758 resultados para Methionine cystine
Resumo:
La dysplasie broncho-pulmonaire (DBP), caractrise par un dfaut de lalvolarisation, est une complication pathologique associe un stress oxydant chez le nouveau-n prmatur. La DBP est prsente chez prs de 50 % des nouveau-ns de moins de 29 semaines de gestation. La nutrition parentrale (NP) que ces nouveau-ns reoivent pour cause dimmaturit gastro-intestinale est une source importante de stress oxydant. En effet, leur NP est contamine par des peroxydes, dont lascorbylperoxyde qui est une forme peroxyde du dshydroascorbate. La gnration des peroxydes est catalyse par la lumire ambiante. La photoprotection de la NP, quoique difficile dapplication en clinique, est associe une diminution de lincidence de la DBP chez les enfants prmaturs. Chez lanimal nouveau-n, la photoprotection de la NP est associe un meilleur dveloppement alvolaire. Ainsi, nous mettons lhypothse que lascorbylperoxide infus avec la NP cause la perte dalvoles suite une apoptose exagre induite par loxydation du potentiel redox du glutathion. Cette oxydation du potentiel redox serait occasionne par linhibition de la transformation hpatique de la mthionine en cystine, menant une diminution de la synthse de glutathion au foie et dans les tissus tels que les poumons. La confirmation de cette hypothse suggrera quun ajout de glutathion dans la NP permettra une meilleure dtoxification de lascorbylperoxide par laction de la glutathion peroxydase, et prviendra loxydation du potentiel redox et ainsi, la perte d'alvoles par apoptose. Objectifs : Le but de mon projet de recherche est de comprendre les mcanismes biochimiques liant la NP et le dveloppement de la DBP chez le nouveau-n prmatur et de proposer une alternative nutritionnelle prvenant le dveloppement de cette complication frquemment observe dans cette population. Les objectifs spcifiques sont : 1) dvaluer limpact, au poumon, de linfusion de lascorbylperoxyde sur laxe mtabolique potentiel redox du glutathion - apoptose - le dveloppement alvolaire; 2) dtudier limpact de lascorbylperoxyde et du potentiel redox sur lactivit hpatique de la mthionine adnosyltransfrase (MAT), premire enzyme de la cascade mtabolique transformant la mthionine en cystine; et 3) de tenter de prvenir limpact ngatif de la NP ou de linfusion dascorbylperoxyde sur le poumon en amliorant le statut en glutathion. Mthodes: Par un cathter fix dans la jugulaire, des cochons dInde de trois jours de vie (n = 8 par groupe) ont reu en continu durant 4 jours une NP ou une solution de base (dextrose + NaCl) enrichie des diffrentes molcules lessai. Le premier objectif a t atteint en enrichissant la solution de base en ascorbylperoxyde 0, 20, 60 et 180 M. Ces solutions contenaient ou non 350 M H2O2 pour se rapprocher des conditions cliniques. Le second objectif a t atteint en investiguant les mcanismes dinhibition de la MAT dans des animaux infuss ou non avec des solutions contenant la solution de base, des peroxydes, du glutathion et la NP (dextrose + acides amins + multivitamines + lipides). Le troisime objectif a t atteint en ajoutant ou non une solution dascorbylperoxide ou la NP 10 M de glutathion (GSSG), afin dobtenir une concentration plasmatique normale de glutathion. Aprs 4 jours, les poumons taient prlevs et traits pour la dtermination de GSH et GSSG par lectrophorse capillaire, le potentiel redox tait calcul selon l'quation de Nernst et le niveau de caspase-3 actif (marqueur dapoptose) par Western blot et lindex dalvolarisation quantifi par le nombre dinterceptes entre des structures histologiques et une droite calibre. Les donnes taient compares par ANOVA, les effets taient considrs comme significatifs si le p tait infrieur 0,05. Rsultats: Linfusion de lascorbylperoxyde, indpendamment du H2O2, a induit une hypoalvolarisation, une activation de la caspase-3 et une oxydation du potentiel redox de manire dose-dpendante. Ces effets ont t empchs par lajout de GSSG la NP ou la solution dascorbylperoxyde (180 M). Lascorbylperoxyde et le H2O2 ont inhib lactivit de MAT tandis quelle tait linairement module par la valeur du potentiel redox hpatique. Conclusion : Nos rsultats suggrent que lascorbylperoxyde est lagent actif de la NP conduisant au dveloppement de la DBP. Ainsi la correction des bas niveaux de glutathion induits par les peroxydes de la NP favorise la dtoxification des peroxydes et la correction du potentiel redox pulmonaire ; ce qui a protg les poumons des effets dltres de la NP en outrepassant linhibition de la MAT hpatique. Nos rsultats sont d'une grande importance car ils donnent de l'espoir pour une prvention possible de la DBP.
Resumo:
La dysplasie broncho-pulmonaire (DBP), caractrise par un dfaut de lalvolarisation, est une complication pathologique associe un stress oxydant chez le nouveau-n prmatur. La DBP est prsente chez prs de 50 % des nouveau-ns de moins de 29 semaines de gestation. La nutrition parentrale (NP) que ces nouveau-ns reoivent pour cause dimmaturit gastro-intestinale est une source importante de stress oxydant. En effet, leur NP est contamine par des peroxydes, dont lascorbylperoxyde qui est une forme peroxyde du dshydroascorbate. La gnration des peroxydes est catalyse par la lumire ambiante. La photoprotection de la NP, quoique difficile dapplication en clinique, est associe une diminution de lincidence de la DBP chez les enfants prmaturs. Chez lanimal nouveau-n, la photoprotection de la NP est associe un meilleur dveloppement alvolaire. Ainsi, nous mettons lhypothse que lascorbylperoxide infus avec la NP cause la perte dalvoles suite une apoptose exagre induite par loxydation du potentiel redox du glutathion. Cette oxydation du potentiel redox serait occasionne par linhibition de la transformation hpatique de la mthionine en cystine, menant une diminution de la synthse de glutathion au foie et dans les tissus tels que les poumons. La confirmation de cette hypothse suggrera quun ajout de glutathion dans la NP permettra une meilleure dtoxification de lascorbylperoxide par laction de la glutathion peroxydase, et prviendra loxydation du potentiel redox et ainsi, la perte d'alvoles par apoptose. Objectifs : Le but de mon projet de recherche est de comprendre les mcanismes biochimiques liant la NP et le dveloppement de la DBP chez le nouveau-n prmatur et de proposer une alternative nutritionnelle prvenant le dveloppement de cette complication frquemment observe dans cette population. Les objectifs spcifiques sont : 1) dvaluer limpact, au poumon, de linfusion de lascorbylperoxyde sur laxe mtabolique potentiel redox du glutathion - apoptose - le dveloppement alvolaire; 2) dtudier limpact de lascorbylperoxyde et du potentiel redox sur lactivit hpatique de la mthionine adnosyltransfrase (MAT), premire enzyme de la cascade mtabolique transformant la mthionine en cystine; et 3) de tenter de prvenir limpact ngatif de la NP ou de linfusion dascorbylperoxyde sur le poumon en amliorant le statut en glutathion. Mthodes: Par un cathter fix dans la jugulaire, des cochons dInde de trois jours de vie (n = 8 par groupe) ont reu en continu durant 4 jours une NP ou une solution de base (dextrose + NaCl) enrichie des diffrentes molcules lessai. Le premier objectif a t atteint en enrichissant la solution de base en ascorbylperoxyde 0, 20, 60 et 180 M. Ces solutions contenaient ou non 350 M H2O2 pour se rapprocher des conditions cliniques. Le second objectif a t atteint en investiguant les mcanismes dinhibition de la MAT dans des animaux infuss ou non avec des solutions contenant la solution de base, des peroxydes, du glutathion et la NP (dextrose + acides amins + multivitamines + lipides). Le troisime objectif a t atteint en ajoutant ou non une solution dascorbylperoxide ou la NP 10 M de glutathion (GSSG), afin dobtenir une concentration plasmatique normale de glutathion. Aprs 4 jours, les poumons taient prlevs et traits pour la dtermination de GSH et GSSG par lectrophorse capillaire, le potentiel redox tait calcul selon l'quation de Nernst et le niveau de caspase-3 actif (marqueur dapoptose) par Western blot et lindex dalvolarisation quantifi par le nombre dinterceptes entre des structures histologiques et une droite calibre. Les donnes taient compares par ANOVA, les effets taient considrs comme significatifs si le p tait infrieur 0,05. Rsultats: Linfusion de lascorbylperoxyde, indpendamment du H2O2, a induit une hypoalvolarisation, une activation de la caspase-3 et une oxydation du potentiel redox de manire dose-dpendante. Ces effets ont t empchs par lajout de GSSG la NP ou la solution dascorbylperoxyde (180 M). Lascorbylperoxyde et le H2O2 ont inhib lactivit de MAT tandis quelle tait linairement module par la valeur du potentiel redox hpatique. Conclusion : Nos rsultats suggrent que lascorbylperoxyde est lagent actif de la NP conduisant au dveloppement de la DBP. Ainsi la correction des bas niveaux de glutathion induits par les peroxydes de la NP favorise la dtoxification des peroxydes et la correction du potentiel redox pulmonaire ; ce qui a protg les poumons des effets dltres de la NP en outrepassant linhibition de la MAT hpatique. Nos rsultats sont d'une grande importance car ils donnent de l'espoir pour une prvention possible de la DBP.
Resumo:
On cover: Du Pont methionine for broiler, starter and turkey rations.
Resumo:
The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.
Resumo:
The aim of the present study was to compare the protein-free diet, guanidinated casein (GuC) and enzyme hydrolysed casein (EHC) methods for the quantification of endogenous amino acid (AA) flow in the avian ileum. Growing broiler chickens (5 weeks old) were used. All three assay diets were based on dextrose, and in the GuC and EHC diets GuC or EHC were the sole source of N. Endogenous AA flows determined with the use of protein-free diet were considerably lower (P < 0.05) than those determined by the GuC and EHC methods. The, total endogenous AA flows determined by the GuC and EHC methods were almost 3-fold greater (P < 0.05) than those determined by the protein-free diet. The endogenous AA values obtained from GuC and EHC methods were similar (P >0.05), except for the flow of arginine, which was lower (P < 0.05) in the EHC method. Glutamic acid, aspartic acid, threonine and glycine were the predominant endogenous AA present in digesta from the distal ileum. The contents of methionine, histidine and cystine were lower compared with other AA. The method of determination had no effect on the AA composition of endogenous protein, except for threonine, glutamic acid, lysine, arginine and cystine. The concentrations of threonine and arginine were lower (P < 0.05) and that of lysine was higher (P < 0.05) with the EHC method compared with the other two methods. The concentration of glutamic acid was greater (P < 0.05) and that of cystine was lower (P < 0.05) in the EHC and GuC methods compared with the protein-free diet method. The results showed that the ileal endogenous flows of N and AA are markedly enhanced by the presence of protein and peptides, above those determined following feeding of a protein-free diet. It is concluded that the use of EHC and GuC methods enables the measurement of ileal endogenous losses in chickens under normal physiological conditions.
Resumo:
The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.
Resumo:
The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides; poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.
Resumo:
Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded - sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and &SIM; 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 m.
Resumo:
The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.
Resumo:
The present study was carried out to determine the ileal digestibility of Arg and Lys in acutely heatstressed broilers using diets varying in Arg:Lys ratio, NaCl concentration, and Met Source. Male broilers were maintained at 22degreesC from 21 to 33 d of age and then at 32degreesC from 33 to 38 d of age. From 28 to 38 d of age, birds were fed a diet with an Arg:Lys ratio of 1.05 and 3 g of supplemental NaCl/kg of diet with or without L-arg free base to increase the Arg:Lys to 1.35, and with or without 3 g/kg of additional NaCl. Methionine was supplied as equimolar amounts of DL-Met or 2-hydroxy-4-(methylthio)-butanoic acid in a 2 x 2 x 2 design. At 38 d of age, digesta were collected from the terminal ileum, and amino acid analyses were conducted on feed and digesta samples and compared with acid-insoluble ash (dietary celite) to calculate the apparent ileal digestibilities of Lys and Arg. Increasing the NaCl concentration and the presence of HMB significantly decreased the digestibility of both Arg and Lys, whereas increasing the Arg:Lys ratio increased the digestibility of only Arg but did increase BW gain (P = 0.08). An interaction between dietary NaCl and Arg:Lys ratio as well as the 3-way interaction suggested that dietary NaCl could affect the apparent ileal digestibility of Arg and Lys at certain Arg:Lys ratios and the response may be influenced by the Met source.
Resumo:
Cyclotides are a large family of mini-proteins that have the distinguishing features of a head-to-tail cyclised backbone and a cystine knot formed by six conserved cysteine residues. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. The unique structural features of the cyclotides make them extremely resistant to chemical, thermal and proteolytic degradation. In this article we review recent Studies from our laboratory that dissect the role of the individual structural elements in defining the stability of cyclotides. The resistance of cyclotides to chemical and proteolytic degradation is in large part due to the cystine knot, whereas the thermal stability is I composite of several features including the cystine knot, the cyclic backbone and the hydrogen bonding network. A range of biological activities of cyclotides is critically dependent oil the presence of the cyclic backbone.
Resumo:
The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is unknown and the discovery of novel sequences may assist in achieving this goal. In the present study, we have isolated a new cyclotide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Mobius and bracelet) of currently known cyclotides. We have determined the three-dimensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Further-more, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure-activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The 'uncoupling' of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide.
Resumo:
The cyclotide family of plant proteins is of interest because of their unique topology, which combines a head-to-tail cyclic backbone with an embedded cystine knot, and because their-remarkable chemical and biological properties make them ideal candidates as grafting templates for biologically active peptide epitopes. The present Study describes the first steps towards exploiting the cyclotide framework by synthesizing and structurally characterizing two grafted analogues of the cyclotide kalata B1. The modified peptides have polar or charged residues substituted for residues that form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kalata B1. Both analogues retain the native cyclotide fold, but lack the undesired haemolytic activity of their parent molecule, kalata B1. This finding confirms the tolerance of the cyclotide framework to residue Substitutions and opens up possibilities for the Substitution of biologically active peptide epitopes into the framework.
