RESUMO
Sulfur mustard (HD) ranks among the alkylating chemical warfare agents. Skin contact with HD produces an inflammatory response that evolves into separation at the epidermal-dermal junction conducting to blistering and epidermis necrosis. Up to now, current treatment strategies of HD burns have solely consisted in symptomatic management of skin damage. Therapeutic efficacy studies are still being conducted; classically using appropriate animal skin toxicity models. In order to substantiate the use of SKH-1 hairless mouse as an appropriate model for HD-induced skin lesions, we investigate the time-dependent quantitative gene expression of various selected transcripts associated to the dorsal skin exposure to HD saturated vapors. Using quantitative real time polymerase chain reaction (RT-qPCR), the expression of interleukins (IL-1ß and IL-6), tumor necrosis factor (TNF)-α, macrophage inflammatory proteins (MIP)-2α (also called Cxcl2) and MIP-1αR (also called Ccr1), matrix metalloproteases (MMP-9 and MMP-2), laminin γ2 monomer (Lamc2) and keratin (K)1 was determined up to 21 days after HD challenge in order to allow enough time for wound repair to begin. Specific transcript RT-qPCR analysis demonstrated that IL-6, IL-1ß, Ccr1, Cxcl2 mRNA levels increased as early as 6 h in HD-exposed skins and remained up-regulated over a 14-day period. Topical application of HD also significantly up-regulated MMP-9, TNF-α, and Lamc2 expression at specific time points. In contrast, MMP-2 mRNA levels remained unaffected by HD over the time-period considered, whereas that long-term study revealed that K1 mRNA level significantly increased only 21 days after HD challenge. Our study hereby provides first-hand evidence to substantiate a long period variation expression in the inflammatory cytokine, MMPs and structural components following cutaneous HD exposure in hairless mouse SKH-1. Our data credit the use of SKH-1 for investigating mechanisms of HD-induced skin toxicity and for the development of pharmacological countermeasures.
Assuntos
Substâncias para a Guerra Química/toxicidade , Gás de Mostarda/toxicidade , Pele/efeitos dos fármacos , Animais , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Queratina-1/genética , Laminina/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Pelados , RNA Mensageiro/metabolismo , Receptores CCR1/genética , Pele/metabolismo , Transcrição GênicaRESUMO
Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.
Assuntos
DNA Mitocondrial/análise , Animais , Fígado/metabolismo , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Wistar , Deleção de SequênciaRESUMO
Studies focusing on the interaction of dietary and acute emotional stress on oxidative stress in cortex frontal and in brain mitochondria are scarce. Dietary-induced insulin resistance, as observed in Western diets, has been associated with increased oxidative stress causing mitochondrial dysfunction. We hypothesized that acute emotional stress could be an aggravating factor by impacting redox status in cortex and brain mitochondria. Thus, the aim of the present study was to evaluate the combination of an insulin resistance inducing high-fat/high-fructose (HF/HFr) diet and acute emotional stress on brain oxidative stress in rats. We measured several oxidative stress parameters (carbonyls, FRAP, TBARS assays, GSH, GSSG, oxidized DNA, mRNA expression of redox proteins (Nrf2), and uric acid). The HF/HFr diet resulted in increased oxidative stress both in the brain mitochondria and in the frontal cortex and decreased expression of the Nrf2 gene. The emotional stress induced an oxidative response in plasma and in brain mitochondria of the control group. In the HF/HFr group it triggered an increase expression of the redox transcription factor Nrf2 and its downstream antioxidant genes. This suggests an improvement of the redox stress tolerance in response to an enhanced production of reactive oxygen species. Accordingly, a blunted oxidative effect on several markers was observed in plasma and brain of HF/HFr-stressed group. This was confirmed in a parallel study using lipopolysaccharide as a stress model. Beside the Nrf2 increase, the stress induced a stronger UA release in HF/HFr which could take a part in the redox stress.
Assuntos
Encéfalo/metabolismo , Dieta Hiperlipídica , Dieta Ocidental , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Angústia Psicológica , Animais , Antioxidantes/metabolismo , Açúcares da Dieta/administração & dosagem , Frutose/administração & dosagem , Regulação da Expressão Gênica , Masculino , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Ácido Úrico/metabolismoRESUMO
Among the different areas of biotechnology, enzyme engineering represents a growing field where major progress has been recently made. Indeed, chemical, pharmaceutical or food industries have increased needs for enzymes. This increase requires enzyme optimization in order to achieve, together or separately, greater operational stability, better specificity, increased solubility or preferential enantioselectivity. Directed and random mutagenesis, the classical methods of enzymatic engineering, have proved to be efficient in some cases, but are quite tricky. Directed evolution is a hybrid method recently developed in order to reproduce the random mechanisms of evolution in vitro. This method has now been used to optimise an increasing number of enzymes. In our research group, a directed evolution project has been initiated on a bacterial phosphotriesterase, a promising enzyme, capable of efficiently detoxifying organophosphorus nerve agents.
Assuntos
Evolução Molecular Direcionada , Enzimas/genética , Enzimas/metabolismo , Animais , Enzimas/química , Humanos , Mutagênese , Hidrolases de Triester Fosfórico/químicaRESUMO
Huntington's disease (HD) is an inherited disorder characterized by cognitive impairments, motor deficits, and progressive dementia. These symptoms result from progressive neurodegenerative changes mainly affecting the neostriatum. This pathology is fatal in 10 to 20 years and there is currently no treatment for HD. Early in the course of the disease, initial clinical manifestations are due to striatal neuronal dysfunction, which is later followed by massive neuronal death. A major therapeutic objective is therefore to reverse striatal dysfunction prior to cell death. Using a primate model reproducing the clinical features and the progressive neuronal degeneration typical of HD, we tested the therapeutic effects of direct intrastriatal infusion of ciliary neurotrophic factor (CNTF). To achieve a continuous delivery of CNTF over the full period of evaluation, we took advantage of the macroencapsulation technique. Baby hamster kidney (BHK) cells previously engineered to produce human CNTF were encapsulated into semipermeable membranes and implanted bilaterally into striata. We show here that intracerebral delivery of low doses of CNTF at the onset of symptoms not only protects neurons from degeneration but also restores neostriatal functions. CNTF-treated primates recovered, in particular, cognitive and motor functions dependent on the anatomofunctional integrity of frontostriatal pathways that were distinctively altered in this HD model. These results support the hypothesis that CNTF infusion into the striatum of HD patients not only could block the degeneration of neurons but also alleviated motor and cognitive symptoms associated with persistent neuronal dysfunction.
Assuntos
Encéfalo/patologia , Fator Neurotrófico Ciliar/genética , Terapia Genética/métodos , Doença de Huntington/terapia , Animais , Encéfalo/metabolismo , Calbindinas , Linhagem Celular , Fator Neurotrófico Ciliar/administração & dosagem , Convulsivantes/farmacologia , Cricetinae , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Humanos , Imuno-Histoquímica , Macaca fascicularis , Imageamento por Ressonância Magnética , Destreza Motora , Manifestações Neurocomportamentais , Nitrocompostos , Propionatos/farmacologia , Putamen/metabolismo , Ratos , Proteína G de Ligação ao Cálcio S100/metabolismo , Succinato Desidrogenase/metabolismo , Fatores de Tempo , Transfecção , TransgenesRESUMO
Positron emission tomography (PET) coupled to 6-[18F]Fluoro-L-Dopa (18F-Dopa) remains the gold standard for assessing dysfunctionality concerning the dopaminergic nigrostriatal pathway in Parkinson's disease and related disorders. The use of ligands of the dopamine transporters (DAT) is an attractive alternative target; consequently, the current aim was to validate one of them, 11C-PE2I, using a multiinjection modeling approach allowing accurate quantitation of DAT densities in the striatum. Experiments were performed in three controls, three MPTP-treated (parkinsonian) baboons, and one reserpine-treated baboon. 11C-PE2I B'max values obtained with this approach were compared with 18F-Dopa input rate constant values (Ki), in vitro Bmax binding of 125I-PE2I, and the number of dopaminergic neurons in the substantia nigra estimated postmortem by stereology. In the caudate nucleus and putamen, control values for 11C-PE2I B'max were 673 and 658 pmol/mL, respectively, whereas it was strongly reduced in the MPTP-treated (B'max = 26 and 36 pmol/mL) and reserpine-treated animals (B'max = 338 and 483 pmol/mL). In vivo 11C-PE2I B'max values correlated with 18F-Dopa Ki values and in vitro 125I-PE2I Bmax values in the striatum and with the number of nigral dopaminergic neurons. Altogether, these data support the use of 11C-PE2I for monitoring striatal dopaminergic disorders and the effect of potential neuroprotective strategies.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/análise , Di-Hidroxifenilalanina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Nortropanos/metabolismo , Doença de Parkinson Secundária/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Animais , Química Encefálica , Radioisótopos de Carbono , Proteínas de Transporte/metabolismo , Núcleo Caudado/química , Núcleo Caudado/metabolismo , Cerebelo/química , Cerebelo/metabolismo , Corpo Estriado/química , Di-Hidroxifenilalanina/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina , Radioisótopos de Flúor , Cinética , Ligantes , Papio , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Putamen/química , Putamen/metabolismo , Reserpina/administração & dosagem , Substância Negra/química , Substância Negra/metabolismo , Tomografia Computadorizada de Emissão , Tirosina 3-Mono-Oxigenase/análiseRESUMO
The possibility of a large-scale acute radiation exposure necessitates the development of new methods that could provide rapid individual dose estimates with high sample throughput. The focus of the study was an intercomparison of laboratories' dose-assessment performances using gene expression assays. Lithium-heparinized whole blood from one healthy donor was irradiated (240 kVp, 1 Gy/min) immediately after venipuncture at approximately 37°C using single X-ray doses. Blood samples to establish calibration curves (0.25-4 Gy) as well as 10 blinded test samples (0.1-6.4 Gy) were incubated for 24 h at 37°C supplemented with an equal volume of medium and 10% fetal calf serum. For quantitative reverse transcription polymerase chain reaction (qRT-PCR), samples were lysed, stored at -20°C and shipped on ice. For the Chemical Ligation Dependent Probe Amplification methodology (CLPA), aliquots were incubated in 2 ml CLPA reaction buffer (DxTerity), mixed and shipped at room temperature. Assays were run in each laboratory according to locally established protocols. The mean absolute difference (MAD) of estimated doses relative to the true doses (in Gy) was calculated. We also merged doses into binary categories reflecting aspects of clinical/diagnostic relevance and examined accuracy, sensitivity and specificity. The earliest reported time on dose estimates was <8 h. The standard deviation of technical replicate measurements in 75% of all measurements was below 11%. MAD values of 0.3-0.5 Gy and 0.8-1.3 Gy divided the laboratories contributions into two groups. These fourfold differences in accuracy could be primarily explained by unexpected variances of the housekeeping gene (P = 0.0008) and performance differences in processing of calibration and blinded test samples by half of the contributing laboratories. Reported gene expression dose estimates aggregated into binary categories in general showed an accuracies and sensitivities of 93-100% and 76-100% for the groups, with low MAD and high MAD, respectively. In conclusion, gene expression-based dose estimates were reported quickly, and for laboratories with MAD between 0.3-0.5 Gy binary dose categories of clinical significance could be discriminated with an accuracy and sensitivity comparable to established cytogenetic assays.
Assuntos
Bioensaio/métodos , Expressão Gênica/efeitos da radiação , Ensaio de Proficiência Laboratorial , Leucócitos/efeitos da radiação , Técnicas de Amplificação de Ácido Nucleico/métodos , Radiometria/métodos , Adulto , Relação Dose-Resposta à Radiação , Eletroforese Capilar/métodos , Humanos , Leucócitos/ultraestrutura , Masculino , Microesferas , Lesões por Radiação/diagnóstico , Lesões por Radiação/genética , Liberação Nociva de Radioativos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Método Simples-Cego , Fatores de Tempo , TriagemRESUMO
Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools.
Assuntos
Bioensaio/métodos , Cromossomos Humanos/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Histonas/metabolismo , Ensaio de Proficiência Laboratorial , Leucócitos/efeitos da radiação , Testes para Micronúcleos , Radiometria/métodos , Adulto , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/efeitos da radiação , Aberrações Cromossômicas , Citocinese/efeitos da radiação , Relação Dose-Resposta à Radiação , Expressão Gênica/efeitos da radiação , Humanos , Leucócitos/ultraestrutura , Masculino , Fosforilação , Processamento de Proteína Pós-Traducional , Lesões por Radiação/diagnóstico , Lesões por Radiação/genética , Liberação Nociva de Radioativos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Método Simples-Cego , Fatores de Tempo , Triagem/métodosRESUMO
Presenilin 1 (PS1) mutations account for the majority of early-onset dominant cases of familial Alzheimer's disease. Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticulum, Golgi apparatus, nuclear region and vesicular structures. These proteins include from seven to nine putative transmembrane domains, with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm. We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11, a small GTPase belonging to the Ras-related superfamily. Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11, a region including the fourth GTP-binding domain. Considering the implication of Rab proteins in vesicular transport pathways, the PS-Rab11 inter-action suggests that PSs might be involved in amyloid precursor protein vesicular routing.