet water content that results in acceptable level of the degradant at the end of shelf-life. A strategy was established for the moisture and degradant control in the tablet based on the understanding of its stability behavior and mathematical models. The control strategy includes a specification limit on the tablet water content and manufacturing process controls that achieve this limit at the time of tablet release testing.

  10. Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase.

    PubMed

    Szymańska-Majchrzak, Jolanta; Pałka, Katarzyna; Kańska, Marianna

    2017-05-01

    Synthesis of 3-fluoro-[2-(2)H]-L-alanine (3-F-[(2)H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H2O - KIE's on Vmax: 1.1; on Vmax/KM: 1.2; for 3-F-L-Ala in (2)H2O - on Vmax: 1.4; on Vmax/KM: 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE's on Vmax: 1.0; on Vmax/KM: 0.87; for 3-F-[2-(2)H]-L-Ala - on Vmax: 1.4; on Vmax/KM: 1.5). Studies explain some details of reaction mechanism.

  11. An "enigmatic" L-carnosine (β-alanyl-L-histidine)? Cell proliferative activity as a fundamental property of a natural dipeptide inherent to traditional antioxidant, anti-aging biological activities: balancing and a hormonally correct agent, novel patented oral therapy dosage formulation for mobility, skeletal muscle power and functional performance, hypothalamic-pituitary- brain relationship in health, aging and stress studies.

    PubMed

    Babizhayev, Mark A; Yegorov, Yegor E

    2015-01-01

    Hypothalamic releasing and inhibiting hormones are major neuroendocrine regulators of human body metabolism being driven directly to the anterior pituitary gland via hypothalamic-hypophyseal portal veins. The alternative physiological or therapeutic interventions utilizing the pharmaco-nutritional boost of imidazole-containing dipeptides (non-hydrolized oral form of carnosine, carcinine, N-acetylcarnosine lubricant eye drops) can maintain health, enhance physical exercise performance and prevent ageing. Carnosine (β-alanyl-L-histidine) is synthesized in mammalian skeletal muscle. There is an evidence that the release of carnosine from the skeletal muscle sarcomeres moieties during physical exercise affects autonomic neurotransmission and physiological functions. Carnosine released from skeletal muscle during exercise acts as a powerful afferent physiological signaling stimulus for hypothalamus, may be transported into the hypothalamic tuberomammillary nucleus (TMN), specifically to TMN-histamine neurons and hydrolyzed herewith via activities of carnosine-degrading enzyme (carnosinase 2) localized in situ. Through the colocalized enzymatic activity of Histidine decarboxylase in the histaminergic neurons, the resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of carnosine on neurotransmission and physiological function. Carnosine and its imidazole-containing dipeptide derivatives are renowned for their anti-aging, antioxidant, membrane protective, metal ion chelating, buffering, anti-glycation/ transglycating activities used to prevent and treat a spectrum of age-related and metabolic diseases, such as neurodegenerative disease, sight threatening eye diseases, Diabetes mellitus and its complications, cancers and other disorders due to their wide spectrum biological activities. The precursor of carnosine (and related imidazole containing compounds) synthesis in skeletal muscles beta-alanine is used as the

  12. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH)

    PubMed Central

    Diab, Houssein; Limami, Anis M.

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  13. Effects of exercise on leukocyte death: prevention by hydrolyzed whey protein enriched with glutamine dipeptide.

    PubMed

    Cury-Boaventura, Maria Fernanda; Levada-Pires, Adriana C; Folador, Alessandra; Gorjão, Renata; Alba-Loureiro, Tatiana C; Hirabara, Sandro M; Peres, Fabiano P; Silva, Paulo R S; Curi, Rui; Pithon-Curi, Tania C

    2008-06-01

    Lymphocyte and neutrophil death induced by exercise and the role of hydrolyzed whey protein enriched with glutamine dipeptide (Gln) supplementation was investigated. Nine triathletes performed two exhaustive exercise trials with a 1-week interval in a randomized, double blind, crossover protocol. Thirty minutes before treadmill exhaustive exercise at variable speeds in an inclination of 1% the subjects ingested 50 g of maltodextrin (placebo) or 50 g of maltodextrin plus 4 tablets of 700 mg of hydrolyzed whey protein enriched with 175 mg of glutamine dipeptide dissolved in 250 mL water. Cell viability, DNA fragmentation, mitochondrial transmembrane potential and production of reactive oxygen species (ROS) were determined in lymphocytes and neutrophils. Exhaustive exercise decreased viable lymphocytes but had no effect on neutrophils. A 2.2-fold increase in the proportion of lymphocytes and neutrophils with depolarized mitochondria was observed after exhaustive exercise. Supplementation of maltodextrin plus Gln (MGln) prevented the loss of lymphocyte membrane integrity and the mitochondrial membrane depolarization induced by exercise. Exercise caused an increase in ROS production by neutrophils, whereas supplementation of MGln had no additional effect. MGln supplementation partially prevented lymphocyte apoptosis induced by exhaustive exercise possibly by a protective effect on mitochondrial function.

  14. Some dipeptides reverse the inhibitory effect of GABA on /sup 35/S-TBPS binding

    SciTech Connect

    Squires, R.F.; Saederup, E.

    1987-05-01

    All known GABA-A receptor blocker reverse the inhibitory effect of GABA on /sup 35/S-t-butylphosphorothionate (TBPS) binding to rat brain membranes in vitro. This system has already been used to identify several novel GABA antagonists. The authors now report that 12 out of 52 dipeptides tested (all containing L-amino acids), at 1 mM, significantly reverse the inhibitory effect of 1 ..mu..M GABA, which inhibits specific /sup 35/S-TBPS binding about 60%. Most of the active dipeptides contain an aromatic and a basic amino acid. Tryptophan usually conferred greater activity than phe or tyr, while arg usually conferred greater activity than lys or his. Several larger peptides containing the HFRW sequence found in ACTH were also GABA antagonists; ACTH(1-24), ACTH(1-18), ACTH(1-13), ACTH(4-10) and ..gamma..-MSH while ACTH(11-24) was inactive. The excitatory effects of these later peptides may be in part due to blockade of GABA-A receptors.

  15. Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport.

    PubMed

    Links, Jennifer L S; Kulkarni, Ashutosh A; Davies, Daryl L; Lee, Vincent H L; Haworth, Ian S

    2007-04-01

    The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.

  16. Adsorption and Transport of Methane Molecules through One-Dimensional Channels in Dipeptide-Based Materials

    NASA Astrophysics Data System (ADS)

    Paradiso, Daniele; Perelli Cippo, Enrico; Gorini, Giuseppe; Rossi, Giorgio; Larese, John Z.

    The development of new materials for use in energy and environmental applications is of great interest, in particular in the areas of gas separation and carbon capture, where molecular transport plays a significant role. The dipeptides are organic molecules that offer an attractive possibility in such areas, because they form open hexagonal crystalline structures (space group P61) with quasi one-dimensional channels of tunable pore diameters in the range 3-6 Å. These molecular crystals exhibit selective adsorption, as well as, water and gas transport properties: these are believed to result from collective vibrations of the crystal structure that are coupled to the motions of the guest molecules within the channels. Current studies focus on characterizing the system methane and L-Isoleucyl-L-Valine (IV): this was initially done with high-resolution adsorption isotherms; then, high-resolution Inelastic Neutron Scattering measurements at the Spallation Neutron Source (BASIS spectrometer) revealed clear rotational tunneling peaks, offering details to unravel the potential energy surface of the system, as well as, evidences that channels flexibility and dynamical motion of the molecules have influence on the dipeptides adsorption properties.

  17. Electrocatalytic Oxidation of Formate with Nickel Diphosphane Dipeptide Complexes. Effect of Ligands Modified with Amino Acids

    SciTech Connect

    Galan, Brandon R.; Reback, Matthew L.; Jain, Avijita; Appel, Aaron M.; Shaw, Wendy J.

    2013-09-03

    A series of nickel bis-diphosphine complexes with dipeptides appended to the ligands were investigated for the catalytic oxidation of formate. Typical rates of ~7 s-1 were found, similar to the parent complex (~8 s-1), with amino acid size and positioning contributing very little to rate or operating potential. Hydroxyl functionalities did result in lower rates, which were recovered by protecting the hydroxyl group. The results suggest that the overall dielectric introduced by the dipeptides does not play an important role in catalysis, but free hydroxyl groups do influence activity suggesting contributions from intra- or intermolecular interactions. These observations are important in developing a fundamental understanding of the affect that an enzyme-like outer coordination sphere can have upon molecular catalysts. This work was funded by the US DOE Basic Energy Sciences, Chemical Sciences, Geoscience and Biosciences Division (BRG, AJ, AMA, WJS), the US DOE Basic Energy Sciences, Physical Bioscience program (MLR). Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

  18. Activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

    SciTech Connect

    Fogler, W.E.; Fidler, I.J.

    1986-03-15

    The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-..gamma.. (IFN-..gamma..)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and (/sup 3/H)nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced its encapsulation within multilamellar vesicles (MLV). Studies with (/sup 3/H)nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-..gamma..-primed monocytes. The intracellular interaction of (/sup 3/H)nor-MDP with IFN-..gamma..-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.

  19. Combination of amino acid/dipeptide with ligustrazine-betulinic acid as antitumor agents.

    PubMed

    Xu, Bing; Yan, Wen-Qiang; Xu, Xin; Wu, Gao-Rong; Zhang, Chen-Ze; Han, Yao-Tian; Chu, Fu-Hao; Zhao, Rui; Wang, Peng-Long; Lei, Hai-Min

    2017-04-21

    The lead compound TBA, 3β-Hydroxy-lup-20(29)-ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester, which exhibited promising antitumor activity and induced tumor cell apoptosis in various cancer cell lines, had previously been reported. Moreover, reports have revealed that the introduction of amino acid to betulinic acid could improve selective cytotoxicity as well as water solubility. Thus, a series of novel TBA amino acid and dipeptide derivatives were designed, synthesized and screened for selective cytotoxic activity against five cancer cell lines (HepG2, HT-29, Hela, BCG-823 and A549) and the not malignant cell line MDCK by standard MTT assay. Most of the tested TBA-amino acid and dipeptide analogues showed stronger anti-proliferative activity against all tested tumor cell lines than TBA. Among them, BA-25 exhibited the greatest cytotoxic activity on tumor cell lines (mean IC50 = 2.31 ± 0.78 μM), that was twofold than the positive drug cisplatin (DDP), while it showed lower cytotoxicity on MDCK cell line than DDP. Further cell apoptosis analyses indicated BA-25-induced apoptosis was associated with loss of mitochondrial membrane potential and increase of intracellular free Ca(2+) concentration.

  20. The Activation of Free Dipeptides Promoted by Strong Activating Agents in Water Does not Yield Diketopiperazines

    NASA Astrophysics Data System (ADS)

    Beaufils, Damien; Jepaul, Sandra; Liu, Ziwei; Boiteau, Laurent; Pascal, Robert

    2016-03-01

    The activation of dipeptides was studied in the perspective of the abiotic formation of oligopeptides of significant length as a requirement for secondary structure formation. The formation of piperazin-2,5-diones (DKP), previously considered as a dead end when activating free dipeptides, was shown in this work to be efficiently suppressed when using strong activating agents (e.g., carbodiimides). This behaviour was explained by the fast formation of a 5(4 H)-oxazolone intermediate at a rate that exceeds the time scale of the rotation of the peptide bond from the predominant trans-conformation into the cis-isomer required for DKP formation. No DKP was observed when using strong activating agents whereas phosphate mixed anhydrides or moderately activated esters were observed to predominantly yield DKP. The DKP side-reaction no longer constitutes a drawback for the C-terminus elongation of peptides. These results are considered as additional evidence that pathways involving strong activation are required to drive the emergence of living entities rather than close to equilibrium processes.

  1. The Activation of Free Dipeptides Promoted by Strong Activating Agents in Water Does not Yield Diketopiperazines.

    PubMed

    Beaufils, Damien; Jepaul, Sandra; Liu, Ziwei; Boiteau, Laurent; Pascal, Robert

    2016-03-01

    The activation of dipeptides was studied in the perspective of the abiotic formation of oligopeptides of significant length as a requirement for secondary structure formation. The formation of piperazin-2,5-diones (DKP), previously considered as a dead end when activating free dipeptides, was shown in this work to be efficiently suppressed when using strong activating agents (e.g., carbodiimides). This behaviour was explained by the fast formation of a 5(4H)-oxazolone intermediate at a rate that exceeds the time scale of the rotation of the peptide bond from the predominant trans-conformation into the cis-isomer required for DKP formation. No DKP was observed when using strong activating agents whereas phosphate mixed anhydrides or moderately activated esters were observed to predominantly yield DKP. The DKP side-reaction no longer constitutes a drawback for the C-terminus elongation of peptides. These results are considered as additional evidence that pathways involving strong activation are required to drive the emergence of living entities rather than close to equilibrium processes.

  2. Dipeptide Prodrug Approach to Evade Efflux Pumps and CYP3A4 Metabolism of Lopinavir

    PubMed Central

    Patel, Mitesh; Sheng, Ye; Mandava, Nanda K.; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity towards P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration PMID:25261710

  3. New diorganotin(IV) derivatives of dipeptides: Synthesis and characteristic spectral studies

    NASA Astrophysics Data System (ADS)

    Nath, Mala; Singh, Hitendra; Eng, George; Song, Xueqing

    2008-11-01

    Some new diorganotin(IV) derivatives of the formulae, R 2SnL, where R = Me, n-Bu, Ph, and n-Oct, and L is the dianion of histidinylalanine (H 2L-1) and histidinylleucine (H 2L-2) have been synthesized by the reaction of R 2SnCl 2 and the preformed sodium salt of the respective dipeptides. The bonding and coordination behaviour in these derivatives are discussed on the basis of FT-IR, multinuclear 1H, 13C and 119Sn NMR and 119Sn Mössbauer spectroscopic studies. These investigations suggest that dipeptides in R 2SnL act as dianionic tridentate coordinating through the COO -, NH 2 and N -peptide groups. The 119Sn Mössbauer studies, together with the NMR data, suggest a trigonal bipyramidal geometry around tin in R 2SnL with the alkyl/aryl groups and N in the equatorial positions, while a carboxylic oxygen and the amino nitrogen atom occupy the axial positions.

  4. Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

    PubMed

    Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

    2015-08-01

    Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed.

  5. Salsolinol, a catechol neurotoxin, induces modification of ferritin: Protection by histidine dipeptide.

    PubMed

    Kang, Jung Hoon

    2010-05-01

    1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin present in the mammalian brain, is known to perform a role in the pathogenesis of Parkinson's disease. In this study, we evaluated oxidative modifications of ferritin occurring after incubation with salsolinol. When ferritin was incubated with salsolinol, protein aggregation increased in a time-dependent manner. Free radical scavengers inhibited this salsolinol-mediated ferritin modification. The exposure of ferritin to salsolinol also results in the generation of protein carbonyl compounds and the formation of dityrosine. The results of this study show that free radicals may perform a pivotal role in salsolinol-mediated ferritin modification. Histidine dipeptides, such as carnosine, have been proposed to function as antioxidant agents in vivo. In this study, we also attempted to determine whether the histidine dipeptides, carnosine and N-acetyl-carnosine, could prevent salsolinol-mediated oxidative modification of ferritin. Our results showed that both carnosine and N-acetyl-carnosine significantly reduced ferritin aggregation. Both compounds effectively inhibited the formation of both carbonyl compounds and dityrosine. These results suggest that carnosine derivatives can, indeed, protect against salsolinol-mediated ferritin modification, as the consequence of free radical-scavenging activity.

  6. Relative response of alanine dosemeters for high-energy electrons determined using a Fricke primary standard.

    PubMed

    Vörös, Sándor; Anton, Mathias; Boillat, Bénédicte

    2012-03-07

    A significant proportion of cancer patients is treated using MeV electron radiation. One of the measurement methods which is likely to furnish reliable dose values also under non-reference conditions is the dosimetry using alanine and read-out via electron spin resonance (ESR). The system has already proven to be suitable for QA purposes for modern radiotherapy involving megavoltage x-rays. In order to render the secondary standard measurement system of the Physikalisch-Technische Bundesanstalt based on alanine/ESR useable for dosimetry in radiotherapy, the dose-to-water (D(W)) response of the dosemeter needs to be known for relevant radiation qualities. For MeV electrons, the D(W) response was determined using the Fricke primary standard of the Swiss Federal Office of Metrology. Since there were no citable detailed publications on the Swiss primary standard available, this measurement system is described in some detail. The experimental results for the D(W) response are compared to results of Monte Carlo simulations which model in detail the beams furnished by the electron accelerator as well as the geometry of the detectors. The agreement between experiment and simulation is very good, as well as the agreement with results published by the National Research Council of Canada which are based on a different primary standard. No significant dependence of the D(W) response was found in the range between 6 and 20 MeV. It is therefore suggested to use a unique correction factor k(E) for alanine for all MeV qualities of k(E) = 1.012 ± 0.010.

  7. Systematic analysis of a dipeptide library for inhibitor development using human dipeptidyl peptidase IV produced by a Saccharomyces cerevisiae expression system.

    PubMed

    Hikida, Aya; Ito, Keisuke; Motoyama, Takayasu; Kato, Ryuji; Kawarasaki, Yasuaki

    2013-01-25

    The inhibition of human dipeptidyl peptidase IV/CD26 (hDPPIV) is an accepted treatment for type 2 diabetes. In this study, an extracellular production system of hDPPIV using Saccharomyces cerevisiae was established to facilitate the screening of hDPPIV inhibitors. As dipeptides that mimic the hDPPIV substrate are candidate inhibitors of this protein, X-Ala or X-Pro dipeptides (in which X represents any amino acid) were tested systematically. Based on the results obtained in the first screening, a second screening was performed for Trp-X dipeptides. To elucidate the manner via which the physicochemical features at the P(1) and P(2) positions contributed to the hDPPIV inhibitory effect, correlations between the inhibitory activity of dipeptides and 13 amino acid indices were analyzed. The most effective inhibitory dipeptide was Trp-Pro (K(i)=0.04 mM). The mode of inhibition of hDPPIV by dipeptides was explained well by some amino acid indices and by the structure of the substrate-binding site of hDPPIV. The information obtained from the systematic analysis of a dipeptide library provides important clues for the development of hDPPIV targeting drugs and functional foods for type 2 diabetes.

  8. Computational Study of Environmental Effects on Torsional Free Energy Surface of N-Acetyl-N'-methyl-L-alanylamide Dipeptide

    ERIC Educational Resources Information Center

    Carlotto, Silvia; Zerbetto, Mirco

    2014-01-01

    We propose an articulated computational experiment in which both quantum mechanics (QM) and molecular mechanics (MM) methods are employed to investigate environment effects on the free energy surface for the backbone dihedral angles rotation of the small dipeptide N-Acetyl-N'-methyl-L-alanylamide. This computation exercise is appropriate for an…

  9. Phenylalanine-containing cyclic dipeptides--the lowest molecular weight hydrogelators based on unmodified proteinogenic amino acids.

    PubMed

    Kleinsmann, Alexander J; Nachtsheim, Boris J

    2013-09-14

    Cyclic dipeptides (diketopiperazines - DKPs) that are based on the proteinogenic amino acid phenylalanine in combination with serine, cysteine, glutamate, histidine and lysine are described as simple and remarkable low molecular weight hydrogelators. Blends of selected DKPs show remarkable pH-dependent properties and can be applied as easy to tune materials in drug delivery.

  10. Transnitrosylation products of the dipeptide cysteinyl-cysteine: an examination by tandem mass spectrometry and density functional theory.

    PubMed

    Butler, Matias; Michael Siu, K W; Hopkinson, Alan C

    2016-02-17

    The fragmentation pathways of protonated mono- and di-nitrosylated derivatives from the dipeptide Cys-Cys obtained by electrospray were examined. Protonated mononitrosylated dipeptide upon loss of ˙NO formed a radical cation, which in turn shows two fragment ions, one from the loss of HS˙ and the other from a neutral loss giving a radical cation of formula C2H5NS˙(+). Protonated dinitrosylated dipeptide dissociated by losing both ˙NO molecules, forming a cyclic structure with a vicinal disulfide bridge whose major dissociation channel was the loss of CO. After CO loss, two pathways were observed (loss of NH3 and C2H3NS) which were preceded by proton exchange occurring between one β-carbon and the nitrogen atom. DFT calculations did not show significant differences in the energies involved for the loss of the NO radical from either of the cysteine residues of the protonated di-nitrosylated dipeptide. Upon loss of the first NO radical, the thiyl radical afforded the vicinal disulfide product with a small barrier through radical substitution of the remaining NO moiety. The calculated relative energy barriers for the different channels are in good agreement with experimental observations. Structures of the ions obtained after dissociation are suggested on the basis of the proposed mechanisms.

  11. Pharmacokinetic studies and LC-MS/MS method development of ganciclovir and dipeptide monoester prodrugs in Sprague Dawley rats.

    PubMed

    Gunda, Sriram; Earla, Ravinder; Cholkar, Kishore; Mitra, Ashim K

    2015-09-01

    Ganciclovir (GCV) is utilized as an anti-herpetic agent. Reports from our laboratory have suggested that dipeptide ester prodrugs of GCV exhibit high affinity towards the oligopeptide transporter hPEPT1 and therefore seem to be promising candidates for the treatment of oral herpes virus infections. In this study, we have examined the bio-availability of a dipeptide prodrug of GCV after oral administration in jugular cannulated Sprague-Dawley rats. A new bio-analytical method was developed with Q-TRAP liquid chromatography tandem mass spectroscopy (LC-MS/MS) for simultaneous analysis of GCV, Valine-GCV (VGCV) and Tyrosine-Valine-GCV (YVGCV). Acyclovir (ACV) was used as an internal standard in the analysis. Area under plasma-concentration time curves for total concentration of GCV after oral administration of YVGCV was found to be approximately 200 % more than that of GCV following intestinal absorption. A complete conversion of the dipeptide prodrug (YVGCV) to parent compound, GCV, by hepatic first-pass metabolism was evident due to the absence of intermediate metabolite VGCV and administered prodrug YVGCV. The dipeptide prodrugs of GCV exhibit higher systemic availability of regenerated GCV upon oral administration and thus seem to be promising drug candidate in the treatment of systemic herpes infections.

  12. Solution-Phase Synthesis of Dipeptides: A Capstone Project That Employs Key Techniques in an Organic Laboratory Course

    ERIC Educational Resources Information Center

    Marchetti, Louis; DeBoef, Brenton

    2015-01-01

    A contemporary approach to the synthesis and purification of several UV-active dipeptides has been developed for the second-year organic laboratory. This experiment exposes students to the important technique of solution-phase peptide synthesis and allows an instructor to highlight the parallel between what they are accomplishing in the laboratory…

  13. Uncertainty in a Markov state model with missing states and rates: Application to a room temperature kinetic model obtained using high temperature molecular dynamics

    NASA Astrophysics Data System (ADS)

    Chatterjee, Abhijit; Bhattacharya, Swati

    2015-09-01

    Several studies in the past have generated Markov State Models (MSMs), i.e., kinetic models, of biomolecular systems by post-analyzing long standard molecular dynamics (MD) calculations at the temperature of interest and focusing on the maximally ergodic subset of states. Questions related to goodness of these models, namely, importance of the missing states and kinetic pathways, and the time for which the kinetic model is valid, are generally left unanswered. We show that similar questions arise when we generate a room-temperature MSM (denoted MSM-A) for solvated alanine dipeptide using state-constrained MD calculations at higher temperatures and Arrhenius relation — the main advantage of such a procedure being a speed-up of several thousand times over standard MD-based MSM building procedures. Bounds for rate constants calculated using probability theory from state-constrained MD at room temperature help validate MSM-A. However, bounds for pathways possibly missing in MSM-A show that alternate kinetic models exist that produce the same dynamical behaviour at short time scales as MSM-A but diverge later. Even in the worst case scenario, MSM-A is found to be valid longer than the time required to generate it. Concepts introduced here can be straightforwardly extended to other MSM building techniques.

  14. Convergent Synthesis of Novel Muramyl Dipeptide Analogues: Inhibition of Porphyromonas gingivalis-Induced Pro-inflammatory Effects by High Doses of Muramyl Dipeptide.

    PubMed

    Cai, Bin; Panek, James S; Amar, Salomon

    2016-07-28

    Porphyromonas gingivalis (P.g.)-induced TNF-α can be affected by muramyl dipeptide (MDP) in a biphasic concentration-dependent manner. We found that in P.g.-exposed macrophages, treatment with 10 μg/mL of MDP (MDP-low) up-regulated TNF-α by 29%, while 100 μg/mL or higher (MDP-high) significantly decreased it (16% to 38%). MDP-high was found to affect the ubiquitin-editing enzyme A20 and activator protein 1 (AP1). An AP1 binding site was found in the promoter region of A20. A20 promoter activity was up-regulated after transfection of AP1 cDNA in cells. Four analogues of MDP (3-6) were prepared through a convergent strategy involving the synthesis of two unique carbohydrate fragments, 7a and 7b, using the peptide coupling reagents, EDCI and HOAt. Analogue 4 improved MDP function and P.g.-induced activities. We propose a new signaling pathway for TNF-α induction activated after exposing macrophages to both P.g. and MDP-high or analogue 4.

  15. Biologic activities of molecular chaperones and pharmacologic chaperone imidazole-containing dipeptide-based compounds: natural skin care help and the ultimate challenge: implication for adaptive responses in the skin.

    PubMed

    Babizhayev, Mark A; Nikolayev, Gennady M; Nikolayeva, Juliana G; Yegorov, Yegor E

    2012-03-01

    Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of photoaged skin and pathogenesis of human cutaneous disease. Growing evidence demonstrates the ability of molecular chaperone proteins and of pharmacologic chaperones to decrease the environmental stress and ameliorate the oxidation stress-related and glycation disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for skin diseases and aging. In this review, we examine the evidence suggesting a role for molecular chaperone proteins in the skin and their inducer and protecting agents: pharmacologic chaperone imidazole dipeptide-based agents (carcinine and related compounds) in cosmetics and dermatology. Furthermore, we discuss the use of chaperone therapy for the treatment of skin photoaging diseases and other skin pathologies that have a component of increased glycation and/or free radical-induced oxidation in their genesis. We examine biologic activities of molecular and pharmacologic chaperones, including strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human skin disease. This allows the protein to function and traffic to the appropriate location in the skin, thereby increasing protein activity and cellular function and reducing stress on skin cells. The benefits of imidazole dipeptide antioxidants with transglycating activity (such as carcinine) in skin care are that they help protect and repair cell membrane damage and help retain youthful, younger-looking skin. All skin types will benefit from daily, topical application of pharmacologic chaperone antioxidants, anti-irritants, in combination with water-binding protein agents that work to mimic the structure and function of healthy skin. General strategies are presented addressing ground techniques to improve absorption of usually active chaperone proteins and dipeptide compounds, include

  16. Ibalizumab-human CD4 receptor interaction: computational alanine scanning molecular dynamics studies.

    PubMed

    Su, Zhi-Yuan

    2014-01-01

    Antibody drugs are used in the treatment of many chronic diseases. Recently, however, patients and doctors have encountered problems with drug resistance, and improving the affinity of antibody drugs has therefore become a pressing issue. Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). In this study, we sought to identify the key residues of the complementaritydetermining regions (CDRs) of ibalizumab. Virtual alanine mutations (complementarity-determining regions of ibalizumab) were also studied using solvated interaction energies derived from molecular dynamics and the explicit water model. Using 1,000 nanosecond molecular dynamic simulations, we identified six residues: Tyr50 [HCDR2], Tyr53 [HCDR3], Asp58 [HCDR2], Glu95 [HCDR2], and Arg95 [LCDR3]. The Robetta alanine-scanning mutagenesis method and crystallographic information were used to verify our simulations. Our simulated binding affinity of -17.33 kcal/mol is close to the experimentally determined value of -16.48 kcal/mol. Our findings may be useful for protein engineering the structure of the ibalizumab-human CD4 receptor complex. Moreover, the six residues that we identified may play a significant role in the development of bioactive antibody analogues.

  17. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  18. The effect of immunonutrition (glutamine, alanine) on fracture healing

    PubMed Central

    Küçükalp, Abdullah; Durak, Kemal; Bayyurt, Sarp; Sönmez, Gürsel; Bilgen, Muhammed S.

    2014-01-01

    Background There have been various studies related to fracture healing. Glutamine is an amino acid with an important role in many cell and organ functions. This study aimed to make a clinical, radiological, and histopathological evaluation of the effects of glutamine on fracture healing. Methods Twenty rabbits were randomly allocated into two groups of control and immunonutrition. A fracture of the fibula was made to the right hind leg. All rabbits received standard food and water. From post-operative first day for 30 days, the study group received an additional 2 ml/kg/day 20% L-alanine L-glutamine solution via a gastric catheter, and the control group received 2 ml/kg/day isotonic via gastric catheter. At the end of 30 days, the rabbits were sacrificed and the fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union. Results Radiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists. In the clinical evaluation, the mean score was 1.875 for the control group and 2.0 for the study group. Histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group. Conclusion One month after orally administered glutamine–alanine, positive effects were observed on fracture healing radiologically, clinically, and histopathologically, although no statistically significant difference was determined.

  19. Formation of chloroform during chlorination of alanine in drinking water.

    PubMed

    Chu, Wen-Hai; Gao, Nai-Yun; Deng, Yang; Dong, Bing-Zhi

    2009-11-01

    Currently, dissolved nitrogenous organic matters in water, important precursors of disinfection by-products (DBPs), are of significant concern. This study was to explore the formation of chloroform (CF) during chlorination of alanine (Ala), an important nitrogenous organic compound commonly present in water sources. Our results indicated that the CF yield reached a maximum value of 0.143% at the molar ratio of chlorine atom to nitrogen atom (Cl/N)=1.0 over a Cl/N range of 0.2-5.0 (pH=7.0, reaction time=5d, and initial Ala=0.1mM). At an acidic-neutral condition (pH 4-7), the formation of CF was suppressed. However, the highest CF yield (0.227%) occurred at weakly alkaline condition (pH 8.0) (initial Ala=0.1mM, and Cl/N=1.0). The increase of Br(-) in water can increase total trihalomethanes (THMs) and bromo-THMs. However, the bromo-THMs level reached a plateau at Br(-)/Cl>0.04. Finally, based on the computation of frontier electron density and identification and measurement of key intermediates during Ala chlorination, we proposed a formation pathway of CF from Ala chlorination: Ala-->monochloro-N-alanine (MC-N-Ala)-->acetaldehyde (AAld)-->monochloroacetaldehyde acetaldehyde (MCAld)-->dichloroacetaldehyde (DCAld)-->trichloroacetaldehyde (TCAld)-->CF.

  20. Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

    PubMed

    Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

    2015-11-01

    Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity.

  1. The hydrophobic dipeptide Leu-Ile inhibits immobility induced by repeated forced swimming via the induction of BDNF.

    PubMed

    Furukawa-Hibi, Yoko; Nitta, Atsumi; Ikeda, Takeshi; Morishita, Koji; Liu, Wenting; Ibi, Daisuke; Alkam, Tursun; Nabeshima, Toshitaka; Yamada, Kiyofumi

    2011-07-07

    Depression has recently become a serious problem in society worldwide. However, we lack appropriate therapeutic tools, since the causes of depression remain unclear. Degeneration of neuronal cells and a decrease in neurogenesis have been suggested recently as two of the factors responsible for depression-like behavior. Furthermore, brain-derived neurotrophic factor (BDNF) is also suggested to be an important factor in recovering from such behavior. We have previously demonstrated that the hydrophobic dipeptide leucyl-isoleucine (Leu-Ile) induces BDNF in cultured neuronal cells. We therefore investigated possible antidepressant-like effects of Leu-Ile in an animal model using the repeated forced swim test (FST). Mice were forced to swim for 6 min once a day in a cylinder containing water. The mice were treated with Leu-Ile s.c. or p.o. immediately after each FST. Five-day repeated Leu-Ile treatment significantly increased BDNF mRNA levels and activated the BDNF/Akt/mTOR signaling pathway in the hippocampi of the mice. While 2-week repeated FST increased immobility time, Leu-Ile treatment for 2 weeks offset this increase. In C57BL/6J-BDNF heterozygous knockout (BDNF(+/-)) mice, Leu-Ile failed to reduce the immobility time increased by repeated FST. We next investigated the extent of cell proliferation in the hippocampus as 5-bromo-2'-deoxy-uridine (BrdU) uptake into hippocampal cells. Repeated FST significantly reduced the number of BrdU-positive cells in the hippocampal dentate gyrus, while this deficit was prevented by repeated Leu-Ile treatment. These results suggest that Leu-Ile has an antidepressant-like effect, at least in part by supporting cell proliferation through the BDNF signaling pathway.

  2. Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

    PubMed

    Venir, Elena; Del Torre, Manuela; Cunsolo, Vincenzo; Saletti, Rosaria; Musetti, Rita; Stecchini, Mara Lucia

    2014-02-01

    The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

  3. The Helical Alanine Controversy: An (Ala)6 Insertion Dramatically Increases Helicity

    PubMed Central

    Lin, Jasper C.; Barua, Bipasha

    2013-01-01

    Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal α-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson–Roig formulation, the calculated value (wAla = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host–guest techniques or in N-terminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present. PMID:15493925

  4. Asymmetric cyanation of imines via dipeptide-derived organophosphine dual-reagent catalysis

    PubMed Central

    Wang, Hong-Yu; Zheng, Chang-Wu; Chai, Zhuo; Zhang, Jia-Xing; Zhao, Gang

    2016-01-01

    Over the past few decades, enantioselective phosphine organocatalysis has evolved rapidly into a highly efficient catalytic strategy for a range of useful reactions. However, as restricted by the traditional catalytic modes, some important reactions, such as asymmetric Strecker-type reactions, have thus far been out of reach of this strategy. Reported herein is an application of enantioselective phosphine organocatalysis for asymmetric Strecker-type reactions, enabled by a dual-reagent catalyst system in which the key organophosphorus zwitterion intermediate, generated in situ by mixing a chiral dipeptide-derived multifunctional organophosphine with methyl acrylate, is used as a highly efficient chiral Lewis base catalyst. The high efficiency of this catalytic system is demonstrated in the asymmetric cyanation of isatin-derived ketimines and azomethine aldimines as well as in the kinetic resolution of racemic 3-substituted azomethines. Mechanistic studies provide experimental evidence for the intermediacy of the putative zwitterion and its role as a catalytically active Lewis base. PMID:27625043

  5. Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment

    PubMed Central

    2016-01-01

    The number of antibiotic-resistant bacterial infections has increased dramatically over the past decade. To combat these pathogens, novel antimicrobial strategies must be explored and developed. We previously reported a strategy based on hapten-modified cell wall analogues to induce recruitment of endogenous antibodies to bacterial cell surfaces. Cell surface remodeling using unnatural single d-amino acid cell wall analogues led to modification at the C-terminus of the peptidoglycan stem peptide. During peptidoglycan processing, installed hapten-displaying amino acids can be subsequently removed by cell wall enzymes. Herein, we disclose a two-step dipeptide peptidoglycan remodeling strategy aimed at introducing haptens at an alternative site within the stem peptide to improve retention and diminish removal by cell wall enzymes. Through this redesigned strategy, we determined size constraints of peptidoglycan remodeling and applied these constraints to attain hapten–linker conjugates that produced high levels of antibody recruitment to bacterial cell surfaces. PMID:27294199

  6. Evaluation of dipeptide nitriles as inhibitors of rhodesain, a major cysteine protease of Trypanosoma brucei.

    PubMed

    Schirmeister, Tanja; Schmitz, Janina; Jung, Sascha; Schmenger, Torsten; Krauth-Siegel, R Luise; Gütschow, Michael

    2017-01-01

    A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8μM) the compounds represent promising starting points for new rhodesain inhibitors.

  7. A novel cyclic dipeptide from deep marine-derived fungus Aspergillus sp. SCSIOW2.

    PubMed

    Zhou, Xiang; Fang, Pingyan; Tang, Jianqiang; Wu, Zhiqin; Li, Xiaofan; Li, Shuiming; Wang, Yong; Liu, Gang; He, Zhendan; Gou, Deming; Yao, Xinsheng; Wang, Liyan

    2016-01-01

    A novel cyclic dipeptide, 14-hydroxy-cyclopeptine (1), was purified from a deep sea derived fungal isolate identified as an Aspergillus sp. The structure was elucidated by detailed spectroscopic analyses using 1D and 2D NMR experiments and high resolution mass spectrometry. The absolute configuration of the amino acid was determined by Marfey's method. Two conformational isomers of 1 were established by ROE analyses. 1 inhibited nitric oxide production with IC50 values at 40.3 μg/mL in a lipopolysaccharide and recombinant mouse interferon-γ -activated macrophage-like cell line, RAW 264.7 and showed no cytotoxic effect in the tested dose range up to 100 μg/mL.

  8. Muramyl-dipeptide and its synthetic analog as possible inducers of interleukin-2 production

    SciTech Connect

    Malaitsev, V.V.; Andronova, T.M.; Bogdanova, I.M.; Sukhikh, G.T.

    1986-01-01

    This paper studies the action of muramyl-dipeptide (MDP) and its synthetic derivative N-acetylglucosaminyl-N-acetyl-muramyl-alanyl-D-isoglutamine (GMDP) on IL2 production in a population of BALB/c mouse spleen cells. Tritium-thymidine was added to the wells in the flat-bottomed culture panes and the radioactivity of the samples was measured on a scintillation beta-counter after deposition of material was precipitated with TCA on the filters. The supernatant of the culture of activated spleen cells, stimulated by MDP or GMDP induced a proliferative response of the con A blast cells. The effectiveness of the stimulating action of MDP and GMDP, and of the classical IL2 inducer, con A, was compared.

  9. Solvent-tunable morphology and emission of pyrene-dipeptide organogels.

    PubMed

    Bartocci, S; Morbioli, I; Maggini, M; Mba, M

    2015-12-01

    Two pyrene based organogelators in which the pyrene moiety has been linked to the diphenylalanine dipeptide have been synthesized. We show how the solvent can tune both the morphology and the optical properties of the organogels: spherical aggregates with quenched emission were obtained in acetonitrile, whereas an entangled fibrillar network with enhanced emission was formed in o-dichlorobenzene. Fourier transform infrared spectroscopy, circular dichroism and nuclear magnetic resonance spectroscopy experiments suggest that both π-π stacking and hydrogen bonding contribute to the formation of the supramolecular networks. Ultraviolet-visible and steady state emission studies demonstrated the formation of I-aggregates in acetonitrile. In contrast, in o-dichlorobenzene, the formation of J-type aggregates leads to assemblies with enhanced emission. These results give some insight into the important role of the gelling solvent in the morphology of the supramolecular gels and may help in the design of new soft-materials.

  10. Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract.

    PubMed

    Yamamoto, Kenji; Hayashi, Miki; Murakami, Yuka; Araki, Yoko; Otsuka, Yuuki; Kashiwagi, Takehiro; Shimamura, Tomoko; Ukeda, Hiroyuki

    2015-01-01

    2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017-0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.

  11. The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

    PubMed

    Gudasheva, T A; Deeva, O A; Mokrov, G V; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-01-01

    On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action.

  12. Asymmetric cyanation of imines via dipeptide-derived organophosphine dual-reagent catalysis

    NASA Astrophysics Data System (ADS)

    Wang, Hong-Yu; Zheng, Chang-Wu; Chai, Zhuo; Zhang, Jia-Xing; Zhao, Gang

    2016-09-01

    Over the past few decades, enantioselective phosphine organocatalysis has evolved rapidly into a highly efficient catalytic strategy for a range of useful reactions. However, as restricted by the traditional catalytic modes, some important reactions, such as asymmetric Strecker-type reactions, have thus far been out of reach of this strategy. Reported herein is an application of enantioselective phosphine organocatalysis for asymmetric Strecker-type reactions, enabled by a dual-reagent catalyst system in which the key organophosphorus zwitterion intermediate, generated in situ by mixing a chiral dipeptide-derived multifunctional organophosphine with methyl acrylate, is used as a highly efficient chiral Lewis base catalyst. The high efficiency of this catalytic system is demonstrated in the asymmetric cyanation of isatin-derived ketimines and azomethine aldimines as well as in the kinetic resolution of racemic 3-substituted azomethines. Mechanistic studies provide experimental evidence for the intermediacy of the putative zwitterion and its role as a catalytically active Lewis base.

  13. Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum.

    PubMed

    Takahashi, Nobuhiro; Sato, T

    2002-02-01

    Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.

  14. Synthesis and characterization of bradykinin B(2) receptor agonists containing constrained dipeptide mimics.

    PubMed

    Amblard, M; Daffix, I; Bergé, G; Calmès, M; Dodey, P; Pruneau, D; Paquet, J L; Luccarini, J M; Bélichard, P; Martinez, J

    1999-10-07

    We have previously shown that substitution of the D-Tic-Oic dipeptide by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety in the bradykinin B(2) receptor antagonist HOE 140 resulted in a full potent and selective bradykinin B(2) receptor agonist (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, JMV1116) exhibiting a high affinity for the human receptor (K(i) 0.7 nM). In the present study, we have investigated the effects of replacement of the D-Tic-Oic moiety by various constrained dipeptide mimetics. The resulting compounds were tested for their binding affinity toward the cloned human B(2) receptor and for their functional interaction with the bradykinin-induced contraction of isolated human umbilical vein. Subsequently, we have designed novel bradykinin B(2) receptor agonists which are likely to be resistant to enzymatic cleavage by endopeptidases and which might represent interesting new pharmacological tools. In an attempt to increase the potency of compound JMV1116, both its N-terminal part and the D-BT moiety were modified. Substitution of the D-arginine residue by a L-lysine residue led to a 10-fold more potent bradykinin B(2) ligand [compound 22 (JMV1465) (K(i) 0.07 nM)], retaining full agonist activity on human umbilical vein. Substitution of the D-BT moiety by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-8-methyl-1, 5-benzothiazepin-4(5H)-one [D-BT(Me)] moiety led to compound 23 (JMV1609) which exhibited a higher agonist activity (pD(2) = 7.4) than JMV1116 (pD(2) = 6.8).

  15. [The effect of leucine and lysine dipeptides on the proliferation of the myocardium and spleen from young and old rats in organotypic culture].

    PubMed

    Chalisova, N I; Zhekalov, A N

    2014-01-01

    The effect of new dipeptides consisting from leucine and lysine was investigated in organotypic tissue culture on the cell proliferation development in spleen and myocardium explants in 3- and 24-months old rats. Dipeptide L-Leu-Lys stimulated the cell proliferation in the young and old rats at concentrations 0,001-10 ng/ml and 0,01-1 ng/ml accordingly. Dipeptide L-Lys-Leu stimulated as well the cell proliferation in the young and old rats at some deviation of diapason of stimulating concentrations. The data obtained aboutthe new dipeptides with high biological activity create a basis for future research at the organism level for their application in geriatrics to enhance regenerative processes in lymphoid and myocard tissue.

  16. Evaluating the Reduced Hydrophobic Taste Sensor Response of Dipeptides by Theasinensin A by Using NMR and Quantum Mechanical Analyses

    PubMed Central

    Guo, Jian; Hirasaki, Naoto; Miyata, Yuji; Tanaka, Kazunari; Tanaka, Takashi; Wu, Xiao; Tahara, Yusuke; Toko, Kiyoshi; Matsui, Toshiro

    2016-01-01

    The current study demonstrated that theasinensin A (TSA) had a potential to form the complex with hydrophobic Trp-containing dipeptides, and to reduce their membrane potential by artificial-lipid membrane taste sensor. At a 1:3 molar ratio of the 6 Trp-containing dipeptides together with TSA, we observed a significant chemical shift of the protons of the dipeptides (Δδ) to a high magnetic field, when analyzed using 1H-nuclear-magnetic resonance (NMR) spectroscopy. The Δδ values were correlated with the hydrophobicity (log P) of the dipeptides and significant correlations were obtained (P = 0.022, R2 = 0.77); e.g., Trp-Leu with the highest log P value of 1.623 among the tested dipeptides showed the highest Δδ value of 0.105 ppm for the H7 proton of Trp-Leu, while less chemical shifts were observed in theasinensin B and epigallocatechin-3-O-gallate. Diffusion-ordered NMR spectroscopy revealed that the diffusion coefficient of 3 mM of Trp-Leu (7.6 × 10−11 m2/s) at a pulse field gradient in the range 0.05–0.3 T/m decreased in the presence of 3 mM TSA (6.6 × 10−11 m2/s), suggesting that Trp-Leu forms a complex with TSA. Quantum mechanical calculations and rotating frame nuclear Overhauser effect-NMR spectroscopy provided configuration information on the geometry of the complex that Trp-Leu formed with TSA (1:1 complex) with a ΔG energy of –8.7 kJ/mol. A sensor analysis using artificial-lipid membranes demonstrated that the changes in membrane potential of 1 mM Trp-Leu (21.8 ± 1.3 mV) and Leu-Trp (5.3 ± 0.9 mV) were significantly (P < 0.001) reduced by 1 mM TSA (Trp-Leu, 13.1 ± 2.4 mV; Leu-Trp, 3.5 ± 0.5 mV; TSA alone, 0.2 ± 0.01 mV), indicating the effective suppression of hydrophobicity of dipeptides by TSA-formed complex. PMID:27309380

  17. Intrinsic α-helical and β-sheet conformational preferences: A computational case study of alanine

    PubMed Central

    Caballero, Diego; Määttä, Jukka; Zhou, Alice Qinhua; Sammalkorpi, Maria; O'Hern, Corey S; Regan, Lynne

    2014-01-01

    A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–Cα–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ. PMID:24753338

  18. Intrinsic α-helical and β-sheet conformational preferences: a computational case study of alanine.

    PubMed

    Caballero, Diego; Määttä, Jukka; Zhou, Alice Qinhua; Sammalkorpi, Maria; O'Hern, Corey S; Regan, Lynne

    2014-07-01

    A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle ( ϕ-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (NCα C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the "bridge" region of ϕ-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between ϕ-ψ conformations and τ.

  19. pH and reduction dual-responsive dipeptide cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

    PubMed

    Liu, Qiang; Su, Rong-Chuan; Yi, Wen-Jing; Zheng, Li-Ting; Lu, Shan-Shan; Zhao, Zhi-Gang

    2017-03-31

    A series of tocopherol-based cationic lipid 3a-3f bearing a pH-sensitive imidazole moiety in the dipeptide headgroup and a reduction-responsive disulfide linkage were designed and synthesized. Acid-base titration of these lipids showed good buffering capacities. The liposomes formed from 3 and co-lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could efficiently bind and condense DNA into nanoparticles. Gel binding and HPLC assays confirmed the encapsulated DNA could release from lipoplexes 3 upon addition of 10 mM glutathione (GSH). MTT assays in HEK 293 cells demonstrated that lipoplexes 3 had low cytotoxicity. The in vitro gene transfection studies showed cationic dipeptide headgroups clearly affected the transfection efficiency (TE), and arginine-histidine based dipeptide lipid 3f give the best TE, which was 30.4 times higher than Lipofectamine 3000 in the presence of 10% serum. Cell-uptake assays indicated that basic amino acid containing dipeptide cationic lipids exhibited more efficient cell uptake than serine and aromatic amino acids based dipeptide lipids. Confocal laser scanning microscopy (CLSM) studies corroborated that 3 could efficiently deliver and release DNA into the nuclei of HeLa cells. These results suggest that tocopherol-based dipeptide cationic lipids with pH and reduction dual-sensitive characteristics might be promising non-viral gene delivery vectors.

  20. Quantum mechanical investigations on the role of C-terminal residue in influencing the structural features of dipeptides containing N-terminal proline.

    PubMed

    Das, Gunajyoti; Mandal, Shilpi

    2014-04-01

    This study investigates the influence of the side chain moiety of C-terminal residue on the structural and molecular properties of seven dipeptides having proline at their N-terminal positions. The C-terminal component of the dipeptides is varied with seven different combinations viz. Ala, Leu, Asp, Thr, Asn, Arg and Sec. The calculations are carried out using B3LYP/6-311++G(d,p) level of theory in gas and implicit aqueous phase. Effects of explicit aqueous environment on the dipeptide structures are also investigated for two systems. The results furnished by this DFT study provide valuable information regarding the role of the side chain groups of C-terminal residues in determining the structural features of the amide planes, values of the ψ and ф dihedrals, geometry about the α-carbon atoms, theoretical IR spectra as well as the number and type of intramolecular H-bond interactions existing in the dipeptides, and extend a fine corroboration to the earlier theoretical and experimental observations. In aqueous phase the dipeptide geometries exhibit larger values of total dipole moments, greater HOMO-LUMO energy gaps and enhanced thermodynamic stability than those in gas phase. The explicit water molecules are found to modify the geometrical parameters related to the amide planes and vibrational spectra of the dipeptides.

  1. Antibacterial Activity of Alanine-Derived Gemini Quaternary Ammonium Compounds.

    PubMed

    Piecuch, Agata; Obłąk, Ewa; Guz-Regner, Katarzyna

    The antibacterial activity of alanine-derived gemini quaternary ammonium salts (chlorides and bromides) with various spacer and alkyl chain lengths was investigated. The studied compounds exhibited a strong bactericidal effect, especially bromides with 10 and 12 carbon alkyl chains and 3 carbon spacer groups (TMPAL-10 Br and TMPAL-12 Br), with a short contact time. Both salts dislodged biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis, and were lethal to adherent cells of S. epidermidis. Bromide with 2 carbon spacer groups and 12 carbon alkyl chains (TMEAL-12 Br) effectively reduced microbial adhesion by coating polystyrene and silicone surfaces. The results obtained suggest that, after further studies, gemini QAS might be considered as antimicrobial agents in medicine or industry.

  2. Charge dependent photodynamic activity of alanine based zinc phthalocyanines.

    PubMed

    Wang, Ao; Li, Yejing; Zhou, Lin; Yuan, Linxin; Lu, Shan; Lin, Yun; Zhou, Jiahong; Wei, Shaohua

    2014-12-01

    In this paper, to minimize the effects of different structure, three alanine-based zinc phthalocyanines (Pcs) of differing charges were engineered and synthesized with the same basic structure. On this premise, the relationship between nature of charge and photodynamic activity was studied. Besides, further verification and explanation of some inconsistent results were also carried out. The results showed that charge can influence the aggregation state, singlet oxygen generation ability and cellular uptake of Pcs, thereby affecting their photodynamic activity. In addition, the biomolecules inside cells may interact with Pcs of differing charges, which can also influence the aggregation state and singlet oxygen generation of the Pcs, and then influence the relationship between nature of charge and photodynamic activity.

  3. Radiolysis of alanine adsorbed in a clay mineral

    SciTech Connect

    Aguilar-Ovando, Ellen Y.; Negron-Mendoza, Alicia

    2013-07-03

    Optical activity in molecules is a chemical characteristic of living beings. In this work, we examine the hypothesis of the influence of different mineral surfaces on the development of a specific chirality in organic molecules when subjected to conditions simulating the primitive Earth during the period of chemical evolution. By using X-ray diffraction techniques and HPLC/ELSD to analyze aqueous suspensions of amino acids adsorbed on minerals irradiated in different doses with a cobalt-60 gamma source, the experiments attempt to prove the hypothesis that some solid surfaces (like clays and meteorite rocks) may have a concentration capacity and protective role against external sources of ionizing radiation (specifically {gamma}-ray) for some organic compounds (like some amino acids) adsorbed on them. Preliminary results show a slight difference in the adsorption and radiolysis of the D-and L-alanine.

  4. First-principles study of fluorination of L-Alanine

    NASA Astrophysics Data System (ADS)

    Sreepad, H. R.; Ravi, H. R.; Ahmed, Khaleel; Dayananda, H. M.; Umakanth, K.; Manohara, B. M.

    2013-02-01

    First-principles calculations based on Density Functional Theory have been done on effect of fluorination of an important amino acid - L-Alanine. Its structure has been simulated. The unit cell is orthorhombic with lattice parameters a=5.90Å, b=13.85Å and c=5.75Å with volume 470 (Å)3. Bond lengths and bond angles have been estimated. Electronic Density of States calculations show that the material has a band gap of 4.47eV. Electronic band structure indicates that the material can be effectively used for NLO applications. The electronic contribution to the dielectric constant has been calculated and its average value comes out to be 2.165.

  5. Alanine Aminotransferase Variants Conferring Diverse NUE Phenotypes in Arabidopsis thaliana

    PubMed Central

    McAllister, Chandra H.; Good, Allen G.

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5’-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed. PMID:25830496

  6. Alanine aminotransferase variants conferring diverse NUE phenotypes in Arabidopsis thaliana.

    PubMed

    McAllister, Chandra H; Good, Allen G

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.

  7. Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

    PubMed

    Zhou, Li; Deng, Can; Cui, Wen-Jing; Liu, Zhong-Mei; Zhou, Zhe-Min

    2016-01-01

    L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

  8. Structure-based design, synthesis, and biological evaluation of Leu-Arg dipeptide analogs as novel hepsin inhibitors.

    PubMed

    Kwon, Hongmok; Kim, YunHye; Park, Kieung; Choi, Soo An; Son, Sang-Hyun; Byun, Youngjoo

    2016-01-15

    Hepsin, a type II transmembrane serine protease, is an attractive protein as a potential therapeutic and diagnostic biomarker for prostate cancer because it is highly up-regulated in prostate cancer and promotes both progression and metastasis. Starting from the reported tetrapeptide hepsin inhibitor Ac-KQLR-ketothiazole (kt) (1), we investigated the minimal structural requirements for hepsin inhibitory activity by truncating amino acids at the N-terminus. The kt and ketobenzothiazole (kbt) dipeptide analogs Ac-LR-kt (3) and Ac-LR-kbt (15) were found to be potent hepsin inhibitors, exhibiting Ki values of 22nM and 3nM, respectively. The present work suggests that LR-containing dipeptide molecules could be useful as lead compounds for the development of novel hepsin inhibitors.

  9. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.

    PubMed

    Lin, Yi; Mori, Eiichiro; Kato, Masato; Xiang, Siheng; Wu, Leeju; Kwon, Ilmin; McKnight, Steven L

    2016-10-20

    Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

  10. Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers

    PubMed Central

    Lin, Yi; Mori, Eiichiro; Kato, Masato; Xiang, Siheng; Wu, Leeju; Kwon, Ilmin; McKnight, Steven L.

    2016-01-01

    Summary Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding, and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein. PMID:27768897

  11. Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

    PubMed Central

    Dugelay, S; Chauvin, M F; Megnin-Chanet, F; Martin, G; Laréal, M C; Lhoste, J M; Baverel, G

    1999-01-01

    Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction. PMID:10477267

  12. Dipeptide-Derived Multifunctional Quaternary Phosphonium Salt Catalyzed Asymmetric Cyclizations via a Tandem Michael Addition/SN 2 Sequence.

    PubMed

    Cao, Dongdong; Zhang, Jiaxing; Wang, Hongyu; Zhao, Gang

    2015-07-06

    A novel family of dipeptide-based multifunctional quaternary phosphonium salts has been developed as chiral phase-transfer catalysts, which feature ready accessibility and structure modularity, allowing easy fine-tunings of activity. They demonstrated high efficiency in catalyzing the tandem asymmetric Michael addition/intramolecular SN 2 reaction between 6 or 7-substituted conjugate enones and malonates, providing synthetically important five or six-membered carbocycles and heterocycles in good yields and with good to excellent enantioselectivities.

  13. C7-prenylation of tryptophanyl and O-prenylation of tyrosyl residues in dipeptides by an Aspergillus terreus prenyltransferase.

    PubMed

    Wunsch, Carsten; Zou, Hui-Xi; Linne, Uwe; Li, Shu-Ming

    2015-02-01

    During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-L-Tyr-L-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-L-Trp-L-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K M value of 204.1 μM and a turnover number of 0.125 s(-1). Cyclo-L-Tyr-L-Tyr was accepted with a K M value of 1,411.3 μM and a turnover number of 0.012 s(-1).

  14. Inhibition of the Arg/N-end rule pathway-mediated proteolysis by dipeptide-mimetic molecules.

    PubMed

    Kitamura, Kenji

    2016-01-01

    Ubr11 in the fission yeast Schizosaccharomyces pombe is an evolutionarily conserved ubiquitin ligase functioning in the Arg/N-end rule pathway, which promotes degradation of substrate proteins via the proteasome. Ubr11 recognizes the N-degron sequence in substrates. The primary N-degron contains a destabilization-inducing N-terminal amino acid, which is either a basic (type 1) or bulky hydrophobic (type 2) residue. Dipeptides are known to inhibit proteolytic degradation via the Arg/N-end rule pathway. Here, I examined the potency of some amino acid- or dipeptide-related molecules in their inhibition of Ubr11/N-end rule-mediated degradation. An amide form of L-arginine and L-tryptophan had weak inhibitory activity for type 1 and type 2 substrates, respectively, although the unmodified amino acid monomer and its carboxymethylated ester were ineffective. Among the naturally occurring dipeptides tested, Lys-Leu and Tyr-Leu showed potent inhibitory activity, but their effect was transient, especially at submillimolar concentrations. L-arginine-β-naphthylamide (Arg-βNA) showed stronger activity than several dipeptides for type 1 substrates, but all Lys-Leu, Tyr-Leu, and Arg-βNA caused growth retardation. The inhibitory activity of the L-phenylalanine carbobenzoxy-hydrazide for type 2 substrates was not very strong, but it prolonged the action of Tyr-Leu at low concentrations and, importantly, did not interfere with cell growth. Apart from their utility, these dipeptidomimetics provide a clue for understanding the determinants of recognition by Ubr ubiquitin ligase and further designing novel inhibitors of the Arg/N-end rule pathway.

  15. Dipeptide-Based Chiral Tertiary Amine-Catalyzed Asymmetric Conjugate Addition Reactions of 5H-Thiazol/Oxazol-4-Ones.

    PubMed

    Li, Jiangtao; Qiu, Shuai; Ye, Xinyi; Zhu, Bo; Liu, Hongjun; Jiang, Zhiyong

    2016-12-02

    Highly enantio- and chemo-selective 1,4-conjugate addition process of 5H-thiazol-4-ones with maleimides or 1,4-naphthoquinones, and 5H-oxazol-4-ones with maleimides were performed under a dipeptide-based tertiary amine (DP-UAA) catalyst. A series of valuable N,S- and N,O-containing heterocyclic compounds with excellent enantio- and disastereo-selectivities (up to >99% ee, > 20:1 dr) were attained.

  16. Excited state electron distribution and role of the terminal amine in acidic and basic tryptophan dipeptide fluorescence

    NASA Astrophysics Data System (ADS)

    Eisenberg, Azaria S.; Nathan, Moshe; Juszczak, Laura J.

    2016-08-01

    The results of quantum yield (QY) study of tryptophanyl glutamate (Trp-Glu), tryptophanyl lysine (Trp-Lys) and lysinyl tryptophan (Lys-Trp) dipeptides over the pH range, 1.5-13, show that the charge state of the N-terminal amine, and not the nominal molecular charge determines the QY. When the terminal amine is protonated, QY is low (10-2) for all three dipeptides. As the terminal amine cation is found proximal to the indole ring in Trp-Glu and Trp-Lys conformers but not in those for Lys-Trp, its effect may lie only in the partitioning of energy between nonradiative processes, not on QY reduction. QY is also low when both the N-terminal amine and indole amine are deprotonated. These two low QY states can be distinguished by fluorescence lifetime measurement. Molecular dynamics simulation shows that the Chi 1 conformers persist for tens of nanoseconds such that 100-101 ns lifetimes may be associated with individual Chi 1 conformers. The ground state electron density or isosurface of high QY (0.30) 3-methyindole has a uniform electron density over the indole ring as do the higher QY Trp dipeptide conformers. This validates the association of ground state isosurfaces with QY. Excited state orbitals from calculated high intensity, low energy absorption transitions are typically centered over the indole ring for higher QY dipeptide species and off the ring in lower QY species. Thus excited state orbitals substantiate the earlier finding that the ground state isosurface charge density pattern on the indole ring can be predictive of QY.

  17. L-histidyl-L-serine 3.7-hydrate: water channels in the crystal structure of a polar dipeptide.

    PubMed

    Görbitz, Carl Henrik

    2010-11-01

    Dipeptides may form nanotubular structures with pore diameters in the range 3.2-10 Å. These compounds normally contain at least one and usually two hydrophobic residues, but L-His-L-Ser hydrate, C(9)H(14)N(4)O(4)·3.7H(2)O, with two hydrophilic residues, forms large polar channels filled with ordered as well as disordered water molecules.

  18. Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins.

    PubMed

    Perez, Romel B; Tischer, Alexander; Auton, Matthew; Whitten, Steven T

    2014-12-01

    Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (R(h)) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PP(II)) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in R(h) were not associated with folding. The experiments showed that changes in local PP(II) structure cause changes in R(h) that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs.

  19. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  20. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  1. Polymerization of alanine in the presence of a non-swelling montmorillonite

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.; Lahav, N.

    1977-01-01

    Alanine, starting from alanine-adenylate, has been polymerized in the presence of non-swelling Al-montmorillonite. The yield of polymerization is much lower than that obtained in the presence of swelling Na-montmorillonite. The possibility that the changing interlayer spacing in Na-montmorillonite might be responsible for its catalytic properties, is discussed.

  2. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  3. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  4. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  5. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  6. Advances in simultaneous DSC-FTIR microspectroscopy for rapid solid-state chemical stability studies: some dipeptide drugs as examples.

    PubMed

    Lin, Shan-Yang; Wang, Shun-Li

    2012-04-01

    The solid-state chemistry of drugs has seen growing importance in the pharmaceutical industry for the development of useful API (active pharmaceutical ingredients) of drugs and stable dosage forms. The stability of drugs in various solid dosage forms is an important issue because solid dosage forms are the most common pharmaceutical formulation in clinical use. In solid-state stability studies of drugs, an ideal accelerated method must not only be selected by different complicated methods, but must also detect the formation of degraded product. In this review article, an analytical technique combining differential scanning calorimetry and Fourier-transform infrared (DSC-FTIR) microspectroscopy simulates the accelerated stability test, and simultaneously detects the decomposed products in real time. The pharmaceutical dipeptides aspartame hemihydrate, lisinopril dihydrate, and enalapril maleate either with or without Eudragit E were used as testing examples. This one-step simultaneous DSC-FTIR technique for real-time detection of diketopiperazine (DKP) directly evidenced the dehydration process and DKP formation as an impurity common in pharmaceutical dipeptides. DKP formation in various dipeptides determined by different analytical methods had been collected and compiled. Although many analytical methods have been applied, the combined DSC-FTIR technique is an easy and fast analytical method which not only can simulate the accelerated drug stability testing but also at the same time enable to explore phase transformation as well as degradation due to thermal-related reactions. This technique offers quick and proper interpretations.

  7. Quantitative mass spectrometric analysis of dipeptides in protein hydrolysate by a TNBS derivatization-aided standard addition method.

    PubMed

    Hanh, Vu Thi; Kobayashi, Yutaro; Maebuchi, Motohiro; Nakamori, Toshihiro; Tanaka, Mitsuru; Matsui, Toshiro

    2016-01-01

    The aim of this study was to establish, through a standard addition method, a convenient quantification assay for dipeptides (GY, YG, SY, YS, and IY) in soybean hydrolysate using 2,4,6-trinitrobenzene sulfonate (TNBS) derivatization-aided LC-TOF-MS. Soybean hydrolysate samples (25.0 mg mL(-1)) spiked with target standards were subjected to TNBS derivatization. Under the optimal LC-MS conditions, five target dipeptides derivatized with TNBS were successfully detected. Examination of the standard addition curves, with a correlation coefficient of r(2) > 0.979, provided a reliable quantification of the target dipeptides, GY, YG, SY, YS, and IY, in soybean hydrolysate to be 424 ± 20, 184 ± 9, 2188 ± 199, 327 ± 16, and 2211 ± 133 μg g(-1) of hydrolysate, respectively. The proposed LC-MS assay is a reliable and convenient assay method, with no interference from matrix effects in hydrolysate, and with no requirement for the use of an isotope labeled internal standard.

  8. [THE REGULATING EFFECT OF DIPEPTIDES ON CELL PROLIFERATION IN NERVE TISSUE CULTURE IN MAMMALS AND ON ASSOCIATIVE LEARNING IN INSECTS].

    PubMed

    Chalisova, N I; Zachepilo, T G; Kamyshev, N G; Lopatina, N G

    2015-01-01

    The effect of dipeptides AspPro and AspSer and of their composing amino acids (asparagine acid--Asp, proline--Pro, serin--Ser) on the proliferative activity in the explants of cortex and subcortical structures of the rat brain and on the functional activity of CNS of the honeybee was studied. The square index defined as a proportion of the whole explant square to the square of its central zone was determined. The number of bees responded with the conditional reaction (proboscis extension in the direction to aromatized solution) after 1 min (short-term memory) and 180 min (long-term memory) was detected after single learning procedure. Both dipeptides, as well as the asparagine acid, stimulated an increase of the growth zone of the subcortical structure explants in rats and of the number of honeybees with retention of conditional reaction in the short-term/long-term memory independently of the effect of the second member of the dipeptide. The unidirectionality of the effect suggests the existence of common mechanisms of reception and signal transduction established during evolution that require the further study.

  9. Medium-dependent regulation of proteinase gene expression in Lactococcus lactis: control of transcription initiation by specific dipeptides.

    PubMed Central

    Marugg, J D; Meijer, W; van Kranenburg, R; Laverman, P; Bruinenberg, P G; de Vos, W M

    1995-01-01

    Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene (gusA) were used to study the medium- and growth-dependent expression of the divergently transcribed genes involved in proteinase production (prtP and prtM) of Lactococcus lactis SK11. The results show that both the prtP and prtM genes are controlled at the transcriptional level by the peptide content of the medium and, to a lesser extent, by the growth rate. A more than 10-fold regulation in beta-glucuronidase activity was observed for both prtP and prtM promoters in batch and continuous cultures. The level of expression of the prtP and prtM promoters was high in whey permeate medium with relatively low concentrations of peptides, whereas at increased concentrations the expression of the promoters was repressed. The lowest level of expression was observed in peptide- and amino acid-rich laboratory media, such as glucose-M17 and MRS. The addition of specific dipeptides, such as leucylproline and prolylleucine, to the growth medium negatively affected the expression of the prtP-gusA fusions. The repression by dipeptides was not observed in mutants defective in the uptake of di-tripeptides, indicating that the internal concentration of dipeptides or derivatives is important in the regulation of proteinase production. PMID:7768792

  10. Conformational distributions of denatured and unstructured proteins are similar to those of 20 × 20 blocked dipeptides.

    PubMed

    Oh, Kwang-Im; Jung, Young-Sang; Hwang, Geum-Sook; Cho, Minhaeng

    2012-05-01

    Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out (1)H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH(2)), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.

  11. Alanylglutamine dipeptide and growth hormone maintain PepT1-mediated transport in oxidatively stressed Caco-2 cells.

    PubMed

    Alteheld, B; Evans, M E; Gu, L H; Ganapathy, V; Leibach, F H; Jones, D P; Ziegler, T R

    2005-01-01

    Reactive oxygen species (ROS) produced by gut mucosal cells during conditions such as inflammatory bowel disease (IBD) may impair mucosal repair and nutrient transport/absorptive function. Absorption of di- and tripeptides in the small intestine and colon is mediated by the H(+)-dependent transporter PepT1, but effects of oxidative stress on di- and tripeptide transport are unknown. We assessed whether exposure to hydrogen peroxide (H(2)O(2)) influences dipeptide transport in human colonic epithelial (Caco-2) cells. Uptake of [(14)C]glycylsarcosine (Gly-Sar) was used to evaluate PepT1-mediated dipeptide transport. Exposure to 1-5 mmol/L H(2)O(2) for 24 h caused a dose-dependent decrease in Gly-Sar transport, which was associated with decreased PepT1 transport velocity (V(max)). Treatment with alanylglutamine (Ala-Gln) or growth hormone (GH) did not alter Caco-2 Gly-Sar transport in the absence of H(2)O(2). However, both Ala-Gln and GH prevented the decrease in dipeptide transport observed with 1 mmol/L H(2)O(2) treatment. Ala-Gln, but not GH, maintained cellular glutathione and prevented the decrease in PepT1 protein expression. Thus, these agents should be further investigated as potential therapies to improve absorption of small peptides in disorders associated with oxidative injury to the gut mucosa.

  12. Global Langevin model of multidimensional biomolecular dynamics

    NASA Astrophysics Data System (ADS)

    Schaudinnus, Norbert; Lickert, Benjamin; Biswas, Mithun; Stock, Gerhard

    2016-11-01

    Molecular dynamics simulations of biomolecular processes are often discussed in terms of diffusive motion on a low-dimensional free energy landscape F ( 𝒙 ) . To provide a theoretical basis for this interpretation, one may invoke the system-bath ansatz á la Zwanzig. That is, by assuming a time scale separation between the slow motion along the system coordinate x and the fast fluctuations of the bath, a memory-free Langevin equation can be derived that describes the system's motion on the free energy landscape F ( 𝒙 ) , which is damped by a friction field and driven by a stochastic force that is related to the friction via the fluctuation-dissipation theorem. While the theoretical formulation of Zwanzig typically assumes a highly idealized form of the bath Hamiltonian and the system-bath coupling, one would like to extend the approach to realistic data-based biomolecular systems. Here a practical method is proposed to construct an analytically defined global model of structural dynamics. Given a molecular dynamics simulation and adequate collective coordinates, the approach employs an "empirical valence bond"-type model which is suitable to represent multidimensional free energy landscapes as well as an approximate description of the friction field. Adopting alanine dipeptide and a three-dimensional model of heptaalanine as simple examples, the resulting Langevin model is shown to reproduce the results of the underlying all-atom simulations. Because the Langevin equation can also be shown to satisfy the underlying assumptions of the theory (such as a delta-correlated Gaussian-distributed noise), the global model provides a correct, albeit empirical, realization of Zwanzig's formulation. As an application, the model can be used to investigate the dependence of the system on parameter changes and to predict the effect of site-selective mutations on the dynamics.

  13. Synthesis and GGCT Inhibitory Activity of N-Glutaryl-L-alanine Analogues.

    PubMed

    Ii, Hiromi; Yoshiki, Tatsuhiro; Hoshiya, Naoyuki; Uenishi, Jun'ichi

    2016-01-01

    γ-Glutamylcyclotransferase (GGCT) is an important enzyme that cleaves γ-glutamyl-amino acid in the γ-glutamyl cycle to release 5-oxoproline and amino acid. Eighteen N-acyl-L-alanine analogues including eleven new compounds have been synthesized and examined for their inhibitory activity against recombinant human GGCT protein. Simple N-glutaryl-L-alanine was found to be the most potent inhibitor for GGCT. Other N-glutaryl-L-alanine analogues having methyl and dimethyl substituents at the 2-position were moderately effective, while N-(3R-aminoglutary)-L-alanine, the substrate having an (R)-amino group at the 3-position or N-(N-methyl-3-azaglutaryl)-L-alanine, the substrate having an N-methyl substituent on the 3-azaglutaryl carbon, in constract, exhibited excellent inhibition properties.

  14. Rotational resonance determination of the structure of an enzyme-inhibitor complex: Phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP

    SciTech Connect

    McDermott, A.E.; Creuzet, F.; Griffin, R.G. ); Zawadzke, L.E.; Ye, Qizhuang; Walsh, C.T. )

    1990-06-19

    The authors have used a newly developed solid-state NMR method, rotational resonance, to establish the structure of an inhibited complex formed upon reaction of D-alanyl-D-alanine ligase, ATP, and the aminoalkyl dipeptide analogue (1(S)-aminoethyl)(2-carboxy-2(R)-methyl-1-ethyl)phosphinic acid (Ib). Analogue Ib was determined to be an ATP-dependent, slow-binding inhibitor of the D-Ala-D-Ala ligase from Salmonella typhimurium, with an enzyme-inhibitor half-life of 17 days at 37{degree}C. The inhibited complex shows a {sup 31}P NMR spectrum which is very different from that which would arise from a mixture of the free inhibitor and ATP. Four well-resolved lines were observed. A rotational resonance the spectrum shows evidence for strong dipolar couplings between the phosphinate phosphorus and a phosphate ester species. The dipolar coupling between the phosphorus signals at 53 and {minus}3 ppm was measured at rotational resonance by use of numerical simulations of both the line shape of the signal and the profile of magnetization transfer between the two sites. The measured coupling, 1.0 {plus minus} 0.2 kHz, indicates that the two species are bridged in a P-O-P linkage, with a P-P through-space distance of 2.7 {plus minus} 0.2 {angstrom}. This proves that the mechanism of inactivation involves phosphorylation of the enzyme-bound inhibitor by ATP to form a phosphoryl-phosphinate adduct.

  15. Cyclic cis-Locked Phospho-Dipeptides Reduce Entry of AβPP into Amyloidogenic Processing Pathway.

    PubMed

    Fisher, Carolyn L; Resnick, Ross J; De, Soumya; Acevedo, Lucila A; Lu, Kun Ping; Schroeder, Frank C; Nicholson, Linda K

    2017-01-01

    The cis/trans isomerization of X-Pro peptide bonds in proteins in some instances acts as a molecular switch in biological pathways. Our prior work suggests that the cis isomer of the phospho-Thr668-Pro669 motif, located in the cytoplasmic domain of the amyloid-β protein precursor (AβPP), is correlated with an increase in amyloidogenic processing of AβPP and production of amyloid-beta (Aβ), the neurotoxic peptide fragment in Alzheimer's disease (AD). We designed a 100% cis-locked cyclic dipeptide composed of cyclized phospho-Thr-Pro (pCDP) as a mimic for this putative pathological conformation, and three phosphate-blocked derivatives (pCDP-diBzl, pCDP-Bzl, and pCDP-diPOM). Two H4 neuroglioma cell lines were established as AD cell models for use in testing these compounds: H4-AβPP695 for stable overexpression of wild-type AβPP695, and H4-BACE1 for stable overexpression of β-site AβPP cleaving enzyme-1 (BACE1). The level of the secreted AβPP fragment resulting from BACE1 activity, sAβPPβ, served as a key proxy for amyloidogenic processing, since cleavage of AβPP by BACE1 is a requisite first step in Aβ production. Of the compounds tested, pCDP-diBzl decreased sAβPPβ levels in both cell lines, while pCDP-diPOM decreased sAβPPβ levels in only H4-BACE1 cells, all with similar dose-dependences and patterns of proteolytic AβPP fragments. Enzymatic assays showed that none of the pCDP derivatives directly inhibit BACE1 catalytic activity. These results suggest a model in which pCDP-diBzl and pCDP-diPOM act at a common point to inhibit entry of AβPP into the amyloidogenic AβPP processing pathway but through different targets, and provide important insights for the development of novel AD therapeutics.

  16. Competing Noncovalent Host-guest Interactions and H/D Exchange: Reactions of Benzyloxycarbonyl-Proline Glycine Dipeptide Variants with ND3

    NASA Astrophysics Data System (ADS)

    Miladi, Mahsan; Olaitan, Abayomi D.; Zekavat, Behrooz; Solouki, Touradj

    2015-11-01

    A combination of density functional theory calculations, hydrogen/deuterium exchange (HDX) reactions, ion mobility-mass spectrometry, and isotope labeling tandem mass spectrometry was used to study gas-phase "host-guest" type interactions of a benzyloxycarbonyl (Z)-capped proline (P) glycine (G) model dipeptide (i.e., Z-PG) and its various structural analogues with ND3. It is shown that in a solvent-free environment, structural differences between protonated and alkali metal ion (Na+, K+, or Cs+)-complexed species of Z-PG affect ND3 adduct formation. Specifically, [Z-PG + H]+ and [Z-PG-OCH3 + H]+ formed gas-phase ND3 adducts ([Z-PG (or Z-PG-OCH3) + H + ND3]+) but no ND3 adducts were observed for [Z-PG + alkali metal]+ or [Z-PG + H - CO2]+. Experimentally measured and theoretically calculated collision cross sections (CCSs) of protonated and alkali metal ion-complexed Z-PG species showed similar trends that agreed with the observed structural differences from molecular modeling results. Moreover, results from theoretical ND3 affinity calculations were consistent with experimental HDX observations, indicating a more stable ND3 adduct for [Z-PG + H]+ compared to [Z-PG + alkali metal]+ species. Molecular modeling and experimental MS results for [Z-PG + H]+ and [Z-PG + alkali metal]+ suggest that optimized cation-π and hydrogen bonding interactions of carbonyl groups in final products are important for ND3 adduct formation.

  17. Epimerization of Alanyl-Alanine Induced by γ-Rays Irradiation in Aqueous Solutions

    NASA Astrophysics Data System (ADS)

    Munegumi, Toratane

    2017-03-01

    Living organisms have homochiral L-amino acids in proteins and homochiral D-mononucleotides in nucleic acids. The chemical evolutionary process to protein homochirality has been discussed for many years. Although many scenarios have been proposed for homochirality in the monomeric compounds, homochirality in amino acids and mononucleotides does not always guarantee homochirality in polypeptides and polynucleotides. Integrated scenarios containing the pathways from monomer to polymer should be proposed because in the pathways oligomers and polymers as well as monomers racemize (or epimerize), degrade, and condense. This research addresses epimerization and degradation of dipeptides under γ-rays irradiation by a cobalt-60 (60Co) radiation source. The different rate constants of epimerization between diastereomeric dipeptides in the research suggest that the potential pathway toward homochirality could be much more complex.

  18. Epimerization of Alanyl-Alanine Induced by γ-Rays Irradiation in Aqueous Solutions.

    PubMed

    Munegumi, Toratane

    2017-03-01

    Living organisms have homochiral L-amino acids in proteins and homochiral D-mononucleotides in nucleic acids. The chemical evolutionary process to protein homochirality has been discussed for many years. Although many scenarios have been proposed for homochirality in the monomeric compounds, homochirality in amino acids and mononucleotides does not always guarantee homochirality in polypeptides and polynucleotides. Integrated scenarios containing the pathways from monomer to polymer should be proposed because in the pathways oligomers and polymers as well as monomers racemize (or epimerize), degrade, and condense. This research addresses epimerization and degradation of dipeptides under γ-rays irradiation by a cobalt-60 ((60)Co) radiation source. The different rate constants of epimerization between diastereomeric dipeptides in the research suggest that the potential pathway toward homochirality could be much more complex.

  19. Chiral selectivity of amino acid adsorption on chiral surfaces—The case of alanine on Pt

    SciTech Connect

    Franke, J.-H.; Kosov, D. S.

    2015-02-07

    We study the binding pattern of the amino acid alanine on the naturally chiral Pt surfaces Pt(531), Pt(321), and Pt(643). These surfaces are all vicinal to the (111) direction but have different local environments of their kink sites and are thus a model for realistic roughened Pt surfaces. Alanine has only a single methyl group attached to its chiral center, which makes the number of possible binding conformations computationally tractable. Additionally, only the amine and carboxyl group are expected to interact strongly with the Pt substrate. On Pt(531), we study the molecule in its pristine as well as its deprotonated form and find that the deprotonated one is more stable by 0.47 eV. Therefore, we study the molecule in its deprotonated form on Pt(321) and Pt(643). As expected, the oxygen and nitrogen atoms of the deprotonated molecule provide a local binding “tripod” and the most stable adsorption configurations optimize the interaction of this “tripod” with undercoordinated surface atoms. However, the interaction of the methyl group plays an important role: it induces significant chiral selectivity of about 60 meV on all surfaces. Hereby, the L-enantiomer adsorbs preferentially to the Pt(321){sup S} and Pt(643){sup S} surfaces, while the D-enantiomer is more stable on Pt(531){sup S}. The binding energies increase with increasing surface density of kink sites, i.e., they are largest for Pt(531){sup S} and smallest for Pt(643){sup S}.

  20. Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.

    PubMed

    Awasthy, Disha; Bharath, Sowmya; Subbulakshmi, Venkita; Sharma, Umender

    2012-02-01

    Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

  1. Porcine alanine transaminase after liver allo-and xenotransplantation

    PubMed Central

    Ekser, Burcin; Gridelli, Bruno; Cooper, David K.C.

    2013-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3 Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3 Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording. PMID:22360753

  2. Porcine alanine transaminase after liver allo-and xenotransplantation.

    PubMed

    Ekser, Burcin; Gridelli, Bruno; Cooper, David K C

    2012-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording.

  3. Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori.

    PubMed

    Wu, Dalei; Zhang, Liang; Kong, Yunhua; Du, Jiamu; Chen, Shuai; Chen, Jing; Ding, Jianping; Jiang, Hualiang; Shen, Xu

    2008-09-01

    D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.

  4. A dipeptide-based superhydrogel: Removal of toxic dyes and heavy metal ions from waste water.

    PubMed

    Nandi, Nibedita; Baral, Abhishek; Basu, Kingshuk; Roy, Subhasish; Banerjee, Arindam

    2017-01-01

    A short peptide-based molecule has been found to form a strong hydrogel at phosphate buffer solution of pH 7.46. The hydrogel has been characterized thoroughly using various techniques including field emission scanning electron microscopy (FE-SEM), wide angle powder X-ray diffraction (PXRD), and rheological analysis. It has been observed from FE-SEM images that entangled nanofiber network is responsible for gelation. Rheological investigation demonstrates that the self-assembly of this synthetic dipeptide results in the formation of mechanically strong hydrogel with storage modulus (G') around 10(4) Pa. This gel has been used for removing both cationic and anionic toxic organic dyes (Brilliant Blue, Congo red, Malachite Green, Rhodamine B) and metal ions (Co(2+) and Ni(2+) ) from waste water. Moreover, only a small amount of the gelator is required (less than 1 mg/mL) for preparation of this superhydrogel and even this hydrogel can be reused three times for dye/metal ion absorption. This signifies the importance of the hydrogel towards waste water management.

  5. The structure of the gas-phase tyrosine-glycine dipeptide

    NASA Astrophysics Data System (ADS)

    Toroz, Dimitrios; van Mourik, Tanja

    The structural preferences of the neutral dipeptide Tyr-Gly have been investigated using a hierarchical selection scheme. This scheme consists of a hierarchy of increasingly more accurate electronic structure methods (single-point HF/3-21G* energy calculation, HF/3-21G* geometry optimization, B3LYP/6-31+G* geometry optimization, MP2/6-31+G* single-point energy calculation, and MP2/6-31+G* geometry optimization). The conformers are sorted according to their single-point or optimized energy, and only the most stable conformers according to one level are taken through to the next level of calculation. The defining structural characteristics in the 20 most stable Tyr-Gly conformers are the presence or absence of a folded arrangement of the peptide backbone ('book') and an OH···O hydrogen bond between the C-terminal hydroxyl group and the carbonyl oxygen of tyrosine ('OHO'). The most stable conformer is of the book/OHO type. MP2 geometry optimization significantly alters the structure of the book-type conformers, increasing their degree of foldedness. Thus, care has to be taken when applying standard density functionals like B3LYP in structural studies of peptides with aromatic side chains.

  6. A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase

    PubMed Central

    Belmondo, Simone; Fiorilli, Valentina; Pérez-Tienda, Jacob; Ferrol, Nuria; Marmeisse, Roland; Lanfranco, Luisa

    2014-01-01

    Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase. PMID:25232358

  7. Dipeptide Mimetic of the Brain-derived Neurotrophic Factor Prevents Impairments of Neurogenesis in Stressed Mice.

    PubMed

    Gudasheva, T A; Povarnina, P Yu; Seredenin, S B

    2017-02-01

    Brain-derived neurotrophic factor (BDNF) plays the central role in the mechanisms of regulation of neurogenesis and neuroplasticity. Impairment of these mechanisms is considered as one of the main etiological factors of depression. Dimeric dipeptide mimetic of BDNF loop 4 bis-(N-monosuccinyl-l-seryl-l-lysine) hexamethylenediamide (GSB-106) was synthesized at the V. V. Zakusov Research Institute of Pharmacology. In vivo experiments revealed significant antidepressant properties of GSB-106 in doses of 0.1-10 mg/kg (intraperitoneally and orally). Effects of GSB-106 on hippocampal neurogenesis were studied in mice subjected to chronic predator stress. Proliferative activity in the subgranular zone of the dental gyrus was assessed immunohistochemically by Ki-67 expression (a marker of dividing cells). It was found that GSB-106 (10 mg/kg, intraperitoneally, 5 days) completely prevents neurogenesis disturbances in stressed mice. These findings suggest that GSB-106 is a promising candidate for the development of antidepressant agents with BDNF-like mechanism of action.

  8. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway

    PubMed Central

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-01-01

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication. PMID:26830977

  9. Stimulation of macrophage protease secretion via liposomal delivery of muramyl dipeptide derivatives to intracellular sites.

    PubMed Central

    Mehta, K; Juliano, R L; Lopez-Berestein, G

    1984-01-01

    We have observed that murine macrophages can be activated for enhanced neutral protease secretion by exposing the cells to muramyl dipeptides (MDPs). A lipophilic derivative of nor-MDP is more efficacious than the parent hydrophilic nor-MDP. The efficacy and potency of the lipophilic and more prominently the hydrophilic drugs can be increased (10-10(3) fold) by encapsulating them in lipid vesicles (liposomes); however the encapsulation of drug causes a delay in the onset of activation. The enhanced effectiveness of liposomal MDPs seems in part, to be due to increased uptake, slow release and thus potentiated action of the drug at intracellular sites as emphasized by studies with [3H]-MDP. Appropriate distribution of the drug to intracellular compartments of the cell also seems to be an important factor in the activation process. The internalization of a relatively large amounts (greater than 5 ng/10(6) cells) of nor-MDP results in 'down regulation', that is reduced protease secretion, as compared to effects produced by internalization of lesser amounts of the drug. The macrophage activating effects of liposomal MDPs do not seem to require the processing of liposomes in the lysosomal compartment; thus lysosome-blocking agents, such as chloroquine and dextran sulphate, do not affect the induction of protease secretion. PMID:6365745

  10. [Study on the interaction of L-cysteine dipeptide with DNA].

    PubMed

    Lu, Kui; Cheng, Hong-li; Zhao, Dong-xin; Yang, Liang-ru; Zhao, Yu-fen

    2010-01-01

    In the present paper the interaction mechanism and mode of biologically active cysteine dipeptide (Cys-Cys) with DNA was studied by UV-Vis spectrophotometry and fluorescence spectroscopy using ethidium bromide as a fluorescence probe. The results showed that in the buffer solution of Tris-HCl (pH 7.20), at low concentration of Cys-Cys, the ultraviolet spectrum of DNA-Cys-Cys system produced hypochromic effect with increasing the concentration of Cys-Cys. When the concentration of Cys-Cys increased to some high extent, the ultraviolet spectrum of DNA-Cys-Cys system produced hyperchromic effect. Salt-effect experiment showed that the interaction is liable to be affected by the ionic strengths, suggesting the existence of electrostatic binding between Cys-Cys and DNA. The fluorescence of EB-DNA had quenching effect with increasing the concentration of Cys-Cys, and the Stern-Volmer equation indicated that the quenching process was a static one. According to the Lineweaver-Burk equation the binding constant was determined to be 1.640 x 10(4) L x mol(-1). From the above results it can be concluded that the interaction mode of Cys-Cys with DNA was mainly electrostatic binding. These findings could contribute to further investigation on the mechanism of oligopeptides interaction with DNA.

  11. Interaction of papain-like cysteine proteases with dipeptide-derived nitriles.

    PubMed

    Löser, Reik; Schilling, Klaus; Dimmig, Elke; Gütschow, Michael

    2005-12-01

    A series of 44 dipeptide nitriles with various amino acids at the P2 position and glycine nitrile at position P1 were prepared and evaluated as inhibitors of cysteine proteinases. With respect to the important contribution of the P2-S2 interaction to the formation of enzyme-inhibitor complexes, it was focused to introduce structural diversity into the P2 side chain. Nonproteinogenic amino acids were introduced, and systematic fluorine, bromine, and phenyl scans for phenylalanine in the P2 position were performed. Moreover, the N-terminal protection was varied. Kinetic investigations were carried out with cathepsin L, S, and K as well as papain. Changes in the backbone structure of the parent N-(tert-butoxycarbonyl)-phenylalanyl-glycine-nitrile (16), such as the introduction of an R-configured amino acid or an azaamino acid into P2 as well as methylation of the P1 nitrogen, resulted in a drastic loss of affinity. Exemplarily, the cyano group of 16 was replaced by an aldehyde or methyl ketone function. Structure-activity relationships were discussed with respect to the substrate specificity of the target enzymes.

  12. Trace Solvent as a Predominant Factor To Tune Dipeptide Self-Assembly.

    PubMed

    Wang, Juan; Liu, Kai; Yan, Linyin; Wang, Anhe; Bai, Shuo; Yan, Xuehai

    2016-02-23

    Solvent molecules such as water are of key importance for tuning self-assembly in biological systems. However, it remains a great challenge to detect the role of different types of noncovalent interactions between trace solvents and biomolecules such as peptides. In this work, we discover a dominant role of trace amounts of solvents for mediation of dipeptide self-assembly, in which solvent-bridged hydrogen bonding is demonstrated as a crucial force in directing fiber formation. Hydrogen-bond-forming solvents (including ethanol, N,N-dimethylformamide, and acetone) can affect the hydrogen bonding of C═O and N-H in diphenylalanine (FF) molecules with themselves, but this does not induce π-π stacking between FF molecules. The directional hydrogen bonding promotes a long-range-ordered arrangement of FF molecules, preferentially along one dimension to form nanofibers or nanobelts. Furthermore, we demonstrate that water with strong hydrogen-bond-forming capability can notably speed up structure formation with long-range order, revealing the importance of water as a trace solvent for regulation of persistent and robust fiber formation.

  13. Immunostimulant activities of a lipophilic muramyl dipeptide derivative and of desmuramyl peptidolipid analogs.

    PubMed Central

    Parant, M A; Audibert, F M; Chedid, L A; Level, M R; Lefrancier, P L; Choay, J P; Lederer, E

    1980-01-01

    The immunostimulant properties of a new muramyl dipeptide (MDP) derivative bearing a lipophilic moiety on the C-terminal end of the peptide chain are described. It is shown, in particular, that 1,O-(acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate had increased immunostimulant activity in comparison with MDP. It induced hypersensitivity even when administered with an antigen in saline, and it gave higher protection against bacterial infections than did MDP. A quite unexpected finding was obtained with the corresponding desmuramyl compound 1,O-(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate, which had no activity in producing humoral antibodies but was just as active as the muramic acid-containing compound in stimulating nonspecific resistance to bacterial infections. It was not pyrogenic. Modifications of the peptide moiety or the lipid moiety of this peptidolipid led to decrease, or even loss, of activity. These results show the importance of the N-acetylmuramyl moiety in MDP for humoral antibody production. The peptidolipid 1,O-(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate is the first member of a new category of nonspecific immunostimulants. PMID:7380554

  14. Prediction of Protein Structural Class Based on Gapped-Dipeptides and a Recursive Feature Selection Approach.

    PubMed

    Liu, Taigang; Qin, Yufang; Wang, Yongjie; Wang, Chunhua

    2015-12-24

    The prior knowledge of protein structural class may offer useful clues on understanding its functionality as well as its tertiary structure. Though various significant efforts have been made to find a fast and effective computational approach to address this problem, it is still a challenging topic in the field of bioinformatics. The position-specific score matrix (PSSM) profile has been shown to provide a useful source of information for improving the prediction performance of protein structural class. However, this information has not been adequately explored. To this end, in this study, we present a feature extraction technique which is based on gapped-dipeptides composition computed directly from PSSM. Then, a careful feature selection technique is performed based on support vector machine-recursive feature elimination (SVM-RFE). These optimal features are selected to construct a final predictor. The results of jackknife tests on four working datasets show that our method obtains satisfactory prediction accuracies by extracting features solely based on PSSM and could serve as a very promising tool to predict protein structural class.

  15. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway.

    PubMed

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-02-02

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication.

  16. Rational Design of Chiral Nanostructures from Self-Assembly of a Ferrocene-Modified Dipeptide.

    PubMed

    Wang, Yuefei; Qi, Wei; Huang, Renliang; Yang, Xuejiao; Wang, Mengfan; Su, Rongxin; He, Zhimin

    2015-06-24

    We report a new paradigm for the rational design of chiral nanostructures that is based on the hierarchical self-assembly of a ferrocene (Fc)-modified dipeptide, ferrocene-L-Phe-L-Phe-OH (Fc-FF). Compared to other chiral self-assembling systems, Fc-FF is unique because of its smaller size, biocompatibility, multiple functions (a redox center), and environmental responsiveness. X-ray and spectroscopic analyses showed that the incorporation of counterions during the hierarchical self-assembly of Fc-FF changed the conformations of the secondary structures from flat β sheets into twisted β sheets. This approach enables chiral self-assembly and the formation of well-defined chiral nanostructures composed of helical twisted β sheets. We identified two elementary forms for the helical twist of the β sheets, which allowed us to create a rich variety of rigid chiral nanostructures over a wide range of scales. Furthermore, through subtle modulations in the counterions, temperature, and solvent, we are able to precisely control the helical pitch, diameter, and handedness of the self-assembled chiral nanostructures. This unprecedented level of control not only offers insights into how rationally designed chiral nanostructures can be formed from simple molecular building blocks but also is of significant practical value for the use in chiroptics, templates, chiral sensing, and separations.

  17. Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

    PubMed

    Boeynaems, Steven; Bogaert, Elke; Kovacs, Denes; Konijnenberg, Albert; Timmerman, Evy; Volkov, Alex; Guharoy, Mainak; De Decker, Mathias; Jaspers, Tom; Ryan, Veronica H; Janke, Abigail M; Baatsen, Pieter; Vercruysse, Thomas; Kolaitis, Regina-Maria; Daelemans, Dirk; Taylor, J Paul; Kedersha, Nancy; Anderson, Paul; Impens, Francis; Sobott, Frank; Schymkowitz, Joost; Rousseau, Frederic; Fawzi, Nicolas L; Robberecht, Wim; Van Damme, Philip; Tompa, Peter; Van Den Bosch, Ludo

    2017-03-16

    Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

  18. Theoretical study of structural and optical properties of noble metal cluster-dipeptide hybrids at defect centers of MgO.

    PubMed

    Kulesza, Alexander; Mitrić, Roland; Bonačić-Koutecký, Vlasta

    2012-07-14

    We present the theoretical investigation of structural and optical properties of silver and gold cluster-dipeptide hybrids bound to the F(S) defect of the MgO (100) surface. We use DFT and its TDDFT variant combined with the polarizable embedded cluster model for the description of the extended MgO environment. As model peptide we have chosen CysTrp since the cysteine residue interacts strongly with metal particles through the sulfur atom and tryptophan is the most important chromophoric amino acid. Our results show that in the case of CysTrp bound to the supported Ag(4) cluster an intense optical signal arises at 400 nm. In contrast, in the case of gold no strongly localized absorption is present since the optical response of supported gold-peptide hybrids is dominated by a large number of low intensity d-electron excitations spread over a broad energy range. Such a localized optical signal which is present in supported silver hybrids can be exploited for the optical detection of peptides and thus can serve as basis for the development of biosensing materials.

  19. Alanine-EPR as a transfer standard dosimetry system for low energy X radiation

    NASA Astrophysics Data System (ADS)

    Khoury, H. J.; da Silva, E. J.; Mehta, K.; de Barros, V. S.; Asfora, V. K.; Guzzo, P. L.; Parker, A. G.

    2015-11-01

    The purpose of this paper is to evaluate the use of alanine-EPR as a transfer standard dosimetry system for low energy X radiation, such as that in RS-2400, which operates in the range from 25 to 150 kV and 2 to 45 mA. Two types of alanine dosimeters were investigated. One is a commercial alanine pellets from Aérial-Centre de Ressources Technologiques, France and one was prepared in our laboratory (LMRI-DEN/UFPE). The EPR spectra of the irradiated dosimeters were recorded in the Nuclear Energy Department of UFPE, using a Bruker EMX10 EPR spectrometer operating in the X-band. The alanine-EPR dosimetry system was calibrated in the range of 20-220 Gy in this X-ray field, against an ionization chamber calibrated at the relevant X-ray energy with traceability to PTB. The results showed that both alanine dosimeters presented a linear dose response the same sensitivity, when the EPR signal was normalized to alanine mass. The total uncertainty in the measured dose was estimated to be about 3%. The results indicate that it is possible to use the alanine-EPR dosimetry system for validation of a low-energy X ray irradiator, such as RS-2400.

  20. Importance of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery

    SciTech Connect

    Wasserman, D.H.; Williams, P.E.; Lacy, D.B.; Green, D.R.; Cherrington, A.D.

    1988-04-01

    These studies were performed to assess the importance of intrahepatic mechanisms to gluconeogenesis in the dog during 150 min of treadmill exercise and 90 min of recovery. Sampling catheters were implanted in an artery and portal and hepatic veins 16 days before experimentation. Infusions of (U-/sup 14/C)alanine, (3-/sup 3/H)glucose, and indocyanine green were used to assess gluconeogenesis. During exercise, a decline in arterial and portal vein plasma alanine and in hepatic blood flow led to a decrease in hepatic alanine delivery. During recovery, hepatic blood flow was restored to basal, causing an increase in hepatic alanine delivery beyond exercise rates but still below resting rates. Hepatic fractional alanine extraction increased from 0.26 +/- 0.02 at rest to 0.64 +/- 0.03 during exercise and remained elevated during recovery. Net hepatic alanine uptake was 2.5 +/- 0.2 mumol.kg-1.min-1 at rest and remained unchanged during exercise but was increased during recovery. The conversion rate of (/sup 14/C)alanine to glucose had increased by 248 +/- 38% by 150 min of exercise and had increased further during recovery. The efficiency with which alanine was channeled into glucose in the liver was accelerated to a rate of 338 +/- 55% above basal by 150 min of exercise but declined slightly during recovery. In conclusion, 1) gluconeogenesis from alanine is accelerated during exercise, due to an increase in the hepatic fractional extraction of the amino acid and through intrahepatic mechanisms that more efficiently channel it into glucose.

  1. Determination of D- and L-alanine concentrations using a pyruvic acid sensor.

    PubMed

    Inaba, Yohei; Hamada-Sato, Naoko; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo

    2003-08-01

    The concentrations of D- and L-alanine in bivalves are useful as indicators of environmental pollution. Amino acid oxidase with a low substrate specificity catalyzes the oxidation of various amino acids. Among the various amino acids, pyruvic acid can be generated from alanine only by the catalytic oxidative reaction of this oxidase. Therefore, in this study, the concentrations of D- and L-alanine were determined from the concentration of pyruvic acid, which was determined from the consumption of oxygen based on the oxidative reaction of pyruvate oxidase. From this point of view, there is a very strong possibility that biosensors utilizing enzymes with a low substrate specificity can be developed. The results obtained were as follows. (1) The optimum conditions for the use of pyruvic acid sensor were as follows: temperature of 25 degrees C, pH of 6.8, flow rate of 0.1 ml/min, thiamin diphosphate concentration of 1.5 mM, and injection volume of 50 microl. (2) D-Alanine and L-alanine optimally reacted with D- and L-amino acid oxidase at 30 degrees C, pH 8.2, for 30 min and at 37 degrees C, pH 7.8, for 90 min, respectively. (3) The linear relationships between the concentrations of D- and L-alanine and the output of the sensor were obtained at 3.56-106.8 microg of D-alanine and 5.34-71.3 microg of L-alanine. (4) The concentrations of D- and L-alanine in Meretrix iusoria, Patinopecten yessonsi, and Corbicula leana obtained by the proposed assay were in good agreement with those determined by a conventional method.

  2. Serum Alanine Aminotransferase Levels, Hematocrit Rate and Body Weight Correlations Before and After Hemodialysis Session

    PubMed Central

    Lopes, Edmundo Pessoa; Sette, Luis Henrique B. C.; Sette, Jorge Bezerra C.; Luna, Carlos F.; Andrade, Amaro M.; Moraes, Maviael; Sette, Paulo C. A.; Menezes, Roberto; Cavalcanti, Rui L.; Conceição, Sergio C.

    2009-01-01

    PURPOSE To evaluate alanine aminotransferase levels before and after a hemodialysis session and to correlate these values with the hematocrit rate and weight loss during hemodialysis. PATIENTS AND METHODS The serum alanine aminotransferase levels, hematocrit rate and body weight were measured and correlated before and after a single hemodialysis session for 146 patients with chronic renal failure. An receiver operating characteristic (ROC) curve for the serum alanine aminotransferase levels collected before and after hemodialysis was plotted to identify hepatitis C virus-infected patients. RESULTS The mean weight loss of the 146 patients during hemodialysis was 5.3% (p < 0.001). The mean alanine aminotransferase levels before and after hemodialysis were 18.8 and 23.9 IU/, respectively, denoting a significant 28.1% increase. An equally significant increase of 16.4% in the hematocrit rate also occurred after hemodialysis. The weight loss was inversely correlated with the rise in both the alanine aminotransferase level (r = 0.3; p < 0.001) and hematocrit rate (r = 0.5; p < 0.001). A direct correlation was found between the rise in alanine aminotransferase levels and the hematocrit during the hemodialysis session (r = 0.4; p < 0.001). Based on the ROC curve, the upper limit of the normal alanine aminotransferase level should be reduced by 40% relative to the upper limit of normal if the blood samples are collected before the hemodialysis session or by 60% if blood samples are collected after the session. CONCLUSION In the present study, significant elevations in the serum alanine aminotransferase levels and hematocrit rates occurred in parallel to a reduction in body weight after the hemodialysis session. These findings suggest that one of the factors for low alanine aminotransferase levels prior to hemodialysis could be hemodilution in patients with chronic renal failure. PMID:19841699

  3. The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

    PubMed

    Rubach, Jon K; Cui, Guanglei; Schneck, Jessica L; Taylor, Amy N; Zhao, Baoguang; Smallwood, Angela; Nevins, Neysa; Wisnoski, David; Thrall, Sara H; Meek, Thomas D

    2012-09-25

    We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 μM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 μM(-1 )s(-1) and 5.3 ± 0.5 μM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) ≫ k(dac) ∼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 μM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and

  4. Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity.

    PubMed

    Walter, Frederik; Grenz, Sebastian; Ortseifen, Vera; Persicke, Marcus; Kalinowski, Jörn

    2016-08-20

    In this work the role of γ-glutamyl transpeptidase in the metabolism of γ-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate γ-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45°C. The KM value was 123μM. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of γ-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five γ-glutamyl dipeptides (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Val, γ-Glu-Leu, γ-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo.

  5. Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes.

    PubMed

    Ansurudeen, Ishrath; Sunkari, Vivekananda Gupta; Grünler, Jacob; Peters, Verena; Schmitt, Claus Peter; Catrina, Sergiu-Bogdan; Brismar, Kerstin; Forsberg, Elisabete Alcantara

    2012-07-01

    Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100 mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.

  6. Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Hori, Hatsuhiro; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2015-08-01

    We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

  7. Progress towards an alanine/ESR therapy level reference dosimetry service at NPL.

    PubMed

    Sharpe, P H; Rajendran, K; Sephton, J P

    1996-01-01

    This paper describes work being carried out at the National Physical Laboratory towards the establishment of an alanine reference dosimetry service for radiotherapy applications. A precision fused quartz holder has been constructed to allow precise positioning of alanine dosimeters in the ESR cavity. A novel method of signal analysis based on spectrum fitting has been developed to minimize the effect of baseline distortions. Data are also presented on the relative response of alanine to 60Co gamma rays and high energy photons (4-12 MeV).

  8. Applicability of EPR/alanine dosimetry for quality assurance in proton eye radiotherapy.

    PubMed

    Michalec, B; Mierzwinska, G; Ptaszkiewicz, M; Sowa, U; Stolarczyk, L; Weber, A

    2014-06-01

    A new quality assurance and quality control method for proton eye radiotherapy based on electron paramagnetic resonance (EPR)/alanine dosimetry has been developed. It is based on Spread-Out Bragg Peak entrance dose measurement with alanine detectors. The entrance dose is well correlated with the dose at the facility isocenter, where, during the therapeutic irradiation, the tumour is placed. The unique alanine detector features namely keeping the dose record in a form of stable radiation-induced free radicals trapped in the material structure, and the non-destructive read-out makes this type of detector a good candidate for additional documentation of the patient's exposure over the therapy course.

  9. Interactions of L-alanine with alumina as studied by vibrational spectroscopy.

    PubMed

    Garcia, Ana R; de Barros, Ricardo Brito; Fidalgo, Alexandra; Ilharco, Laura M

    2007-09-25

    The interactions of L-alanine with gamma- and alpha-alumina have been investigated by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). L-alanine/alumina samples were dried from aqueous suspensions, at 36.5 degrees C, with two amino acid concentrations (0.4 and 0.8 mmol g-1) and at different pH values (1, 6, and 13). The vibrational spectra proved that the nature of L-alanine interactions with both aluminas is the same (hydrogen bonding), although the groups involved depend on the L-alanine form and on alumina surface groups, both controlled by the pH. For samples prepared at pH 1, cationic L-alanine [CH3CH(NH3+)COOH] displaces physisorbed water from alumina, and strong hydrogen bonds are established between the carbonyl groups of alanine, as electron donors, and the surface Al-OH2+ groups of alumina. This occurs at the expense of alanine dimer dissociation and breaking of intramolecular bonds. When samples are prepared at pH 6, the interacting groups are Al-OH2+ and the carboxylate groups of zwitterionic L-alanine [CH3CH(NH3+)COO-]. The affinity of L-alanine toward alumina decreases, as the strong NH3+...-OOC intermolecular hydrogen bonds prevail over the interactions with alumina. Thus, for a load of 0.8 mmol g-1, phase segregation is observed. On alpha-alumina, crystal deposition is even observed for a load of 0.4 mmol g-1. At pH 13, the carboxylate groups of anionic L-alanine [CH3CH(NH2)COO-] are not affected by alumina. Instead, hydrogen bond interactions occur between NH2 and the Al-OH surface groups of the substrate. Complementary N2 adsorption-desorption isotherms showed that adsorption of L-alanine occurs onto the alumina pore network for samples prepared at pH 1 and 13, whereas at pH 6 the amino acid/alumina interactions are not strong enough to promote adsorption. The mesoporous structure and the high specific surface area of gamma-alumina make it a more efficient substrate for adsorption of L-alanine. For each alumina, however, it is

  10. Temperature dependences of piezoelectric, elastic and dielectric constants of L-alanine crystal

    NASA Astrophysics Data System (ADS)

    Tylczyński, Z.; Sterczyńska, A.; Wiesner, M.

    2011-09-01

    Temperature changes in the components of piezoelectric, elastic and dielectric tensors were studied in L-alanine crystals in the range 100-300 K. A jumpwise increase in the c55 component of the elastic stiffness accompanied by maxima in damping of all face-shear modes observed at 199 K in L-alanine crystal were interpreted as a result of changes in the NH3+ vibrations occurring through electron-phonon coupling. All components of the piezoelectric tensor show small anomalies in this temperature range. The components of the electromechanical coupling coefficient determined indicate that L-alanine is a weak piezoelectric.

  11. Optical and Spectral Studies on β Alanine Metal Halide Hybrid Crystals

    NASA Astrophysics Data System (ADS)

    Sweetlin, M. Daniel; Selvarajan, P.; Perumal, S.; Ramalingom, S.

    2011-10-01

    We have synthesized and grown β alanine metal halide hybrid crystals viz. β alanine cadmium chloride (BACC), an amino acid transition metal halide complex crystal and β alanine potassium chloride (BAPC), an amino acid alkali metal halide complex crystal by slow evaporation method. The grown crystals were found to be transparent and have well defined morphology. The optical characteristics of the grown crystals were carried out with the help of UV-Vis Spectroscopy. The optical transmittances of the spectrums show that BAPC is more transparent than BACC. The Photoluminescence of the materials were determined by the Photoluminescent Spectroscopy

  12. Temperature dependences of piezoelectric, elastic and dielectric constants of L-alanine crystal.

    PubMed

    Tylczyński, Z; Sterczyńska, A; Wiesner, M

    2011-09-07

    Temperature changes in the components of piezoelectric, elastic and dielectric tensors were studied in L-alanine crystals in the range 100-300 K. A jumpwise increase in the c(55) component of the elastic stiffness accompanied by maxima in damping of all face-shear modes observed at 199 K in L-alanine crystal were interpreted as a result of changes in the NH(3)(+) vibrations occurring through electron-phonon coupling. All components of the piezoelectric tensor show small anomalies in this temperature range. The components of the electromechanical coupling coefficient determined indicate that L-alanine is a weak piezoelectric.

  13. GMXPBSA 2.0: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2014-11-01

    provisions: GNU General Public License, version 3 No. of lines in distributed program, including test data, etc.: 185 937 No. of bytes in distributed program, including test data, etc.: 7 074 217 Distribution format: tar.gz Programming language: Bash, Perl. Computer: Any computer. Operating system: Linux, Unix OS. RAM: ˜2 GB Classification: 3. External routines: APBS (http://www.Poissonboltzmann.org/apbs/) and GROMACS installations (http://www.gromacs.org). Optionally LaTeX. Nature of problem: Calculates the Molecular Mechanics (MM) data (Lennard-Jones and Coulomb terms) and the solvation energy terms (polar and nonpolar terms respectively) from an ensemble of structures derived from GROMACS molecular dynamics simulation trajectory. These calculations are performed for each single component of the simulated complex, including protein and ligand. In order to cancel out artefacts an identical grid setup for each component, including complex, protein and ligand, is required. Performs statistical analysis on the extracted data and comparison with wild-type complex in case of either computational alanine scanning or calculations on a set of simulations. Evaluates possible outliers in the frames extracted from the simulations during the binding free energy calculations. Solution method: extract frames from a single or multiple complex molecular dynamics (MD) simulation, allowing comparison between multiple trajectories; split the complex frames in the single components including complex, protein and ligand; calculate the Lennard-Jones and Coulomb energy values (MM terms); calculate the polar solvation energy values using the implicit solvation Poisson-Boltzmann model (PB); calculate the nonpolar solvation energy value based on the solvent accessible surface area (SASA); combine all the calculated terms into the final binding free energy value; repeat the same procedure from point 1 to 6 for each simulation in case of computational alanine scanning (CAS) or simultaneous comparison

  14. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  15. Rapid Ti(III) reduction of perchlorate in the presence of beta-alanine: kinetics, pH effect, complex formation, and beta-alanine effect.

    PubMed

    Wang, Chao; Huang, Zhengdao; Lippincott, Lee; Meng, Xiaoguang

    2010-03-15

    Ti(III) reduction of perchlorate might be a useful method for the treatment of highly perchlorate-contaminated water. Though the reaction rate was usually low, we observed that beta-alanine (HOOCCH(2)CH(2)NH(2)) could significantly promote the reaction. A complete (>99.9%) perchlorate removal was obtained in a solution containing [ClO(4)(-)]=1.0mM, [Ti(III)]=40 mM, and [beta-alanine]=120 mM after 2.5h of reaction under 50 degrees C. The effects of both pH and complex formation on the reaction were then studied. The results showed that without beta-alanine the optimal pH was 2.3. When pH increased from 1.6 to 2.3, the reduction rate increased remarkably. In the pH range >2.3, however, the reduction was significantly inhibited, attributed to the formation of Ti(III) precipitate. The presence of beta-alanine at a molar ratio of [beta-alanine]:[Ti(III)]=3:1 significantly increased the reduction rate of perchlorate even at near neutral pH. This is because beta-alanine formed complexes with Ti(III), which greatly improved the total soluble [Ti(III)] in the pH range between 3.5 and 6. The findings may lead to the development of rapid treatment methods for intermittent and small stream of highly perchlorate-contaminated water, which are resulted from the manufacturing, storage, handling, use and/or disposal of large quantities of perchlorate salts.

  16. Reactions of 1,3-Diketones with a Dipeptide Isothiazolidin-3-one: Toward Agents That Covalently Capture Oxidized Protein Tyrosine Phosphatase 1B.

    PubMed

    Ruddraraju, Kasi Viswanatharaju; Parsons, Zachary D; Llufrio, Elizabeth M; Frost, Natasha L; Gates, Kent S

    2015-12-18

    Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of type 2 diabetes; however, the enzyme has been classified by some as an "undruggable target". Here we describe studies directed toward the development of agents that covalently capture the sulfenyl amide "oxoform" of PTP1B generated during insulin signaling events. The sulfenyl amide residue found in oxidized PTP1B presents a unique electrophilic sulfur center that may be exploited in drug and probe design. Covalent capture of oxidized PTP1B could permanently disable the intracellular pool of enzyme involved in regulation of insulin signaling. Here, we employed a dipeptide model of oxidized PTP1B to investigate the nucleophilic capture of the sulfenyl amide residue by structurally diverse 1,3-diketones. All of the 1,3-diketones examined here reacted readily with the electrophilic sulfur center in the sulfenyl amide residue to generate stable covalent attachments. Several different types of products were observed, depending upon the substituents present on the 1,3-diketone. The results provide a chemical foundation for the development of agents that covalently capture the oxidized form of PTP1B generated in cells during insulin signaling events.

  17. Chelation behavior of various flavonols and transfer of flavonol-chelated zinc(II) to alanylaspartic dipeptide: A PCM/DFT investigation

    NASA Astrophysics Data System (ADS)

    Yasarawan, Nuttawisit; Thipyapong, Khajadpai; Ruangpornvisuti, Vithaya

    2016-03-01

    Alanylaspartic dipeptide (AlaAsp) and zinc(II)-flavonol complex could represent a metal-binding site in proteins and a metal-ion releasing agent, respectively. Chelation of zinc(II) by either AlaAsp or flavonol ligands in aqueous solution has been examined using DFT methods with polarizable continuum model (PCM/DFT). Coordination geometry, complexation stoichiometry, coordination bond strength, preferable metal-binding site on ligands and effect of water coordination on the stability of complexes have been addressed. In several cases, the long-range corrected density functional CAM-B3LYP allows the most accurate prediction of both structural and spectroscopic data. The preferential transfer of flavonol-chelated zinc(II) to AlaAsp under solvation is attainable through the ligand-exchange reaction. The energy barrier of such reaction is significantly dependent on the degree of hydrogen bonding within the transition state. In summary, either hydroxylation or methoxylation at particular positions on the 3-hydroxyflavone backbone significantly affects the reactivity of flavonol chelates in the metal-ion transfer.

  18. Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study.

    PubMed

    Thanigaimalai, Pillaiyar; Konno, Sho; Yamamoto, Takehito; Koiwai, Yuji; Taguchi, Akihiro; Takayama, Kentaro; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Naser-Tavakolian, Aurash; Schön, Arne; Freire, Ernesto; Hayashi, Yoshio

    2013-07-01

    This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.

  19. A comparative study on the growth and characterization of nonlinear optical amino acid crystals: L-alanine (LA) and L-alanine alaninium nitrate (LAAN).

    PubMed

    Aravindan, A; Srinivasan, P; Vijayan, N; Gopalakrishnan, R; Ramasamy, P

    2008-11-15

    A comparative study on the properties of L-alanine and LAAN crystals has been made and discussed. It may be concluded that the protonation of the amino group in the L-alanine molecule is the key factor in increasing the relative SHG efficiency of LAAN. The protonation is justified by the crystal structure analysis, FTIR and photoluminescence studies. The factor group vibrations are compared and found that there is an increase in vibrational modes of LA when reacted with nitric acid forming LAAN.

  20. Determination of the carbon, hydrogen and nitrogen contents of alanine and their uncertainties using the certified reference material L-alanine (NMIJ CRM 6011-a).

    PubMed

    Itoh, Nobuyasu; Sato, Ayako; Yamazaki, Taichi; Numata, Masahiko; Takatsu, Akiko

    2013-01-01

    The carbon, hydrogen, and nitrogen (CHN) contents of alanine and their uncertainties were estimated using a CHN analyzer and the certified reference material (CRM) L-alanine. The CHN contents and their uncertainties, as measured using the single-point calibration method, were 40.36 ± 0.20% for C, 7.86 ± 0.13% for H, and 15.66 ± 0.09% for N; the results obtained using the bracket calibration method were also comparable. The method described in this study is reasonable, convenient, and meets the general requirement of having uncertainties ≤ 0.4%.

  1. Computational analysis of binding affinity and neural response at the l-alanine receptor

    NASA Astrophysics Data System (ADS)

    Venanzi, Thomas J.; Bryant, Bruce P.; Venanzi, Carol A.

    1995-10-01

    A model of analogue-receptor binding is developed for the l-alanine receptor in the channel catfish using the AM1-SM2 and ab initio SCRF computational methods. Besides interactions involving the zwitterionic moiety of the amino acid analogue and complementary subsites on the receptor, the model suggests the presence of a hydrophobic pocket with dispersion interactions between the receptor and the residue on the amino acid analogue. Conformational analysis suggests not only a small compact active site on the receptor, but also that the analogues with the highest affinity occupy nearly identical regions of space. Although the binding interaction is dominated by the ionic terms, AM1-SM2 calculations indicate that free energy terms associated with cavity formation, solvent reorganization, and dispersion interactions can be correlated to activation and neural response. From a consideration of this model, molecular features of the analogues that are important for binding and neural response were deduced and other analogues or ligands were developed and tested.

  2. The dipeptide H-Trp-Arg-OH (WR) Is a PPARα agonist and reduces hepatic lipid accumulation in lipid-loaded H4IIE cells.

    PubMed

    Jia, Yaoyao; Kim, Jong-Ho; Nam, Bora; Kim, Jiyoung; Lee, Ji Hae; Kim, Kyung Ok; Hwang, Kwang Yeon; Lee, Sung-Joon

    2015-01-01

    Dipeptides absorbed by the intestinal epithelium are delivered to circulation, but their metabolic roles are not yet clearly understood. We investigated the biological activities of a dietary dipeptide, H-Trp-Arg-OH (WR), on the regulation of peroxisome proliferator-activated receptor (PPAR) α activity. Reporter gene assays revealed that WR dose-dependently induced PPARα transactivation. Surface plasmon resonance experiments demonstrated that WR interacts directly with the PPARα ligand binding domain, and time-resolved fluorescence energy transfer analyses revealed recruitment of a co-activator peptide, fluorescein-PGC1α, to PPARα, confirming the direct binding of WR to PPARα and occurrence of conformational changes. WR induced cellular fatty acid uptake and the expression of PPARα response genes in fatty acid oxidation, thus reducing intracellular triglyceride accumulation in lipid-loaded hepatocytes. In conclusion, the dietary dipeptide WR activates PPARα and reduces hepatic lipid accumulation in lipid-loaded hepatocytes.

  3. Titration of Alanine Monitored by NMR Spectroscopy: A Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Waller, Francis J.; And Others

    1977-01-01

    The experiment described here involves simultaneous monitoring of pH and NMR chemical shifts during an aqueous titration of alpha- and beta-alanine. This experiment is designed for use in an undergraduate biochemistry course. (MR)

  4. Second harmonic generation studies in L-alanine single crystals grown from solution

    NASA Astrophysics Data System (ADS)

    Boomadevi, Shanmugam; Pandiyan, Krishnamoorthy

    2014-01-01

    Single crystals of L-alanine of dimensions 2×1.1×0.5 cm3 were grown by evaporation method using deionised water as a solvent. The morphology of the grown crystals had (1 2 0) and (0 1 1) as their prominent faces. UV-vis-near IR spectrum shows the transparency range of L-alanine crystal available for frequency doubling from 250 to 1400 nm. Phase-matched second harmonic generation was observed in L-alanine sample by using 7 ns Q-switched Nd:YAG laser with OPO set up. In the present work, phase matching was achieved by angle and wavelength tuning. The angular and spectral phase-matching bandwidths were determined experimentally for a 1.5 mm thick L-alanine crystal and the results have been compared with their theoretical results. Further the possible reasons for the broadening of SHG spectrum have been discussed.

  5. Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice

    PubMed Central

    Vallejo, Julian; Spence, Madoka; Cheng, An-Lin; Brotto, Leticia; Edens, Neile K.; Garvey, Sean M.; Brotto, Marco

    2016-01-01

    There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice. PMID

  6. Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization ▿

    PubMed Central

    Sallam, Ahmed; Kast, Alene; Przybilla, Simon; Meiswinkel, Tobias; Steinbüchel, Alexander

    2009-01-01

    A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphEal) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter−1 citrate, pH 6.5, and 37°C are ideal parameters for CphEal production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter−1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter−1 induced CphEal production with only about 30% efficiency in comparison to that with CGP. CphEal was purified utilizing its affinity for the substrate and its specific binding to CGP. CphEal turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphEal preparation. Optimum degradation parameters were 100 g liter−1 CGP, 10 g liter−1 crude CphEal powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphEal powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale. PMID:18978083

  7. SU-E-T-643: Pure Alanine Dosimeter for Verification Dosimetry in IMRT

    SciTech Connect

    Al-Karmi, Anan M.; Zraiqat, Fadi

    2015-06-15

    Purpose: The objective of this study was evaluation of accuracy of pure alanine dosimeters measuring intensity-modulated radiation therapy (IMRT) dose distributions in a thorax phantom. Methods: Alanine dosimeters were prepared in the form of 110 mg pure L-α-alanine powder filled into clear tissue-equivalent polymethylmethacrylate (PMMA) plastic tubes with the dimensions 25 mm length, 3 mm inner diameter, and 1 mm wall thickness. A dose-response calibration curve was established for the alanine by placing the dosimeters at 1.5 cm depth in a 30×30×30 cm{sup 3} solid water phantom and then irradiating on a linac with 6 MV photon beam at 10×10 cm{sup 2} field size to doses ranging from 1 to 5 Gy. Electron paramagnetic resonance (EPR) spectroscopy was used to determine the absorbed dose in alanine. An IMRT treatment plan was designed for a commercial heterogeneous CIRS thorax phantom and the dose values were calculated at three different points located in tissue, lung, and bone equivalent materials. A set of dose measurements was carried out to compare measured and calculated dose values by placing the alanine dosimeters at those selected locations inside the thorax phantom and delivering the IMRT to the phantom. Results: The alanine dose measurements and the IMRT plan dose calculations were found to be in agreement within ±2%. Specifically, the deviations were −0.5%, 1.3%, and −1.7% for tissue, lung, and bone; respectively. The slightly large deviations observed for lung and bone may be attributed to tissue inhomogeneity, steep dose gradients in these regions, and uncontrollable changes in spectrometer conditions. Conclusion: The results described herein confirmed that pure alanine dosimeter was suitable for in-phantom dosimetry of IMRT beams because of its high sensitivity and acceptable accuracy. This makes the dosimeter a promising option for quality control of the therapeutic beams, complementing the commonly used ionization chambers, TLDs, and films.

  8. The effects of the EW dipeptide optical and chemical isomers on the CFU-S population in intact and irradiated mice.

    PubMed

    Deigin, V I; Semenets, T N; Zamulaeva, I A; Maliutina, Ya V; Selivanova, E I; Saenko, A S; Semina, O V

    2007-03-01

    The influence of Glu-Trp (EW) synthetic dipeptide isomers on hemopoietic progenitor cells and certain immune response reactions is determined by their optical and chemical properties. Thus, the all L-amino acid containing dipeptides L-Glu-L-Trp and L-gammaGlu-L-Trp have no effect on proliferation of committed and pluripotent CFU-S in intact bone marrow. The optical isomers of the Glu residue are an essential determinant of the EW dipeptide biological activity. The inversion of the amino acid optical form imparts suppressor properties: D-Glu-D-Trp,D--gammaGlu-D-Trp, D-Glu-L-Trp and D-gammaGlu-L-Trp inhibit proliferation of hemopoietic progenitors in intact bone marrow. The type of the peptide bond between L-Glu and Trp is another important factor for the biological activity of the L-Glu-containing peptides. Unlike L-Glu-D-Trp with alpha-peptide bond, the dipeptide L-gammaGlu-D-Trp with gamma-peptide bond stimulates CFU-S-8 proliferation in intact bone marrow. The diverse effects of the EW optical isomers on hemopoietic progenitors underlie the radioprotective properties of the D-Glu-containing dipeptides and the radiotherapeutic ones of the L-Glu dipeptides. In animals, pre-irradiation injection of D-Glu-D-Trp, D-gammaGlu-D-Trp, D-Glu-L-Trp, D-gammaGlu-L-Trp, or post-irradiation injection of L-Glu-L-Trp, L-gammaGlu-L-Trp promoted regeneration of the hemopoietic progenitor population.

  9. Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept

    PubMed Central

    Vakhitova, Y. V.; Sadovnikov, S. V.; Borisevich, S. S.; Ostrovskaya, R. U.; A.Gudasheva, T.; Seredenin, S. B.

    2016-01-01

    This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide. PMID:27099787

  10. Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum.

    PubMed

    Coon, Steven D; Schwartz, John H; Rajendran, Vazhaikkurichi M; Jepeal, Lisa; Singh, Satish K

    2013-11-15

    Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes. The present study investigated the effect of GIP on proton-peptide cotransporter-1 (PepT1) that mediates di- and tripeptide absorption as well as peptidomimetic drugs. Immunohistochemistry studies localized both GIP receptor (GIPR) and PepT1 proteins on the basolateral and apical membranes of normal mouse jejunum, respectively. Anti-GIPR antibody detected 50-, 55-, 65-, and 70-kDa proteins, whereas anti-PepT1 detected a 70-kDa proteins in mucosal homogenates of mouse jejunum. RT-PCR analyses established the expression of GIPR- and PepT1-specific mRNA in mucosal cells of mouse jejunum. Absorption of Gly-Sar (a nondigestible dipeptide) measured under voltage-clamp conditions revealed that the imposed mucosal H(+) gradient-enhanced Gly-Sar absorption as an evidence for the presence of PepT1-mediated H(+):Gly-Sar cotransport on the apical membranes of mouse jejunum. H(+):Gly-Sar absorption was completely inhibited by cephalexin (a competitive inhibitor of PepT1) and was activated by GIP. The GIP-activated Gly-Sar absorption was completely inhibited by RP-cAMP (a cAMP antagonist). In contrast to GIP, the ileal L cell secreting glucagon-like peptide-1 (GLP-1) did not affect the H(+):Gly-Sar absorption in mouse jejunum. We conclude from these observations that GIP, but not GLP-1, directly activates PepT1 activity by a cAMP-dependent signaling pathway in jejunum.

  11. Increased production of superoxide anion by macrophages exposed in vitro to muramyl dipeptide or lipopolysaccharide

    PubMed Central

    1980-01-01

    After in vitro exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP), cultured resident mouse peritoneal macrophages were primed to display enhanced generation of superoxide anion (O2-) in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Priming with LPS (1 microgram/ml) produced a sevenfold enhancement of PMA-stimulated O2- generation; priming was detected within 30 min and persisted for at least 4 d. Exposure to MDP (1 muM) primed the macrophages to double their O2- release; the response was first observed after 4 h and persisted for at least 3 d. The priming response was not observed with stereoisomers of MDP, which are inactive as adjuvants. LPS and MDP appeared to work directly on the macrophages rather than indirectly by interacting with adherent lymphocytes: (a) Addition of nonadherent cell populations that contained lymphocytes had no effect on the response. (b) The response was normal with cells from nude mice, which lack mature T lymphocytes. (c) Macrophages from C3H/HeJ mice, whose B lymphocytes fail to respond to LPS, were weak in their response to priming LPS; the addition of normal (C3Heb/FeJ) nonadherent cells had no effect on this weak response. (d) The macrophage-like cell line J774.1 also showed enhanced O2--generating capacity after a 4-h exposure to LPS or MDP. The O2--generating capacity of macrophages primed with LPS in vitro was equivalent to that previously observed with cells elicited in vivo by injection of LPS or activated by infection with Bacille Calmette-Guerin. The data suggest that previous exposure to bacterial products could prime macrophages to respond with increased production of toxic oxygen metabolites on contact with invading microorganisms or tumor cells. PMID:7350246

  12. Acetazolamide serves as selective delivery vehicle for dipeptide-linked drugs to renal cell carcinoma

    PubMed Central

    Cazzamalli, Samuele; Dal Corso, Alberto; Neri, Dario

    2016-01-01

    In most cases, cytotoxic drugs do not preferentially accumulate at the tumor site, causing unwanted toxicities and preventing dose escalation to therapeutically active regimens. Here, we show that acetazolamide derivatives, which bind to carbonic anhydrase IX (CAIX) on the surface of kidney cancer cells, selectively deliver payloads at the site of disease, sparing normal organs. Biodistribution studies, performed in tumor-bearing mice with acetazolamide derivatives bearing a technetium-99m chelator complex or a red fluorophore as payload, revealed a preferential tumor accumulation of the compound at doses up to 560 nmol/Kg. The percentage of injected dose per gram in the tumor was dose-dependent and revealed optimal tumor:organ ratios at 140 nmol/Kg, with a tumor:blood ratio of 80:1 at 6 h. Acetazolamide, coupled to potent cytotoxic drugs via a dipeptide linker, exhibited a potent antitumor activity in nude mice bearing SKRC-52 renal cell carcinomas, while drug derivatives devoid of the acetazolamide moiety did not exhibit any detectable anticancer activity at the same doses. The observation of tumor regression with a noninternalizing ligand and with different cytotoxic moieties (MMAE and PNU-159682) indicates a general mechanism of action, based on the selective accumulation of the product on tumor cells, followed by the extracellular proteolytic release of the cytotoxic payload at the neoplastic site and the subsequent drug internalization into tumor cells. Acetazolamide-based drug conjugates may represent a promising class of targeted agents for the treatment of metastatic kidney cancer, as the majority of human clear cell renal cell carcinomas are strongly positive for CAIX. PMID:27609641

  13. Partial purification and characterization of an enzyme involved in the formation of beta-aspartyl dipeptides in rat kidney.

    PubMed

    Tanaka, T; Hirai, M; Nakajima, T

    1978-11-01

    The formation of beta-aspartyl-glycine from asparagine and glycine was demonstrated in the supernatant of rat kidney. The enzyme involved in this process was partially purified. Based on the properties of the enzyme reaction and the coincidence of purification rates of this activity and asparaginase, it can be speculated that the enzyme is a kind of asparaginase. Examination of the preference for beta-aspartyl donors and acceptors showed that asparagine and glycine were the preferred donor and acceptor, respectively. beta-Aspartyl dipeptides also transferred their aspartyl residues to amino acids. Amino acids other than glycine also accepted the aspartyl moiety from the donors.

  14. Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

    PubMed

    Ettari, Roberta; Pinto, Andrea; Previti, Santo; Tamborini, Lucia; Angelo, Ilenia C; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Schirmeister, Tanja; Zappalà, Maria; Grasso, Silvana; De Micheli, Carlo; Conti, Paola

    2015-11-01

    Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

  15. Dipeptide vinyl sultams: synthesis via the Wittig-Horner reaction and activity against papain, falcipain-2 and Plasmodium falciparum.

    PubMed

    Valente, Cláudia; Guedes, Rita C; Moreira, Rui; Iley, Jim; Gut, Jiri; Rosenthal, Philip J

    2006-08-01

    The synthesis of phosphonate derivatives of N-phenyl- and N-benzyl-gamma- and delta-sultams, and their application in the Wittig-Horner reaction with N-Boc-L-phenylalanine aldehyde to afford E- and Z-isomers, are described. These compounds were further processed to provide five dipeptide vinyl sultams, which were found to be inactive against papain at concentrations up to 50 microM. In contrast, vinyl sultams demonstrated weak activity against recombinant falcipain-2 and Plasmodium falciparum W2.

  16. New potent αvβ3 integrin ligands based on azabicycloalkane (γ,α)-dipeptide mimics.

    PubMed

    Pilkington-Miksa, M; Araldi, E M V; Arosio, D; Belvisi, L; Civera, M; Manzoni, L

    2016-03-28

    We have designed a new synthetic strategy for the preparation of a new class of cyclic RGD integrin ligands in which the azabicycloalkane scaffold can be envisaged as a (γ,α) dipeptide mimic. The synthesis and in vitro biological evaluation of these RGD derivatives, as well as the computational study of their conformational properties and binding modes to αVβ3 integrin are described. Compound has shown to be a promising candidate as αVβ3 integrin antagonist able to interfere with both cell adhesion and movement on vitronectin with no evidence of cytotoxic effects.

  17. [The pharmacokinetics of the dipeptide analog of piracetam with nootropic activity GVS-111 and of its basic metabolites].

    PubMed

    Boĭko, S S; Zherdev, V P; Dvorianinov, A A; Gudasheva, T A; Ostrovskaia, R U; Voronina, T A; Rozantsev, G G; Seredenin, S B

    1997-01-01

    The pharmacokinetics of a new nootropic dipeptide analog of piracetam-N-phenylacetyl-L-prolylglycine (GWS-111) and its main metabolites were studied in rats by means of high performance liquid chromatography and gas-liquid chromatography. The compound under study showed a greater resistance to an enzymatic effect than natural neuropeptides. In addition to an unchanged compound three of its metabolites were found in the blood plasma of the rats. One of them, cyclo-Pro-Gly was an active metabolite of GWS-111.

  18. Synthesis and antimycobacterial activity of N-(2-aminopurin-6-yl) and N-(purin-6-yl) amino acids and dipeptides.

    PubMed

    Krasnov, Victor P; Vigorov, Alexey Yu; Musiyak, Vera V; Nizova, Irina A; Gruzdev, Dmitry A; Matveeva, Tatyana V; Levit, Galina L; Kravchenko, Marionella A; Skornyakov, Sergey N; Bekker, Olga B; Danilenko, Valery N; Charushin, Valery N

    2016-06-01

    Synthetic routes to novel N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates with amino acids and glycine-containing dipeptides were developed. In vitro testing of 42 new and known compounds made it possible to reveal a series of N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates exhibiting significant antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant M. tuberculosis strain isolated from tuberculosis patients in the Ural region (Russia). N-(2-Aminopurin-6-yl)- and N-(purin-6-yl)-glycyl-(S)-glutamic acids were the most active compounds.

  19. Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep.

    PubMed

    Obitsu, T; Bremner, D; Milne, E; Lobley, G E

    2000-08-01

    The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0.75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0.75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0.040) and urea (P = 0.009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0.024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0.011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15%). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

  20. [Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

    PubMed

    Li, Su-Bo; Zhang, Xue; Zhang, Yin-Ze; Tan, Ying-Xia; Bao, Guo-Qiang; Wang, Ying-Li; Ji, Shou-Ping; Gong, Feng; Gao, Hong-Wei

    2014-06-01

    The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

  1. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    PubMed

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  2. Expression, crystallization and preliminary X-ray crystallographic analysis of D-alanine-D-alanine ligase from OXA-23-producing Acinetobacter baumannii K0420859.

    PubMed

    Huynh, Kim-Hung; Tran, Huyen-Thi; Pham, Tan-Viet; Ngo, Ho-Phuong-Thuy; Cha, Sun-Shin; Chung, Kyung Min; Lee, Sang Hee; Kang, Lin-Woo

    2014-04-01

    Acinetobacter baumannii causes bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producing A. baumannii K0420859 (A. baumannii OXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug against A. baumannii OXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene from A. baumannii OXA-23 was cloned and expressed, and the AbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug against A. baumannii OXA-23. The AbDdl crystal diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 113.4, b = 116.7, c = 176.5 Å, a corresponding VM of 2.8 Å(3) Da(-1) and a solvent content of 56.3%, and six protomers in the asymmetric unit.

  3. Dipeptide Piracetam Analogue Noopept Improves Viability of Hippocampal HT-22 Neurons in the Glutamate Toxicity Model.

    PubMed

    Antipova, T A; Nikolaev, S V; Ostrovskaya, P U; Gudasheva, T A; Seredenin, S B

    2016-05-01

    Effect of noopept (N-phenylacetyl-prolylglycine ethyl ester) on viability of neurons exposed to neurotoxic action of glutamic acid (5 mM) was studied in vitro in immortalized mouse hippocampal HT-22 neurons. Noopept added to the medium before or after glutamic acid improved neuronal survival in a concentration range of 10-11-10-5 M. Comparison of the effective noopept concentrations determined in previous studies on cultured cortical and cerebellar neurons showed that hippocampal neurons are more sensitive to the protective effect of noopept.

  4. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides.

    PubMed

    Zhang, Yanan; Li, Dan; Li, Yan

    2017-01-20

    L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10(-4) mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10(-5) mol/L.

  5. Salsolinol, a tetrahydroisoquinoline-derived neurotoxin, induces oxidative modification of neurofilament-L protection by histidyl dipeptides.

    PubMed

    Kang, Jung Hoon

    2012-02-01

    Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (•OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of •OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.

  6. [N6-dipeptide derivatives of the N12-ribosyl-indolo[2,3-a]carbazole].

    PubMed

    Goryunova, O V; Zakharchuk, G M; Zhukova, O S; Fetisova, L V; Kuzmina, N E

    2014-01-01

    N6-derivatives of N12-ribosyl-indolo[2,3-a]pirrolo[3,4-c]carbazole-5,7-dione are synthesized as potential antitumor agents, in which an atom of N6-pyrrole part of heterocycle is included into the dipeptide residual of the general formula >N6-(CH2)n-CO-Ala/βAla-OMe (n = 2 or 3). These compounds are derived by reacting of 13-methyl-12-(2,3,4-three-O-acetyl-β-D-ribopyranosyl)indolo[2,3-a]furano[3,4-c] carbazole-5,7-dione with dipeptides, having an unreplaced N-amino end-group, in DMF at 130°C, wherein the nitrogen atom of peptide amino group replaces oxygen O6 in furan ring of heterocycle and is embedded in imide nitrogen atom of pyrrole N6. The ability of the obtained compounds to inhibit growth of SKOV3 human ovarian carcinoma cells was studied, only derivative with radical >N6-(CH2)3-CO-L-Ala-OMe showed cytotoxic activity with an inhibitory concentration of IC50 = 8 μM.

  7. Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

    SciTech Connect

    Ernst, Heidi A.; Pham, Antony; Hald, Helle; Kastrup, Jette S.; Rahman, Moazur; Mirza, Osman

    2009-11-06

    Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC{sub 50} value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC{sub 50} values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.

  8. Hydrophobic dipeptide crystals: a promising Ag-free class of ultramicroporous materials showing argon/oxygen adsorption selectivity.

    PubMed

    Afonso, R; Mendes, A; Gales, L

    2014-09-28

    The adsorption isotherms of nitrogen, oxygen and argon in four VA-class hydrophobic dipeptides are presented. Isotherms were determined at 5, 20 and 35 °C, for a pressure range of 0-6 bar. Under these conditions, adsorption is still in the Henry region. For all materials and temperatures, the sequence of preferential adsorption is Ar > O2 > N2, a highly abnormal result. At 5 °C, the dipeptide with the smallest pores, VI, has Ar/O2 adsorption equilibrium selectivities up to 1.30, the highest ever measured in Ag-free adsorbents. Gas uptakes, at 1 bar and 20 °C, are ∼0.05 mol kg(-1), very low relative values that are partially explained by the low porosity of the solids (<10%). The significance of these results for the development of new materials for the process of O2 generation by pressure swing adsorption (PSA) is discussed. The results indicate some of the structural and chemical properties that prospective Ag-free adsorbents should have in order to have Ar/O2 selectivity, hydrophobic pores, less than 0.5 nm-wide, and porosity of, at least, 20%.

  9. Terminal functionalized thiourea-containing dipeptides as multidrug-resistance reversers that target 20S proteasome and cell proliferation.

    PubMed

    Qin, Jian-Mei; Huang, Ri-Zhen; Yao, Gui-Yang; Liao, Zhi-Xin; Pan, Ying-Ming; Wang, Heng-Shan

    2017-01-27

    A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S proteasome and the effects of multidrug-resistance reversers. These compounds exhibited significant selectivity to the β5-subunit of the human 20S proteasome with IC50 values at submicromolar concentrations. A docking study of the most active compound 6i revealed key interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and a nitro group were important for improving activity. In particular, compound 6i appeared to be the most potent compound against the NCI-H460 cell line, and displayed similar efficiency in drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of 20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp. Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome with the ability to reverse multidrug resistance.

  10. Enantioseparation of glycyl-dipeptides by CEC using particle-loaded monoliths prepared by ring-opening metathesis polymerization (ROMP).

    PubMed

    Gatschelhofer, Christina; Schmid, Martin G; Schreiner, Karin; Pieber, Thomas R; Sinner, Frank M; Gübitz, Gerald

    2006-11-30

    Novel particle-loaded monolithic capillary electrochromatography (CEC) phases for chiral separations were prepared via ring-opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8,exo,endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens, RuCl2(PCy3)2(CHPh) as initiator and silica-based particles containing the chiral selector. By suspending silica particles bearing the chiral selector in the polymerization mixture, particle-based monoliths are easily prepared. This approach has several advantages compared to particle-based separation media: (i) the concept of particle-based monoliths is broadly applicable, as any silica-based chiral phase can be used; (ii) they are inexpensive to prepare; and (iii) the manufacturing process is very simple, no sophisticated packing procedures or the preparation of end frits are required. To show the usefulness of this concept for chiral CEC, the chiral separation performance of particle-loaded CEC monoliths bearing teicoplanin aglycone, chemically bonded to 3 microm silica gel, was investigated for a set of glycyl-dipeptides. Particle-loaded ROMP CEC monoliths showed good separation performance for glycyl-dipeptides.

  11. Molecular-Level Thermodynamic Switch Controls Chemical Equilibrium in Sequence-Specific Hydrophobic Interaction of 35 Dipeptide Pairs

    PubMed Central

    Chun, Paul W.

    2003-01-01

    Applying the Planck-Benzinger methodology, the sequence-specific hydrophobic interactions of 35 dipeptide pairs were examined over a temperature range of 273–333 K, based on data reported by Nemethy and Scheraga in 1962. The hydrophobic interaction in these sequence-specific dipeptide pairs is highly similar in its thermodynamic behavior to that of other biological systems. The results imply that the negative Gibbs free energy change minimum at a well-defined stable temperature, 〈Ts〉, where the bound unavailable energy, TΔSo = 0, has its origin in the sequence-specific hydrophobic interactions, are highly dependent on details of molecular structure. Each case confirms the existence of a thermodynamic molecular switch wherein a change of sign in ΔCpo(T)reaction (change in specific heat capacity of reaction at constant pressure) leads to true negative minimum in the Gibbs free energy change of reaction, ΔGo(T)reaction, and hence a maximum in the related equilibrium constant, Keq. Indeed, all interacting biological systems examined to date by Chun using the Planck-Benzinger methodology have shown such a thermodynamic switch at the molecular level, suggesting its existence may be universal. PMID:12547816

  12. FTIR spectra and conformational structure of deutero-β-alanine isolated in argon matrices

    NASA Astrophysics Data System (ADS)

    Stepanian, Stepan G.; Ivanov, Alexander Yu; Adamowicz, Ludwik

    2016-02-01

    Low temperature FTIR spectra of β-alanine-d3 isolated in argon matrices are used to determine the conformational composition of this compound. UV irradiation of the matrix samples is found to change the relative populations of the β-alanine-d3 conformers. The populations of conformers I and II with an Nsbnd D⋯O intramolecular H-bond decrease after the UV irradiation while the populations of conformer V with an N⋯Dsbnd O H-bond and conformer IV which has no intramolecular H-bonds increase. This behavior of the β-alanine-d3 conformers are used to separate the bands of the different conformers. The analysis of the experimental FTIR spectra is based on the calculated harmonic B3LYP/6-311++G(df,pd) frequencies and on the MP2/aug-cc-pVDZ frequencies calculated with a method that includes anharmonic effects. Polynomial scaling of the calculated frequencies is used to achieve better agreement with the experimental data. The observation of the wide band of the OD stretching vibration at 2201 cm-1 is a direct evidence of the presence of the β-alanine-d3 conformer V in the Ar matrix. In total ten bands of conformer V are detected. The influence of the matrix environment on the structures and the IR spectra of the β-alanine and β-alanine-d3 conformers is investigated. This involves performing calculations of the β-alanine conformers embedded in argon clusters containing from 163 to 166 argon atoms using the M06-2X and B3LYP(GD3BJ) density-functional methods. Good agreement between the calculated and the experimental matrix splitting is demonstrated.

  13. Glucose and Alanine Metabolism in Children with Maple Syrup Urine Disease

    PubMed Central

    Haymond, Morey W.; Ben-Galim, Ehud; Strobel, Karen E.

    1978-01-01

    In vitro studies have suggested that catabolism of branched chain amino acids is linked with alanine and glutamine formed in, and released from, muscle. To explore this possibility in vivo, static and kinetic studies were performed in three patients with classical, and one patient with partial, branched chain α-ketoacid decarboxylase deficiency (maple syrup urine disease, MSUD) and compared to similar studies in eight age-matched controls. The subjects underwent a 24-30-h fast, and a glucose-alanine flux study using stable isotopes. Basal plasma leucine concentrations were elevated (P <0.001) in patients with MSUD (1,140±125 μM vs. 155±18 μM in controls); and in contrast to the controls, branched chain amino acid concentrations in plasma increased during the fast in the MSUD patients. Basal plasma alanine concentrations were lower (P <0.01) in patients with classical MSUD (153±8 μM vs. 495±27 μM in controls). This discrepancy was maintained throughout the fast despite a decrease in alanine concentrations in both groups. Plasma alanine and leucine concentrations in the patient with partial MSUD were intermediate between those of the controls and the subjects with the classical form of the disease. Circulating ketone bodies and glucoregulatory hormones concentrations were similar in the MSUD and normal subjects during the fast. Alanine flux rates in two patients with classical MSUD (3.76 and 4.00 μmol/Kg per min) and the patient with partial MSUD (5.76 μmol/Kg per min) were clearly lower than those of the controls (11.72±2.53 [SD] μmol/Kg per min). After short-term starvation, glucose flux and fasting concentrations were similar in the MSUD patients and normal subjects. These data indicate that branched chain amino acid catabolism is an important rate limiting event for alanine production in vivo. PMID:670400

  14. Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

    PubMed

    Maharana, Jitendra; Swain, Banikalyan; Sahoo, Bikash R; Dikhit, Manas R; Basu, Madhubanti; Mahapatra, Abhijit S; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-08-01

    In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

  15. Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase*

    PubMed Central

    Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Álvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

    2010-01-01

    Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism. PMID:20056599

  16. Lack of behavioral and neuropathological effects of dietary beta-methylamino-L-alanine (BMAA) in mice.

    PubMed

    Cruz-Aguado, Reyniel; Winkler, Daniella; Shaw, Christopher A

    2006-06-01

    Beta-methylamino-L-alanine (BMAA) is an excitotoxin allegedly involved in ALS-parkinsonism-dementia complex (ALS-PDC), a neurological disorder found in Guam and its surrounding islands, in which motor neuron disease symptoms can present alone or can co-occur with parkinsonism and dementia. Although in vitro experiments have shown BMAA's neurotoxic properties, studies using adult animals and systemic administration which better model the case of environmentally-induced human neurodegenerative diseases have not supported the involvement of BMAA in these disorders. In order to better test the hypothesized role of BMAA in neurodegeneration, we fed adult mice BMAA at a dose (28 mg/kg body weight, daily for 30 days) that reproduces the natural levels and tested the animals with a battery of behavioural tests, the latter including the evaluation of motor coordination, motor neuron-mediated reflexes, locomotion, muscular strength and memory. We also assessed whether BMAA exposure triggers cell death in the central nervous system (CNS) of mice by examining neuronal numbers and glial response in the spinal cord and the brain. No motor, cognitive or neuropathological outcome resulted from this feeding paradigm. Our findings support neither the causal role of BMAA in neurodegeneration nor the specific involvement of this amino acid in ALS-PDC.

  17. β-N-Methylamino-L-alanine Induces Neurological Deficits and Shortened Life Span in Drosophila

    PubMed Central

    Zhou, Xianchong; Escala, Wilfredo; Papapetropoulos, Spyridon; Zhai, R. Grace

    2010-01-01

    The neurotoxic non-protein amino acid, β-N-methylamino-L-alanine (BMAA), was first associated with the high incidence of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) in Guam. Recently, BMAA has been implicated as a fierce environmental factor that contributes to the etiology of Alzheimer’s and Parkinson’s diseases, in addition to ALS. However, the toxicity of BMAA in vivo has not been clearly demonstrated. Here we report our investigation of the neurotoxicity of BMAA in Drosophila. We found that dietary intake of BMAA reduced life span, locomotor functions, and learning and memory abilities in flies. The severity of the alterations in phenotype is correlated with the concentration of BMAA detected in flies. Interestingly, developmental exposure to BMAA had limited impact on survival rate, but reduced fertility in females, and caused delayed neurological impairment in aged adults. Our studies indicate that BMAA exposure causes chronic neurotoxicity, and that Drosophila serves as a useful model in dissecting the pathogenesis of ALS/PDC. PMID:22069570

  18. The Cyanobacteria Derived Toxin Beta-N-Methylamino-L-Alanine and Amyotrophic Lateral Sclerosis

    PubMed Central

    Banack, Sandra Anne; Caller, Tracie A.; Stommel, Elijah W.

    2010-01-01

    There is mounting evidence to suggest that environmental factors play a major role in the development of neurodegenerative diseases like ALS (Amyotrophic Lateral Sclerosis). The non-protein amino acid beta-N-methylamino-L-alanine (BMAA) was first associated with the high incidence of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) in Guam, and has been implicated as a potential environmental factor in ALS, Alzheimer’s disease, and other neurodegenerative diseases. BMAA has a number of toxic effects on motor neurons including direct agonist action on NMDA and AMPA receptors, induction of oxidative stress, and depletion of glutathione. As a non-protein amino acid, there is also the strong possibility that BMAA could cause intraneuronal protein misfolding, the hallmark of neurodegeneration. While an animal model for BMAA-induced ALS is lacking, there is substantial evidence to support a link between this toxin and ALS. The ramifications of discovering an environmental trigger for ALS are enormous. In this article, we discuss the history, ecology, pharmacology and clinical ramifications of this ubiquitous, cyanobacteria-derived toxin. PMID:22069578

  19. Deuterium NMR study of methyl group dynamics in L-alanine

    NASA Astrophysics Data System (ADS)

    Beshah, Kebede; Olejniczak, Edward T.; Griffin, Robert G.

    1987-05-01

    Deuterium quadrupole echo spectroscopy is used to study the dynamics of the CD3 group in polycrystalline L-alanine-d3. Temperature-dependent quadrupole echo line shapes, their spectral intensities and τ dependence, and the anisotropy of the 2H spin-lattice relaxation were employed to determine the rate and mechanism of the -CD3 group motion. The rigid lattice Pake pattern observed at low temperature (T<-120 °C) transforms to a triplet spectrum characteristic of threefold jumps in the intermediate exchange regime (-120 to -70 °C) and this in turn to a Pake pattern of reduced breadth at higher temperatures (T>-70 °C). The quadrupole echo line shapes and their τ dependence, which are especially sensitive to the rate and mechanism of the motion, can be simulated quantitatively with the threefold jump model. We find Ea=20.0 kJ/mol for this process which is higher than is observed for most methyl groups, probably because of steric crowding in the L-Ala molecule. Finally, we observe a line shape due to the presence of multiple crystallographic forms which suggests that this technique can be extended to studies of the dynamics of more complex systems.

  20. Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities.

    PubMed

    Nishanth Kumar, S; Nath, Vishnu Sukumari; Pratap Chandran, R; Nambisan, Bala

    2014-02-01

    The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, (1)H NMR, (13)C NMR, (1)H-(1)H COSY, (1)H-(13)C HMBC) and Marfey's method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(L-Pro-L-Trp), cyclo(L-Leu-L-Val), cyclo(D-Pro-D-Met), and cyclo(D-Pro-D-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(L-Leu-L-Val). Cyclo(L-Leu-L-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(L-Pro-L-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(L-Pro-L-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete's foot, jock itch, and ringworm. The activity of cyclo(L-Pro-L-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial

  1. Ex vivo response to mucosal bacteria and muramyl dipeptide in inflammatory bowel disease

    PubMed Central

    Loganes, Claudia; Valencic, Erica; Pin, Alessia; Marini, Elisa; Martelossi, Stefano; Naviglio, Samuele; De Leo, Luigina; Not, Tarcisio; Monasta, Lorenzo; Tommasini, Alberto; Marcuzzi, Annalisa

    2016-01-01

    AIM To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). METHODS Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Two tissue samples were taken from inflamed mucosal segments (in patients with active disease) or from non-inflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo. For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS We demonstrated that subjects with CD display a spontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in

  2. Multi-chelation approach towards natural product-like skeletons: one-pot access to a nitrogen-containing tetracyclic framework from AlaAla dipeptide.

    PubMed

    Vellaisamy, Kasipandi; Napoleon, John Victor; Venkatachalam, Ramkumar; Manheri, Muraleedharan Kannoth

    2010-12-28

    Reductive transformation of the dipeptide BocAlaAlaOMe to a complex, internally charge-stabilized, natural product-like skeleton in one synthetic step is discussed. Stepwise replacement of the B-H bonds in borane by B-N or B-O resulted in incorporation of three boron atoms in a tetracyclic framework whereby one is stereogenic!

  3. Quantification of cyclic dipeptides from cultures of Lactobacillus brevis R2Δ by HRGC/MS using stable isotope dilution assay.

    PubMed

    Axel, Claudia; Zannini, Emanuele; Arendt, Elke K; Waters, Deborah M; Czerny, Michael

    2014-04-01

    Lactic acid bacteria (LAB) play an important role as natural preservatives in fermented food and beverage systems, reducing the application of chemical additives. Thus, investigating their antifungal compounds, such as cyclic dipeptides, has gained prominence. Previous research has primarily focussed on isolation of these compounds. However, their precise quantification will provide further information regarding their antifungal performance in a complex system. To address this, deuterated labelled standards of the cyclic dipeptides cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) were synthesized, and stable isotope dilution assays were developed, enabling an accurate quantification of cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) in MRS-broth and wort. Quantitative results showed that, in the Lactobacillus brevis R2Δ fermented MRS-broth, the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro) and cyclo(Phe-Pro) were significantly higher (P < 0.05), than in wort for cyclo (Leu-Pro) when compared with their controls. This indicates that the formation of these three cyclic dipeptides is related to L. brevis R2Δ metabolism. Furthermore, this represents the first report of cyclic dipeptides quantification using stable isotope dilution assays in LAB cultures both in vitro and in a food system.

  4. Enantioseparation of dansyl amino acids and dipeptides by chiral ligand exchange capillary electrophoresis based on Zn(II)-L-hydroxyproline complexes coordinating with γ-cyclodextrins.

    PubMed

    Mu, Xiaoyu; Qi, Li; Qiao, Juan; Yang, Xinzheng; Ma, Huimin

    2014-10-10

    A chiral ligand exchange capillary electrophoresis (CLE-CE) method using Zn(II) as the central ion and L-4-hydroxyproline as the chiral ligand coordinating with γ-cyclodextrin (γ-CD) was developed for the enantioseparation of amino acids (AAs) and dipeptides. The effects of various separation parameters, including the pH of the running buffer, the ratio of Zn(II) to L-4-hydroxyproline, the concentration of complexes and cyclodextrins (CDs) were systematically investigated. After optimization, it has been found that eight pairs of labeled AAs and six pairs of labeled dipeptides could be baseline-separated with a running electrolyte of 100.0mM boric acid, 5.0mM ammonium acetate, 3.0mM Zn(II), 6.0mM L-hydroxyproline and 4.0mM γ-CD at pH 8.2. The quantitation of AAs and dipeptides was conducted and good linearity (r(2)≥0.997) and favorable repeatability (RSD≤3.6%) were obtained. Furthermore, the proposed method was applied in determining the enantiomeric purity of AAs and dipeptides. Meanwhile, the possible enantiorecognition mechanism based on the synergistic effect of chiral metal complexes and γ-CD was explored and discussed briefly.

  5. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

    PubMed

    Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

    2013-02-01

    Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

  6. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

  7. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams.

    PubMed

    von Voigts-Rhetz, P; Anton, M; Vorwerk, H; Zink, K

    2016-02-07

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range [Formula: see text] up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction [Formula: see text] depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of [Formula: see text] on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  8. Theoretical and experimental study of valence photoelectron spectrum of D,L-alanine amino acid.

    PubMed

    Farrokhpour, H; Fathi, F; De Brito, A Naves

    2012-07-05

    In this work, the He-I (21.218 eV) photoelectron spectrum of D,L-alanine in the gas phase is revisited experimentally and theoretically. To support the experiment, the high level ab initio calculations were used to calculate and assign the photoelectron spectra of the four most stable conformers of gaseous alanine, carefully. The symmetry adapted cluster/configuration interaction (SAC-CI) method based on single and double excitation operators (SD-R) and its more accurate version, termed general-R, was used to separately calculate the energies and intensities of the ionization bands of the L- and D-alanine conformers. The intensities of ionization bands were calculated based on the monopole approximation. Also, natural bonding orbital (NBO) calculations were employed for better spectral band assignment. The relative electronic energy, Gibbs free energy, and Boltzmann population ratio of the conformers were calculated at the experimental temperature (403 K) using several theoretical methods. The theoretical photoelectron spectrum of alanine was calculated by summing over the spectra of individual D and L conformers weighted by different population ratios. Finally, the population ratio of the four most stable conformers of alanine was estimated from the experimental photoelectron spectrum using theoretical calculations for the first time.

  9. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams

    NASA Astrophysics Data System (ADS)

    von Voigts-Rhetz, P.; Anton, M.; Vorwerk, H.; Zink, K.

    2016-02-01

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range {{n}e}/{{n}e,\\text{w}}=0.20 up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction {{k}\\text{env}} depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of {{k}\\text{env}} on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  10. Effect of 10 week beta-alanine supplementation on competition and training performance in elite swimmers.

    PubMed

    Chung, Weiliang; Shaw, Greg; Anderson, Megan E; Pyne, David B; Saunders, Philo U; Bishop, David J; Burke, Louise M

    2012-10-09

    Although some laboratory-based studies show an ergogenic effect with beta-alanine supplementation, there is a lack of field-based research in training and competition settings. Elite/Sub-elite swimmers (n = 23 males and 18 females, age = 21.7 ± 2.8 years; mean ± SD) were supplemented with either beta-alanine (4 weeks loading phase of 4.8 g/day and 3.2 g/day thereafter) or placebo for 10 weeks. Competition performance times were log-transformed, then evaluated before (National Championships) and after (international or national selection meet) supplementation. Swimmers also completed three standardized training sets at baseline, 4 and 10 weeks of supplementation. Capillary blood was analyzed for pH, bicarbonate and lactate concentration in both competition and training. There was an unclear effect (0.4%; ± 0.8%, mean, ± 90% confidence limits) of beta-alanine on competition performance compared to placebo with no meaningful changes in blood chemistry. While there was a transient improvement on training performance after 4 weeks with beta-alanine (-1.3%; ± 1.0%), there was an unclear effect at ten weeks (-0.2%; ± 1.5%) and no meaningful changes in blood chemistry. Beta-alanine supplementation appears to have minimal effect on swimming performance in non-laboratory controlled real-world training and competition settings.

  11. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence

    PubMed Central

    Giffin, Michelle M.; Shi, Lanbo; Gennaro, Maria L.; Sohaskey, Charles D.

    2016-01-01

    Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation. PMID:27203084

  12. Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.

    PubMed

    Arai, Toshinobu; Noguchi, Atsushi; Takano, Eriko; Kino, Kuniki

    2013-04-01

    Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.

  13. Using Position-Specific 13C and 14C Labeling and 13C-PLFA Analysis to Assess Microbial Transformations of Free Versus Sorbed Alanine

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Herschbach, J.; Bore, E. K.; Kuzyakov, Y.; Dippold, M. A.

    2015-12-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model substance for the pool of LMWOS. To assess transformations of sorbed alanine, we added position-specific and uniformly 13C and 14C labeled alanine tracer to soil that had previously been sterilized by γ-radiation. The labeled soil was added to non-sterilized soil from the same site and incubated. Soil labeled with the same tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time intervals. The incorporation of 14C into microbial biomass was determined by chloroform fumigation extraction (CFE), and utilization of individual C positions by distinct microbial groups was evaluated by 13C-phospholipid fatty acid analysis (PLFA). A dual peak in the respired CO2 revealed two sorption mechanisms. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we applied the divergence index (DI). The DI reveals the convergent or divergent behavior of C from individual molecule positions during microbial utilization. Alanine C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFA. This indicates that sorption by the COOH group does not protect this group from preferential oxidation. Microbial metabolism was determinative for the preferential oxidation of individual molecule positions. The use of position-specific labeling revealed mechanisms and kinetics of microbial utilization of sorbed and non

  14. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    PubMed Central

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  15. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    PubMed

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

  16. Altered Strand Transfer Activity of a Multi-drug-resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant with a Dipeptide Fingers Domain Insertion

    PubMed Central

    Nguyen, Laura A.; Daddacha, Waaqo; Rigby, Sean; Bambara, Robert A.; Kim, Baek

    2013-01-01

    Prolonged highly active anti-retroviral therapy (HAART) with multiple nucleoside reverse transcriptase inhibitors (NRTI) for the treatment of human immunodeficiency virus type 1 (HIV-1) infected patients can induce the development of an HIV-1 RT harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation (TAM). This mutation renders viral resistance to multiple NRTIs. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during pre- and post-treatment of an infected patient, termed pre-drug RT without the dipeptide insertion and post-drug RT with the Ser-Gly insertion mutation, respectively. First, the post-drug RT displayed elevated strand transfer activity, compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT, but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate for template binding (kon) and lower equilibrium binding constant KD to template, leading to the tighter template binding affinity than the pre-drug RT. The koff rates for pre-drug RT and post-drug RTs were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity in addition to the loss of AZT resistance. These biochemical data suggests that the dipeptide insertion mutation elevates strand transfer activity by increasing the interaction of the RT with RNA donor template, promoting cleavage that generates more invasion site for the acceptor template during DNA synthesis. PMID:22100453

  17. Organogelation and hydrogelation of low-molecular-weight amphiphilic dipeptides: pH responsiveness in phase-selective gelation and dye removal.

    PubMed

    Kar, Tanmoy; Debnath, Sisir; Das, Dibyendu; Shome, Anshupriya; Das, Prasanta Kumar

    2009-08-04

    The search for efficient low-molecular-weight gelators (LMWGs) with possible structure-activity correlation is on the rise. The present work reports a novel set of amphiphilic dipeptide-based carboxylic acids capable of efficiently gelating organic solvents. More interestingly, their sodium salts showed enhanced efficiency in organogelation with the additional ability to gelate water. Electrostatic interactions present in the aggregation of the sodium carboxylates of amphiphilic dipeptides seem to be important because some of the nongelator carboxylic acids turned out to be excellent gelators upon salt formation. The combinations and sequence of the amino acids in the dipeptide moiety were systematically altered to understand the collective importance of the nonpolar aliphatic/aromatic substitution in amino acids in the self-assembling behavior of amphiphiles. Almost a 20-fold enhancement in the gelation ability was observed on reversing the sequence of the amino acid residues, and in some cases, nongelators were transformed to efficient gelators. Spectroscopic and microscopic studies of these thermoreversible organo/hydrogels revealed that balanced participation of the noncovalent interactions including hydrogen bonding and van der Waals interactions are crucial for organo/hydrogelation. These dipeptides selectively gelate organic solvents from their mixtures with water, and the xerogels prepared from these organogels showed time-dependent adsorption of dyes such as crystal violet. The most remarkable feature of these gelators is the pH responsiveness, which was aptly utilized for the pH-dependent phase-selective gelation of either solvent in a biphasic mixture of oil and water. The dissimilar gelation ability of the acid and its salt originating from the pH responsiveness of the amphiphilic dipeptide was employed in the instant removal of large amounts of dyes for wastewater treatment.

  18. Sensitivity of alanine dosimeters with gadolinium exposed to 6 MV photons at clinical doses.

    PubMed

    Marrale, M; Longo, A; Spanò, M; Bartolotta, A; D'Oca, M C; Brai, M

    2011-12-01

    In this study we analyzed the ESR signal of alanine dosimeters with gadolinium exposed to 6 MV linear accelerator photons. We observed that the addition of gadolinium brings about an improvement in the sensitivity to photons because of its high atomic number. The experimental data indicated that the addition of gadolinium increases the sensitivity of the alanine to 6 MV photons. This enhancement was better observed at high gadolinium concentrations for which the tissue equivalence is heavily reduced. However, information about the irradiation setup and of the radiation beam features allows one to correct for this difference. Monte Carlo simulations were carried out to obtain information on the expected effect of the addition of gadolinium on the dose absorbed by the alanine molecules inside the pellets. These results are compared with the experimental values, and the agreement is discussed.

  19. Antimicrobial activity of antihypertensive food-derived peptides and selected alanine analogues.

    PubMed

    McClean, Stephen; Beggs, Louise B; Welch, Robert W

    2014-03-01

    This study evaluated four food-derived peptides with known antihypertensive activities for antimicrobial activity against pathogenic microorganisms, and assessed structure-function relationships using alanine analogues. The peptides (EVSLNSGYY, barley; PGTAVFK, soybean; TTMPLW, α-casein; VHLPP, α-zein) and the six alanine substitution peptides of PGTAVFK were synthesised, characterised and evaluated for antimicrobial activity using the bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus and the yeast, Candida albicans. The peptides TTMPLW and PGTAVFK inhibited growth of all four microorganisms tested, with activities of a similar order of magnitude to ampicillin and ethanol controls. EVSLNSGYY inhibited the growth of the bacteria, but VHLPP showed no antimicrobial activity. The alanine analogue, PGAAVFK showed the highest overall antimicrobial activity and PGTAVFA showed no activity; overall, the activities of the analogues were consistent with their structures. Some peptides with antihypertensive activity also show antimicrobial activity, suggesting that food-derived peptides may exert beneficial effects via a number of mechanisms.

  20. Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

    PubMed

    Masola, B; Devlin, T M

    1995-12-01

    The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.