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Structural Requirements (structural + requirement)
Selected AbstractsChemInform Abstract: Probing the Structural Requirement of 7-Azabicyclo[2.2.1]hept-2-ene that Functions as Wip1 Inhibitor.CHEMINFORM, Issue 6 2009Suhkmann Kim Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: Tripeptides of the Type H-D-Pro-Pro-Xaa-NH2 as Catalysts for Asymmetric 1,4-Addition Reactions: Structural Requirements for High Catalytic Efficiency.CHEMINFORM, Issue 6 2010Markus Wiesner Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Sequential Kinetic Resolution of C2 -Symmetric Compounds as a Key Step in Two-Directional Synthesis: Structural Requirements for Efficient Resolution of Difuryl Diols.CHEMINFORM, Issue 14 2003Michael Harding Abstract For Abstract see ChemInform Abstract in Full Text. [source] Tripeptides of the Type H- D -Pro-Pro-Xaa-NH2 as Catalysts for Asymmetric 1,4-Addition Reactions: Structural Requirements for High Catalytic EfficiencyCHEMISTRY - A EUROPEAN JOURNAL, Issue 39 2009Markus Wiesner Dr. Abstract Analysis of the structural and functional requirements within the asymmetric peptidic catalyst H- D -Pro-Pro-Asp-NH2 led to the development of the closely related peptide H- D -Pro-Pro-Glu-NH2 as an even more efficient catalyst for asymmetric conjugate addition reactions of aldehydes to nitroolefins. In the presence of as little as 1,mol,% of H- D -Pro-Pro-Glu-NH2, a broad range of aldehydes and nitroolefins react readily with each other. The resulting ,-nitroaldehydes were obtained in excellent yields and stereoselectivities at room temperature. Within the structure of the peptidic catalysts, the D -Pro-Pro motif is the major contributor to the high stereoselectivities. The C-terminal amide and the spacer to the carboxylic acid in the side-chain of the C-terminal amino acid are responsible for the fine-tuning of the stereoselectivity. The peptidic catalysts not only allow for highly effective asymmetric catalysis under mild conditions, but also function in the absence of additives. Die sorgfältige Analyse der strukturellen und funktionalen Erfordernisse des peptidischen Katalysators H- D -Pro-Pro-Asp-NH2 führte zur Entwicklung des verwandten Peptids H- D -Pro-Pro-Glu-NH2, das einen noch effizienteren Katalysator für asymmetrische konjugierte Additionsreaktionen von Aldehyden an Nitroolefine darstellt. In Gegenwart von nur 1,mol,% von H- D -Pro-Pro-Glu-NH2 reagiert eine große Auswahl verschiedenster Aldehyde und Nitroolefine unter milden Bedingungen bereitwillig miteinander. Die entstehenden ,-Nitroaldehyde bilden sich in exzellenten Ausbeuten und Stereoselektivitäten bei Raumtemperatur. Innerhalb der Struktur des peptidischen Katalysators trägt das D -Pro-Pro Motiv am meisten zu den hohen Stereoselektivitäten bei. Das C-terminale Amid und der Linker vom Peptidrückgrat zur Carbonsäure in der Seitenkette der C-terminalen Aminosäure sind für die Feineinstellung der Stereoselektivitäten verantwortlich. Die peptidischen Katalysatoren sind nicht nur höchst effiziente asymmetrische Katalysatoren sondern benötigen im Gegensatz zu vielen anderen chiralen Katalysatoren auch keine Additive für ihre katalytische Effizienz. [source] Gene Delivery by Aminofullerenes: Structural Requirements for Efficient TransfectionCHEMISTRY - AN ASIAN JOURNAL, Issue 1-2 2006Hiroyuki Isobe Dr. Abstract A series of aminofullerenes that share a common structural motif have been synthesized and subjected to a systematic investigation of structure activity relationship regarding their ability for transient transfection and cytotoxicity. DNA-binding tests indicated that any water-soluble fullerene-bearing amino group would bind to double-stranded DNA. For these molecules to be effective transfection reagents, however, they require additional structural features. First, the molecule must be capable of producing submicrometer-sized fullerene/DNA aggregates that can be internalized into mammalian cells through endocytosis. Second, the molecule must be capable of releasing DNA as the aggregates are transferred into the cytoplasm. This can be achieved in at least two ways: by loss of the DNA-binding amino groups from the fullerene core, and by transformation of the amino groups to neutral groups such as amides. The screening experiments led us to identify the best reagent, a tetrapiperidinofullerene, that can be synthesized in two steps from fullerene, piperazine, and molecular oxygen, and that is more efficient at transfection than a commonly used lipid-based transfection reagent. [source] Structural requirements for initiation of cross-reactivity and CNS autoimmunity with a PLP139,151 mimic peptide derived from murine hepatitis virusEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2006Ludovic Croxford Abstract MS is an autoimmune CNS demyelinating disease in which infection appears to be an important pathogenic factor. Molecular mimicry, the cross-activation of autoreactive T cells by mimic peptides from infectious agents, is a possible explanation for infection-induced autoimmunity. Infection of mice with a non-pathogenic strain of Theiler's murine encephalomyelitis virus (TMEV) engineered to express an epitope from Haemophilus influenzae (HI) sharing 6/13 amino acids with the dominant proteolipid protein (PLP) epitope, PLP139,151, can induce CNS autoimmune disease. Here we demonstrate that another PLP139,151 mimic sequence derived from murine hepatitis virus (MHV) which shares only 3/13 amino acids with PLP139,151 can also induce CNS autoimmune disease, but only when delivered by genetically engineered TMEV, not by immunization with the MHV peptide. Further, we demonstrate the importance of proline at the secondary MHC class,II contact residue for effective cross-reactivity, as addition of this amino acid to the native MHV sequence increases its ability to cross-activate PLP139,151 -specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. This study describes a structural requirement for potential PLP139,151 mimic peptides, and provides further evidence for infection-induced molecular mimicry in the pathogenesis of autoimmune disease. [source] Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)FEBS JOURNAL, Issue 7 2002Anne T. Nies The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin,Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP,MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP,MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells. [source] Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor bindingJOURNAL OF PEPTIDE SCIENCE, Issue 10 2002Michiaki Kawano Abstract Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure,activity studies were carried out. The result of Ala-scanning indicated that the N -terminal tripeptide RYY(=Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N - or C -terminus revealed the special importance of the N -terminal Arg residue. The removal of protecting groups indicated that the N -terminal acetyl group is essential, but the C -terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8,13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the µ opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1,7) or -(14,17) binds. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Structural requirements for initiation of cross-reactivity and CNS autoimmunity with a PLP139,151 mimic peptide derived from murine hepatitis virusEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2006Ludovic Croxford Abstract MS is an autoimmune CNS demyelinating disease in which infection appears to be an important pathogenic factor. Molecular mimicry, the cross-activation of autoreactive T cells by mimic peptides from infectious agents, is a possible explanation for infection-induced autoimmunity. Infection of mice with a non-pathogenic strain of Theiler's murine encephalomyelitis virus (TMEV) engineered to express an epitope from Haemophilus influenzae (HI) sharing 6/13 amino acids with the dominant proteolipid protein (PLP) epitope, PLP139,151, can induce CNS autoimmune disease. Here we demonstrate that another PLP139,151 mimic sequence derived from murine hepatitis virus (MHV) which shares only 3/13 amino acids with PLP139,151 can also induce CNS autoimmune disease, but only when delivered by genetically engineered TMEV, not by immunization with the MHV peptide. Further, we demonstrate the importance of proline at the secondary MHC class,II contact residue for effective cross-reactivity, as addition of this amino acid to the native MHV sequence increases its ability to cross-activate PLP139,151 -specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. This study describes a structural requirement for potential PLP139,151 mimic peptides, and provides further evidence for infection-induced molecular mimicry in the pathogenesis of autoimmune disease. [source] Binding of rat brain hexokinase to recombinant yeast mitochondriaFEBS JOURNAL, Issue 10 2000Identification of necessary physico-chemical determinants The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane. [source] Extrapolation of the W7-X Magnet System to Reactor SizeCONTRIBUTIONS TO PLASMA PHYSICS, Issue 8 2010F. Schauer Abstract The fusion experiment Wendelstein 7-X (W7-X), presently under construction at the Greifswald branch institute of IPP, shall demonstrate the reactor potential of a HELIAS stellarator. HELIAS reactors with three, four and five periods have been studied at IPP since many years. With a plasma axis induction of 5 T, corresponding to about 10 T maximal induction at the coil, it was shown that such reactors are feasible. Now the possibility is being investigated to increase the conductor induction up to the 12 T , range, corresponding to > 5.5 T at the plasma axis. This improves the stellarator confinement properties but does not change the basic physics with respect to the previously analyzed machines. In particular the 5periodic HELIAS type, HSR5, is considered which evolves from W7-X by linear scaling of the main dimensions by a factor of four. Recent progress in superconductor technology and the extensive development work performed for ITER are taken into account. The latter is particularly relevant since by coincidence the circumferences of the HSR5 and the ITER toroidal field coils are practically the same. For the presented 12 T reactor version, the HSR50a, also the conductor and structural requirements are comparable to the corresponding ITER specifications. Therefore, advantage can be taken of these similarities for the stellarator reactor magnet design. The input was provided by the new code "MODUCO" which was developed for interactive coil layout. It is based on Bézier curve approximations and includes the computation of magnetic surfaces and forces (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] TRITURUS NEWTS DEFY THE RUNNING-SWIMMING DILEMMAEVOLUTION, Issue 10 2006Lumíl Gl Abstract Conflicts between structural requirements for carrying out different ecologically relevant functions may result in a compromise phenotype that maximizes neither function. Identifying and evaluating functional trade-offs may therefore aid in understanding the evolution of organismal performance. We examined the possibility of an evolutionary trade-off between aquatic and terrestrial locomotion in females of European species of the newt genus Triturus. Biomechanical models suggest a conflict between the requirements for aquatic and terrestrial locomotion. For instance, having an elongate, slender body, a large tail, and reduced limbs should benefit undulatory swimming, but at the cost of reduced running capacity. To test the prediction of an evolutionary trade-off between swimming and running capacity, we investigated relationships between size-corrected morphology and maximum locomotor performance in females of ten species of newts. Phylogenetic comparative analyses revealed that an evolutionary trend of body elongation (increasing axilla-groin distance) is associated with a reduction in head width and forelimb length. Body elongation resulted in reduced maximum running speed, but, surprisingly, also led to a reduction in swimming speed. The evolution of longer tails was associated with an increase in maximal swimming speed. We found no evidence for an evolutionary trade-off between aquatic and terrestrial locomotor performance, probably because of the unexpected negative effect of body elongation on swimming speed. We conclude that the idea of a design conflict between aquatic and terrestrial locomotion, mediated through antagonistic effects of body elongation, does not apply to our model system. [source] Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chainFEBS JOURNAL, Issue 9 2010Andrew M. James The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial respiration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ (,CoQ) are viable, but cannot grow on nonfermentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a ,CoQ strain on the nonfermentable substrate glycerol [James AM et al. (2005) J Biol Chem280, 21295,21312]. In the present study we investigated the structural requirements of exogenously supplied CoQ2 for growth on glycerol and found that the first double bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ2 analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ6) is extremely hydrophobic and transported to mitochondria via the endocytic pathway when supplied exogenously. We found that CoQ2 does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of ,CoQ yeast in the presence of exogenous CoQ2. Using a ,CoQ double-knockout library we identified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a signal for growth during the shift to respiration. [source] Crystal structure of the parasite inhibitor chagasin in complex with papain allows identification of structural requirements for broad reactivity and specificity determinants for target proteasesFEBS JOURNAL, Issue 3 2009Izabela Redzynia A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and papain, a classic family C1 cysteine protease, has been crystallized. Kinetic studies revealed that inactivation of papain by chagasin is very fast (kon = 1.5 × 106 m,1·s,1), and results in the formation of a very tight, reversible complex (Ki = 36 pm), with similar or better rate and equilibrium constants than those for cathepsins L and B. The high-resolution crystal structure shows an inhibitory wedge comprising three loops, which forms a number of contacts responsible for the high-affinity binding. Comparison with the structure of papain in complex with human cystatin B reveals that, despite entirely different folding, the two inhibitors utilize very similar atomic interactions, leading to essentially identical affinities for the enzyme. Comparisons of the chagasin,papain complex with high-resolution structures of chagasin in complexes with cathepsin L, cathepsin B and falcipain allowed the creation of a consensus map of the structural features that are important for efficient inhibition of papain-like enzymes. The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors. As papain exhibits high structural similarity to the catalytic domain of the T. cruzi enzyme cruzipain, the present chagasin,papain complex provides a reliable model of chagasin,cruzipain interactions. Such information, coupled with our identification of specificity-conferring interactions, should be important for the development of drugs for treatment of the devastating Chagas disease caused by this parasite. [source] Tissue Repair: Wet-Spun Biodegradable Fibers on Conducting Platforms: Novel Architectures for Muscle Regeneration (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Mater. Bio-synthetic platforms, consisting of a conducting polymer substrate overlaid with aligned biodegradable fibers promote the linear growth (ex vivo) of partially differentiated muscle fibers, consistent with the structural requirements of skeletal muscle in vivo, as described by J. M. Razal et al. on page 3381. The conducting surface facilitates development of electrical stimulation paradigms for optimizing muscle growth and development ex vivo that may potentially be applied to repair diseased or damaged muscle. [source] Wet-Spun Biodegradable Fibers on Conducting Platforms: Novel Architectures for Muscle RegenerationADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Joselito M. Razal Abstract Novel biosynthetic platforms supporting ex vivo growth of partially differentiated muscle cells in an aligned linear orientation that is consistent with the structural requirements of muscle tissue are described. These platforms consist of biodegradable polymer fibers spatially aligned on a conducting polymer substrate. Long multinucleated myotubes are formed from differentiation of adherent myoblasts, which align longitudinally to the fiber axis to form linear cell-seeded biosynthetic fiber constructs. The biodegradable polymer fibers bearing undifferentiated myoblasts can be detached from the substrate following culture. The ability to remove the muscle cell-seeded polymer fibers when required provides the means to use the biodegradable fibers as linear muscle-seeded scaffold components suitable for in vivo implantation into muscle. These fibers are shown to promote differentiation of muscle cells in a highly organized linear unbranched format in vitro and thereby potentially facilitate more stable integration into recipient tissue, providing structural support and mechanical protection for the donor cells. In addition, the conducting substrate on which the fibers are placed provides the potential to develop electrical stimulation paradigms for optimizing the ex vivo growth and synchronization of muscle cells on the biodegradable fibers prior to implantation into diseased or damaged muscle tissue. [source] Identification of a novel REV1-interacting motif necessary for DNA polymerase , functionGENES TO CELLS, Issue 2 2009Eiji Ohashi When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Pol,, Pol,, Pol, and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Pol,, Pol,, and Pol, and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Pol,. We have identified novel REV1-interacting regions (RIRs) present in Pol,, Pol, and Pol,. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Pol, mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk -null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Pol, function in vivo. [source] Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides,JOURNAL OF MOLECULAR RECOGNITION, Issue 1 2005Johannes Oehlke Abstract In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure,activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg3,Orn7 -SP and its D -amino acid analog, all- D -Arg3,Orn7 -SP, as well as by electron microscopic autoradiography using 3H-labelled SP and 125I-labelled all- D -SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature. Copyright © 2004 John Wiley & Sons, Ltd. [source] Structure,Activity Relationships Among N -Arachidonylethanolamine (Anandamide) Head Group Analogues for the Anandamide TransporterJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Abbas Jarrahian Abstract: Two putative endocannabinoids, N -arachidonylethanolamine (AEA) and 2-arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein-mediated facilitated diffusion. We have synthesized and studied 22 N-linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB1 cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4-hydroxybenzyl results in a high-affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3-pyridinyl also results in a high-affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB1 receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements. [source] Analysis of the interactions between the peptides from secreted human CKLF1 and heparin using capillary zone electrophoresisJOURNAL OF PEPTIDE SCIENCE, Issue 8 2008Yi Liu Abstract The Chemokine-like factor 1 (CKLF1) is a novel human cytokine and exhibits chemotactic activities on leukocytes. Two peptides named CKLF1-C27 and CKLF1-C19, were obtained from secreted CKLF1. In this study, a selective high-performance analytical method based on capillary zone electrophoresis (CZE) to investigate interactions between heparin and CKLF1-C27/CKLF1-C19 was developed. Samples containing CKLF1-C27/CKLF1-C19 and heparin at various ratios were incubated at room temperature and then separated by CZE with Tris-acetate buffer at pH 7.2. Both qualitative and quantitative characterizations of the binding were determined. The binding constants of the interactions between CKLF1-C27/CKLF1-C19 and heparin were calculated as (3.38 ± 0.49) × 105M,1 and (1.10 ± 0.02) × 105M,1 by Scatchard analysis. To study structural requirements, CKLF1-C19pm and CKLF1-C19km have been synthesized, and their interactions with heparin have been studied by CZE. We found that the Pro or Lys to Ala substitution within the residues of CKLF1-C19 (CKLF1-C19pm or CKLF1-C19km) strongly decreased or abolished its interaction with heparin, suggesting that the residues of Pro affect the affinity of CKLF1-C19 for heparin, and the residues of Lys of CKLF1-C19 play the important role for the interaction of CKLF1-C19 and heparin, respectively. The methodology presented should be generally applicable to study peptides and heparin interactions quantitatively and qualitatively. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor bindingJOURNAL OF PEPTIDE SCIENCE, Issue 10 2002Michiaki Kawano Abstract Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure,activity studies were carried out. The result of Ala-scanning indicated that the N -terminal tripeptide RYY(=Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N - or C -terminus revealed the special importance of the N -terminal Arg residue. The removal of protecting groups indicated that the N -terminal acetyl group is essential, but the C -terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8,13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the µ opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1,7) or -(14,17) binds. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Probing structural requirements of fMLP receptor: On the size of the hydrophobic pocket corresponding to residue 2 of the tripeptideJOURNAL OF PEPTIDE SCIENCE, Issue 2 2002Susanna Spisani Abstract The conformationally constrained f- L -Met-Acnc- L -Phe-OMe (n = 4,9,12) tripeptides, analogues of the chemoattractant f- L -Met- L -Leu- L -Phe-OH, were synthesized in solution by classical methods and fully characterized. These compounds and the published f- L -Met-Xxx- L -Phe-OMe (Xxx = Aib and Acnc where n = 3, 5,8) analogues were compared to determine the combined effect of backbone preferred conformation and side-chain bulkiness at position 2 on the relation of 3D-structure to biological activity. A conformational study of all the analogues was performed in solution by FT-IR absorption and 1H-NMR techniques. In parallel, each peptide was tested for its ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The biological and conformational data are discussed in relation to the proposed model of the chemotactic receptor on neutrophils, in particular of the hydrophobic pocket accommodating residue 2 of the tripeptide. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Specific GABAA agonists and partial agonistsTHE CHEMICAL RECORD, Issue 6 2002Povl Krogsgaard-Larsen Abstract The GABAA receptor system is implicated in a number of neurological and psychiatric diseases, making GABAA receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the hetero-pentameric GABAA receptor complex, reflecting the very strict structural requirements for GABAA receptor recognition and activation. Within the series of compounds showing agonist activity at the GABAA receptor site that have been developed, most of the ligands are structurally derived from the GABAA agonists muscimol, THIP, or isoguvacine, which we developed in the initial stages of the project. Using recombinant GABAA receptors, functional selectivity was demonstrated for a number of compounds, including THIP, showing highly subunit-dependent potency and maximal response. In light of the interest in partial GABAA receptor agonists as potential therapeutics, structure,activity studies of a number of analogs of 4-PIOL, a low-efficacy partial GABAA agonist derived from THIP, have been performed. In this connection, a series of GABAA ligands has been developed that exhibit pharmacological profiles from moderately potent low-efficacy partial GABAA agonist activity to potent and selective antagonist effects. Very little information is available on direct-acting GABAA receptor agonists in clinical studies. However, the results of clinical studies on the effect of the partial GABAA agonist THIP on human sleep patterns show that the functional consequences of a direct-acting agonist are different from those seen after the administration of GABAA receptor modulators, such as benzodiazepines and barbiturates. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc., Chem Rec 2: 419,430; 2002: Published online in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/tcr.10040 [source] Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene productTHE PLANT JOURNAL, Issue 5 2000Wim Reidt Summary The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis -motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis -element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis -motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development. [source] Hantzsch 1,4-dihydropyridine esters and analogs: candidates for generating reproducible one-dimensional packing motifsACTA CRYSTALLOGRAPHICA SECTION B, Issue 3 2009R. S. Rathore Examination of the symmetric Hantzsch 1,4-dihydropyridine ester derivatives of the prototypical nifedipine molecule indicates the tendency of this class of molecule to form a common packing motif. Crystal structure analysis of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic diesters and analogs reveals that they form extended chains, characterized as the C(6) packing motif, via intermolecular (amine) N,H...O=C (C3,C5 carbonyl) hydrogen bonds. In addition, all the prepared derivatives also satisfy the basic structural requirements for their high binding efficiency to the receptor. The reproducible C(6) packing motif observed among these compounds has a use in the design of solid-state materials. [source] Purification, crystallization and X-ray diffraction analysis of the extracellular part of the human Fc receptor for IgA, Fc,RI (CD89)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Katja Wenig Fc,RI is the predominant receptor for IgA in the serum. Nevertheless, the interaction between the molecules that finally leads to an immune response is poorly understood. To investigate the structural requirements for IgA binding, the extracellular region of Fc,RI was cloned and overexpressed in Escherichia coli. The resulting inclusion-body protein was refolded and purified. Despite its deglycosylated state, this recombinant Fc,RI retained its ability to bind human IgA. The protein crystallized spontaneously as microcrystalline needles. Recrystallization yielded crystals belonging to a primitive monoclinic space group. A complete 2.8,Å resolution X-ray diffraction data set was collected using synchrotron radiation. [source] Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site ABIOPOLYMERS, Issue 2 2007Peter C. Anderson Abstract We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC50 = 0.28 ,M), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 95,111, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2007Nazzareno Dimasi IREM-1 is an inhibitory receptor involved in the functional regulation of myeloid cells. The expression, in vitro folding, purification, crystallization and X-ray data collection of the Ig-V like domain of IREM-1 are reported. X-ray data were collected from a microcrystal (300 × 10 × 10,µm) at 100,K and a diffraction pattern was obtained to 2.6,Å resolution on microfocus beamline ID23-2 at the ESRF. The crystal belongs to space group P3121, with unit-cell parameters a = b = 54.23, c = 72.02,Å, , = , = 90, , = 120°. Assuming the presence of one molecule per asymmetric unit, VM (the Matthews coefficient) was calculated to be 1.96,Å3,Da,1 and the solvent content was estimated to be 37.27%. Determination of the IREM-1 structure will provide insights into its structural requirements for ligand discrimination and binding. [source] Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this studyBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2004Lidia Ruiz-Llorente The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7±0.2 ,M and Vmax=3.3±0.3 pmol min,1 (106 cells),1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1,8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)- N -(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3±0.5 ,M, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator. British Journal of Pharmacology (2004) 141, 457,467. doi:10.1038/sj.bjp.0705628 [source] Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitroBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2003Rachel A Brown Neutrophil-derived elastase is an enzyme implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Heparin inhibits the enzymatic activity of elastase and here we provide evidence for the first time that heparin can inhibit the release of elastase from human neutrophils. Unfractionated and low molecular weight heparins (UH and LMWH, 0.01,1000 U ml,1) and corresponding concentrations (0.06,6000 ,g ml,1) of nonanticoagulant O-desulphated heparin (ODH), dextran sulphate (DS) and nonsulphated poly- L -glutamic acid (PGA) were compared for their effects on both elastase release from and aggregation of neutrophils. UH, ODH and LMWH inhibited (P<0.05) the homotypic aggregation of neutrophils, in response to both N -formyl-methionyl-leucyl-phenylalanine (fMLP, 10,6M) and platelet-activating factor (PAF, 10,6M), as well as elastase release in response to these stimuli, in the absence and presence of the priming agent tumour necrosis factor-alpha (TNF- ,, 100 U ml,1). DS inhibited elastase release under all the conditions of cellular activation tested (P<0.05) but had no effect on aggregation. PGA lacked efficacy in either assay, suggesting general sulphation to be important in both effects of heparin on neutrophil function and specific patterns of sulphation to be required for inhibition of aggregation. Further investigation of the structural requirements for inhibition of elastase release confirmed the nonsulphated GAG hyaluronic acid and neutral dextran, respectively, to be without effect, whereas the IP3 receptor antagonist 2-aminoethoxydiphenylborate (2-APB) mimicked the effects of heparin, itself an established IP3 receptor antagonist, suggesting this to be a possible mechanism of action. British Journal of Pharmacology (2003) 139, 845,853. doi:10.1038/sj.bjp.0705291 [source] | |||||||||||||||||||||||||||||||