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1.
Commun Biol ; 2: 144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044169

RESUMO

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3 - and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Animais , Bicarbonatos/química , Tampões (Química) , Células CACO-2 , Proliferação de Células , HEPES/química , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Laboratórios , Mamíferos , Pesquisadores/educação , Cloreto de Sódio/química
2.
J Mol Cell Cardiol ; 130: 184-196, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30986378

RESUMO

Maladaptive hypertrophy of cardiac myocytes increases the risk of heart failure. The underlying signaling can be triggered and interrogated in cultured neonatal ventricular myocytes (NRVMs) using sophisticated pharmacological and genetic techniques. However, the methods for quantifying cell growth are, by comparison, inadequate. The lack of quantitative, calibratable and computationally-inexpensive high-throughput technology has limited the scope for using cultured myocytes in large-scale analyses. We present a ratiometric method for quantifying the hypertrophic growth of cultured myocytes, compatible with high-throughput imaging platforms. Protein biomass was assayed from sulforhodamine B (SRB) fluorescence, and image analysis calculated the quotient of signal from extra-nuclear and nuclear regions. The former readout relates to hypertrophic growth, whereas the latter is a reference for correcting protein-independent (e.g. equipment-related) variables. This ratiometric measure, when normalized to the number of cells, provides a robust quantification of cellular hypertrophy. The method was tested by comparing the efficacy of various chemical agonists to evoke hypertrophy, and verified using independent assays (myocyte area, transcripts of markers). The method's high resolving power and wide dynamic range were confirmed by the ability to generate concentration-response curves, track the time-course of hypertrophic responses with fine temporal resolution, describe drug/agonist interactions, and screen for novel anti-hypertrophic agents. The method can be implemented as an end-point in protocols investigating hypertrophy, and is compatible with automated plate-reader platforms for generating high-throughput data, thereby reducing investigator-bias. Finally, the computationally-minimal workflow required for obtaining measurements makes the method simple to implement in most laboratories.


Assuntos
Cardiomegalia , Processamento de Imagem Assistida por Computador , Miócitos Cardíacos , Rodaminas/química , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley
3.
EMBO Mol Med ; 9(10): 1398-1414, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28729482

RESUMO

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.


Assuntos
Acetaldeído/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Front Pharmacol ; 8: 215, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28588481

RESUMO

Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.

5.
Nat Struct Mol Biol ; 23(8): 755-757, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27322732

RESUMO

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses.


Assuntos
Proteína BRCA2/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Replicação do DNA , Genoma Humano , Instabilidade Genômica , Células HEK293 , Humanos , Ftalazinas/farmacologia , Piperazinas/farmacologia
6.
EMBO J ; 35(9): 909-23, 2016 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-27037238

RESUMO

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error-free pathway for double-strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication-associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.


Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Predisposição Genética para Doença , Recombinação Homóloga , Neoplasias/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Redes e Vias Metabólicas
7.
J Nat Prod ; 79(1): 30-7, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26706944

RESUMO

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.


Assuntos
Aristolochia/química , Ácidos Aristolóquicos/isolamento & purificação , Ácidos Aristolóquicos/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Metabolômica , Ressonância Magnética Nuclear Biomolecular/métodos , Plantas Medicinais/química , Aristolochia/genética , Ácidos Aristolóquicos/química , Cromatografia Líquida , Medicamentos de Ervas Chinesas/química , Humanos , Nefropatias/induzido quimicamente , Estrutura Molecular
8.
Eur Neuropsychopharmacol ; 24(9): 1463-74, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25044049

RESUMO

The vast majority of approved antidepressants and antipsychotics exhibit a complex pharmacology. The mechanistic understanding of how these psychotropic medications are related to adverse drug reactions (ADRs) is crucial for the development of novel drug candidates and patient adherence. This study aims to associate in vitro assessed binding affinity profiles (39 compounds, 24 molecular drug targets) and ADRs (n=22) reported in clinical trials of antidepressants and antipsychotics (n>59.000 patients) by the use of robust multivariate statistics. Orthogonal projection to latent structures (O-PLS) regression models with reasonable predictability were found for several frequent ADRs such as nausea, diarrhea, hypotension, dizziness, headache, insomnia, sedation, sleepiness, increased sweating, and weight gain. Results of the present study support many well-known pharmacological principles such as the association of hypotension and dizziness with α1-receptor or sedation with H1-receptor antagonism. Moreover, the analyses revealed novel or hardly investigated mechanisms for common ADRs including the potential involvement of 5-HT6-antagonism in weight gain, muscarinic receptor antagonism in dizziness, or 5-HT7-antagonism in sedation. To summarize, the presented study underlines the feasibility and value of a multivariate data mining approach in psychopharmacological development of antidepressants and antipsychotics.


Assuntos
Antidepressivos/efeitos adversos , Antipsicóticos/efeitos adversos , Tontura/induzido quimicamente , Hipertensão/induzido quimicamente , Análise Multivariada , Ensaios Clínicos como Assunto/efeitos adversos , Ensaios Clínicos como Assunto/estatística & dados numéricos , Bases de Dados Factuais/estatística & dados numéricos , Feminino , Humanos , Técnicas In Vitro , Masculino , Ligação Proteica
9.
Nat Prod Rep ; 31(5): 676-93, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24691743

RESUMO

Covering: 1960 to 2013. Aristolochic acids are known for causing aristolochic acid nephropathy, a renal fibrosis often associated with urothelial carcinoma. Aristolochic acid I and II are considered to be the cause of these nephrotoxic and carcinogenic effects. However a variety of aristolochic acid analogues, including aristolactams and 4,5-dioxoaporphines have been reported. Their implications in aristolochic acid nephropathy have possibly been overlooked. In this report, in vivo and in vitro toxicity and mutagenicity of these three classes of compounds are discussed. Furthermore, the review gives an update of aristolochic acids, aristolactams and 4,5-dioxoaporphines reported between 2003 and 2013 and their biological activities.


Assuntos
Ácidos Aristolóquicos , Produtos Biológicos , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/isolamento & purificação , Ácidos Aristolóquicos/toxicidade , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/toxicidade , Humanos , Estrutura Molecular
10.
J Ethnopharmacol ; 149(1): 235-44, 2013 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-23806867

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Aristolochia are associated with aristolochic acid nephropathy (AAN), a renal interstitial fibrosis and upper urinary tract cancer (UUC). Aristolochic acid nephropathy has been reported in ten countries but its true incidence is unknown and most likely underestimated. By combining an ethnobotanical and phytochemical approach we provide evidence for the risk of AAN occurring in Bangladesh. More specifically, we assess the intra-specific variation of aristolochic acid analogues in medicinally used Aristolochia indica samples from Bangladesh. MATERIALS AND METHODS: Ethnobotanical information was collected from 16 kavirajes (traditional healers) in different study locations in Bangladesh. Plant samples were obtained from native habitats, botanical gardens, herbal markets and pharmaceutical companies. The samples were extracted using 70% methanol and were analysed using LC-DAD-MS and (1)H-NMR. RESULTS: Roots as well as leaves are commonly used for symptoms such as snake bites and sexual problems. Among the informants knowledge about toxicity or side effects is very limited and Aristolochia indica is often administered in very high doses. Replacement of Aristolochia indica with other medicinal plants such as Rauvolfia serpentina (L.) Benth. ex Kurz was common. Aristolochia indica samples contained a variety of aristolochic acid analogues such as aristolochic acid I, aristolochic acid II, cepharadione A and related compounds. CONCLUSIONS: AAN cases are likely to occur in Bangladesh and more awareness needs to be raised about the health risks associated with the use of Aristolochia indica and other species of Aristolochia as herbal medicines.


Assuntos
Aristolochia/química , Ácidos Aristolóquicos/toxicidade , Países em Desenvolvimento , Etnobotânica , Etnofarmacologia , Nefropatias/induzido quimicamente , Ácidos Aristolóquicos/isolamento & purificação , Ácidos Aristolóquicos/uso terapêutico , Bangladesh/epidemiologia , Humanos , Nefropatias/epidemiologia , Risco
11.
J Agric Food Chem ; 59(18): 10388-93, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-21842884

RESUMO

Identification of toxic or harmful agents continues to be a key goal in agricultural chemistry. This paper reports a metabolomic analysis of Ranunculus repens and related species, which were recently postulated to be cocausative agents in the etiology of equine grass sickness (EGS). Specifically, samples collected at EGS sites were compared with those from non-EGS sites. Furthermore, interspecific and seasonal variations and the species' response to edaphic and climatic factors were investigated. (1)H NMR spectroscopy in combination with multivariate data analysis was applied to the crude methanol extracts of the Ranunculus samples, as well as their chloroform fractions. Samples from EGS sites were significantly different from control samples. The metabolite composition varied greatly between different Ranunculus species. No significant changes could be observed between samples collected in different seasons. This work provides strong evidence that Ranunculus is involved in the etiology of EGS and has implications for agricultural management of pastures.


Assuntos
Doenças dos Cavalos/etiologia , Metabolômica , Ranunculus/metabolismo , Animais , Cavalos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Ranunculus/química , Estações do Ano , Reino Unido
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