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1.
Free Radic Res ; 53(9-10): 979-992, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31476923

RESUMO

Elevated intracellular levels of reactive oxygen species (ROS), e.g. resulting from exposure to xenobiotics, can cause severe damages. Antioxidant defence mechanisms, which involve regulation of enzyme activities, protect cells to a certain extent. Nevertheless, continuous or increased exposure can overwhelm this system resulting in an adverse cellular state. To simulate exposure scenarios and to investigate the transition to an adverse cellular state, a mathematical model for the dynamics of ROS in response to xenobiotic-induced oxidative stress has been developed. It is based on exposure experiments of human urothelial cells (RT4) to the nitrated polycyclic aromatic hydrocarbon 3-nitrobenzanthrone (3-NBA), a component of diesel engine exhaust, and takes into account the following metabolic pathways of the antioxidant defence system: glutathione redox cycle scavenging directly ROS, the pentose phosphate pathway and the gluconate shunt as NADPH supplier and the beginning of glycolysis. In addition, ROS generation due to the bioactivation of 3-NBA has been implemented. The regulation of enzyme activities plays an important role in the presented mathematical model. The in silico model consists of ordinary differential equations on the basis of enzyme kinetics and mass action for the metabolism of 3-NBA. Parameters are either estimated from performed in vitro experiments via least-squares fitting or obtained from the literature. The results underline the importance of the pentose phosphate pathway to cope with oxidative stress and suggest an important role of the gluconate shunt during low-dose exposure.

2.
Arch Toxicol ; 93(9): 2593-2602, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31342136

RESUMO

Exposure to xenobiotic such as benzo[a]pyrene (B[a]P) induces metabolic changes, which have a considerable impact on the cellular response. Nevertheless, we are just in the beginning to reach an understanding of these processes. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was applied to distinguish the metabolic changes that bladder epithelia cells undergo upon B[a]P exposure. To closely reflect the epithelia cell conditions in vivo, freshly isolated primary porcine urinary bladder epithelial cells (PUBEC) were utilized for the current study. An untargeted metabolomics approach was used to characterize the time- (6 h, 24 h, 48 h) and dose-dependent (0.5 µM, 5 µM, 10 µM B[a]P) changes in the metabolome of PUBEC upon B[a]P exposure, which led to the profiling of more than 200 metabolites that differed significantly between control and exposed samples. Multivariate analysis of the data highlighted that in the experimental setup/model used other than the exposure concentration, it is the exposure time which seems to be most important for distinguishing between different groups and hence may have a bigger role in B[a]P-mediated toxicity but may be specific for cell model used and hence requires further investigations. Further, enrichment and pathway analysis using MetaboAnalyst highlighted that exposure to B[a]P mainly alters the cellular amino acid metabolism. Particularly, 1-pyrroline-5-carboxylic acid (P5C), an intermediate of the cycling of the amino acid proline, was identified as a differentially altered metabolite at all concentrations and exposure times used in the experiment. An increase in the activity of proline dehydrogenase/proline oxidase (PRODH/POX), which oxidizes proline to P5C, was also observed, further supporting our metabolomic data. Our findings contribute to an improved knowledge about the reprogramming of metabolism which is a fundamental element of the cellular response to B[a]P and draw attention to the role of proline in this context.

3.
J Clin Lab Anal ; : e22627, 2018 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-30058083

RESUMO

BACKGROUND: Impaired levels or function of C1 inhibitor (C1-INH) results in angioedema due to increased bradykinin. It is important to distinguish between angioedema related to C1-INH deficiency and that caused by other mechanisms, as treatment options are different. In hereditary (HAE) and acquired (AAE) angioedema, C1-INH concentration is measured to aid patient diagnosis. Here, we describe an automated turbidimetric assay to measure C1-INH concentration on the Optilite® analyzer. METHODS: Linearity, precision, and interference were established over a range of C1-INH concentrations. The 95th percentile reference interval was generated from 120 healthy adult donors. To compare the Optilite C1-INH assay with a predicate assay used in a clinical laboratory, samples sent for C1-INH investigation were used. The predicate results were provided to allow comparison. RESULTS: The Optilite C1-INH assay was linear across the measuring range at the standard sample dilution. Intra and interassay variability was <6%. The 95th percentile adult reference interval for the assay was 0.21-0.38 g/L. There was a strong correlation between the Optilite concentrations and those generated with the predicate assay (R2  = 0.94, P < 0.0001, slope y = 0.83x). All patients with Type I HAE (n = 24) and AAE (n = 3) tested had concentrations below the measuring range in both assays, while all patients with unspecified angioedema (UAE), not diagnosed with HAE or AAE had values within the reference range. CONCLUSION: The Optilite assay allows the automated and precise quantification of C1-INH concentrations in patient samples. It could therefore be used as a tool to aid the investigation of patients with angioedema.

4.
Chem Res Toxicol ; 30(10): 1855-1864, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-28922594

RESUMO

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 µM (environmentally relevant) to 80 µM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 µM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).


Assuntos
Benzo(a)Antracenos/administração & dosagem , Benzo(a)Antracenos/farmacologia , Neoplasias da Bexiga Urinária/induzido quimicamente , Benzo(a)Antracenos/química , Benzo(a)Antracenos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Via de Pentose Fosfato/efeitos dos fármacos , Relação Estrutura-Atividade , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
5.
Sci Rep ; 7(1): 9773, 2017 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-28851999

RESUMO

Benzo[a]pyrene (B[a]P), a well-known polyaromatic hydrocarbon, is known for its lung carcinogenicity, however, its role in bladder cancer development is still discussed. Comparative two-dimensional blue native SDS-PAGE analysis of protein complexes isolated from subcellular fractions of 0.5 µM B[a]P-exposed cells indicated a differential regulation of proteins involved in carbohydrate, fatty acid, and nucleotide metabolism, suggesting a possible metabolic flux redistribution. It appeared that B[a]P exposure led to a repression of enzymes (fructose-bisphosphate aldolase A, glucose-6-phosphate isomerase, lactate dehydrogenase) involved in glycolysis, and an up-regulation of proteins (glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconolactonase) catalyzing the pentose phosphate pathway and one carbon metabolism (10-formyltetrahydrofolate dehydrogenase, bifunctional purine biosynthesis protein). Untargeted metabolomics further supported the proteomic data, a lower concentration of glycolytic metabolite was observed as compared to glutamine, xylulose and fatty acids. The analysis of the glutathione and NADPH/NADP+ content of the cells revealed a significant increase of these cofactors. Concomitantly, we did not observe any detectable increase in the production of ROS. With the present work, we shed light on an early phase of the metabolic stress response in which the urothelial cells are capable of counteracting oxidative stress by redirecting the metabolic flux from glycolysis to pentose phosphate pathway.

6.
J Allergy Clin Immunol Pract ; 5(4): 938-945, 2017 Jul - Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28351785

RESUMO

A proportion of people living with common variable immunodeficiency disorders develop granulomatous-lymphocytic interstitial lung disease (GLILD). We aimed to develop a consensus statement on the definition, diagnosis, and management of GLILD. All UK specialist centers were contacted and relevant physicians were invited to take part in a 3-round online Delphi process. Responses were graded as Strongly Agree, Tend to Agree, Neither Agree nor Disagree, Tend to Disagree, and Strongly Disagree, scored +1, +0.5, 0, -0.5, and -1, respectively. Agreement was defined as greater than or equal to 80% consensus. Scores are reported as mean ± SD. There was 100% agreement (score, 0.92 ± 0.19) for the following definition: "GLILD is a distinct clinico-radio-pathological ILD occurring in patients with [common variable immunodeficiency disorders], associated with a lymphocytic infiltrate and/or granuloma in the lung, and in whom other conditions have been considered and where possible excluded." There was consensus that the workup of suspected GLILD requires chest computed tomography (CT) (0.98 ± 0.01), lung function tests (eg, gas transfer, 0.94 ± 0.17), bronchoscopy to exclude infection (0.63 ± 0.50), and lung biopsy (0.58 ± 0.40). There was no consensus on whether expectant management following optimization of immunoglobulin therapy was acceptable: 67% agreed, 25% disagreed, score 0.38 ± 0.59; 90% agreed that when treatment was required, first-line treatment should be with corticosteroids alone (score, 0.55 ± 0.51).


Assuntos
Imunodeficiência de Variável Comum , Granuloma , Doenças Pulmonares Intersticiais , Instituições de Caridade , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/diagnóstico por imagem , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunodeficiência de Variável Comum/patologia , Consenso , Granuloma/diagnóstico , Granuloma/diagnóstico por imagem , Granuloma/tratamento farmacológico , Granuloma/patologia , Humanos , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/patologia , Sociedades Médicas , Reino Unido
7.
Blood ; 129(11): 1458-1468, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28159733

RESUMO

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Antígeno CTLA-4/genética , Mutação , Antígeno CTLA-4/metabolismo , Linhagem Celular , Imunodeficiência de Variável Comum/genética , Fatores de Transcrição Forkhead/análise , Humanos , Fenômenos do Sistema Imunológico/genética , Ligantes , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
8.
Lancet Respir Med ; 3(8): 651-60, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26188881

RESUMO

This Review summarises current knowledge on the pulmonary manifestations of primary antibody deficiency (PAD) syndromes in adults. We describe the major PAD syndromes, with a particular focus on common variable immunodeficiency (CVID). Respiratory infection is a common presenting feature of PAD syndromes. Respiratory complications are frequent and responsible for much of the morbidity and mortality associated with these syndromes. Respiratory complications include acute infections, the sequelae of infection (eg, bronchiectasis), non-infectious immune-mediated manifestations (notably the development of granulomatous-lymphocytic interstitial lung disease in CVID), and an increased risk of lymphoma. Although minor abnormalities are detectable in the lungs of most patients with CVID by CT scanning, not all patients develop lung complications. Mechanisms associated with the maintenance of lung health versus lung disease, and the development of bronchiectasis versus immune-mediated complications, are now being dissected. We review the investigation, treatment, and management strategies for PAD syndromes, and include key research questions relating to both infectious and non-infectious complications of PAD in the lung.


Assuntos
Bronquiectasia/imunologia , Imunodeficiência de Variável Comum/complicações , Pneumopatias/imunologia , Adulto , Humanos , Infecções Respiratórias/imunologia , Fatores de Risco
9.
J Proteome Res ; 14(1): 202-13, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25348606

RESUMO

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.


Assuntos
Cálcio/metabolismo , Poluentes Ambientais/toxicidade , Ferro/metabolismo , Óxido Nítrico/fisiologia , Dibenzodioxinas Policloradas/toxicidade , Proteoma/metabolismo , Calmodulina/metabolismo , Fracionamento Celular , Linhagem Celular , Núcleo Celular/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Homeostase , Humanos , Eletroforese em Gel de Poliacrilamida Nativa , Proteômica , Bexiga Urinária/citologia
10.
Arch Toxicol ; 88(4): 913-34, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24464499

RESUMO

Epidemiological studies suggest that environmental exposure to airborne particulate matter may promote cardiovascular diseases; however, it is not clear whether this observation actually reflects exposure to nanosized particles in the environment. In the present study, the human endothelial cell line EA.hy926 was exposed to pure carbon black and, to mimic exposure to diesel exhaust, carbon black loaded with benzo[a]pyrene to ascertain effects of these particles on the cell proteome and metabolom. Particular emphasis was laid on an extended exposure period (14 days) and a low particle concentration (100 ng/mL). While ROS production essentially remained unaffected, exposure of the cells to the particles resulted in a significantly enhanced cell proliferation. Evaluation of the obtained proteomic and phosphoproteomic data revealed modulations of proteins involved in catalytic processes and cytoskeleton maintenance. The bioinformatic evaluation of the data revealed the possible involvement of the transcription factor peroxisome proliferator-activated receptor gamma. The further analysis of the cytoskeleton indicated changes of the cell motility, which is in agreement with an observed increase in the cellular migration and invasion, and macroscopic changes of the cytoskeleton of the exposed cells.


Assuntos
Benzo(a)pireno/toxicidade , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolômica , Proteômica , Fuligem/toxicidade , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Metabolômica/métodos , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Integração de Sistemas , Fatores de Tempo
12.
Nat Neurosci ; 16(8): 1000-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23817546

RESUMO

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.


Assuntos
Fatores de Ribosilação do ADP/fisiologia , Cílios/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neuroglia/ultraestrutura , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Anormalidades Múltiplas , Animais , Axonema/ultraestrutura , Divisão Celular , Polaridade Celular , Doenças Cerebelares/enzimologia , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Cerebelo/anormalidades , Córtex Cerebral/anormalidades , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Ventrículos Cerebrais/anormalidades , Cílios/fisiologia , Epitélio/ultraestrutura , Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Humanos , Doenças Renais Císticas/enzimologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/ultraestrutura , Neurogênese/genética , Neuroglia/fisiologia , Retina/anormalidades , Retina/enzimologia , Retina/patologia , Telencéfalo/embriologia , Telencéfalo/ultraestrutura
13.
J Proteomics ; 85: 53-64, 2013 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-23624345

RESUMO

UNLABELLED: The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 µM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis. BIOLOGICAL SIGNIFICANCE: Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen.


Assuntos
Apoptose/efeitos dos fármacos , Benzo(a)pireno/toxicidade , Dano ao DNA , Células Epiteliais/metabolismo , Proteoma/metabolismo , Bexiga Urinária/metabolismo , Animais , Catepsina D/metabolismo , Células Cultivadas , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Histonas/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Fatores de Tempo , Bexiga Urinária/patologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo
14.
Phys Chem Chem Phys ; 15(18): 6990-7, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23552642

RESUMO

Half-Heusler thermoelectrics offer the possibility to choose from a variety of non-toxic and earth-abundant elements. TiNiSn is of particular interest and - with its relatively high electrical conductivity and Seebeck coefficient - allows for optimization of its thermoelectric figure of merit, reaching values of up to 1 in heavily-doped and/or phase-segregated systems. In this contribution, we used an energy- and time-efficient process involving solid-state preparation in a commercial microwave oven and a fast consolidation technique, Spark Plasma Sintering, to prepare a series of Ni-rich TiNi1+xSn with small deviations from the half-Heusler composition. Spark Plasma Sintering plays an important role in the process by being a part of the synthesis of the material rather than solely a densification technique. Synchrotron powder X-ray diffraction and microprobe data confirm the presence of a secondary TiNi2Sn full-Heusler phase within the half-Heusler matrix. We observe a clear correlation between the amount of full-Heusler phase and the lattice thermal conductivity of the samples, resulting in decreasing total thermal conductivity with increasing TiNi2Sn fraction. This trend shows that phonons are scattered effectively as a result of the microstructure of the materials with full-Heusler inclusions in the size range of microns to tens of microns. The best performing samples with around 5% of TiNi2Sn phase exhibit maximum figures of merit of almost 0.6 between 750 K and 800 K which is an increase of ca. 35% compared to the zT of the parent compound TiNiSn.

15.
Drugs Aging ; 30(7): 503-12, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23605785

RESUMO

Primary immunodeficiency disease (PID) has traditionally been viewed as a group of illnesses seen in the paediatric age group. New advances in diagnosis and treatment have led to an increase in the number of elderly PID patients. However, there is lack of research evidence on which to base clinical management in this group of patients. Management decisions often have to be based therefore on extrapolations from other patient cohorts or from younger patients. Data from the European Society for Immunodeficiencies demonstrates that the vast majority of elderly patients suffer from predominantly antibody deficiency syndromes. We review the management of PID disease in the elderly, with a focus on antibody deficiency disease.


Assuntos
Agamaglobulinemia/tratamento farmacológico , Imunodeficiência de Variável Comum/tratamento farmacológico , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Etários , Idoso , Antibacterianos/uso terapêutico , Transplante de Medula Óssea , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/terapia , Terapia Genética , Humanos
16.
Electrophoresis ; 34(4): 501-4, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23192385

RESUMO

The recent introduction of the La(3+) precipitation method for the enrichment of phosphoproteins allows a gel-based analysis of these posttranslationally modified proteins. However, if this method is applied to cell lysates stored in urea-containing lysis buffer for an extended period of time, incomplete phosphoprotein recovery is observed. We ascribe this effect to the presence of urea in the lysis buffer. To overcome this problem various strategies were tested, where cell lysates stored at least for one year were utilized. By applying an optimized protocol approximately 250 proteins could be observed following separation by 2DE.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Lantânio/química , Fosfoproteínas/isolamento & purificação , Tioureia/química , Ureia/química , Precipitação Química , Células Endoteliais/química , Humanos , Concentração de Íons de Hidrogênio , Fosfoproteínas/análise , Redobramento de Proteína
17.
Arch Toxicol ; 86(12): 1861-71, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22790669

RESUMO

More than 90 % of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5 µM BaP for up to 24 h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70 µM in cells exposed to 0.5 µM BaP and from 29.9 to 406.64 µM in cells exposed to 10 µM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.


Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Células Epiteliais/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Animais , Benzo(a)pireno/metabolismo , Carcinógenos/metabolismo , Linhagem Celular , Fragmentação do DNA , Cromatografia Gasosa-Espectrometria de Massas , Marcação In Situ das Extremidades Cortadas , Indicadores e Reagentes , Cinética , Microscopia Confocal , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Suínos , Urotélio/citologia
18.
Proteomics ; 12(11): 1731-55, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22623321

RESUMO

Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.


Assuntos
Benzo(a)pireno/metabolismo , Benzo(a)pireno/toxicidade , Poluentes Ambientais/toxicidade , Animais , Benzo(a)pireno/química , Dano ao DNA , Exposição Ambiental , Peixes , Humanos , Camundongos , Órgãos em Risco , Proteômica , Ratos
19.
Electrophoresis ; 32(24): 3600-11, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22180210

RESUMO

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases.


Assuntos
Fosfoproteínas/análise , Proteoma/análise , Bexiga Urinária/química , Urotélio/química , Animais , Células Cultivadas , Eletroforese em Gel Bidimensional , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Bexiga Urinária/citologia , Bexiga Urinária/metabolismo , Urotélio/citologia , Urotélio/metabolismo
20.
J Proteomics ; 75(2): 375-83, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21871587

RESUMO

Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell functions. In many signal transduction processes, PTM is a pivotal step. Various analytical methods have been proposed for the identification of phosphoproteins; however, most of these methods require sophisticated equipment. Here we present an easily applicable method of phosphoprotein enrichment. This method is based on single-step precipitation by lanthanum chloride and allows subsequent protein identification by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS). The method proved its suitability for the isolation of phosphoproteins from frozen tissue and cultured cells samples after cell lysis in various buffer systems (urea/thiourea and EGTA/EDTA). The tests revealed that the isolation of phosphoproteins can be achieved with high efficiency even from complex protein mixtures. Our results indicate that lanthanum-based enrichment of phosphoproteins can be a useful tool in phosphoproteomic studies.


Assuntos
Lantânio/química , Fosfoproteínas/isolamento & purificação , Proteômica/métodos , Animais , Bovinos , Proteínas do Ovo/isolamento & purificação , Proteínas do Leite/isolamento & purificação , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Suínos
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