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1.
Bol. latinoam. Caribe plantas med. aromát ; 18(4): 359-377, jul. 2019. tab
Artigo em Inglês | LILACS | ID: biblio-1008174

RESUMO

Plant species have been used for therapeutic purposes since ancient times and are still in use today since these products represent a source of raw material for the production of phytotherapeutic formulations. Screening and investigation of plants with pharmacological potential require the evaluation of characteristics related to their action, efficacy and safety in different steps. Among these steps, pre- clinical trials are used to evaluate the properties of the test product in in vitro experiments, such as cytotoxicity assays. Within this context, this study consists of a bibliometric analysis of some in vitro cytotoxicity and toxicity assays in erythrocytes used during bioprospecting of medicinal plants. The results demonstrated the wide application of erythrocytes to evaluate the biological effects of medicinal plant extracts. The methods were found to be valid and effective for the preliminary investigation of the in vitro cytotoxicity and toxicity of plant products.


El uso de especies vegetales para fines terapéuticos es una práctica histórica y todavía bastante actual, ya que estos productos pueden representar una fuente de materia prima para la producción de formulaciones fitoterápicas. En investigación de plantas con potencial farmacológico requiere la evaluación de su acción, eficacia y seguridad, a través de diferentes etapas. Entre estas, en los ensayos preclínicos se evalúan las propiedades del producto-prueba en experimentos in vitro, tales como ensayos de citotoxicidad, entre otros. En este aspecto, el presente estudio consiste en un análisis bibliométrico acerca de algunas pruebas de citotoxicidad y toxicidad in vitro en eritrocitos realizados en los ensayos de bioprospección de plantas medicinales. Los resultados evidencian la amplia utilización de eritrocitos para la evaluación de los efectos biológicos de extractos de plantas medicinales, apuntándolos como métodos válidos y eficaces para la investigación preliminar de la citotoxicidad y toxicidad in vitro de productos vegetales.


Assuntos
Bioensaio/métodos , Extratos Vegetais/toxicidade , Eritrócitos/efeitos dos fármacos , Antioxidantes/toxicidade , Fragilidade Osmótica , Estresse Oxidativo , Eritrócitos/citologia , Bioprospecção , Hemólise/efeitos dos fármacos
2.
Chem Commun (Camb) ; 55(50): 7211-7214, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31165808

RESUMO

A label-free and universal photosensitization colorimetric assay based on target-induced dsDNA termini switching has been developed for the analysis of nucleic acids, proteins, small molecules and metal ions. This strategy is highly sensitive and versatile, the principle of which will be desirable for various biosensor developments and applications.


Assuntos
Colorimetria/métodos , DNA/química , Metais/química , Ácidos Nucleicos/química , Proteínas/química , Bioensaio/métodos , Conformação de Ácido Nucleico , Conformação Proteica
3.
Anal Chim Acta ; 1074: 142-149, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159934

RESUMO

A simple proximity hybridization-induced on particle DNA walker was designed for ultrasensitive detection of proteins, for example platelet-derived growth factor (PDGF-BB) secreted by cancer cells, in which the DNA walker was activated by specific target binding and powered by an enzymatic cleavage to produce amplified signal. High-density FAM-labeled hairpin oligonucleotides (FAM-DNA1) were functionalized on AuNPs to construct three-dimensional (3D) DNA tracks. The specific binding of PDGF-BB with two aptamer probes (DNA3 and DNA4) led to the proximity hybridization-induced DNA displacement and the free of DNA walker (DNA2) to perform movement on the 3D tracks by an enzymatic cleavage, resulting in the release of massive FAM-DNA1 fragments from the AuNPs and the generation of fluorescent signal. This DNA walker based sensing strategy could detect PDGF-BB in a concentration range of 4 orders of magnitude with a detection limit down to sub-pM level. The practical applicability of the assay was demonstrated by detecting PDGF-BB secreted from MCF-7 cells with satisfactory results. The proposed DNA walker based assay could conveniently detect PDGF-BB with high sensitivity and good accuracy, along with the good extensibility of the assay, showing promise for practical diagnosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Becaplermina/análise , DNA/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Bioensaio/métodos , DNA/genética , Ouro/química , Humanos , Limite de Detecção , Células MCF-7 , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico
4.
J Microbiol Biotechnol ; 29(7): 1117-1123, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216609

RESUMO

Control of pine wilt disease, which is caused by pine wilt nematode, Bursaphelenchus xylophilus, is heavily dependent on the use of chemicals such as abamectin. Although such chemicals are highly effective, demands for alternatives that are derived preferentially from natural sources, are increasing out of environmental concerns. One of the challenges to discovery of alternative control agents is lack of fast and efficient screening method that can be used in high-throughput manner. Here we described the development of colorimetric assay for the rapid and accurate screening of candidate nematicidal compounds/biologics targeting B. xylophilus. Contrary to the conventional method, which relies on laborious visual inspection and counting of nematode population under microscope, our method utilizes a redox dye that changes its color in response to metabolic activity of nematode population in a given sample. In this work, we optimized parameters of our colorimetric assay including number of nematodes and amount of redox dye, and tested applicability of our assay for screening of chemicals and biologics. We demonstrated that our colorimetric assay can applied to rapid and accurate quantification of nematode viability/mortality in a nematode population treated with candidate chemicals/biologics. Application of our method would facilitate high-throughput endeavors aiming at finding environment-friendly control agents for deadly disease of pine trees.


Assuntos
Bioensaio/métodos , Nematoides/fisiologia , Pinus , Doenças das Plantas/parasitologia , Animais , Antinematódeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colorimetria , Indicadores e Reagentes/metabolismo , Nematoides/metabolismo , Oxazinas/metabolismo , Oxirredução , Tylenchida/metabolismo , Tylenchida/fisiologia , Xantenos/metabolismo
5.
Malar J ; 18(1): 153, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039788

RESUMO

BACKGROUND: Insecticide-treated net (ITN) durability, measured through physical integrity and bioefficacy, must be accurately assessed in order to plan the timely replacement of worn out nets and guide procurement of longer-lasting, cost-effective nets. World Health Organization (WHO) guidance advises that new intervention class ITNs be assessed 3 years after distribution, in experimental huts. In order to obtain information on whole-net efficacy cost-effectively and with adequate replication, a new bioassay, the Ifakara Ambient Chamber Test (I-ACT), a semi-field whole net assay baited with human host, was compared to established WHO durability testing methods. METHODS: Two experiments were conducted using pyrethroid-susceptible female adult Anopheles gambiae sensu stricto comparing bioefficacy of Olyset®, PermaNet® 2.0 and NetProtect® evaluated by I-ACT and WHO cone and tunnel tests. In total, 432 nets (144/brand) were evaluated using I-ACT and cone test. Olyset® nets (132/144) that did not meet the WHO cone test threshold criteria (≥ 80% mortality or ≥ 95% knockdown) were evaluated using tunnel tests with threshold criteria of ≥ 80% mortality or ≥ 90% feeding inhibition for WHO tunnel and I-ACT. Pass rate of nets tested by WHO combined standard WHO bioassays (cone/tunnel tests) was compared to pass in I-ACT only by net brand and time after distribution. RESULTS: Overall, more nets passed WHO threshold criteria when tested with I-ACT than with standard WHO bioassays 92% vs 69%, (OR: 4.1, 95% CI 3.5-4.7, p < 0.0001). The proportion of Olyset® nets that passed differed if WHO 2005 or WHO 2013 LN testing guidelines were followed: 77% vs 71%, respectively. Based on I-ACT results, PermaNet® 2.0 and NetProtect® demonstrated superior mortality and non-inferior feeding inhibition to Olyset® over 3 years of field use in Tanzania. CONCLUSION: Ifakara Ambient Chamber Test may have use for durability studies and non-inferiority testing of new ITN products. It measures composite bioefficacy and physical integrity with both mortality and feeding inhibition endpoints, using fewer mosquitoes than standard WHO bioassays (cone and tunnel tests). The I-ACT is a high-throughput assay to evaluate ITN products that work through either contact toxicity or feeding inhibition. I-ACT allows mosquitoes to interact with a host sleeping underneath a net as encountered in the field, without risk to human participants.


Assuntos
Bioensaio/métodos , Mosquiteiros Tratados com Inseticida/normas , Animais , Anopheles , Bioensaio/normas , Feminino , Humanos , Mosquiteiros Tratados com Inseticida/economia , Malária/prevenção & controle , Controle de Mosquitos/métodos , Piretrinas/farmacologia , Tanzânia , Organização Mundial da Saúde
6.
Exp Parasitol ; 202: 1-6, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077732

RESUMO

Neospora caninum is an apicomplexan parasite distributed worldwide. Although a positive association between the presence of birds and abortions in cattle associated to N. caninum has been reported, the role of the birds in the epidemiologic cycle of the parasite is unknown. To the best knowledge, no experimental studies have evaluated N. caninum in the eared dove, Zenaida auriculata. Therefore, we aimed to determine whether Z. auriculat can act as intermediate host for N. caninum. Eighteen birds were divided into four groups, G1, G2, G3, and G4 (control); G1, G2 and G3 received 2 × 106 tachyzoites of NC-1 strain via different routes: subcutaneous, intramuscular, and intraperitoneal, respectively. G4 composed of three birds. Serum samples were collected weekly, and one bird each from G1, G2 and G3 was euthanized on the 7th and 14th day post-inoculation (dpi). The remaining birds were euthanized after the 28th dpi. Tissues from the doves were evaluated using histopathological analysis, PCR and dog bioassay to detect the parasite. Dogs were fed with tissues from the birds and monitored for 30 days. Serum samples were collected weekly from the dogs for serological analysis, and feces samples were collected daily until the end of the experiment for coproparasitological examinations. No dove showed clinical signs of the infection; however, all of them seroconverted after the inoculation, with stronger immunological response in the G3 birds. The lung tissue of one G3 bird showed positive PCR results; it was euthanized on the 7th dpi, and an inflammatory infiltrate was observed in the lung and kidney from this dove. The dogs did not shed oocysts or seroconverted. Our results indicate that the intraperitoneal route induced infection in the doves; however, the parasite may have been eliminated by the host, and the doves may be resistant to chronic infection.


Assuntos
Doenças das Aves/parasitologia , Coccidiose/veterinária , Columbidae/parasitologia , Neospora/isolamento & purificação , Animais , Anticorpos Antiprotozoários/análise , Bioensaio/métodos , Bioensaio/veterinária , Coccidiose/parasitologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Cães , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunoglobulina G/análise , Imuno-Histoquímica/veterinária , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Músculo Esquelético/patologia , Neospora/genética , Neospora/imunologia , Reação em Cadeia da Polimerase/veterinária
7.
Sci Total Environ ; 679: 45-51, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31078774

RESUMO

One risk of growing Bacillus thuringiensis (Bt) crops is the potential nontarget effects which are likely related to the environmental behavior of crystal (Cry) toxins. Bt rice residues left in field after harvest constitute a main source of Cry toxins entering the environment. To our knowledge, very few studies have simultaneously evaluated the persistence of Cry toxins in Bt rice residues under field conditions using different methods. Here, we established a bioassay method with a target insect: the striped stem borer (SSB), Chilo suppressalis Walker. The reaction limit of the SSB to Cry toxins ranged from 5.4 to 12.7 ng g-1 in artificial diet, indicating that the detection limit of the bioassay ranged from 54 to 127 ng g-1 rice residues. A field decomposition experiment lasting for 210 d was conducted with the straw of two Bt rice lines transformed with either cry1Ab/1Ac or cry2A. Enzyme-linked immunosorbent assays (ELISAs) revealed that the Cry toxins in the Bt rice residues experienced rapid degradation to below 25% of the initial level in the first 42 d, and then decreased to below 100 ng g-1 rice residues within 100 to 140 d. Flooded conditions accelerated the degradation in the beginning compared with buried conditions. The Cry toxins were still detectable by ELISA, although at levels below 10 ng g-1 rice residues (<0.3% of the initial level) 210 d after harvest. However, the bioassay revealed that the SSB no longer had a significant reaction to Bt rice residues added into artificial diets 16 to 18 d after harvest under both conditions, which indicated that the level of bioactive Cry toxins had declined to below the detection limit. Our results suggest that ELISA overestimate the persistence of Cry toxins and that the potential risks mediated by Cry toxins may be much smaller than originally expected.


Assuntos
Bacillus thuringiensis/isolamento & purificação , Proteínas de Bactérias/análise , Toxinas Bacterianas/análise , Bioensaio/métodos , Monitoramento Ambiental/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Animais , China , Inseticidas/análise , Mariposas , Oryza/química , Controle Biológico de Vetores , Fatores de Tempo
8.
Planta Med ; 85(11-12): 925-933, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127604

RESUMO

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 ß 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 ß 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.


Assuntos
Fluorometria/métodos , Extratos Vegetais/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Alcaloides/farmacologia , Animais , Benzodioxóis/farmacologia , Bioensaio/métodos , Compostos de Bifenilo/farmacologia , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetulus , Indenos/farmacologia , Lignanas/farmacologia , Magnolia/química , Oócitos/metabolismo , Piper nigrum/química , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Sesquiterpenos/farmacologia , Valeriana/química , Xenopus laevis
9.
Methods Mol Biol ; 1966: 175-192, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041747

RESUMO

Nuclear receptors act as ligand-activated transcription factors translating ligand signals into changes in gene expression. The 48 members of the superfamily of nuclear receptors identified so far are involved amongst others in maintenance of metabolic balance, inflammation, and cancer. Thus, they hold enormous potential for drug discovery and some nuclear receptors are experiencing considerable academic and industrial interest. Nuclear receptor modulator discovery requires reliable, robust, and economic test systems that allow for high throughput. In this chapter, we discuss the principle, strengths, and advantages of hybrid reporter gene assays for nuclear receptor focused drug discovery and describe how they can be developed, established, and validated.


Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Genes Reporter , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular Transformada , Escherichia coli , Humanos , Ligantes
10.
Methods Mol Biol ; 1966: 193-202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041748

RESUMO

Here, we describe several assays to analyze the transcriptional activity of retinoic acid-related orphan receptors (RORs) and the effect of inverse agonists on their activity. One assay measures the effect of an inverse agonist on the transcriptional activation of a luciferase reporter by RORs in a Tet-On cell system. A mammalian two-hybrid assay analyzes the interaction of the ROR ligand binding domain with a coactivator peptide. Two additional assays examine the effect of an inverse agonist on the activation of a luciferase reporter under control of the promoter of the ROR target gene, IL17, and on ROR-mediated activation using a mammalian monohybrid assay.


Assuntos
Bioensaio/métodos , Genes Reporter , Receptores do Ácido Retinoico/metabolismo , Ativação Transcricional , Animais , Células CHO , Cricetulus/metabolismo , Receptores do Ácido Retinoico/agonistas , Tretinoína/metabolismo
11.
Pestic Biochem Physiol ; 156: 96-104, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027587

RESUMO

Aedes aegypti is a vector of viruses that negatively impact human health. Insecticide resistance complicates mosquito control efforts, but understanding the mechanisms of resistance can help to improve management practices. This study examined different factors that could influence the interpretation of toxicity bioassays and gene expression studies in A. aegypti, including sex and age, in the context of resistance to pyrethroids. Bioassays using a pyrethroid-resistant strain, Puerto Rico (PR), and a pyrethroid-susceptible strain, Rockefeller (Rock), of A. aegypti were conducted with females and males of three age groups to determine differences in mortality induced by deltamethrin. Overall, strain was the only factor with a significant effect on the LD50. Enzyme assays showed that cytochrome P450 monooxygenase activity in PR was constitutively higher than in Rock, and that pretreatment with the cytochrome P450 inhibitor piperonyl butoxide (PBO) followed by a topical application of deltamethrin (LD25) significantly increased mortality in both strains. Evaluation of the expression levels of seven CYP9J genes previously reported to be involved in pyrethroid resistance revealed that CYP9J10, CYP9J19, and CYP9J28 were more highly expressed in PR than in Rock at all ages of females and males, indicating that they may be essential for resistance. The expression of CYP9J24, CYP9J26, CYP9J27, and CYP9J32 was higher in PR males compared to other groups, including PR females. Significant differences in expression between sexes and strains were also observed as a result of age.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inseticidas/farmacologia , Nitrilos/farmacologia , Piretrinas/farmacologia , Aedes , Animais , Bioensaio/métodos , Feminino , Resistência a Inseticidas , Masculino , Porto Rico
12.
Toxicol Lett ; 311: 80-90, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029752

RESUMO

In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD. Moreover, we established sodium dodecyl sulfate (SDS) as an external positive control in this assay. To measure reduction in animal use to estimate the starting dose for the ATC method, we evaluated 10 chemicals representing Globally Harmonized System of Classification and Labeling of Chemicals (GHS) toxicity categories 1-5 and unclassified toxicity and determined the dose-response curves for percentage and area of cell differentiation by using the Hill function with an R2 ≥ 0.85. The resulting IC50 values were used for LD50 prediction and for estimating the starting dose for the ATC method. Our results indicated that use of the inhibition of adipogenesis assay to estimate the starting dose for the ATC method would decrease animal use for 7 out of 10 tested substances, possibly all substances if we consider the more toxic test substances in GHS categories 1, 2, and 3. We can conclude that the present assay is a suitable alternative to reduce animal testing in the first steps of predicting highly toxic substances. Moreover, this method also presents internal and external controls as differentials, which guarantee the quality of the assay as well as the results. These features are important for suggesting a methodology for regulatory purposes.


Assuntos
Adipogenia/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Alternativas aos Testes com Animais/métodos , Bioensaio/métodos , Células-Tronco/efeitos dos fármacos , Testes de Toxicidade Aguda/métodos , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Alternativas aos Testes com Animais/normas , Bioensaio/normas , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Dose Letal Mediana , Fenótipo , Reprodutibilidade dos Testes , Células-Tronco/imunologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Tempo , Testes de Toxicidade Aguda/normas
13.
Medicine (Baltimore) ; 98(14): e14972, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30946323

RESUMO

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) assays are widely used for complementary or companion diagnostic purposes during treatment with immune checkpoint inhibitors. However, limited information is available on the clinical reliability of the PD-L1 IHC assay using small biopsy samples.Participants included 46 patients with nonsmall cell lung cancer who underwent PD-L1 testing using 3 PD-L1 IHC assays (22C3, SP142, and SP263) for both small biopsy samples and surgical specimens from November 2017 to June 2018. The PD-L1 IHC assay results were analyzed with cut-off values of 1%, 5%, 10%, and 50%. The PD-L1 IHC results obtained from the surgical specimens were regarded as the reference values.The 22C3, SP142, and SP263 PD-L1 IHC assays were performed in 26 (57%), 20 (43%), and 46 (100%) patients, respectively. Biopsy methods included radial probe endobronchial ultrasound using a guide sheath, endobronchial ultrasound-guided transbronchial needle aspiration, bronchoscopic biopsy, and percutaneous needle aspiration in 26 (57%), 4 (9%), 12 (25%), and 4 (9%) patients, respectively. The 22C3, SP142, and SP263 PD-L1 assays had concordance rates of 73-96, 65-80, and 72%-91%, respectively, compared with the reference values.PD-L1 testing with 3 commercial PD-L1 IHC assays using small biopsy samples is reliable in patients with nonsmall cell lung cancer.


Assuntos
Bioensaio/métodos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Idoso , Biópsia , Biópsia por Agulha/métodos , Broncoscopia/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Testes Imunológicos/instrumentação , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Manejo de Espécimes/métodos
14.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965663

RESUMO

Small fish are an excellent experimental model to screen endocrine-disrupting compounds, but current fish-based assays to detect endocrine disruption have not been standardized yet, meaning that there is not consensus on endpoints and biomarkers to be measured. Moreover, exposure conditions may vary depending on the species used as the experimental model and the endocrine pathway evaluated. At present, a battery of a wide range of assays is usually needed for the complete assessment of endocrine activities. With the aim of providing a simple, robust, and fast assay to assess endocrine-disrupting potencies for the three major endocrine axes, i.e., estrogens, androgens, and thyroid, we propose the use of a panel of eight gene expression biomarkers in zebrafish larvae. This includes brain aromatase (cyp19a1b) and vitellogenin 1 (vtg1) for estrogens, cytosolic sulfotransferase 2 family 2 (sult2st3) and cytochrome P450 2k22 (cyp2k22) for androgens, and thyroid peroxidase (tpo), transthyretin (ttr), thyroid receptor α (trα), and iodothyronine deiodinase 2 (dio2) for thyroid metabolism. All of them were selected according to their responses after exposure to the natural ligands 17ß-estradiol, testosterone, and 3,3',5-triiodo-L-thyronine (T3), respectively, and subsequently validated using compounds reported as endocrine disruptors in previous studies. Cross-talk effects were also evaluated for all compounds.


Assuntos
Bioensaio/métodos , Disruptores Endócrinos/toxicidade , Transcriptoma/genética , Androgênios/análise , Animais , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Estrogênios/análise , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Peixe-Zebra
15.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970583

RESUMO

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.


Assuntos
Bioensaio/métodos , Hormônio do Crescimento Humano/análise , Fragmentos Fc das Imunoglobulinas/análise , Proteínas Recombinantes de Fusão/análise , Células HEK293 , Hormônio do Crescimento Humano/farmacocinética , Hormônio do Crescimento Humano/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia
16.
Methods Mol Biol ; 1947: 137-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969414

RESUMO

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Hidrazinas/química , Fragmentos de Peptídeos/química , Receptores Acoplados a Proteínas-G/metabolismo , Acilação , Fluorescência , Humanos , Ligantes , Fragmentos de Peptídeos/metabolismo , Receptores Acoplados a Proteínas-G/química
17.
Methods Mol Biol ; 1947: 151-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969415

RESUMO

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.


Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Transdução de Sinais
18.
Methods Mol Biol ; 1947: 257-267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969421

RESUMO

Intracellular signal transduced by G protein-coupled receptors (GPCRs) is tightly controlled by a guanine nucleotide-binding complex made of G protein Gα, Gß, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent ß-arrestin recruitment at GPCRs is universally accepted as the canonical interactor system and it has been found to be a powerful tracker of most GPCRs activation. Pharmacological concepts have evolved remarkably after the finding that different ligands, binding at the same receptor, can selectively activate specific subsets of signaling pathways among all pathways activated by balanced ligands. This new paradigm referred to as functional selectivity or biased signaling, has opened new avenues for the design of tailored drugs with enhanced therapeutic efficacies and reduced side effects. Here, we describe a unique platform for the interrogation of GPCR using a transcriptional-based assay to measure transient ß-arrestin recruitment called Tango.


Assuntos
Bioensaio/métodos , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Bleomicina/farmacologia , Células HEK293 , Humanos , Ligantes , Transdução de Sinais , Ativação Transcricional
19.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
20.
Chemosphere ; 227: 334-344, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999174

RESUMO

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.


Assuntos
Estrogênios/análise , Receptores Estrogênicos/efeitos dos fármacos , Animais , Compostos Benzidrílicos/farmacologia , Bioensaio/métodos , Linhagem Celular , Estrogênios/química , Feminino , Genisteína/farmacologia , Humanos , Ligantes , Fígado/citologia , Fenóis/farmacologia , Receptores Estrogênicos/antagonistas & inibidores , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
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