RESUMEN
A validated method has been developed for the simultaneous measurement of reduced and oxidized glutathione in de-proteinised cellular extracts. This has been used to compare models of malignant and non-malignant human prostate cell lines. Analysis of LNCaP and DU145 cells showed a glutathione to glutathione disulfide ratio of 8:1 and 32:1 respectively, whilst the control cell line, PZ-HPV7 displayed a ratio of 93:1. Results indicate that the more aggressive phenotype displays adaptation to increased oxidative stress via up regulation of glutathione turnover. It was also noted that in the LNCaP and DU145 cell line, glutathione was only responsible for ca. 60% and 79% respectively, of the total cellular reduced thiol; indicating the presence of other biological thiols.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía Líquida de Alta Presión , Electroquímica , Disulfuro de Glutatión/análisis , Glutatión/análisis , Neoplasias de la Próstata/química , Línea Celular Tumoral , Humanos , Masculino , Oxidación-Reducción , Estrés Oxidativo , Reproducibilidad de los ResultadosRESUMEN
In this review, we examine the state-of-the-art technologies (gas and liquid chromatography, mass spectroscopy and nuclear magnetic resonance, etc.) in the well-established area of metabolomics especially as they relate to protozoan parasites.
Asunto(s)
Eucariontes/metabolismo , Metaboloma , Metabolómica/métodos , Proteínas Protozoarias/metabolismo , Animales , Cromatografía de Gases , Eucariontes/clasificación , Eucariontes/genética , Humanos , Imagen por Resonancia Magnética , Espectrometría de Masas , Infecciones por Protozoos/parasitología , Proteínas Protozoarias/genéticaRESUMEN
Previous studies [1,2] have reported the existence of two conformers of TATP. This study demonstrates the ability of LC-NMR to separate and characterise the individual conformers. The NMR data is consistent with the proposed structures for the two conformers. Re-equilibration can be followed by NMR and the kinetics of the process studied. Over the past decade the use of the explosive TATP in terrorist devices has increased. Therefore, the ability to analyse and characterise this material has assumed greater importance.
RESUMEN
Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.
Asunto(s)
Ácido Hipocloroso/farmacología , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/metabolismo , 5-Hidroxitriptófano/metabolismo , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/metabolismo , Oxidación-Reducción/efectos de los fármacos , Péptidos/química , Proteínas/química , Análisis de Secuencia de Proteína , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.
Asunto(s)
Rhodococcus/metabolismo , Tiazoles/metabolismo , Benzotiazoles , Biodegradación Ambiental , Contaminantes Ambientales/metabolismo , Residuos Industriales , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Rhodococcus/crecimiento & desarrollo , Rhodococcus/aislamiento & purificación , Aguas del Alcantarillado/microbiología , Espectrometría de Masa por Ionización de Electrospray , Tiazoles/químicaRESUMEN
The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.