RESUMEN
This chapter is focussing on the interaction of arsenic, mercury and selenium with plans. Aspects of biotransformations are discussed, before the analytical methodologies are listed and critically appraised in the second part. A holistic view is given, starting from the soil environment and continuing to the plant roots and the translocations into the upper part of the plants. Under different soil conditions, different kinds of elemental species are identified, which have an impact on how the elemental species are taken up by the plant. The uptake mechanisms of these elemental species are explained and compared before the biotransformation reactions of all elemental species in the plant root; their transport into the vacuoles and translocation to the leaves and grains are discussed. Here in particular the interaction with sulphur-rich phytochelatins is described for all three elemental species. Since the sulphur chemistry is so important for the uptake, bioaccumulation and translocation of the metals and metalloids, a subchapter about sulphur chemistry in plants has been added. All aspects of biotransformation dealt with in this chapter is finally rounded up by a thorough description of the analytical methodology given with a focus on the use of HPLC-ICPMS/ESI-MS for both quantitative and molecular analysis.
Asunto(s)
Arsénico/metabolismo , Mercurio/metabolismo , Plantas/metabolismo , Selenio/metabolismo , Azufre/metabolismo , BiotransformaciónRESUMEN
Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1-controlled polyamine export.
Asunto(s)
Antiportadores/metabolismo , Puntos de Control del Ciclo Celular , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Catión Orgánico/metabolismo , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Espermina/metabolismo , Antiportadores/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Peróxido de Hidrógeno/toxicidad , Proteínas de Transporte de Catión Orgánico/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factores de TiempoRESUMEN
Occupational exposure to veterinary antibiotics in hen houses at poultry feeding farms was demonstrated by biomonitoring campaigns in the past. The objective of this study was to investigate pharmacokinetics of three uptake routes: dermal, oral and inhaled. In an open-label cross-over study, six healthy volunteers were exposed to single occupational relevant doses of enrofloxacin. Plasma and urine samples were analysed for enrofloxacin and ciprofloxacin. Physiologically based pharmacokinetic (PBPK) modelling based on bioanalysis data showed underestimation for the elimination rate in comparison to experimental data pointing towards a lack of sufficient ADME information and limitations of available physico-chemical properties of the parent drug. The data obtained in this study indicate that oral uptake with its various sources, e.g. airborne enrofloxacin, direct hand-mouth contact, is the major source for occupational exposure to enrofloxacin in hen houses. Dermal exposure was considered negligible.
Asunto(s)
Antibacterianos , Exposición Profesional , Humanos , Ciprofloxacina , Estudios Cruzados , EnrofloxacinaRESUMEN
The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin (As(III)PC) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2% of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83% of arsenic was bound to peptides, mainly as As(III)PC(3) and (GS)As(III)PC(2). Key analytical factors were identified which destabilise the As(III)PCs. The low pH of the mobile phase (0.1% formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As(III)(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although As(III)PC(3) showed a ten times higher half-life (23 h) in a plant extract, the pre-cleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90% of an As(III)PC(3) complex and smaller free peptides such as PC(2) and PC(3) can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of As(III)PC(3) in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As(v) L(-1) was found when the fresh plant was stored for 21 days at 193 K.
Asunto(s)
Acanthaceae/química , Arsénico/análisis , Fitoquelatinas/química , Extractos Vegetales/química , Manejo de Especímenes/métodos , Arsénico/química , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Semillas/química , Factores de TiempoRESUMEN
The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.
Asunto(s)
Acanthaceae/química , Arsénico/análisis , Espectrometría de Masas/métodos , Fitoquelatinas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Líquida de Alta Presión/métodos , Raíces de Plantas/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis Espectral/métodos , Factores de Tiempo , Rayos XRESUMEN
Amino acids are the building blocks for protein synthesis and the precursors for many biomolecules, such as glutathione and S-adenosylmethionine. Their intracellular concentrations provide valuable information about the overall metabolic state of the cell, as they are closely connected to carbon and nitrogen metabolism and are tightly regulated to meet cellular demands in ever-changing environments. Here, we describe a fast and simple method enabling metabolic profiling for free amino acids for large numbers of yeast strains. Metabolites are extracted with boiling ethanol and, without further conditioning, analyzed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS). Several hundred samples can be prepared in a single day with an analytical runtime of 3.25 min. This method is valuable for functional characterization, identification of metabolic regulators and processes, or monitoring of biotechnological processes.
Asunto(s)
Aminoácidos/análisis , Cromatografía Liquida/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas en Tándem/métodos , Metaboloma , Estándares de ReferenciaRESUMEN
A series of laboratory simulations were conducted in order to determine the airborne protection that might be afforded by different combinations of workplace exposure controls typically encountered when handling volatile solvents (e.g. solvent transfer). These conditions, referred to as risk management measures (RMMs) under the Registration, Evaluation and Authorisation of Chemicals Regulation (REACH), are typically described using standard phrases in safety data sheets [and specifically those of the European Phrase Catalogue (EUPhraC)]. Ethanol was used as a model compound and its emissions were monitored continuously with a portable IR spectrometer at 3000 cm-1. The average emission reduction performance of the investigated RMMs (e.g. containment, extract ventilation, drum pump) exceeded 90%. They present suitable ways to reduce airborne solvent exposure in a workplace and confirmed the initial expectations derived at by the European Solvents Industry Group (ESIG) and the European Centre For Ecotoxicology and toxicology of Chemicals (ECETOC) Targeted Risk Assessment (TRA) model.
Asunto(s)
Contención de Riesgos Biológicos/métodos , Exposición Profesional/prevención & control , Gestión de Riesgos/métodos , Solventes/análisis , Ventilación/métodos , Ecotoxicología , Etanol/análisis , Humanos , Modelos Biológicos , Exposición Profesional/análisis , Medición de RiesgoRESUMEN
Posttranslational modifications (PTM) have been shown to be essential for protein function and signaling. Here we report the identification of a novel modification, protein transfer of histamine, and provide evidence for its function in G protein signaling. Histamine, known as neurotransmitter and mediator of the inflammatory response, was found incorporated into mastocytoma proteins. Histaminylation was dependent on transglutaminase II. Mass spectrometry confirmed histamine modification of the small and heterotrimeric G proteins Cdc42, Gαo1 and Gαq. The modification was specific for glutamine residues in the catalytic core, and triggered their constitutive activation. TGM2-mediated histaminylation is thus a novel PTM that functions in G protein signaling. Protein αmonoaminylations, thus including histaminylation, serotonylation, dopaminylation and norepinephrinylation, hence emerge as a novel class of regulatory PTMs.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de Unión al GTP/metabolismo , Histamina/metabolismo , Procesamiento Proteico-Postraduccional , Transglutaminasas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular Tumoral , Escherichia coli/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Proteínas de Unión al GTP/genética , Expresión Génica , Glutamina/genética , Glutamina/metabolismo , Histamina/genética , Espectrometría de Masas , Mastocitoma/enzimología , Mastocitoma/genética , Ratones , Datos de Secuencia Molecular , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Transglutaminasas/genética , Proteína de Unión al GTP cdc42/genéticaRESUMEN
The glycolytic enzyme pyruvate kinase (PK) is required for cancer development, and has been implicated in the metabolic transition from oxidative to fermentative metabolism, the Warburg effect. However, the global metabolic response that follows changes in PK activity is not yet fully understood. Using shotgun proteomics, we identified 31 yeast proteins that were regulated in a PK-dependent manner. Selective reaction monitoring confirmed that their expression was dependent on PK isoform, level and activity. Most of the PK targets were amino acid metabolizing enzymes or factors of protein translation, indicating that PK plays a global regulatory role in biosynthethic amino acid metabolism. Indeed, we found strongly altered amino acid profiles when PK levels were changed. Low PK levels increased the cellular glutamine and glutamate concentrations, but decreased the levels of seven amino acids including serine and histidine. To test for evolutionary conservation of this PK function, we quantified orthologues of the identified PK targets in thyroid follicular adenoma, a tumor characterized by high PK levels and low respiratory activity. Aminopeptidase AAP-1 and serine hydroxymethyltransferase SHMT1 both showed PKM2- concentration dependence, and were upregulated in the tumor. Thus, PK expression levels and activity were important for maintaining cellular amino acid homeostasis. Mediating between energy production, ROS clearance and amino acid biosynthesis, PK thus plays a central regulatory role in the metabolism of proliferating cells.
Asunto(s)
Adenoma/enzimología , Aminoácidos/metabolismo , Piruvato Quinasa/metabolismo , Neoplasias de la Tiroides/enzimología , Adenoma/genética , Adenoma/patología , Aminopeptidasas/metabolismo , Procesos de Crecimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Glicina Hidroximetiltransferasa/metabolismo , Homeostasis , Humanos , Isoenzimas/metabolismo , Proteoma/metabolismo , Piruvato Quinasa/biosíntesis , Piruvato Quinasa/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Regulación hacia ArribaRESUMEN
Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.
Asunto(s)
Extractos Celulares/química , Cromatografía Liquida/métodos , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation.However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon,and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form.Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue.
Asunto(s)
Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Neoplasias/enzimología , Isoformas de Proteínas/metabolismo , Piruvato Quinasa/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Glucólisis/genética , Humanos , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Espectrometría de Masas , Neoplasias/genética , Fosforilación Oxidativa , Isoformas de Proteínas/genética , Piruvato Quinasa/genética , Glándula Tiroides/metabolismo , Glándula Tiroides/patologíaRESUMEN
In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in yeast, we discovered that central metabolism is self-adapting to synchronize redox metabolism when respiration is activated. Low PYK activity activated yeast respiration. However, levels of reactive oxygen species (ROS) did not increase, and cells gained resistance to oxidants. This adaptation was attributable to accumulation of the PYK substrate phosphoenolpyruvate (PEP). PEP acted as feedback inhibitor of the glycolytic enzyme triosephosphate isomerase (TPI). TPI inhibition stimulated the pentose phosphate pathway, increased antioxidative metabolism, and prevented ROS accumulation. Thus, a metabolic feedback loop, initiated by PYK, mediated by its substrate and acting on TPI, stimulates redox metabolism in respiring cells. Originating from a single catalytic step, this autonomous reconfiguration of central carbon metabolism prevents oxidative stress upon shifts between fermentation and respiration.
Asunto(s)
Respiración de la Célula/fisiología , Retroalimentación Fisiológica , Glucólisis/fisiología , Fosfoenolpiruvato/metabolismo , Piruvato Quinasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Triosa-Fosfato Isomerasa/metabolismo , Proliferación Celular , Cromatografía Liquida , Galactosa/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Oxidación-Reducción , Estrés Oxidativo/genética , Vía de Pentosa Fosfato , Reacción en Cadena de la Polimerasa , Piruvato Quinasa/genética , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem , Triosa-Fosfato Isomerasa/genéticaRESUMEN
AIMS: A shift in primary carbon metabolism is the fastest response to oxidative stress. Induced within seconds, it precedes transcriptional regulation, and produces reducing equivalents in form of NADPH within the pentose phosphate pathway (PPP). RESULTS: Here, we provide evidence for a regulatory signaling function of this metabolic transition in yeast. Several PPP-deficiencies caused abnormal accumulation of intermediate metabolites during the stress response. These PPP-deficient strains had strong growth deficits on media containing oxidants, but we observed that part of their oxidant-phenotypes were not attributable to the production of NADPH equivalents. This pointed to a second, yet unknown role of the PPP in the antioxidant response. Comparing transcriptome profiles obtained by RNA sequencing, we found gene expression profiles that resembled oxidative conditions when PPP activity was increased. Vice versa, deletion of PPP enzymes disturbed and delayed mRNA and protein expression during the antioxidant response. INNOVATION: Thus, the transient activation of the PPP is a metabolic signal required for balancing and timing gene expression upon an oxidative burst. CONCLUSION: Consequently, dynamic rearrangements in central carbon metabolism seem to be of major importance for eukaryotic redox sensing, and represent a novel class of dynamic gene expression regulators.
Asunto(s)
Antioxidantes/metabolismo , Regulación de la Expresión Génica , Vía de Pentosa Fosfato , Transcripción Genética , Transporte de Electrón , Perfilación de la Expresión Génica , Glucólisis , Mutación , NADP/metabolismo , Oxidación-ReducciónRESUMEN
Ribose 5-phosphate isomerase (RPI) deficiency is an enzymopathy of the pentose phosphate pathway. It manifests with progressive leukoencephalopathy and peripheral neuropathy and belongs, with one sole diagnosed case, to the rarest human disorders. The single patient was found compound heterozygous for a RPI frameshift and a missense (RPI(Ala61Val)) allele. Here, we report that two patient-derived cell lines differ in RPI enzyme activity, enzyme concentration, and mRNA expression. Furthermore, we present a transgenic yeast model, which exhibits metabolite- and enzyme-activity changes that correspond to the human syndrome and show that the decrease in RPI activity in patient cells is not fully attributable to the residue exchange. Taken together, our results demonstrate that RPI deficiency is caused by the combination of a RPI null allele with an allele that encodes for a partially active enzyme which has, in addition, cell-type-dependent expression deficits. We speculate that a low probability for comparable traits accounts for the rareness of RPI deficiency.
Asunto(s)
Isomerasas Aldosa-Cetosa/deficiencia , Isomerasas Aldosa-Cetosa/genética , Fibroblastos/metabolismo , Humanos , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Vía de Pentosa Fosfato , ARN Mensajero/metabolismoRESUMEN
The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.