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1.
Mem Inst Oswaldo Cruz ; 114: e180593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31433004

RESUMO

BACKGROUND: Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES: Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS: We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS: Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in ß-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION: The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Miócitos Cardíacos/parasitologia , Paxilina/metabolismo , Talina/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Mecanotransdução Celular/fisiologia , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
2.
Exp Parasitol ; 204: 107723, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299265

RESUMO

Toxoplasmosis, caused by apicomplexan parasite Toxoplasma gondii, is a common food-borne disease in humans. Undercooked meat is a potential source of T. gondii infection. As meat of chicken or rabbit is consumed worldwide, tools such as ELISA for the detection of infection of this parasite in rabbits and chickens are much-needed. To search diagnostic antigens of T. gondii special for rabbits and chickens, we conducted two dimensional electrophoresis (2-DE), Western blotting and mass spectrometry (MS) with T. gondii tachyzoite proteins. When probed with rabbit or chicken anti-T. gondii sera, about 60 positive spots among over 500 visible protein spots were detected. In subsequent mass spectrometric analysis, microneme 4 (MIC4) and a putative rhoptry protein are of diagnositic value among the 13 spots selectively picked from the equivalent gel. This study encourages further validation of these candidate antigens for the development of immunologic tools for the detection of T. gondii infection in chickens and rabbits.


Assuntos
Antígenos de Protozoários/análise , Parasitologia de Alimentos , Carne/parasitologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/diagnóstico , Animais , Western Blotting , Galinhas , Biologia Computacional , Soros Imunes/imunologia , Immunoblotting , Proteínas de Membrana/imunologia , Distribuição Normal , Coelhos , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Toxoplasmose Animal/imunologia , Eletroforese em Gel Diferencial Bidimensional
3.
J Med Microbiol ; 68(9): 1341-1352, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355743

RESUMO

Introduction. Timely detection of invasive aspergillosis (IA) caused by fungal pathogens, i.e. Aspergillus fumigatus and Aspergillus flavus, in immunocompromised patients is crucial in preventing high mortality.Aim. To develop a simple immunoassay for the detection of galactomannan (GM), an IA biomarker.Methodology. GM from A. fumigatus and A. flavus clinical strains was purified and characterized by X-ray diffraction, IR spectroscopy and 13C/1H nuclear magnetic resonance (NMR) for polyclonal antibody (pAb) production in rabbits. An enzyme-linked immunosorbent assay (ELISA) was standardized using concanavalin A to capture Aspergillus GM and pAbs to detect it. Gold nanoparticles (AuNPs) were synthesized and conjugated to pAbs for the development of a dot-blot immunoassay. The developed dot-blot was evaluated with 109 clinical serum and bronchoalveolar lavage samples.Results. Spectroscopy studies characterized the d-galactofuranosyl groups of GM responsible for the immune response and generation of pAbs. The ELISA employing pAbs showed a sensitivity of 1 ng ml-1 for Aspergillus GM. Furthermore, a sensitive, visual, rapid dot-blot assay developed by the conjugation of pAbs to AuNPs (~24±5 nm size, -36±2 mV zeta potential) had a detection limit of 1 pg ml-1 in serum. The pAbs interacted with Aspergillus spp. but did not cross-react with other fungal pathogen genera such as Penicillium and Candida. Evaluation of the dot-blot with 109 clinical samples showed high sensitivity (80 %) and specificity (93.2 %), with an overall assay accuracy of 89%.Conclusion. The developed nano-gold immunodiagnostic assay has immense potential for practical use in rapid, specific and sensitive on-site diagnosis of IA, even under resource-limited settings.


Assuntos
Aspergilose/diagnóstico , Ouro/química , Testes Imunológicos/métodos , Nanopartículas Metálicas/química , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Antígenos de Fungos/sangue , Antígenos de Fungos/imunologia , Aspergillus/imunologia , Aspergillus/isolamento & purificação , Aspergillus flavus/imunologia , Aspergillus flavus/isolamento & purificação , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/isolamento & purificação , Humanos , Immunoblotting , Mananas/análise , Mananas/imunologia , Testes Imediatos , Coelhos , Sensibilidade e Especificidade
4.
Rev Bras Parasitol Vet ; 28(2): 298-302, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31188947

RESUMO

The aim of this study was to determine the prevalence of infection by Sarcocystis neurona in horses and identify potential risk factors. Were analyzed 427 samples from 36 farms in 21 municipalities in the Alagoas State, Brazil. Presence of anti-S. neurona antibodies was diagnosed by indirect immunofluorescence antibody test (IFAT) and was confirmed using the immunoblot test. Risk factors were assessed through investigative questionnaires on animal management on the farms. The prevalence of anti-S.neurona antibodies was 2.8% (confidence interval, CI: 1.5-4.9%) from IFAT and 1.6% (CI:0.8-3.34%) from immunoblot, and there were positive horses on 16.6% of the studied farms. None of the variables studied presented associations with serological status for S. neurona. This is the first report on infection by S. neurona in horses reared in Alagoas, Brazil showing a low exposure to S. neurona in this region, but with significant numbers of foci.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças dos Cavalos/epidemiologia , Sarcocystis/imunologia , Sarcocistose/veterinária , Animais , Brasil/epidemiologia , Estudos Transversais , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Doenças dos Cavalos/diagnóstico , Cavalos , Immunoblotting , Masculino , Prevalência , Fatores de Risco , Sarcocistose/diagnóstico , Sarcocistose/epidemiologia , Estudos Soroepidemiológicos
5.
Rev Soc Bras Med Trop ; 52: e20180465, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31038622

RESUMO

INTRODUCTION: We evaluated the anti-hepatitis E virus (HEV) antibody prevalence and HEV-RNA in archived serum samples of non-A-C hepatitis, or suspected cases of HEV infection from the Eastern Brazilian Amazon from 1993 to 2014. METHODS: Serum samples (n = 318) were tested using ELISA and immunoblotting, and screened for HEV-RNA by RT-qPCR. RESULTS: Anti-HEV IgM and IgG were detected in 3.4% (11/318) and 5.9% (19/318) of the samples, respectively. All samples were HEV-RNA negative. CONCLUSIONS: HEV was detected at a low prevalence. Broader serological and molecular evaluation of HEV infection in the Amazon region should be carried out.


Assuntos
Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/imunologia , Hepatite E/epidemiologia , Brasil , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite E/diagnóstico , Humanos , Immunoblotting , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Prevalência , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Estudos Soroepidemiológicos
6.
Nat Plants ; 5(5): 505-511, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036912

RESUMO

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Troca/genética , Engenharia de Proteínas , Transgenes/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética
7.
Graefes Arch Clin Exp Ophthalmol ; 257(8): 1751-1758, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31065846

RESUMO

PURPOSE: To compare the clinical characteristics of Vogt-Koyanagi-Harada (VKH) disease patients with and without anti-retinal antibodies (ARAs) that are frequently detected in autoimmune retinopathy. METHODS: Using immunoblot analyses, serum autoantibodies for recoverin, carbonic anhydrase II, and α-enolase were examined in 20 treatment-naïve patients with VKH disease. Clinical factors before and after systemic corticosteroid therapy, including best-corrected visual acuity (BCVA) and macular outer retinal morphology, were statistically compared between patients with VKH disease with and without ARAs. RESULTS: Serum ARAs were detected in 50.0% of patients with VKH disease. There were no significant differences in clinical factors between the two groups, including final BCVA, frequency of uveitis recurrence, and recovery of the macular ellipsoid zone after systemic corticosteroid therapy. CONCLUSIONS: Our results suggest that the detected ARAs did not influence visual outcomes, the chronicity of uveitis, or outer retinal morphology in patients with VKH disease.


Assuntos
Autoanticorpos/imunologia , Retina/imunologia , Síndrome Uveomeningoencefálica/imunologia , Acuidade Visual , Adolescente , Adulto , Autoanticorpos/sangue , Anidrase Carbônica II/sangue , Anidrase Carbônica II/imunologia , Criança , Feminino , Glucocorticoides/uso terapêutico , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Fosfopiruvato Hidratase/sangue , Fosfopiruvato Hidratase/imunologia , Prognóstico , Recoverina/sangue , Recoverina/imunologia , Retina/diagnóstico por imagem , Estudos Retrospectivos , Tomografia de Coerência Óptica , Síndrome Uveomeningoencefálica/tratamento farmacológico , Adulto Jovem
8.
Invest Ophthalmol Vis Sci ; 60(6): 1943-1952, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31050722

RESUMO

Purpose: Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods: The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results: Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-ß, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions: These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.


Assuntos
Neovascularização de Coroide/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Neovascularização Retiniana/tratamento farmacológico , Vasos Retinianos/metabolismo , Triterpenos/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Angiofluoresceinografia , Fundo de Olho , Humanos , Immunoblotting , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Tomografia de Coerência Óptica
9.
Methods Mol Biol ; 1966: 7-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041735

RESUMO

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays a central role in the response and adaptation to environmental and nutritional stimuli by initiating tissue-specific transcriptional reprogramming. Since its discovery in 1998, the field of PGC-1α biology has grown exponentially and a large body of research has elucidated the diverse roles of PGC-1α in brown adipose tissue thermogenesis, fatty acid oxidation, muscle fiber type switching, hepatic gluconeogenesis, and circadian clock regulation, etc. In addition, recent research has identified a splice variant(s) of PGC-1α in humans and rodents. The common misconception relating to PGC-1α is that it migrates at a predicted molecular weight of ~90 kDa by SDS-PAGE gel electrophoresis. However, several recent studies have provided solid evidence that the biologically relevant molecular weight of PGC-1α is ~110 kDa. In this chapter, we describe an optimized immunoblotting protocol that is developed to detect the low abundance protein PGC-1α and its alternatively spliced isoform named NT-PGC-1α in various rodent tissues. We also describe an optimized immunoprecipitation protocol that can isolate and concentrate endogenous PGC-1α and NT-PGC-1α. The protocols presented here will hopefully allow investigators to report accurate and reliable data regarding PGC-1α isoforms.


Assuntos
Immunoblotting/métodos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/análise , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Processamento Alternativo , Animais , Camundongos , Isoformas de Proteínas/análise , Ratos , Roedores/metabolismo
10.
Exp Parasitol ; 200: 92-98, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30991039

RESUMO

Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Antígenos de Helmintos/imunologia , Brugia Malayi/imunologia , Filariose/imunologia , Proteínas de Helminto/imunologia , Soros Imunes/imunologia , Animais , Biologia Computacional , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Filariose/sangue , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Immunoblotting , Proteínas de Filamentos Intermediários/imunologia , Microfilárias/imunologia , Fosfoglicerato Mutase/imunologia , Fosfopiruvato Hidratase/imunologia , Proteômica/métodos
11.
Int J Mol Med ; 43(6): 2267-2278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017262

RESUMO

Among a number of mRNA modifications, N6­methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear­replicating viruses. m6A has a significant role in numerous cancer types, including leukemia, brain tumors, liver cancer, breast cancer and lung cancer. Although m6A methyltransferases are essential during RNA modifications, the biological functions of m6A and the underlying mechanisms remain to be fully elucidated, predominantly due to the limited detection methods for m6A. In the present review, the currently available m6A detection methods and the respective scope of their applications are presented to facilitate the further investigation of the roles of m6A in biological process.


Assuntos
Adenosina/análogos & derivados , RNA/química , Adenosina/análise , Adenosina/genética , Animais , Técnicas Biossensoriais/métodos , Northern Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Metilação , Neoplasias/genética , RNA/genética , Análise de Sequência de RNA/métodos
12.
Int Arch Allergy Immunol ; 179(3): 165-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30970365

RESUMO

BACKGROUND: Artemisia pollens have a high potential to induce allergic symptoms. Seven allergen components have been identified, but only Art v 7 has been localized in the pollen grain. This study aimed to localize the allergens in the pollen grains of 4 Artemisia spp. METHODS: Pollen extracts from 2 Chinese Artemisia spp., A. argyi and A. annua, were used to immunize BALB/c mice. Recombinant Art v 1 and Art v 3 allergens were used to select specific monoclonal antibodies (mAbs). Three mAbs were used to purify the natural allergens and were then analyzed by mass spectrometry. As reported previously, polyclonal antibodies were obtained from rabbits immunized with 3 synthesized peptides of Art an 7. Using conventional histology procedures with pollens from 4 Artemisia spp. (A. argyi, A. annua, A. capilaris, and A. sieversiana), allergen images were observed and recorded by fluorescence and confocal laser microscopy. RESULTS: We obtained 2 specific mAbs against Art v 1, 1 against Art v 2, and 4 against Art v 3 homologs. The Art v 1 and Art v 3 homologs were mainly located on the pollen walls, and the Art v 7 homologous protein was localized intracellularly around nuclei. The location of the Art v 2 homologous protein varied across species, being intracellular around nuclei for A. annua and A. argyi, and in both the pollen wall and around nuclei for A. capilaris and A. sieversiana. CONCLUSIONS: Four mugwort allergens were localized in the pollen, and the major Art v 1 and Art v 3 allergens were located mainly in the pollen wall.


Assuntos
Alérgenos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Plantas/imunologia , Artemisia/imunologia , Pólen/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Immunoblotting
13.
Invest Ophthalmol Vis Sci ; 60(5): 1538-1546, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30994864

RESUMO

Purpose: Recent evidence suggests that retinal photoreceptor cells have an important role in the pathogenesis of retinal microvascular lesions in diabetes. We investigated the role of rod cell phototransduction on the pathogenesis of early diabetic retinopathy (DR) using Gnat1-/- mice (which causes permanent inhibition of phototransduction in rod cells without degeneration). Methods: Retinal thickness, oxidative stress, expression of inflammatory proteins, electroretinograms (ERG) and optokinetic responses, and capillary permeability and degeneration were evaluated at up to 8 months of diabetes. Results: The diabetes-induced degeneration of retinal capillaries was significantly inhibited in the Gnat1-/- diabetics. The effect of the Gnat1 deletion on the diabetes-induced increase in permeability showed a nonuniform accumulation of albumin in the neural retina; the defect was inhibited in diabetic Gnat1-/- mice in the inner plexiform layer (IPL), but neither in the outer plexiform (OPL) nor inner nuclear (INL) layers. In Gnat1-deficient animals, the diabetes-induced increase in expression of inflammatory associated proteins (iNOS and ICAM-1, and phosphorylation of IĸB) in the retina, and the leukocyte mediated killing of retinal endothelial cells were inhibited, however the diabetes-mediated induction of oxidative stress was not inhibited. Conclusions: In conclusion, deletion of transducin1 (and the resulting inhibition of phototransduction in rod cells) inhibits the development of retinal vascular pathology in early DR.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/fisiopatologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Deleção de Genes , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transducina/genética , Visão Ocular/fisiologia , Animais , Permeabilidade Capilar , Retinopatia Diabética/metabolismo , Eletrorretinografia , Proteínas I-kappa B/metabolismo , Immunoblotting , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Óxido Nítrico Sintase Tipo II/metabolismo , Nistagmo Optocinético/fisiologia , Estresse Oxidativo , Fosforilação , Vasos Retinianos/patologia , Estreptozocina , Tomografia de Coerência Óptica
14.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987270

RESUMO

Acute lung injury (ALI) is a life-threatening syndrome characterized by acute and severe hypoxemic respiratory failure. Visfatin, which is known as an obesity-related cytokine with pro-inflammatory activities, plays a role in regulation of inflammatory cytokines. The mechanisms of ALI remain unclear in critically ill patients. Survival in ALI patients appear to be influenced by the stress generated by mechanical ventilation and by ALI-associated factors that initiate the inflammatory response. The objective for this study was to understand the mechanisms of how visfatin regulates inflammatory cytokines and promotes ALI. The expression of visfatin was evaluated in ALI patients and mouse sepsis models. Moreover, the underlying mechanisms were investigated using human bronchial epithelial cell lines, BEAS-2B and NL-20. An increase of serum visfatin was discovered in ALI patients compared to normal controls. Results from hematoxylin and eosin (H&E) and immunohistochemistry staining also showed that visfatin protein was upregulated in mouse sepsis models. Moreover, lipopolysaccharide (LPS) induced visfatin expression, activated the STAT3/NFκB pathway, and increased the expression of pro-inflammatory cytokines, including IL1-ß, IL-6, and TNF-α in human bronchial epithelial cell lines NL-20 and BEAS-2B. Co-treatment of visfatin inhibitor FK866 reversed the activation of the STAT3/NFκB pathway and the increase of pro-inflammatory cytokines induced by LPS. Our study provides new evidence for the involvement of visfatin and down-stream events in acute lung injury. Further studies are required to confirm whether the anti-visfatin approaches can improve ALI patient survival by alleviating the pro-inflammatory process.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Colo/patologia , Lipopolissacarídeos/toxicidade , Nicotinamida Fosforribosiltransferase/metabolismo , Peritonite/metabolismo , Stents/efeitos adversos , Acrilamidas , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Imunoensaio , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas , Sepse , Transdução de Sinais/efeitos dos fármacos
15.
Anticancer Res ; 39(4): 2069-2076, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30952751

RESUMO

BACKGROUND/AIM: This study aimed to examine whether the semi-dry dot-blot (SDB) method can correctly identify metastasis to lymph nodes in colorectal cancer. MATERIALS AND METHODS: A total of 200 dissected lymph nodes from 83 patients with colorectal cancer who underwent surgery between November 2013 and May 2016 were examined. Each lymph node was first examined by SDB using anti-pancytokeratin antibody (AE1/AE3). Pathological Stage I/II patients with a negative reaction were further analyzed by SDB using anti-cytokeratin 20 antibody (CK-20) to detect micrometastasis or isolated tumor cells. RESULTS: The sensitivity, specificity, and accuracy of SDB using AE1/AE3 were 91.3%, 100%, and 98.0%, respectively. Five of 99 lymph nodes of pathological Stage I/II patients had a negative reaction to AE1/AE3, but were positive to CK-20, while 3 showed isolated tumor cells. CONCLUSION: The SDB is a useful diagnostic tool to detect lymph node metastases in colorectal cancer.


Assuntos
Neoplasias Colorretais/diagnóstico , Immunoblotting , Metástase Linfática/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Queratina-20/imunologia , Metástase Linfática/imunologia , Masculino , Pessoa de Meia-Idade
16.
Invest Ophthalmol Vis Sci ; 60(5): 1478-1490, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30973576

RESUMO

Purpose: In the eye, chronic hypoxia/reoxygenation (H/R) contributes to the development of a number of ocular disorders. H/R induces the production of reactive oxygen species (ROS), leading to poly(ADP-ribose) polymerase-1 (PARP1) activation that promotes inflammation, cell death, and disease progression. Here, we analyzed the protective effects of the PARP1 inhibitor olaparib in H/R-induced retina injury and investigated the signaling mechanisms involved. Methods: A rat retinal H/R model was used to detect histologic and biochemical changes in the retina. Results: H/R induced reductions in the thickness of most retinal layers, which were prevented by olaparib. Furthermore, H/R caused increased levels of Akt and glycogen synthase kinase-3ß phosphorylation, which were further increased by olaparib, contributing to retina protection. By contrast, H/R-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinases (MAPK) phosphorylation and activation were reduced by olaparib, via mitogen-activated protein kinase phosphatase 1 (MKP-1) expression. In addition, H/R-induced hypoxia-inducible factor 1α (HIF1α) levels were decreased by olaparib, which possibly contributed to reduced VEGF expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was slightly increased by H/R and was further activated by olaparib. Nuclear factor-κB (NFκB) was also activated by H/R through phosphorylation (Ser536) and acetylation (Lys310) of the p65 subunit, although this was significantly reduced by olaparib. Conclusions: Olaparib reduced H/R-induced degenerative changes in retinal morphology. The protective mechanisms of olaparib most probably involved Nrf2 activation and ROS reduction, as well as normalization of HIF1α and related VEGF expression. In addition, olaparib reduced inflammation by NFκB dephosphorylation/inactivation, possibly via the PARP1 inhibition-MKP-1 activation-p38 MAPK inhibition pathway. PARP inhibitors represent potential therapeutics in H/R-induced retinal disease.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Doenças Retinianas/prevenção & controle , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Hipóxia/complicações , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Estresse Oxidativo , Oxigênio/toxicidade , Fosforilação , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Doenças Retinianas/metabolismo
17.
Mediators Inflamm ; 2019: 6217548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944547

RESUMO

Liver X receptors (LXRs) have emerged as important regulators of inflammatory gene expression. Previously, we had reported that an LXRα gene promoter polymorphism (-1830 T > C) is associated with systemic lupus erythematosus (SLE). Therefore, we assessed cytokine expression in relation to LXRα polymorphism in monocyte-derived macrophages from patients with SLE. Macrophages were obtained after 72 hours of culture of human monocytes supplemented with phorbol 12-myristate 13-acetate. Cells were transfected with LXRα promoter constructs. Additionally, peripheral blood mononuclear cell- (PBMC-) derived macrophages from the patients were evaluated for proinflammatory cytokines in relation to the genotypes of LXRα -1830 T > C. The expression of LXRα was increased in macrophages; levels of proinflammatory cytokines were decreased with LXRα expression. Production of proinflammatory cytokines varied depending on LXRα -1830 T > C genotype. In particular, expression of LXRα was decreased and that of proinflammatory cytokines was increased for LXRα -1830 TC genotype compared to that for TT genotype. The data were consistent in PBMC-derived macrophages from patients with SLE. Increased proinflammatory cytokines is related to TLR7 and TLR9 expression. These data suggest that the expression levels of LXRα, according to LXRα -1830 T > C genotype, may contribute to the inflammatory response by induction of inflammatory cytokines in SLE.


Assuntos
Leucócitos Mononucleares/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Genótipo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Immunoblotting , Leucócitos Mononucleares/efeitos dos fármacos , Receptores X do Fígado/agonistas , Macrófagos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
18.
Plant Mol Biol ; 100(3): 231-246, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30911876

RESUMO

KEY MESSAGE: GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm. The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.


Assuntos
Grão Comestível/metabolismo , Glutens/classificação , Glutens/metabolismo , Immunoblotting/métodos , Oryza/metabolismo , Anticorpos , Grão Comestível/genética , Endosperma/metabolismo , Epitopos/imunologia , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glutens/genética , Oryza/citologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Sementes/metabolismo
19.
J Plant Physiol ; 236: 7-14, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30840921

RESUMO

Previous studies have documented mitochondrial dysfunction during the critical node (CN) of rice (Oryza sativa) seed aging, including a decrease in the capacity of NADH dependent O2 consumption. This raises the hypothesis that changes in the activity of NADH:ubiquinone oxidoreductase (complex I) may play a role in seed aging. The composition and activity of complex I was investigated at the CN of aged rice seeds. Using BN-PAGE and SWATH-MS 52 complex I subunits were identified, nineteen for the first time to be experimentally detected in rice. The subunits of the matrix arm (N and Q modules) were reduced in abundance at the CN, in accordance with a reduction in the capacity to oxidise NADH, reducing substrate oxidation and increase ROS accumulation. In contrast, subunits in the P module increased in abundance that contains many mitochondrial encoded subunits. It is proposed that the changes in complex I abundance subunits may indicate a premature re-activation of mitochondrial biogenesis, as evidenced by the increase in mitochondrial encoded subunits. This premature activation of mitochondrial biogenesis may under-pin the decreased viability of aged seeds, as mitochondrial biogenesis is a crucial event in germination to drive growth before autotrophic growth of the seedling is established.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Oryza/metabolismo , Sementes/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Complexo I de Transporte de Elétrons/fisiologia , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Immunoblotting , Mitocôndrias/fisiologia , Oryza/fisiologia , Sementes/fisiologia
20.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897851

RESUMO

BACKGROUND: Many food components influence intestinal epithelial barrier properties and might therefore also affect susceptibility to the development of food allergies. Such allergies are triggered by increased antibody production initiated in Peyer's patches (PP). Usually, the presentation of antigens in the lumen of the gut to the immune cells of the PP is strongly regulated by the follicle-associated epithelium (FAE) that covers the PP. As the food component caprate has been shown to impede barrier properties in villous epithelium, we hypothesized that caprate also affects the barrier function of the PP FAE, thereby possibly contributing a risk factor for the development of food allergies. METHODS: In this study, we have focused on the effects of caprate on the barrier function of PP, employing in vitro and ex vivo experimental setups to investigate functional and molecular barrier properties. Incubation with caprate induced an increase of transepithelial resistance, and a marked increase of permeability for the paracellular marker fluorescein in porcine PP to 180% of control values. These effects are in accordance with changes in the expression levels of the barrier-forming tight junction proteins tricellulin and claudin-5. CONCLUSIONS: This barrier-affecting mechanism could be involved in the initial steps of a food allergy, since it might trigger unregulated contact of the gut lumen with antigens.


Assuntos
Epitélio/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Animais , Linhagem Celular , Claudinas/metabolismo , Immunoblotting , Proteína 2 com Domínio MARVEL/metabolismo , Suínos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
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