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1.
Allergy ; 68(7): 899-905, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23751122

RESUMO

BACKGROUND: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4(+) T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4(+) T lymphocytes in healthy donors. METHODS: Co-cultures were established with CD4(+) T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4(+) T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4(+) T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4(+) T lymphocytes was then calculated using the Poisson distribution law. RESULTS: Results showed the presence of benzylpenicillin-specific CD4(+) T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4(+) T cells. Experiments performed on naive (CD45RA(+) ) and on memory (CD45RO(+) ) CD4(+) T lymphocytes showed that these benzylpenicillin-specific CD4(+) T lymphocytes belonged to the naive T-cell subpopulation. CONCLUSION: This study showed for the first time the existence of naive CD4(+) T lymphocytes specific to benzylpenicillin in healthy donors.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/efeitos dos fármacos , Penicilina G/farmacologia , Linfócitos T CD4-Positivos/citologia , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Hipersensibilidade a Drogas/imunologia , Hipersensibilidade a Drogas/fisiopatologia , ELISPOT , Humanos , Memória Imunológica/imunologia , Memória Imunológica/fisiologia , Interferon gama/imunologia , Interferon gama/metabolismo , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Penicilina G/imunologia , Valores de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Anal Chem ; 84(10): 4383-95, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22509742

RESUMO

Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of "unique" ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.


Assuntos
Proteínas/química , Proteoma , Saliva/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Histatinas/química , Humanos , Dados de Sequência Molecular
3.
Anal Chem ; 83(19): 7306-15, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21800924

RESUMO

Overexpression of a protein in a foreign host is often the only route toward an exhaustive characterization, especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable microheterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top-down tandem mass spectrometry (MSMS) requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top-down MSMS approach combined with traveling wave ion mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle microheterogeneities that were hitherto only accessible with Fourier-transform ion cyclotron resonance-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both the wild type and K496C variant of the recombinant X domain (XD; aa's 459-507) of the measles virus phosphoprotein expressed in Escherichia coli . Using top-down MSMS combined with TWIM, we show that XD samples occasionally exhibit a microheterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, a 1-oxyl-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl methanethiosulfonate nitroxide probe that was grafted onto the K496C XD variant was shown to undergo oxidation and/or protonation in the electrospray ionization source, leading to artifactual mass increases.


Assuntos
Fosfoproteínas/química , Vírus do Sarampo/genética , Modelos Moleculares , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem
4.
Acc Chem Res ; 34(5): 399-408, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11352718

RESUMO

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.


Assuntos
Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Catálise , Cetol-Ácido Redutoisomerase , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Frações Subcelulares/enzimologia
5.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 4): 389-97, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10739911

RESUMO

Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn(2+) and NADPH. The 1.6 A resolution electron-density map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP-ribose instead of the whole NADP(+). This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an R(free) of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni(2+) and Zn(2+), for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the -cleavage of nicotinamide.


Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Manganês/metabolismo , Spinacia oleracea/enzimologia , Valeratos/metabolismo , Adenosina Difosfato Ribose/metabolismo , Cristalografia por Raios X , Dimerização , Cetol-Ácido Redutoisomerase , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
Biochemistry ; 38(19): 6025-34, 1999 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-10320328

RESUMO

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an Mr of approximately 114000 which in the presence of Mg2+ ions catalyzes an unusual alkyl migration followed by an NADPH-dependent reduction. Prior binding of NADPH and Mg2+ to the enzyme was shown to be required for substrate or competitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic data for the enzyme-NADPH-Mg2+-IpOHA complex [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] have shown that IpOHA was completely buried inside the active site. These observations raised the question of how the reaction intermediate analogue inhibitor can reach the active site and implied that conformational changes occurred during the binding process. With a view of characterizing these conformational changes, H-D exchange experiments combined with mass spectrometry were performed. Results demonstrated that Mg2+ ions and NADPH binding led to an initial conformational change at the interface of the two domains of each monomer. Binding of the two cofactors to isomeroreductase alters the structure of the active site to promote inhibitor (substrate) binding, in agreement with the ordered mechanism of the enzyme. Structural changes remote from the active site were also found. They were interpreted as long-range structural effects on the two domains and on the two monomers in the time course of the ligand binding process.


Assuntos
Oxirredutases do Álcool/química , Inibidores Enzimáticos/química , Magnésio/química , NADP/química , Sequência de Aminoácidos , Cristalografia por Raios X , Cetol-Ácido Redutoisomerase , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Spinacia oleracea/enzimologia
7.
Eur J Biochem ; 259(1-2): 356-9, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9914514

RESUMO

The macromolecular complexes formed by the enzyme acetohydroxy acid isomeroreductase with NADPH, magnesium ions and the competitive inhibitor N-hydroxy-N-isopropyloxamate (IpOHA) were analysed by electrospray mass spectrometry. Each ligand was added successively to a protein solution, allowing the stoichiometry of the whole macromolecular edifice (115 583 Da) to be unambiguously determined. The combination of an electrospray ion source with the high mass range magnetic instrument used in the present studies proved to be a very powerful tool for characterizing, in a specific manner, the quaternary structures of proteins by single mass measurements.


Assuntos
Oxirredutases do Álcool/química , Dimerização , Inibidores Enzimáticos , Ácidos Hidroxâmicos/química , Cetol-Ácido Redutoisomerase , Magnésio/química , Espectrometria de Massas , NADP/química , Conformação Proteica , Proteínas Recombinantes , Spinacia oleracea/enzimologia
8.
Biochemistry ; 37(14): 4773-81, 1998 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-9537993

RESUMO

Plant acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the branched chain amino acid biosynthetic pathway, has been submitted to high-throughput screening for herbicide discovery. We report here the discovery of a new class of compounds belonging to the thiadiazole family, which exhibit a strong inhibitory effect on this plant enzyme. Kinetic analyses revealed that these compounds act as either reversible or irreversible noncompetitive inhibitors of the plant enzyme. Reversibility or irreversibility of these compounds can be attributed to the nature of the additional groups of the thiadiazole ring favoring or not favoring the formation of a covalent adduct. Mass spectrometric experiments on the complex between an irreversible compound belonging to the thiadiazole family and the plant enzyme identified Cys498 as the binding site of the inhibitor.


Assuntos
Oxirredutases do Álcool/química , Inibidores Enzimáticos/química , Plantas/enzimologia , Tiadiazóis/química , Oxirredutases do Álcool/antagonistas & inibidores , Sequência de Aminoácidos , Inibidores Enzimáticos/farmacologia , Cetol-Ácido Redutoisomerase , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Tiadiazóis/farmacologia
9.
Protein Sci ; 7(1): 185-92, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9514273

RESUMO

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.


Assuntos
Muramidase/química , Animais , Galinhas , Cristalização , Cristalografia por Raios X , Clara de Ovo , Espectrometria de Massas , Modelos Moleculares , Muramidase/análise , Ribonuclease Pancreático/química , Perus
10.
FEBS Lett ; 380(1-2): 44-8, 1996 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-8603744

RESUMO

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild-type = K32E > K12D = K14E > or = K52E > K14E/K32E > W67Y > P35A > I57N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135-1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.


Assuntos
Grupo dos Citocromos c/química , Espectrometria de Massas/métodos , Mutação , Rhodobacter capsulatus/química , Grupo dos Citocromos c/genética , Deutério/química , Hidrogênio/química , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica
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