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1.
Chemosphere ; 277: 130254, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34384174

RESUMO

Liver S9 fractions from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were incubated with seven pesticides (fenamidone, fenoxaprop-p-ethyl, penflufen, spirotetramat, tebuconazole, tembotrione and trifloxystrobin) and the metabolic pathways of the applied chemicals were determined by HPLC-high-resolution mass spectrometry. Five of the seven pesticides (fenamidone, penflufen, spirotetramat, trifloxystrobin and fenoxaprop-p-ethyl) revealed a higher metabolic capacity of rainbow trout liver fractions compared to carp liver fractions. The other two pesticides (tebuconazole and tembotrione) showed a similar and marginal biotransformation for liver S9 fractions of both species. Furthermore, four compounds (penflufen, spirotetramat, tembotrione and tebuconazole) were incubated with cryo-preserved hepatocytes of rainbow trout showing additional conjugated metabolites compared to liver S9 fractions. The incubations were performed with concentrations of 1 and 10 µM for experiments with liver S9 fractions and 5 µM with hepatocytes for up to 120 (liver S9 fractions) or 240 min (hepatocytes). A set of positive controls was used to confirm the metabolic capability of the in vitro systems. The comparison of the in vitro results from hepatocyte assays of penflufen and tebuconazole with the data from corresponding in vivo studies performed according to OECD (Organisation for Economic Co-operation and Development) guideline 305 exhibited a similar metabolic behavior for these pesticides and emphasizes the reliability of the in vitro assays. Besides investigation of the metabolism of plant protection products for research purposes, inter-species comparison by in vitro assays and the use of PBTK modelling approaches will allow improved environmental and dietary risk assessments.


Assuntos
Carpas , Oncorhynchus mykiss , Praguicidas , Animais , Biotransformação , Fígado/metabolismo , Praguicidas/metabolismo , Praguicidas/toxicidade , Reprodutibilidade dos Testes
2.
Ecotoxicol Environ Saf ; 217: 112247, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33901780

RESUMO

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.


Assuntos
4-Butirolactona/análogos & derivados , Abelhas/fisiologia , Fungicidas Industriais/toxicidade , Inseticidas/toxicidade , Piridinas/toxicidade , 4-Butirolactona/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Imidazóis , Inseticidas/metabolismo , Neonicotinoides , Toxicogenética , Triazóis
3.
Insect Biochem Mol Biol ; 111: 103171, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31136794

RESUMO

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides.


Assuntos
Abelhas/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Neonicotinoides/metabolismo , Tiazinas/metabolismo , Animais , Animais Geneticamente Modificados , Abelhas/genética , Abelhas/metabolismo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Resistência a Inseticidas/genética , Mariposas
4.
PLoS Genet ; 15(2): e1007903, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716069

RESUMO

The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.


Assuntos
Abelhas/efeitos dos fármacos , Abelhas/genética , Neonicotinoides/farmacologia , Animais , Evolução Biológica , Sistema Enzimático do Citocromo P-450/genética , Europa (Continente) , Genômica/métodos , Inseticidas/farmacologia , Polinização/efeitos dos fármacos , Polinização/genética , Tiazinas/farmacologia
5.
Respir Res ; 19(1): 40, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29514648

RESUMO

BACKGROUND: Smoking represents a significant risk factor for many chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). METHODS: To identify dysregulation of specific proteins and pathways in bronchoalveolar lavage (BAL) cells associated with smoking, isobaric tags for relative and absolute quantitation (iTRAQ)-based shotgun proteomics analyses were performed on BAL cells from healthy never-smokers and smokers with normal lung function from the Karolinska COSMIC cohort. Multivariate statistical modeling, multivariate correlations with clinical data, and pathway enrichment analysis were performed. RESULTS: Smoking exerted a significant impact on the BAL cell proteome, with more than 500 proteins representing 15 molecular pathways altered due to smoking. The majority of these alterations occurred in a gender-independent manner. The phagosomal- and leukocyte trans endothelial migration (LTM) pathways significantly correlated with FEV1/FVC as well as the percentage of CD8+ T-cells and CD8+CD69+ T-cells in smokers. The correlations to clinical parameters in healthy never-smokers were minor. CONCLUSION: The significant correlations of proteins in the phagosome- and LTM pathways with activated cytotoxic T-cells (CD69+) and the level of airway obstruction (FEV1/FVC) in smokers, both hallmarks of COPD, suggests that these two pathways may play a role in the molecular events preceding the development of COPD in susceptible smokers. Both pathways were found to be further dysregulated in COPD patients from the same cohort, thereby providing further support to this hypothesis. Given that not all smokers develop COPD in spite of decades of smoking, it is also plausible that some of the molecular pathways associated with response to smoking exert protective mechanisms to smoking-related pathologies in resilient individuals. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02627872 ; Retrospectively registered on December 9, 2015.


Assuntos
Líquido da Lavagem Broncoalveolar , Proteoma/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumantes , Fumar/genética , Espirometria/tendências , Idoso , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/fisiopatologia , Fatores de Tempo
6.
Respir Res ; 19(1): 39, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29514663

RESUMO

BACKGROUND: Smoking is the main risk factor for chronic obstructive pulmonary disease (COPD). Women with COPD who smoke experienced a higher risk of hospitalization and worse decline of lung function. Yet the mechanisms of these gender-related differences in clinical presentations in COPD remain unknown. The aim of our study is to identify proteins and molecular pathways associated with COPD pathogenesis, with emphasis on elucidating molecular gender difference. METHOD: We employed shotgun isobaric tags for relative and absolute quantitation (iTRAQ) proteome analyses of bronchoalveolar lavage (BAL) cells from smokers with normal lung function (n = 25) and early stage COPD patients (n = 18). Multivariate modeling, pathway enrichment analysis, and correlation with clinical characteristics were performed to identify specific proteins and pathways of interest. RESULTS: More pronounced alterations both at the protein- and pathway- levels were observed in female COPD patients, involving dysregulation of the FcγR-mediated phagocytosis-lysosomal axis and increase in oxidative stress. Alterations in pathways of the phagocytosis-lysosomal axis associated with a female-dominated COPD phenotype correlated well with specific clinical features: FcγR-mediated phagocytosis correlated with FEV1/FVC, the lysosomal pathway correlated with CT < -950 Hounsfield Units (HU), and regulation of actin cytoskeleton correlated with FEV1 and FEV1/FVC in female COPD patients. Alterations observed in the corresponding male cohort were minor. CONCLUSION: The identified molecular pathways suggest dysregulation of several phagocytosis-related pathways in BAL cells in female COPD patients, with correlation to both the level of obstruction (FEV1/FVC) and disease severity (FEV1) as well as emphysema (CT < -950 HU) in women. TRIAL REGISTRATION: No.: NCT02627872 , retrospectively registered on December 9, 2015.


Assuntos
Perfilação da Expressão Gênica/métodos , Pulmão/imunologia , Fagócitos/imunologia , Proteômica/métodos , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Estudos Retrospectivos , Caracteres Sexuais , Transdução de Sinais/genética , Transdução de Sinais/imunologia
7.
Curr Biol ; 28(7): 1137-1143.e5, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29576476

RESUMO

The impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e., N-nitroguanidine compounds such as imidacloprid and thiamethoxam, are as intrinsically toxic to bees as to the insect pests they target. However, this is not the case for all neonicotinoids, with honeybees orders of magnitude less sensitive to N-cyanoamidine compounds such as thiacloprid [2]. Although previous work has suggested that this is due to rapid metabolism of these compounds [2-5], the specific gene(s) or enzyme(s) involved remain unknown. Here, we show that the sensitivity of the two most economically important bee species to neonicotinoids is determined by cytochrome P450s of the CYP9Q subfamily. Radioligand binding and inhibitor assays showed that variation in honeybee sensitivity to N-nitroguanidine and N-cyanoamidine neonicotinoids does not reside in differences in their affinity for the receptor but rather in divergent metabolism by P450s. Functional expression of the entire CYP3 clade of P450s from honeybees identified a single P450, CYP9Q3, that metabolizes thiacloprid with high efficiency but has little activity against imidacloprid. We demonstrate that bumble bees also exhibit profound differences in their sensitivity to different neonicotinoids, and we identify CYP9Q4 as a functional ortholog of honeybee CYP9Q3 and a key metabolic determinant of neonicotinoid sensitivity in this species. Our results demonstrate that bee pollinators are equipped with biochemical defense systems that define their sensitivity to insecticides and this knowledge can be leveraged to safeguard bee health.


Assuntos
Abelhas/fisiologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Animais , Abelhas/efeitos dos fármacos , Abelhas/metabolismo
8.
Curr Biol ; 28(2): 268-274.e5, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29337073

RESUMO

Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Duplicação Gênica , Hemípteros/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Animais , Dosagem de Genes , Hemípteros/efeitos dos fármacos
9.
Mol Cell Proteomics ; 15(9): 3017-29, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27452731

RESUMO

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Antígenos HLA-DR/metabolismo , Peptídeos/análise , Sarcoidose/terapia , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Sarcoidose/imunologia
10.
Adv Exp Med Biol ; 845: 121-31, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25355575

RESUMO

Exercise-induced proteinuria has been observed and studied for more than a century. It was found that different sport disciplines alter the urinary proteome in different ways. Moderate-intensity exercise results in increased glomerular filtration, meaning that medium-sized proteins are excreted in higher amounts, while high-intensity exercise of short duration also increases the excretion of low molecular weight proteins as a result of tubular dysfunction. Exhaustive exercise may lead to the excretion of hemoglobin or myoglobin, which changes the urinary proteome considerably. Studies comparing protein maps of different sport types compared to a control group showed that quality and quantity of urinary proteins are interindividually different. In addition, urine samples collected before and after exercise exhibit substantially different protein patterns even from the same person. Therefore, further studies investigating the urinary proteome are desirable. As the variation of protein content and composition in urine are generally much higher than in other matrices, respective studies need to be well controlled and homogenous groups of volunteers should be chosen. In addition to the sport-related physiological and biochemical interest, exercise-induced protein changes also need to be considered for biomarker measurements from urine samples for kidney or other diseases.


Assuntos
Exercício Físico/fisiologia , Nefropatias/urina , Proteinúria/urina , Proteoma/análise , Adulto , Biomarcadores/urina , Feminino , Humanos , Masculino , Proteinúria/fisiopatologia , Proteoma/fisiologia , Esportes/fisiologia
11.
J Venom Res ; 4: 13-20, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24163732

RESUMO

Intraspecific variation in venom composition and activity has been reported from a wide range of snakes. Geographical origin can be one cause for this variation and has recently been documented from Acanthophis antarcticus death adders sampled across four different Australian states. The present study examined whether a narrower sampling range of A. antarcticus from four collection sites within one Australian state (i.e., South Australia) would also exhibit variation in venom composition and/or activity. The present LC-MS results reveal marked differences in the venom composition from different collection sites. The most striking difference was the reduced venom complexity found in the only venom originating from a mallee scrub habitat in comparison to the venoms from coastal heath scrub habitats. Interestingly, the pharmacological activity of all venoms was found to be the same, independent of the collection site.

12.
Drug Test Anal ; 5(11-12): 853-60, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23913913

RESUMO

Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls.


Assuntos
RNA Interferente Pequeno/sangue , RNA Interferente Pequeno/urina , Detecção do Abuso de Substâncias/métodos , Animais , Cromatografia Líquida/métodos , Dopagem Esportivo , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Espectrometria de Massas/métodos , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Sensibilidade e Especificidade
13.
Proteomics Clin Appl ; 7(7-8): 561-70, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23670890

RESUMO

PURPOSE: Gamma irradiation of red blood cell (RBC) concentrates is routinely used to prevent transfusion-associated graft-versus-host disease. So far, the effects of ionizing radiation on RBC structure and function and especially the proteome are not fully understood. EXPERIMENTAL DESIGN: RBC concentrates were irradiated with 30 Gy and stored for 1 or 15 days at 4 ± 2°C. Following cell lysis and hemoglobin depletion, 2D-DIGE was used to examine the changes of the cytosolic RBC proteome. Significantly altered spots were analyzed using bottom-up proteomic approaches and selected marker proteins validated by western blotting. RESULTS: Gamma irradiation was found to enhance conventional RBC storage lesions. Following 15 days of postirradiation storage, the abundances of a total of 27 spots were significantly altered and 3 out of 13 identified proteins were selected and validated as potential marker proteins for the assessment of irradiation-induced cytosolic RBC lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Gamma irradiation and subsequent ex vivo storage according to the Council of Europe guidelines were found to affect RBC protein structures. The validated marker proteins can serve as a basis for the development of a screening assay to monitor the quality of irradiated RBC concentrates during ex vivo storage.


Assuntos
Citosol/metabolismo , Citosol/efeitos da radiação , Eritrócitos/metabolismo , Eritrócitos/efeitos da radiação , Raios gama/efeitos adversos , Proteômica/métodos , Manejo de Espécimes/métodos , Adulto , Biomarcadores/sangue , Cromatografia Líquida , Eritrócitos/citologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Nanotecnologia , Fatores de Tempo , Eletroforese em Gel Diferencial Bidimensional , Adulto Jovem
14.
J Allergy Clin Immunol ; 131(3): 743-51, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23146379

RESUMO

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and is increasing, primarily among women. Underdiagnosis is common, and because of the heterogeneous disease characteristics, molecular markers of specific disease phenotypes and more efficacious treatment regimens are urgently needed. OBJECTIVE: In this study the soluble proteome of bronchoalveolar lavage cells, primarily consisting of macrophages, was investigated with the aim of identifying phenotypic differences in early disease development. METHODS: Two-dimensional difference gel electrophoresis was used for relative quantification of protein levels, and multivariate modeling was applied to identify proteins of interest that were subsequently identified by means of liquid chromatography-mass spectrometry. RESULTS: Significant gender differences were unveiled, with numerous alterations in the bronchoalveolar lavage cell proteome occurring in female but not male patients with COPD. Specifically, a subset of 19 proteins provided classification of female healthy smokers from female patients with COPD with 78% predictive power. Subsequent pathway analyses linked the observed alterations to downregulation of the lysosomal pathway and upregulation of the oxidative phosphorylation pathway, possibly linking dysregulation of macroautophagy to a female-dominated COPD disease phenotype. CONCLUSION: This investigation makes an important contribution to the elucidation of putative molecular mechanisms underlying gender-based differences in the pathophysiology of COPD, linking alterations of specific molecular pathways to previously observed gender differences in clinical COPD phenotypes. Furthermore, these results stress the importance of the gender-specific search for biomarkers, diagnosis, and treatment in COPD.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Proteoma , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumar/metabolismo , Idoso , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fatores Sexuais
15.
Proteomics ; 12(21): 3263-72, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22965759

RESUMO

The storage of packed red blood cells (RBCs) is associated with the development of morphological and biochemical changes leading to a reduced posttransfusion functionality and viability of the cells. Within this study, 2D DIGE and high-resolution/high-accuracy Orbitrap MS were used to analyze the storage-induced changes of the cytosolic RBC proteome and identify characteristic protein patterns and potential marker proteins for the assessment of RBC storage lesions. Leukodepleted RBC concentrates were stored according to standard blood bank conditions for 0, 7, 14, 28, and 42 days and analyzed by using a characterized and validated protocol. Following statistical evaluation, a total of 14 protein spots were found to be significantly altered after 42 days of ex vivo storage. Protein identification was accomplished by tryptic digestion and LC-MS/MS and three proteins potentially useful as biomarkers for RBC aging comprising transglutaminase 2, beta actin, and copper chaperone for superoxide dismutase were selected and validated by western blot analysis. These can serve as a basis for the development of a screening assay to detect RBC storage lesions and autologous blood doping in sports.


Assuntos
Preservação de Sangue/métodos , Eritrócitos/metabolismo , Proteoma/metabolismo , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Western Blotting , Eritrócitos/química , Eritrócitos/citologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Análise de Componente Principal , Proteoma/análise , Reprodutibilidade dos Testes , Eletroforese em Gel Diferencial Bidimensional
16.
Electrophoresis ; 33(16): 2537-45, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22899261

RESUMO

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.


Assuntos
Proteínas Sanguíneas/análise , Eritrócitos/química , Hemoglobinas/isolamento & purificação , Proteoma/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/classificação , Eletroforese em Gel Bidimensional/métodos , Hemoglobinas/química , Humanos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/classificação , Inibidores de Proteases/química , Reprodutibilidade dos Testes , Temperatura
17.
Drug Test Anal ; 3(10): 661-7, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22031503

RESUMO

RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports. Myostatin down-regulation was shown to result in increased performance and muscle growth and the regulation of several other proteins could be relevant for performance enhancement. This mini-review summarizes current approaches for the mass spectrometric analysis of siRNA and antisense oligonucleotides from biological matrices and the available data on biodistribution, metabolism, and half-life of relevant substances are discussed.


Assuntos
Dopagem Esportivo/métodos , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Antissenso/farmacologia , Interferência de RNA , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/farmacologia , Animais , Humanos , Espectrometria de Massas/métodos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacocinética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética
18.
Analyst ; 136(5): 1003-12, 2011 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-21157622

RESUMO

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.


Assuntos
Cromatografia Líquida/métodos , Fator de Crescimento Insulin-Like II/urina , Fator de Crescimento Insulin-Like I/urina , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Dopagem Esportivo , Feminino , Humanos , Limite de Detecção , Masculino , Reprodutibilidade dos Testes , Extração em Fase Sólida/métodos , Fatores de Tempo , Ultrafiltração/métodos
19.
Proteomics Clin Appl ; 4(5): 568-76, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-21137074

RESUMO

PURPOSE: Exercise-induced proteinuria is a well-known phenomenon and the influence of parameters such as intensity and duration was studied extensively. Usually, total protein or albumin was measured for diagnosis of a proteinuria, and the present study was performed to search for qualitative differences in the urinary proteome before and after endurance exercise. EXPERIMENTAL DESIGN: Urine samples were concentrated and proteins separated by means of 2-D PAGE. Proteins differing in the investigated groups were identified by nano-UPLC-Orbitrap MS after trypsin digestion. RESULTS: The study yielded several proteins such as hemopexin, albumin, orosomucoid 1, transferrin or carbonic anhydrase 1 that were elevated after a marathon run in comparison to a control group. These are linked to physiological changes resulting from endurance exercise such as destruction of erythrocytes or increased fat metabolism. On the contrary, 2-D PAGE profiles of athletes at rest did not differ from those of control samples. CONCLUSIONS AND CLINICAL RELEVANCE: The study is a starting point to build up individual 2-D PAGE protein maps of athletes. Further studies will investigate intra-individual differences and further exercise parameters, which potentially lead to a physiological monitoring system for athletes in training and competition and may also complement the blood passport in doping control.


Assuntos
Exercício Físico/fisiologia , Resistência Física/fisiologia , Proteinúria/urina , Adulto , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Espectrometria de Massas , Proteoma , Corrida/fisiologia
20.
Drug Test Anal ; 2(3): 144-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20878896

RESUMO

Black market products of a pharmaceutical nature and nutritional supplements have received substantial and increasing attention because of potential performance enhancement in elite and non-professional sports. In addition, improved general health is claimed for non-competing individuals. The risks and foreseeable dangers of the uncontrolled use of highly potent and non-approved pharmaceutical compounds in healthy individuals are of considerable concern. In the present case report, the emerging drug candidate GHRP-2 with verified growth-hormone-releasing properties was identified and quantified in tablets offered as an over-the-counter nutritional supplement. The impact of this orally active peptide on the hGH/IGF-axis has been established for several years and its illicit use in elite sports has been assumed. As a releasing factor for hGH, GHRP-2 belongs to the list of substances prohibited by the World Anti-Doping Agency (WADA). Unfortunately, to date there is no routinely performed assay for the determination of these peptides potentially occurring in biological fluids of competing athletes, but the present data will facilitate the implementation by providing principle analytical information on liquid chromatographic and mass spectrometric behaviour. Qualitative identification of the target analyte after extraction from the tablet matrix was performed by high resolution/high accuracy mass spectrometry after liquid chromatographic separation under consideration of the accurate masses and the ratios of the protonated molecules and their fragment ions derived from their collisionally induced dissociation. Quantitative results were obtained by means of liquid chromatography coupled to a triple quadrupole mass spectrometer and linear regression using an external calibration curve (with GHRP-2 reference compound) adjusted via internal standard (Hexarelin). Hereby, the content of GHRP-2 was determined with approximately 50 µg per tablet.


Assuntos
Suplementos Nutricionais/análise , Oligopeptídeos/análise , Calibragem , Cromatografia Líquida , Dopagem Esportivo , Espectrometria de Massas por Ionização por Electrospray , Comprimidos/análise
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