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1.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203212

RESUMO

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.


Assuntos
Genes Reporter/fisiologia , Isoflavonas/metabolismo , Luciferases de Renilla/metabolismo , Animais , Vaga-Lumes , Genes Reporter/genética , Isoflavonas/química , Luciferases de Renilla/química , Estrutura Secundária de Proteína
2.
Endocrinology ; 162(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33508122

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) activation is used in the treatment of diabetes and obesity; however, GLP-1 induces many other physiological effects with unclear mechanisms of action. To identify the cellular targets of GLP-1 and GLP-1 analogues, we generated a Glp1r.tdTomato reporter mouse expressing the reporter protein, tdTomato, in Glp1r-expressing cells. The reporter signal is expressed in all cells where GLP-1R promoter was ever active. To complement this, we histologically mapped tdTomato-fluorescence, and performed Glp-1r mRNA in situ hybridization and GLP-1R immunohistochemistry on the same tissues. In male mice, we found tdTomato signal in mucus neck, chief, and parietal cells of the stomach; Brunner's glands; small intestinal enteroendocrine cells and intraepithelial lymphocytes; and myenteric plexus nerve fibers throughout the gastrointestinal tract. Pancreatic acinar-, ß-, and δ cells, but rarely α cells, were tdTomato-positive, as were renal arteriolar smooth muscle cells; endothelial cells of the liver, portal vein, and endocardium; aortal tunica media; and lung type 1 and type 2 pneumocytes. Some thyroid follicular and parafollicular cells displayed tdTomato expression, as did tracheal cartilage chondrocytes, skin fibroblasts, and sublingual gland mucus cells. In conclusion, our reporter mouse is a powerful tool for mapping known and novel sites of GLP-1R expression in the mouse, thus enhancing our understanding of the many target cells and effects of GLP-1 and GLP-1R agonists.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Animais , Imunofluorescência , Expressão Gênica , Genes Reporter/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distribuição Tecidual
3.
Exp Cell Res ; 395(2): 112211, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755554

RESUMO

In this study, we noninvasively assessed whether M2-like macrophages accelerate the progression of ovarian cancer by performing molecular imaging of ovarian cancer cells expressing enhanced firefly luciferase (Effluc) in living mice. First, murine ovarian cancer ID8 cells expressing Effluc (ID8/Effluc cells) were established by retroviral infection. Subsequently, macrophages were isolated from the peritoneal exudate of mice injected with thioglycollate medium and differentiated into M2-like macrophages by adding interleukin 4. To characterize these M2-like macrophages, F4/80 and cluster of differentiation 206 expression levels were determined. Then, the M2-like macrophages were co-cultured with the ID8/Effluc cells and bioluminescence imaging (BLI) of signals from the ID8/Effluc cells was completed. Additionally, migration and wound healing were assessed to evaluate the effects of conditioned medium (CM) from M2-like macrophages on ID8/Effluc cell motility. In the in vivo study, mice were first given either liposome-phosphate-buffered saline or liposome-clodronate (lipo-clodronate). After 24 h, ID8/Effluc cells were intraperitoneally injected into the mice and BLI was completed at the designed time points. Next, histological analysis was conducted to characterize the infiltrated tumor. Flow cytometric analysis revealed high levels of CD206 expression in the differentiated M2-like macrophages. Meanwhile, ID8/Effluc cells co-cultured with these M2-like macrophages proliferated rapidly in an M2-like macrophage, number-dependent manner. The migration of the ID8/Effluc cells was also increased by the application of CM from M2-like macrophages. In vivo BLI revealed that the growth rate of intraperitoneally injected ovarian cancer cells was inhibited following macrophage depletion by treatment with lipo-clodronate. M2-like macrophages accelerated the progression of ovarian cancer, suggesting they are a new therapeutic target for ovarian cancer and that ovarian cancer could be managed by altering the nature of communication between ovarian cancer and macrophages.


Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Diferenciação Celular/fisiologia , Macrófagos/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Meios de Cultivo Condicionados/metabolismo , Feminino , Genes Reporter/genética , Genes Reporter/fisiologia , Humanos , Camundongos , Imagem Molecular/métodos , Neoplasias Ovarianas/patologia
4.
J Med Chem ; 63(15): 8179-8197, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32605372

RESUMO

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.


Assuntos
Vírus da Dengue/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/química , Replicação Viral/efeitos dos fármacos , Vírus do Nilo Ocidental/efeitos dos fármacos , Animais , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Relação Dose-Resposta a Droga , Genes Reporter/fisiologia , Células HeLa , Humanos , Células Vero , Replicação Viral/fisiologia , Vírus do Nilo Ocidental/fisiologia
5.
Viruses ; 12(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486283

RESUMO

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Genes Reporter/fisiologia , Vírus de RNA/efeitos dos fármacos , Replicon/fisiologia , Animais , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Vírus de RNA/genética , Transfecção , Células Vero
6.
Toxicol Lett ; 326: 23-30, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32109534

RESUMO

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter/efeitos dos fármacos , Proteínas de Fluorescência Verde/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/fisiologia , Linhagem Celular Tumoral/fisiologia , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde/fisiologia , Células HeLa/fisiologia , Humanos
7.
Br Poult Sci ; 60(6): 724-728, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31267766

RESUMO

1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs)transfection in vivo and a germline-specific promoter.2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB)were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2-3 or via blood vessel at HH stage 13-14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13-14 were significantly higher than that at HH stage 2-3.3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13-14 may be a more efficient route for PGCs transfection in vivo.


Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Células Germinativas/fisiologia , Proteínas Nucleares/genética , Transfecção/veterinária , Animais , Embrião de Galinha , Elementos de DNA Transponíveis/genética , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética
8.
Food Chem Toxicol ; 130: 68-78, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31082460

RESUMO

The mycotoxins zearalenone (ZEN) and alpha-zearalenone (α-ZOL), which are common contaminants of agri-food products, are known for their oestrogenic potential. In addition to mycotoxins, food may also contain pesticides with oestrogenic properties such as 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (p,p'-DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE), raising the question on the potential effects of individual and combinations of these xeno-oestrogens on the action of natural oestrogens. The present study employed a mammalian reporter gene assay to assess the effects individual and binary combinations of these environmental and food-borne contaminants on oestrogen nuclear receptor (ER) transactivation. As expected, α-ZOL and ZEN exhibited the strongest oestrogenic potency (EC50: 0.27 ±â€¯0.121 nM and 1.32 ±â€¯0.0956 nM, respectively) whereas p,p'-DDT and p,p'-DDE had weak ER agonistic activity with the maximal response of 28.70 ±â€¯2.97% and 18.65 ±â€¯1.77%, respectively. Concurrent treatment of the mycotoxins and/or pesticides, individually or in binary combination, with 17ß-oestradiol (E2) showed either additive, synergistic or antagonistic interactive effects on E2-mediated ER response, depending on the combination ratios, the concentration range of xeno-oestrogens, and the concentration of E2. This study highlights the importance of assessing the mixture effects of chemical contaminants in risk assessment, especially in the area of reproductive and developmental toxicity.


Assuntos
Genes Reporter/fisiologia , Hidrocarbonetos Clorados/toxicidade , Micotoxinas/toxicidade , Praguicidas/toxicidade , Ativação Transcricional/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Estrogênio/metabolismo
9.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30541830

RESUMO

Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).IMPORTANCE Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.


Assuntos
Genes Reporter/genética , Rotavirus/genética , Rotavirus/metabolismo , Linhagem Celular , Expressão Gênica/genética , Técnicas de Transferência de Genes , Genes Reporter/fisiologia , Luciferases/genética , Plasmídeos , Infecções por Rotavirus/virologia , Replicação Viral/genética
10.
Ann Am Thorac Soc ; 15(Suppl 3): S149-S153, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30431351

RESUMO

Dysregulation of gel-forming mucins is associated with many airway diseases. Better knowledge of the pathophysiological mechanisms linking mucins and respiratory diseases will advance the understanding of their pathogenesis and should provide opportunities to develop new therapeutic compounds for treatment. MUC5B and MUC5AC are the two main gel-forming mucins in the respiratory tract. The organization in domains and the expression profile of mouse Muc5b are very similar to those in humans, which makes the mouse a relevant model for studies of the translational activities of human mucins. To assess the in vivo biological functions of Muc5b, a mouse reporter tagged in frame with the green fluorescent protein marker has been engineered by homologous recombination. The proof of concept that this reporter model may be informative for translational studies was confirmed by the finding that interleukin-13 administration in living mice upregulated Muc5b production.


Assuntos
Modelos Animais de Doenças , Pneumopatias/etiologia , Mucina-5B/fisiologia , Animais , Genes Reporter/fisiologia , Interleucina-13/fisiologia , Pneumopatias/diagnóstico , Pneumopatias/terapia , Camundongos , Camundongos Transgênicos
11.
Int J Parasitol ; 48(13): 1023-1033, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30266591

RESUMO

Here we investigated the gene of a transforming growth factor (TGF)-ß type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-ß family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-ß type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-ß type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.


Assuntos
Haemonchus/crescimento & desenvolvimento , Receptor do Fator de Crescimento Transformador beta Tipo I/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Clonagem Molecular , Biologia Computacional , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Feminino , Regulação da Expressão Gênica , Genes Reporter/fisiologia , Teste de Complementação Genética , Cabras , Haemonchus/genética , Haemonchus/fisiologia , Masculino , Conformação Molecular , Filogenia , Pirazóis/farmacologia , Quinolinas/farmacologia , RNA de Helmintos/isolamento & purificação , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/química , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transdução de Sinais , Organismos Livres de Patógenos Específicos
12.
Am J Pathol ; 188(10): 2155-2163, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30031726

RESUMO

ß-Catenin-dependent Wnt signaling controls numerous aspects of skeletal development and postnatal bone repair. Currently available transgenic Wnt reporter mice allow for visualization of global canonical Wnt signaling activity within skeletal tissues, without delineation of cell type. This is particularly important in a bone repair context, in which the inflammatory phase can obscure the visualization of mesenchymal cell types of interest. To tackle the issue of tissue-specific Wnt signaling, we have generated and characterized a transgenic mouse strain [termed paired related homeobox 1 (Prx1)-Wnt-green fluorescent protein (GFP), by crossing a previously validated Prx1-Cre strain with a nuclear fluorescent reporter driven by T-cell factor/lymphoid enhancer factor activity (Rosa26-Tcf/Lef-LSL-H2B-GFP)]. Prx1-Wnt-GFP animals were subject to three models of long bone and membranous bone repair (displaced forelimb fracture, tibial cortical defect, and frontal bone defect). Results showed that, irrespective of bone type, locoregional mesenchymal cell activation of Wnt signaling occurs in a defined temporospatial pattern among Prx1-Wnt-GFP mice. In summary, Prx1-Wnt-GFP reporter animals allow for improved visualization, spatial discrimination, and facile quantification of Wnt-activated mesenchymal cells within models of adult bone repair.


Assuntos
Consolidação da Fratura/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Ossos da Extremidade Superior/fisiologia , Feminino , Osso Frontal/fisiologia , Genes Reporter/fisiologia , Proteínas de Homeodomínio/fisiologia , Masculino , Mesoderma/citologia , Camundongos Transgênicos , Osteogênese/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/fisiologia , Microtomografia por Raio-X
13.
J Mol Cell Cardiol ; 120: 31-41, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29777691

RESUMO

The therapeutic potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is limited by immature functional features including low impulse propagation and reduced cell excitability. Key players regulating electrical activity are voltage-gated Na+ channels (Nav1.5) and gap junctions built from connexin-43 (Cx43). Here we tested the hypothesis that enhanced Cx43 expression increases intercellular coupling and influences excitability by modulating Nav1.5. Using transgenic approaches, Cx43 and Nav1.5 localization and cell coupling were studied by confocal imaging. Nav1.5 currents and action potentials (APs) were measured using the patch-clamp technique. Enhanced sarcolemmal Cx43 expression significantly improved intercellular coupling and accelerated dye transfer kinetics. Furthermore, Cx43 modulated Nav1.5 function leading to significantly higher current and enhanced AP upstroke velocities, thereby improving electrical activity as measured by microelectrode arrays. These findings suggest a mechanistic link between cell coupling and excitability controlled by Cx43 expression in iPSC-CMs. Therefore, we propose Cx43 as novel molecular target for improving electrical properties of iPSC-CMs to match the functional properties of native myocytes.


Assuntos
Potenciais de Ação/fisiologia , Conexina 43/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Estimulação Elétrica , Imunofluorescência , Junções Comunicantes/metabolismo , Genes Reporter/fisiologia , Camundongos , Microscopia Confocal , Técnicas de Patch-Clamp , Plasmídeos , Sarcolema/metabolismo , Transdução Genética , Transfecção
14.
Mol Pharmacol ; 94(1): 700-712, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29735582

RESUMO

Levothyroxine replacement therapy forms the cornerstone of hypothyroidism management. Variability in levothyroxine oral absorption may contribute to the well-recognized large interpatient differences in required dose. Moreover, levothyroxine-drug pharmacokinetic interactions are thought to be caused by altered oral bioavailability. Interestingly, little is known regarding the mechanisms contributing to levothyroxine absorption in the gastrointestinal tract. Here, we aimed to determine whether the intestinal drug uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1) may be involved in facilitating intestinal absorption of thyroid hormones. We also explored whether thyroid hormones regulate OATP2B1 gene expression. In cultured Madin-Darby Canine Kidney II/OATP2B1 cells and in OATP2B1-transfected Caco-2 cells, thyroid hormones were found to inhibit OATP2B1-mediated uptake of estrone-3-sulfate. Competitive counter-flow experiments evaluating the influence on the cellular accumulation of estrone-3-sulfate in the steady state indicated that thyroid hormones were substrates of OATP2B1. Additional evidence that thyroid hormones were OATP2B1 substrates was provided by OATP2B1-dependent stimulation of thyroid hormone receptor activation in cell-based reporter assays. Bidirectional transport studies in intestinal Caco-2 cells showed net absorptive flux of thyroid hormones, which was attenuated by the presence of the OATP2B1 inhibitor, atorvastatin. In intestinal Caco-2 and LS180 cells, but not in liver Huh-7 or HepG2 cells, OATP2B1 expression was induced by treatment with thyroid hormones. Reporter gene assays revealed thyroid hormone receptor α-mediated transactivation of the SLCO2B1 1b and the SLCO2B1 1e promoters. We conclude that thyroid hormones are substrates and transcriptional regulators of OATP2B1. These insights provide a potential mechanistic basis for oral levothyroxine dose variability and drug interactions.


Assuntos
Transportadores de Ânions Orgânicos/metabolismo , Hormônios Tireóideos/metabolismo , Transcrição Genética/fisiologia , Animais , Atorvastatina/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Cães , Interações Medicamentosas/fisiologia , Estrona/análogos & derivados , Estrona/farmacologia , Genes Reporter/efeitos dos fármacos , Genes Reporter/fisiologia , Células HeLa , Células Hep G2 , Humanos , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , Células Madin Darby de Rim Canino , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Transcrição Genética/efeitos dos fármacos
15.
Artigo em Inglês | MEDLINE | ID: mdl-28861396

RESUMO

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Porinas/metabolismo , Regulon/genética , Regulon/fisiologia , Proteínas Repressoras/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano , Detergentes/farmacologia , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Genes Reporter/genética , Genes Reporter/fisiologia , Testes de Sensibilidade Microbiana , Óperon/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plasmídeos/genética , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Porinas/genética , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Deleção de Sequência , Transcrição Genética , Yersinia enterocolitica/genética , beta-Galactosidase/metabolismo
16.
Biochem Biophys Res Commun ; 487(2): 241-246, 2017 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-28412349

RESUMO

Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies.


Assuntos
Avena/metabolismo , Luz , Medições Luminescentes/métodos , Fototropinas/metabolismo , Proteólise/efeitos da radiação , Relação Dose-Resposta à Radiação , Genes Reporter/fisiologia , Células HEK293 , Humanos , Doses de Radiação
17.
J Gerontol A Biol Sci Med Sci ; 72(8): 1033-1037, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28369388

RESUMO

Animals, particularly poikilotherms, exhibit distinct physiologies at different environmental temperatures. Here, we hypothesized that temperature-based differences in physiology could affect the amount of variation in complex quantitative traits. Specifically, we examined, in Caenorhabditis elegans, how different temperatures (15°C, 20°C, and 25°C) affected the amount of interindividual variation in life span and also expression of three reporter genes-transcriptional reporters for vit-2, gpd-2, and hsp-16.2 (a life-span biomarker). We found the expected inverse relationship between temperature and average life span. Surprisingly, we found that at the highest temperature, there were fewer differences between individuals in life span and less interindividual variation in expression of all three reporters. We suggest that growth at 25°C might canalize (reduce interindividual differences in) life span and expression of some genes by eliciting a small constitutive heat shock response. Growth at 25°C requires wild-type hsf-1, which encodes the main heat shock response transcriptional activator. We speculate that increased chaperone activity at 25°C may reduce interindividual variation in gene expression by increasing protein folding efficiency. We hypothesize that reduced variation in gene expression may ultimately cause reduced variation in life span.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Expressão Gênica/fisiologia , Genes Reporter/fisiologia , Chaperonas Moleculares/fisiologia , Temperatura , Fatores de Transcrição/genética , Análise de Variância , Animais , Caenorhabditis elegans , Meio Ambiente , Interação Gene-Ambiente , Marcadores Genéticos/fisiologia , Longevidade/genética , Termotolerância/fisiologia
18.
Cytometry A ; 91(7): 721-729, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28375558

RESUMO

Flow cytometry is a powerful multiparametric technology, widely used for the identification, quantification, and isolation of defined populations of cells based on the expression of target proteins. It also allows for the use of surrogate reporters, either enzymatic or fluorescent, to indirectly monitor the expression of these target proteins. In this work, we optimised the dissociation protocol for the detection of the enzymatic reporter LacZ using the FACS-Gal detection system with the fluorogenic substrate FDG to compare cis- versus trans-positioned reporters efficiency. Particularly, for the FACS-Gal optimization, we studied lung and haematopoietic tissues, focusing on cell recovery, viability, FDG loading conditions and distribution of cellular populations. Reporter genes such as LacZ can be placed together with the gene of interest in the same polycistronic mRNA (in cis), or in independent alleles (in trans), which can strongly affect the correlation with the reporter readout. To address this issue, we generated a mouse model containing both types of reporters for the same gene, and compared them. Our results clearly indicate that trans-positioned reporters can be misleading, and that using a reporter gene in cis rather than trans is a much more specific method to sort for cells undergoing Cre-mediated recombination. © 2017 International Society for Advancement of Cytometry.


Assuntos
Citometria de Fluxo , Expressão Gênica/fisiologia , Genes Reporter/fisiologia , Animais , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Supressoras da Sinalização de Citocina/análise
19.
IEEE Trans Biomed Eng ; 64(3): 557-568, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28221990

RESUMO

OBJECTIVE: The ability to record and to control action potential firing in neuronal circuits is critical to understand how the brain functions. The objective of this study is to develop a monolithic integrated circuit (IC) to record action potentials and simultaneously control action potential firing using optogenetics. METHODS: A low-noise and high input impedance (or low input capacitance) neural recording amplifier is combined with a high current laser/light-emitting diode (LED) driver in a single IC. RESULTS: The low input capacitance of the amplifier (9.7 pF) was achieved by adding a dedicated unity gain stage optimized for high impedance metal electrodes. The input referred noise of the amplifier is [Formula: see text], which is lower than the estimated thermal noise of the metal electrode. Thus, the action potentials originating from a single neuron can be recorded with a signal-to-noise ratio of at least 6.6. The LED/laser current driver delivers a maximum current of 330 mA, which is adequate for optogenetic control. The functionality of the IC was tested with an anesthetized Mongolian gerbil and auditory stimulated action potentials were recorded from the inferior colliculus. Spontaneous firings of fifth (trigeminal) nerve fibers were also inhibited using the optogenetic protein Halorhodopsin. Moreover, a noise model of the system was derived to guide the design. SIGNIFICANCE: A single IC to measure and control action potentials using optogenetic proteins is realized so that more complicated behavioral neuroscience research and the translational neural disorder treatments become possible in the future.


Assuntos
Potenciais de Ação/fisiologia , Eletrodos Implantados , Neurônios/fisiologia , Optogenética/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Imagens com Corantes Sensíveis à Voltagem/instrumentação , Amplificadores Eletrônicos , Animais , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Genes Reporter/fisiologia , Gerbillinae , Iluminação/instrumentação , Fibras Ópticas , Optogenética/métodos , Reprodutibilidade dos Testes , Semicondutores , Sensibilidade e Especificidade , Razão Sinal-Ruído , Integração de Sistemas , Imagens com Corantes Sensíveis à Voltagem/métodos
20.
J Vis Exp ; (120)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28190076

RESUMO

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.


Assuntos
Genes Reporter/fisiologia , Vírus da Hepatite B/fisiologia , Replicação Viral/fisiologia , DNA Viral , Genoma Viral/fisiologia , Células Hep G2 , Vírus da Hepatite B/crescimento & desenvolvimento , Humanos , Transfecção , Vírion/fisiologia
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