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1.
Int J Nanomedicine ; 16: 725-740, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33542627

RESUMO

Purpose: As a dental material, polyetheretherketone (PEEK) is bioinert that does not induce cellular response and bone/gingival tissues regeneration. This study was to develop bioactive coating on PEEK and investigate the effects of coating on cellular response. Materials and Methods: Tantalum pentoxide (TP) coating was fabricated on PEEK surface by vacuum evaporation and responses of rat bone marrow mesenchymal stem (RBMS) cells/human gingival epithelial (HGE) were studied. Results: A dense coating (around 400 nm in thickness) of TP was closely combined with PEEK (PKTP). Moreover, the coating was non-crystalline TP, which contained many small humps (around 10 nm in size), exhibiting a nanostructured surface. In addition, the roughness, hydrophilicity, surface energy, and protein adsorption of PKTP were remarkably higher than that of PEEK. Furthermore, the responses (adhesion, proliferation, and osteogenic gene expression) of RBMS cells, and responses (adhesion and proliferation) of HGE cells to PKTP were remarkably improved in comparison with PEEK. It could be suggested that the nanostructured coating of TP on PEEK played crucial roles in inducing the responses of RBMS/HGE cells. Conclusion: PKTP with elevated surface performances and outstanding cytocompatibility might have enormous potential for dental implant application.


Assuntos
Células Epiteliais/citologia , Gengiva/citologia , Cetonas/farmacologia , Células-Tronco Mesenquimais/citologia , Nanoestruturas/química , Óxidos/farmacologia , Polietilenoglicóis/farmacologia , Tantálio/farmacologia , Adsorção , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Nanoestruturas/ultraestrutura , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Difração de Raios X
2.
Nat Commun ; 12(1): 1140, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602902

RESUMO

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5ß1 and vitronectin-αvß1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.


Assuntos
/fisiologia , Infecções por Clostridium/microbiologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Intestinos/microbiologia , Intestinos/patologia , Esporos Bacterianos/fisiologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Linhagem Celular , /ultraestrutura , Colágeno/metabolismo , Endocitose , Células Epiteliais/ultraestrutura , Feminino , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Nistatina/farmacologia , Ligação Proteica/efeitos dos fármacos , Recidiva , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/ultraestrutura , Ácido Taurocólico/farmacologia , Vitronectina/metabolismo
3.
Am J Physiol Renal Physiol ; 320(3): F262-F272, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33356954

RESUMO

Mitochondrial damage in renal tubular epithelial cells (RTECs) is a hallmark of endotoxin-induced acute kidney injury (AKI). Forkhead box O1 (FOXO1) is responsible for regulating mitochondrial function and is involved in several kidney diseases. Here, we investigated the effect of FOXO1 on endotoxin-induced AKI and the related mechanism. In vivo, FOXO1 downregulation in mouse RTECs and mitochondrial damage were found in endotoxin-induced AKI. Overexpression of FOXO1 by kidney focal adeno-associated virus (AAV) delivery improved renal function and reduced mitochondrial damage. Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), a master regulator of mitochondrial biogenesis and function, was reduced in endotoxin-induced AKI, but the reduction was reversed by FOXO1 overexpression. In vitro, exposure to LPS led to a decline in HK-2 cell viability, mitochondrial fragmentation, and mitochondrial superoxide accumulation, as well as downregulation of FOXO1, PGC1-α, and mitochondrial complex I/V. Moreover, overexpression of FOXO1 in HK-2 cells increased HK-2 cell viability and PGC1-α expression, and it alleviated the mitochondrial injury and superoxide accumulation induced by LPS. Meanwhile, inhibition of FOXO1 in HK-2 cells by siRNA treatment decreased PGC1-α expression and HK-2 cell viability. Chromatin immunoprecipitation assays and PCR analysis confirmed that FOXO1 bound to the PGC1-α promoter in HK-2 cells. In conclusion, downregulation of FOXO1 in RTECs mediated endotoxin-induced AKI and mitochondrial damage. Overexpression of FOXO1 could improve renal injury and mitochondrial dysfunction, and this effect occurred at least in part as a result of PGC1-α signaling. FOXO1 might be a potential target for the prevention and treatment of endotoxin-induced AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Endotoxemia/complicações , Células Epiteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Túbulos Renais/metabolismo , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Células Epiteliais/ultraestrutura , Proteína Forkhead Box O1/genética , Humanos , Túbulos Renais/ultraestrutura , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais
5.
Toxicol Lett ; 333: 242-250, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32841739

RESUMO

The Buccal Micronucleus Cytome Assay (BMCyt) has become an important biomonitoring tool for assessing cytogenetic damage in many studied populations. Each laboratory applies protocols that vary according to the method of collecting and preparing samples. Besides, Brazil is a country of great territorial extensions that received immigrants from various parts of the world with different genetic backgrounds. Therefore, the present study aimed to evaluate the inter-laboratory variation in scoring the same set of slides using the more comprehensive scoring criteria, to standardize the BMCyt protocol, to observe the basal alterations in populations of different Brazilian regions and to compare it with other places around the world. Our results showed that a valuable number of laboratories participated, ten laboratories from different regions of the country, for the validation of the BMCyt in human biomonitoring studies, resulting in the 804 healthy individuals. This was possible because we observed: a range of measures needs to be considered, such as the baseline frequency of DNA damage and cell death in non-exposed individuals; age when grouped showed an influence on DNA damage, although when evaluated by group we did not see an influence; association between smoking habit and all endpoints of the BMCyt (except karyolytic cells) was evident; the basal MN frequency, in the majority of groups, follows those around the world; and the BMCyt was confirmed as a good health status biomarker. We emphasize the need for constant discussions on the parameters of cell death due to greater difficulty among the analyzers.


Assuntos
Bioensaio/normas , Núcleo Celular/genética , Células Epiteliais/ultraestrutura , Laboratórios/normas , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos/normas , Mucosa Bucal/citologia , Adolescente , Adulto , Bioensaio/métodos , Brasil , Morte Celular/genética , Núcleo Celular/ultraestrutura , Dano ao DNA , Feminino , Humanos , Masculino , Micronúcleos com Defeito Cromossômico/estatística & dados numéricos , Testes para Micronúcleos/métodos , Pessoa de Meia-Idade , Mucosa Bucal/ultraestrutura , Adulto Jovem
6.
Ecotoxicol Environ Saf ; 201: 110828, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32531576

RESUMO

Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC50 = 252.23 µg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata.


Assuntos
Sistema Digestório/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Microvilosidades/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Animais , Sistema Digestório/ultraestrutura , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Transmissão , Microvilosidades/ultraestrutura , Mariposas/crescimento & desenvolvimento
7.
Am J Physiol Renal Physiol ; 318(5): F1246-F1251, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249613

RESUMO

Podocytes are highly specialized cells with a clear cell polarity. It is known that in health and disease, microvilli protrude from the apical surface of the podocytes into the urinary space. As a basis to better understand the podocyte microprojections/microvilli, the present study analyzed their spatial localization, extension, and contact site with parietal epithelial cells (PECs). Using different electron microscopic (EM) techniques, we analyzed renal corpuscles of healthy young adult male C57BL/6 mice fixed by vascular perfusion. Serial block-face scanning EM was used to visualize entire corpuscles, focused ion beam scanning EM was performed to characterize microprojection/microvilli-rich regions at higher magnification, and transmission EM of serial sections was used to analyze the contact zone between podocyte microprojections and PECs. Numerous microprojections originating from the primary processes of podocytes were present in the urinary space in all regions of the corpuscle. They often reached the apical surface of the PEC but did not make junctional contacts. At high resolution, it was observed that the glycocalyx of both cells was in contact. Depending on the distance between podocytes and PECs, these microprojections had a stretched or coiled state. The present study shows that microprojections/microvilli of podocytes are a physiological feature of healthy mouse kidneys and are frequently in contact with the apical surface of PECs, thus spanning the urinary space. It is proposed that podocyte microprojections serve mechanosensory or communicative functions between podocytes and PECs.


Assuntos
Comunicação Celular , Extensões da Superfície Celular/ultraestrutura , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Podócitos/ultraestrutura , Animais , Masculino , Camundongos Endogâmicos C57BL
8.
PLoS One ; 15(4): e0231423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302323

RESUMO

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.


Assuntos
Colo/citologia , Células Epiteliais/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Desmossomos/metabolismo , Cães , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Membranas Artificiais , Microvilosidades/fisiologia , Mucinas/metabolismo , Nanoporos , Células-Tronco/citologia , Células-Tronco/metabolismo , Junções Íntimas/metabolismo
9.
Am J Physiol Cell Physiol ; 318(6): C1107-C1122, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267718

RESUMO

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Túbulos de Malpighi/metabolismo , Tetraspaninas/metabolismo , Junções Íntimas/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Impedância Elétrica , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Túbulos de Malpighi/embriologia , Túbulos de Malpighi/ultraestrutura , Via Secretória , Transdução de Sinais , Tetraspaninas/genética , Junções Íntimas/genética , Junções Íntimas/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
10.
Chem Biol Interact ; 323: 109063, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32224134

RESUMO

Exposure to TiO2 NPs induces several cellular alterations after NPs uptake including disruption of cytoskeleton that is crucial for lung physiology but is not considered as a footprint of cell damage. We aimed to investigate cytoskeleton disturbances and the impact on cell migration induced by an acute TiO2 NPs exposure (24 h) and the recovery capability after 6 days of NPs-free treatment, which allowed investigating if cytoskeleton damage was reversible. Exposure to TiO2 NPs (10 µg/cm2) for 24 h induced a decrease 20.2% and 25.1% in tubulin and actin polymerization. Exposure to TiO2 NPs (10 µg/cm2) for 24 h followed by 6 days of NPs-free had a decrease of 26.6% and 21.3% in tubulin and actin polymerization, respectively. The sustained exposure for 7 days to 1 µg/cm2 and 10 µg/cm2 induced a decrease of 22.4% and 30.7% of tubulin polymerization respectively, and 28.7% and 46.2% in actin polymerization. In addition, 24 h followed 6 days of NPs-free exposure of TiO2 NPs (1 µg/cm2 and 10 µg/cm2) decreased cell migration 40.7% and 59.2%, respectively. Cells exposed (10 µg/cm2) for 7 days had a decrease of 65.5% in cell migration. Ki67, protein surfactant B (SFTPB) and matrix metalloprotease 2 (MMP2) were analyzed as genes related to lung epithelial function. The results showed a 20% of Ki67 upregulation in cells exposed for 24 h to 10 µg/cm2 TiO2 NPs while a downregulation of 20% and 25.8% in cells exposed to 1 µg/cm2 and 10 µg/cm2 for 24 h followed by 6 days of NPs-free exposure. Exposure to 1 µg/cm2 and 10 µg/cm2 for 24 h and 7 days upregulates SFTPB expression in 53% and 59% respectively, MMP2 expression remain unchanged. In conclusion, exposure of TiO2 NPs affected cytoskeleton of lung epithelial cells irreversibly but this damage was not cumulative.


Assuntos
Citoesqueleto/patologia , Células Epiteliais/patologia , Pulmão/patologia , Nanopartículas/toxicidade , Titânio/toxicidade , Células A549 , Actinas/metabolismo , Movimento Celular/efeitos dos fármacos , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Endocitose , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Nanopartículas/ultraestrutura , Polimerização , Precursores de Proteínas/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Tubulina (Proteína)/metabolismo
11.
Sci Adv ; 6(10): eaax1909, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32181337

RESUMO

Transduction of extracellular matrix mechanics affects cell migration, proliferation, and differentiation. While this mechanotransduction is known to depend on the regulation of focal adhesion kinase phosphorylation on Y397 (FAKpY397), the mechanism remains elusive. To address this, we developed a mathematical model to test the hypothesis that FAKpY397-based mechanosensing arises from the dynamics of nanoscale integrin clustering, stiffness-dependent disassembly of integrin clusters, and FAKY397 phosphorylation within integrin clusters. Modeling results predicted that integrin clustering dynamics governs how cells convert substrate stiffness to FAKpY397, and hence governs how different cell types transduce mechanical signals. Existing experiments on MDCK cells and HT1080 cells, as well as our new experiments on 3T3 fibroblasts, confirmed our predictions and supported our model. Our results suggest a new pathway by which integrin clusters enable cells to calibrate responses to their mechanical microenvironment.


Assuntos
Microambiente Celular/genética , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Integrinas/metabolismo , Mecanotransdução Celular , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Cães , Células Epiteliais/ultraestrutura , Matriz Extracelular/ultraestrutura , Quinase 1 de Adesão Focal/genética , Expressão Gênica , Humanos , Integrinas/genética , Células Madin Darby de Rim Canino , Camundongos , Células NIH 3T3 , Fosforilação , Especificidade da Espécie
12.
Parasit Vectors ; 13(1): 143, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188507

RESUMO

BACKGROUND: The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS: Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS: Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8 ± 0.9 µm) and macrogamonts (16.6 ± 1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5 ± 0.5 µm) and two flagella (11.2 ± 0.5 µm). Macrogametes were spherical with a diameter of 12.1 ± 0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS: The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.


Assuntos
Células Epiteliais/parasitologia , Merozoítos/crescimento & desenvolvimento , Merozoítos/genética , Sarcocystidae/crescimento & desenvolvimento , Sarcocystidae/genética , Animais , Células Cultivadas , Células Epiteliais/ultraestrutura , Feminino , Intestinos/citologia , Estágios do Ciclo de Vida , Masculino , Microscopia Eletrônica , Suínos
13.
Int J Mol Sci ; 21(4)2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32075009

RESUMO

Salivary gland aquaporins (AQPs) are essential for the control of saliva production and maintenance of glandular structure. However, little is known of their role in salivary gland neoplasia. Salivary gland tumors comprise a heterogeneous group of lesions, featuring variable histological characteristics and diverse clinical behaviors. Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. The aim of this study was to evaluate the expression of AQP1, AQP3, and AQP5 in 24 MEC samples by immunohistochemistry. AQP1 expression was observed in vascular endothelium throughout the tumor stroma. AQP3 was expressed in epidermoid and mucosal cells and AQP5 was expressed in mucosal cells of MEC. These proteins were expressed in the human MEC cell line UH-HMC-3A. Cellular ultrastructural aspects were analyzed by electron microscopy to certificate the tumor cell phenotype. In summary, our results show that, despite the fact that these molecules are important for salivary gland physiology, they may not play a distinct role in tumorigenesis in MEC. Additionally, the in vitro model may offer new possibilities to further investigate mechanisms of these molecules in tumor biology and their real significance in prognosis and possible target therapies.


Assuntos
Aquaporina 1/metabolismo , Aquaporina 3/metabolismo , Aquaporina 5/metabolismo , Carcinoma Mucoepidermoide/patologia , Neoplasias das Glândulas Salivares/patologia , Adulto , Carcinoma Mucoepidermoide/metabolismo , Carcinoma Mucoepidermoide/mortalidade , Linhagem Celular Tumoral , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Fenótipo , Projetos Piloto , Neoplasias das Glândulas Salivares/metabolismo , Neoplasias das Glândulas Salivares/mortalidade , Taxa de Sobrevida
14.
Int J Mol Sci ; 21(3)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012692

RESUMO

Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD). METHODS: Cultured ARPE-19 cells were exposed to 10 and 50 µM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively. RESULTS: Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells. CONCLUSIONS: Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD.


Assuntos
Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Resveratrol/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Células Epiteliais/ultraestrutura , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
15.
Nat Commun ; 11(1): 472, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980653

RESUMO

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.


Assuntos
Junções Aderentes/fisiologia , Junções Aderentes/efeitos da radiação , Actomiosina/química , Animais , Antígenos CD/química , Fenômenos Biomecânicos , Caderinas/química , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/efeitos da radiação , Células Epiteliais/ultraestrutura , Humanos , Luz , Microscopia de Força Atômica , Fenômenos Ópticos , Processos Fotoquímicos , Xenopus laevis/embriologia , alfa Catenina/química
16.
Microsc Res Tech ; 83(5): 531-540, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31943532

RESUMO

The current work gives concern to study the morphology of the Merluccius merluccius gills by using gross morphology, scanning electron microscopy (SEM), and light microscopy. The findings of the present study revealed that the gill system consisted of four pairs of gill arches which carry the gill filaments on the convex border and gill rakers on the concave border of them. SEM results revealed that the rakers and the spines distribution on the first gill arch differed from that of the other three gill arches on the lateral and medial surfaces. On the surface the gill filaments, there were longitudinal ridges that carried pores of chloride cells and mucous cells. The histological examination revealed that, the gill arch composed of hyaline cartilage that presented in the form of cups. Each cup consisted of central cartilagenous core and peripheral cartilagenous matrix. The gill filaments composed of cartilaginous bar of peripheral cartilaginous matrix and central cartilaginous core extended from the gill arches and covered by an epithelial layers with a few mucous cells permeate it, and chloride cells were straggly in the interlamellar epithelium. Each gill filament carried several leaves like secondary lamellae on both sides of it. The epithelium, which lined the secondary lamellae, composed of epithelial pavement cells, some mucous cells, and pillar cells.


Assuntos
Gadiformes/anatomia & histologia , Brânquias/anatomia & histologia , Brânquias/ultraestrutura , Adaptação Fisiológica , Animais , Células Epiteliais/ultraestrutura , Brânquias/citologia , Microscopia , Microscopia Eletrônica de Varredura , Membrana Mucosa/ultraestrutura , Papilas Gustativas
17.
PLoS One ; 15(1): e0227950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978129

RESUMO

Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Corantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Azul Tripano/farmacologia , Morte Celular/fisiologia , Rastreamento de Células/métodos , Células Epiteliais/ultraestrutura , Humanos , Iodetos/farmacologia , Células Jurkat , Imagem Óptica , Coloração e Rotulagem/métodos
18.
Analyst ; 145(2): 667-674, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31799546

RESUMO

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.


Assuntos
Células Epiteliais/ultraestrutura , Dispositivos Lab-On-A-Chip , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Animais , Cães , Células Madin Darby de Rim Canino , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microtúbulos/ultraestrutura , Mitocôndrias/ultraestrutura , Junções Íntimas/ultraestrutura
19.
Exp Cell Res ; 386(2): 111727, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31759054

RESUMO

Following mating, leukocytes are recruited to the uterine epithelium where they phagocytose spermatozoa and mediate maternal immune tolerance as well as a mild inflammatory response. In this ultrastructural study we utilised array tomography, a high-resolution volume scanning electron microscopy approach to 3D reconstruct the cellular relationships formed by leukocytes recruited to the luminal uterine epithelium 12 h post-mating in the rat. We report that following mating, neutrophils and macrophages are internalised by the luminal uterine epithelium, with multiple leukocytes internalised via contortion through a small tunnel in the apical membrane into a large membrane-bound vacuole within the cytoplasm of luminal uterine epithelial cells (UECs). Once internalised within the UECs, recruited leukocytes appear to phagocytose material within the membrane-bound vacuole and most ultimately undergo a specialised cell death, including vacuolisation and loss of membrane integrity. As these observations involve ultrastructurally normal leukocytic cells internalised within non-phagocytic epithelial cells, these observations are consistent with the formation of cell-in-cell structures via entosis, rather than phagocytic engulfment by UECs. Although cell-in-cell structures have been reported in normal and pathological conditions elsewhere, the data collected herein represents the first evidence of the formation of cell-in-cell structures within the uterine epithelium as a novel component of the maternal inflammatory response to mating.


Assuntos
Copulação/fisiologia , Entose/imunologia , Células Epiteliais/ultraestrutura , Epitélio/ultraestrutura , Leucócitos/ultraestrutura , Útero/citologia , Animais , Morte Celular , Células Epiteliais/imunologia , Epitélio/imunologia , Feminino , Tolerância Imunológica , Leucócitos/imunologia , Masculino , Fagocitose , Gravidez , Ratos , Ratos Wistar , Espermatozoides/citologia , Espermatozoides/imunologia , Útero/imunologia , Vacúolos/imunologia , Vacúolos/ultraestrutura
20.
Microsc Res Tech ; 83(3): 232-238, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31769117

RESUMO

The accessory glands of male reproductive system in insects play a significant role in the reproduction process by protecting sperm in spermatheca, preventing female to accept other males after mating and stimulating oviposition. The number, structure, and arrangement of the tubules of accessory glands can change from species to species. In this study, the accessory glands belonging the male reproductive system in Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) were examined with stereomicroscope, light microscope, scanning (SEM), and transmission (TEM) electron microscopes at Gazi University, Faculty of Science in 2017-2019. P. parallelus parallelus is a widespread species that is located at the extending areas from Italy to the Northern Europe and also in Turkey. The accessory glands of P. parallelus parallelus' male reproductive system are composed of about 10 tubules. The tubules can be classified into two groups according to the thickness of their muscle tissues. Both groups have single layered epithelial cells with mitochondria, well-developed endoplasmic reticulum, spherical nucleus with electron dense chromatin, secretory vesicles and multivesicular bodies in their cytoplasm. In addition, apocrine type secretion is seen in epithelial cells.


Assuntos
Células Epiteliais/ultraestrutura , Genitália Masculina/anatomia & histologia , Genitália Masculina/ultraestrutura , Gafanhotos/anatomia & histologia , Gafanhotos/ultraestrutura , Animais , Genitália Masculina/citologia , Masculino , Microscopia , Microscopia Eletrônica de Transmissão , Turquia
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