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1.
Cell Mol Biol Lett ; 24: 38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182966

RESUMO

Background: Exploration of the genes with abnormal expression during the development of breast cancer is essential to provide a deeper understanding of the mechanisms involved. Transcriptome sequencing and bioinformatics analysis of invasive ductal carcinoma and paracancerous tissues from the same patient were performed to identify the key genes and signaling pathways related to breast cancer development. Methods: Samples of breast tumor tissue and paracancerous breast tissue were obtained from 6 patients. Sequencing used the Illumina HiSeq platform. All. Only perfectly matched clean reads were mapped to the reference genome database, further analyzed and annotated based on the reference genome information. Differentially expressed genes (DEGs) were identified using the DESeq R package (1.10.1) and DEGSeq R package (1.12.0). Using KOBAS software to execute the KEGG bioinformatics analyses, enriched signaling pathways of DEGs involved in the occurrence of breast cancer were determined. Subsequently, quantitative real time PCR was used to verify the accuracy of the expression profile of key DEGs from the RNA-seq result and to explore the expression patterns of novel cancer-related genes on 8 different clinical individuals. Results: The transcriptomic sequencing results showed 937 DEGs, including 487 upregulated and 450 downregulated genes in the breast cancer specimens. Further quantitative gene expression analysis was performed and captured 252 DEGs (201 downregulated and 51 upregulated) that showed the same differential expression pattern in all libraries. Finally, 6 upregulated DEGs (CST2, DRP2, CLEC5A, SCD, KIAA1211, DTL) and 6 downregulated DEGs (STAC2, BTNL9, CA4, CD300LG, GPIHBP1 and PIGR), were confirmed in a quantitative real time PCR comparison of breast cancer and paracancerous breast tissues from 8 clinical specimens. KEGG analysis revealed various pathway changes, including 20 upregulated and 21 downregulated gene enrichment pathways. The extracellular matrix-receptor (ECM-receptor) interaction pathway was the most enriched pathway: all genes in this pathway were DEGs, including the THBS family, collagen and fibronectin. These DEGs and the ECM-receptor interaction pathway may perform important roles in breast cancer. Conclusion: Several potential breast cancer-related genes and pathways were captured, including 7 novel upregulated genes and 76 novel downregulated genes that were not found in other studies. These genes are related to cell proliferation, movement and adhesion. They may be important for research into breast cancer mechanisms, particularly CST2 and CA4. A key signaling pathway, the ECM-receptor interaction signal pathway, was also identified as possibly involved in the development of breast cancer.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Regulação para Baixo/genética , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcriptoma/genética , Regulação para Cima/genética
2.
Int J Nanomedicine ; 14: 3413-3425, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190800

RESUMO

Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.


Assuntos
Produtos Biológicos/metabolismo , Exossomos/metabolismo , Inflamação/metabolismo , Células HEK293 , Humanos , Nanopartículas , Domínios Proteicos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
3.
Parasit Vectors ; 12(1): 317, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234897

RESUMO

Glycophorins are heavily glycosylated sialoglycoproteins of human and animal erythrocytes. In humans, there are four glycophorins: A, B, C and D. Glycophorins play an important role in the invasion of red blood cells (RBCs) by malaria parasites, which involves several ligands binding to RBC receptors. Four Plasmodium falciparum merozoite EBL ligands have been identified: erythrocyte-binding antigen-175 (EBA-175), erythrocyte-binding antigen-181 (EBA-181), erythrocyte-binding ligand-1 (EBL-1) and erythrocyte-binding antigen-140 (EBA-140). It is generally accepted that glycophorin A (GPA) is the receptor for P. falciparum EBA-175 ligand. It has been shown that α(2,3) sialic acid residues of GPA O-glycans form conformation-dependent clusters on GPA polypeptide chain which facilitate binding. P. falciparum can also invade erythrocytes using glycophorin B (GPB), which is structurally similar to GPA. It has been shown that P. falciparum EBL-1 ligand binds to GPB. Interestingly, a hybrid GPB-GPA molecule called Dantu is associated with a reduced risk of severe malaria and ameliorates malaria-related morbidity. Glycophorin C (GPC) is a receptor for P. falciparum EBA-140 ligand. Likewise, successful binding of EBA-140 depends on sialic acid residues of N- and O-linked oligosaccharides of GPC, which form a cluster or a conformational structure depending on the presence of peptide fragment encompassing amino acids (aa) 36-63. Evaluation of the homologous P. reichenowi EBA-140 unexpectedly revealed that the chimpanzee homolog of human glycophorin D (GPD) is probably the receptor for this ligand. In this review, we concentrate on the role of glycophorins as erythrocyte receptors for Plasmodium parasites. The presented data support the long-lasting idea of high evolutionary pressure exerted by Plasmodium on the human glycophorins, which emerge as important receptors for these parasites.


Assuntos
Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Glicoforina/metabolismo , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas de Transporte/genética , Glicoforina/genética , Humanos , Ligantes , Merozoítos , Pan troglodytes , Ligação Proteica , Proteínas de Protozoários/genética , Receptores de Superfície Celular/genética
4.
Cell Mol Life Sci ; 76(16): 3229-3248, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197404

RESUMO

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.


Assuntos
Encéfalo/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/química , Humanos , Ácido Hialurônico/metabolismo , Proteólise , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/metabolismo , Sinapses/metabolismo , Tenascina/metabolismo
5.
Parasit Vectors ; 12(1): 205, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060579

RESUMO

BACKGROUND: Vitellogenin (Vg), a key molecule for oocyte development synthesized in the fat body during blood-feeding, is released into the hemolymph and then taken into the oocytes via Vg receptor (VgR) in ticks. Previously, we showed that VgR mRNA is expressed in the ovary at the adult stage of parthenogenetic Haemaphysalis longicornis ticks and its expression increases after blood-feeding. However, intracellular localization of VgR mRNA and protein at each developmental stage of oocytes during oogenesis remains largely unclear. METHODS: mRNA and protein expression profiles of H. longicornis VgR (HlVgR) in the oocytes from the unfed to oviposition periods were analyzed by real-time PCR, in situ hybridization, and immunostaining. To elucidate the timing of the onset of Vg uptake, RNA interference (RNAi)-mediated gene silencing of HlVgR was performed. RESULTS: In situ hybridization revealed that HlVgR mRNA was detected in the cytoplasm of stage I-III oocytes, and weaker positive signals for HlVgR mRNA were found in the cell periphery of stage IV and V oocytes. Likewise, HlVgR protein was detected by immunostaining in the cytoplasm of stage I-III oocytes and in the cell periphery of stage IV and V oocytes. Each developmental stage of the oocytes showed distinct patterns of mRNA and protein expression of HlVgR. Moreover, RNAi of HlVgR caused delayed or arrested development in the oocytes. The ovaries of control ticks showed all developmental stages of oocytes, whereas stage I-III oocytes were found in the ovaries of HlVgR-RNAi ticks at 5 days after engorgement. CONCLUSIONS: These results suggest that active uptake of Vg is required for development from stage III to stage IV during oogenesis. Our data clearly revealed an apparent shift in the intracellular localization of VgR for both mRNA and protein level in oocytes during oogenesis.


Assuntos
Proteínas do Ovo/metabolismo , Ixodidae/metabolismo , Oogênese/fisiologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Vitelogeninas/metabolismo , Animais , Proteínas do Ovo/genética , Feminino , Ixodidae/genética , Ixodidae/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/genética , Transcriptoma
6.
Int J Nanomedicine ; 14: 3203-3220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118632

RESUMO

Background: Tumor-associated macrophages (TAMs) are critical in tumor progression and metastasis. Selective targeting of TAMs holds great potential to ameliorate the immunosuppressive tumor microenvironment and enhance the efficacy of antitumor therapy. Various liposomes have been developed to target TAMs via cell-specific surface receptors either to deplete or re-educate TAMs. Since immuno-stimulation often initiates with the interaction of nanocarriers with the innate immunity cells such as macrophages, the intrinsic impact of drug-free liposomes on macrophage activation and polarization via cell interaction is one of the most critical issues in nanomedicine for promoting effective immunotherapy. Methods: In this study, conventional bare liposomes, PEGylated liposomes, and mannosylated liposomes were developed and the cytotoxicity, cellular internalization, immunostimulatory activity, targeting efficiency, antitumor efficacy, and mechanism were evaluated in vitro and in vivo. Results: All liposomes displayed an ideal particle size, good biocompatibility, and controlled release behavior. Mannosylated liposomes exhibited superior in vitro cellular internalization and tumor spheroid penetration with the aid of the mannose receptor-mediated TAMs-targeting effects. In particular, mannosylated liposomes promoted the polarization of both M0 and M2 to the M1 phenotype by enhancing the expression ratio of CD86/CD206 in vitro. Of note, mannosylated liposomes could inhibit G422 glioma tumor growth, which may be attributed to the polarization of TAMs, as evidenced by the reduction in expression level of the TAMs surface marker. Conclusion: These results indicate the potential value of mannosylated liposomes in the design of a rational delivery system to enhance the antitumor immune efficacy of immunomodulators by inducing a shift from the M2 to the M1 phenotype.


Assuntos
Polaridade Celular , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Lectinas de Ligação a Manose/metabolismo , Neoplasias/patologia , Receptores de Superfície Celular/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Cumarínicos/química , Liberação Controlada de Fármacos , Endocitose , Feminino , Humanos , Lipossomos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Tiazóis/química , Distribuição Tecidual , Microambiente Tumoral
7.
Int J Nanomedicine ; 14: 2829-2846, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114197

RESUMO

Background: Vitamin D3 possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers. Purpose: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo. Methods: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo. Results: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver. Conclusion: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Calcitriol/administração & dosagem , Calcitriol/farmacologia , Lipídeos/química , Macrófagos/metabolismo , Nanopartículas/química , Animais , Anticorpos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Calcitriol/farmacocinética , Quimiocinas/metabolismo , Composição de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Distribuição Tecidual/efeitos dos fármacos
8.
Planta ; 250(1): 381-390, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31062160

RESUMO

MAIN CONCLUSION: Ethylene receptor is crucial for PCD and aerenchyma formation in Typha angustifolia leaves. Not only does it receive and deliver the ethylene signal, but it probably can determine the cell fate during aerenchyma morphogenesis, which is due to the receptor expression quantity. Aquatic plant oxygen delivery relies on aerenchyma, which is formed by a programmed cell death (PCD) procedure. However, cells in the outer edge of the aerenchyma (palisade cells and septum cells) remain intact, and the mechanism is unclear. Here, we offer a hypothesis: cells that have a higher content of ethylene receptors do not undergo PCD. In this study, we investigated the leaf aerenchyma of the aquatic plant Typha angustifolia. Ethephon and pyrazinamide (PZA, an inhibitor of ACC oxidase) were used to confirm that ethylene is an essential hormone for PCD of leaf aerenchyma cells in T. angustifolia. That the ethylene receptor was an indispensable factor in this PCD was confirmed by 1-MCP (an inhibitor of the ethylene receptor) treatment. Although PCD can be avoided by blocking the ethylene receptor, excessive ethylene receptors also protect cells from PCD. TaETR1, TaETR2 and TaEIN4 in the T. angustifolia leaf were detected by immunofluorescence (IF) using polyclonal antibodies. The result showed that the content of ethylene receptors in PCD-unsusceptible cells was 4-14 times higher than that one in PCD-susceptible cells, suggesting that PCD-susceptible cells undergo the PCD programme, while PCD-unsusceptible cells do not due to the content difference in the ethylene receptor in different cells. A higher level of ethylene receptor content makes the cells insensitive to ethylene, thereby avoiding cell death and degradation.


Assuntos
Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Typhaceae/fisiologia , Aminoácido Oxirredutases/antagonistas & inibidores , Apoptose/genética , Diferenciação Celular/genética , Ciclopropanos/farmacologia , Etilenos/metabolismo , Compostos Organofosforados/farmacologia , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Pirazinamida/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Typhaceae/efeitos dos fármacos , Typhaceae/enzimologia , Typhaceae/crescimento & desenvolvimento
9.
Bioelectrochemistry ; 128: 263-273, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31051432

RESUMO

This study aimed to explore the interaction between bombykol and BmOR1 and also provide a paradigm for agroforestry pest control. The electrochemical biosensor signal amplification system was used: nanogold with horseradish peroxidase. An electrochemical bilayer nanogold membrane receptor sensor was developed using the following schemes and processes: twice self-assembly of nanogold and succeeding absorption of Bombyx mori olfactory receptor 1 (BmOR1); sex pheromone-binding protein; spectral scanning and transmission electron microscope to characterize nanogold sol; and atomic force microscope, cyclic voltammetry, and AC impedance methods to characterize individual processes of sensor assembly. The amperometric I-T curve was adopted to measure the response current upon interaction with different concentrations of bombykol (diluted in phosphate-buffered saline) and BmOR1. The results demonstrated the receptor-ligand interaction pattern, which was similar to enzymatic reaction kinetics, with the activation constant Ka of up to 8.57 × 10-20 mol/L and signal magnification of about 10,000-fold. In this study, the simulation of intracellular receptor signaling cascade by an electrochemical signal amplification system helped in directly measuring BmOR1-bombykol ligand interaction and exploring the kinetics after the self-assembly of BmOR1 on the biosensor. It provided a novel platform for future studies on receptor-ligand interaction.


Assuntos
Técnicas Eletroquímicas/métodos , Álcoois Graxos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Técnicas Biossensoriais , Bombyx , Proteínas de Ligação ao GTP/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Cinética , Limite de Detecção
10.
J Med Microbiol ; 68(6): 940-951, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107199

RESUMO

PURPOSE: This study aimed to characterize 82 atypical enteropathogenic Escherichia coli (aEPEC) isolates, obtained from patients with diarrhea in Brazil, regarding their adherence patterns on HeLa cells and attaching and effacing (AE) lesion pathways. METHODOLOGY: The adherence and fluorescence-actin staining (FAS) assays were performed using HeLa cells. AE lesion pathways were determined through the detection of tyrosine residue 474 (Y474) phosphorylation in the Tir protein, after its translocation to host cells, and by PCR assays for tir genotyping and detection of Tir-cytoskeleton coupling protein (tccP) genes. RESULTS: Regarding the adherence pattern, determined in the presence of d-mannose, 12 isolates (14.6 %) showed the localized adherence (LA)-like pattern, 3 (3.7  %) the aggregative adherence pattern and 4 (4.9  %) a hybrid LA/diffuse adherence pattern. In addition, 36 (43.9  %) isolates displayed an undefined adherence, and 26 (31.7  %) were non-adherent (NA), while one (1.2 %) caused cell detachment. Among the 26 NA aEPEC isolates, 11 showed a type 1 pilus-dependent adherence in assays performed without d-mannose, while 15 remained NA. Forty-eight (58.5 %) aEPEC were able to trigger F-actin accumulation underneath adherent bacteria (FAS-positive), which is an important feature of AE lesions. The majority (58.3 %) of these used the Tir-Nck pathway, while 39.6  % may use both Tir-Nck and Tir-TccP pathways to induce AE lesions. CONCLUSION: Our results reveal the diversity of strategies used by aEPEC isolates to interact with and damage epithelial host cells, thereby causing diarrheal diseases.


Assuntos
Aderência Bacteriana , Escherichia coli Enteropatogênica/fisiologia , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Actinas/metabolismo , Diarreia/microbiologia , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/isolamento & purificação , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Genótipo , Células HeLa , Humanos , Fenótipo , Fosforilação , Receptores de Superfície Celular/metabolismo
11.
Plant Physiol Biochem ; 139: 660-671, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31048123

RESUMO

In Arabidopsis, the serine/threonine protein kinase Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 2 polypeptide (EIN2) functions are key negative and positive components, respectively, in the ethylene signalling route. Here, we report on an in silico study of members of the CTR1-like and EIN2-like polypeptide families from poplars. The expression of CTR1-like and EIN2-like genes such as Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a was studied in Populus tremula buds and leaves in response to dehydration, various light conditions and under senescence. In buds under dehydration, the maximal fold-change of the Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a expression level recorded almost identical values. This suggests that maintenance of a constant ratio between the transcript levels of genes encoding positive and negative ethylene signalling components is required under stress. The expression of the studied genes was 1.4-to 3-fold higher in response to darkness, but 4.5- to 51.2-fold and 21.6- to 51.2-fold higher under the early and moderate leaf senescence, respectively. It is worth noting that the senescence-related Ptre-EIN2a and Ptre-CTR3a expression profiles were very similar. Using in vitro assays, we demonstrated the ability of the catalytic domain of Ptre-CTR1 to phosphorylate the Ptre-EIN2a-like polypeptide, which is similar to that in Arabidopsis. The target substrate, the Ptre-CEND2a polypeptide (C-terminal part of Ptre-EIN2a), was only phosphorylated by the protein kinase Ptre-CTR1 and not by Ptre-CTR3. Moreover, the addition of Ptre-CTR3 polypeptides (-CTR3a or -CTR3b forms) to the reaction mixture had an inhibitory effect on Ptre-CTR1 auto- and trans-phosphorylation. In contrast to Ptre-CTR1, Ptre-CTR3 may act as a positive regulator in ethylene signalling in poplar; however, this hypothesis requires in vivo confirmation. Thus, the ethylene signalling route in poplar seems to be under the control of certain additional mechanisms which have not been reported in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Populus/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , Fosforilação , Folhas de Planta/metabolismo , Populus/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
12.
Phys Chem Chem Phys ; 21(22): 11554-11563, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31134261

RESUMO

Biological membranes are characterized by lateral inhomogeneities, termed as membrane domains, which are regions enriched with specific types of lipids and proteins. While the functional consequences of membrane domains are well understood, the physicochemical study of domains has proved to be elusive, mainly due to varying spatiotemporal scales associated with them. In this perspective, we provide an overview of representative experimental approaches based on dynamic fluorescence microscopy to analyze organization and dynamics of membrane lipids and proteins. We further elucidate variation of dynamics as a function of area of observation, a unique feature of biological membranes, and its modulation with membrane components such as cholesterol and the actin cytoskeleton. In terms of spatial resolution, we provide examples from super resolution techniques that overcome the diffraction limit encountered in conventional optical microscopes. We conclude that judicious use of a combination of approaches of varying spatiotemporal resolutions, commensurate with spatiotemporal scales of a given membrane process, would provide a comprehensive dynamic model of the biological membrane in terms of membrane organization, dynamics and function.


Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Difusão , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana , Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/química , Espectrometria de Fluorescência/métodos , Fatores de Tempo
13.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091675

RESUMO

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, ß2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, ß2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-ß2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.


Assuntos
Albuminas/metabolismo , Cádmio/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Lipocalina-2/metabolismo , Microglobulina beta-2/metabolismo , Animais , Cádmio/farmacologia , Intoxicação por Cádmio , Linhagem Celular , Túbulos Renais Proximais/citologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Metalotioneína/metabolismo , Ligação Proteica , Ratos , Receptores de Superfície Celular/metabolismo
14.
Oxid Med Cell Longev ; 2019: 4546975, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049135

RESUMO

Alcoholic cardiomyopathy (ACM) caused by alcohol consumption manifests mainly as by maladaptive myocardial function, which eventually leads to heart failure and causes serious public health problems. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR on ACM remains unclear. The purpose of this study was to determine the role of PRR in myocardial fibrosis and the deterioration of cardiac function in alcoholic cardiomyopathy. Wistar rats were fed a liquid diet containing 9% v/v alcohol to establish an alcoholic cardiomyopathy model. Eight weeks later, rats were injected with 1 × 109v.g./100 µl of recombinant adenovirus containing EGFP (scramble-shRNA), PRR, and PRR-shRNA via the tail vein. Cardiac function was assessed by echocardiography. Cardiac histopathology was measured by Masson's trichrome staining, immunohistochemical staining, and dihydroethidium staining. In addition, cardiac fibroblasts (CFs) were cultured to evaluate the effects of alcohol stimulation on the production of the extracellular matrix and their underlying mechanisms. Our results indicated that overexpression of PRR in rats with alcoholic cardiomyopathy exacerbates myocardial oxidative stress and myocardial fibrosis. Silencing of PRR expression with short hairpin RNA (shRNA) technology reversed the myocardial damage mediated by PRR. Additionally, PRR activated phosphorylation of ERK1/2 and increased NOX4-derived reactive oxygen species and collagen expression in CFs with alcohol stimulation. Administration of the ERK kinase inhibitor (PD98059) significantly reduced NOX4 protein expression and collagen production, which indicated that PRR increases collagen production primarily through the PRR-ERK1/2-NOX4 pathway in CFs. In conclusion, our study demonstrated that PRR induces myocardial fibrosis and deteriorates cardiac function through ROS from the PRR-ERK1/2-NOX4 pathway during ACM development.


Assuntos
Cardiomiopatia Alcoólica/metabolismo , Sistema de Sinalização das MAP Quinases , Miocárdio/metabolismo , NADPH Oxidase 4/metabolismo , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina , Remodelação Ventricular , Animais , Cardiomiopatia Alcoólica/patologia , Modelos Animais de Doenças , Fibrose , Masculino , Miocárdio/patologia , Ratos , Ratos Wistar
15.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(2): 171-176, 2019 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-31106534

RESUMO

OBJECTIVE: To investigate the effects of interleukin (IL)-1ß preteated adipose tissue-derived mesenchymal stem cells (ADMSCs) on vascular endothelial growth factor (VEGF) secretion and macrophages M2 polarization. METHODS: After IL-1ß pretreated ADMSC for 24 h, the expression of cyclooxygenase-2 (COX-2) was detected using Western blot, and the secretions of prostaglandin E2 (PGE2) and VEGF were measured by ELISA method. Conditioned media collected from ADMSCs was used for culturing PMA-U937 macrophages for 72 h, and the expressions of CD163 and IL-10 mRNAs were detected using qRT-PCR. After inhibition of COX-2 expression in ADMSCs by Lentivirus silencing, secretion of VEGF and CD163 and IL-10 mRNA expressions were detected. RESULTS: IL-1ß pretreating ADMSCs increased the expression level of COX-2, enhanced PGE2 and VEGF secretions (P<0.05). In addition, the mRNA levels of CD163 and IL-10 were upregulated when ADMSCs were pretreated by IL-1ß in PMA-U937 cells. After downregulation of COX-2 in ADMSCs, the secretion of VEGF was suppressed, and the mRNA levels of CD163 and IL-10 were also significantly down-regulated. CONCLUSION: IL-1ß pretreatment enhanced the secretion of PGE2 and VEGF, and induced macrophage M2 polarization, which depended on COX-2-PGE2 signal pathway.


Assuntos
Polaridade Celular , Interleucina-1beta/farmacologia , Macrófagos/citologia , Células-Tronco Mesenquimais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo/citologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Interleucina-10/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Células U937
16.
Int J Med Microbiol ; 309(3-4): 252-257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31079999

RESUMO

Plants are always found together with bacteria and other microbes. Although plants can be attacked by phytopathogenic bacteria, they are more often engaged in neutral or mutualistic bacterial interactions. In the soil, plants associate with rhizobia or other plant growth promoting rhizosphere bacteria; above ground, bacteria colonise plants as epi- and endophytes. For mounting appropriate responses, such as permitting colonisation by beneficial symbionts while at the same time fending off pathogenic invaders, plants need to distinguish between the "good" and the "bad". Plants make use of proteins containing the lysin motif (LysM) for perception of N-acetylglucosamine containing carbohydrate structures, such as chitooligosaccharides functioning as symbiotic nodulation factors or bacterial peptidoglycan. Moreover, plant hydrolytic enzymes of the chitinase family, which are able to cleave bacterial peptidoglycan or chitooligosaccharides, are essential for cellular signalling induced by rhizobial nodulation factors during symbiosis as well as bacterial peptidoglycan during pathogenesis. Hence, LysM receptors seem to work in concert with hydrolytic enzymes that fine-tune ligand availability to either allow symbiotic interactions or trigger plant immunity.


Assuntos
Quitinases/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Receptores de Superfície Celular/metabolismo , Bactérias/química , Bactérias/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Lisina , Peptidoglicano/metabolismo , Proteínas de Plantas/química , Receptores de Superfície Celular/química , Transdução de Sinais
17.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075901

RESUMO

The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3ß in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3ß inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.


Assuntos
Moléculas de Adesão Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Células Epiteliais Alveolares/metabolismo , Animais , Bleomicina , Moléculas de Adesão Celular/genética , Camundongos , Agonistas do Receptor Purinérgico P2X/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores Purinérgicos P2X7/deficiência
18.
Emerg Microbes Infect ; 8(1): 734-748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130074

RESUMO

Many pathogens infect hosts through various immune evasion strategies. However, the molecular mechanisms by which pathogen proteins modulate and evade the host immune response remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a pathological strain that can induce mitogen-activated protein (MAP) kinase (Erk, Jnk and p38 MAPK) and NF-κB pathway activation and proinflammatory cytokine production, which then causes diarrheal diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Transforming growth factor ß-activated kinase-1 (TAK1) is a key regulator involved in distinct innate immune signalling pathways. Here we report that EHEC translocated intimin receptor (Tir) protein inhibits the expression of EHEC-induced proinflammatory cytokines by interacting with the host tyrosine phosphatase SHP-1, which is dependent on the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). Mechanistically, the association of EHEC Tir with SHP-1 facilitated the recruitment of SHP-1 to TAK1 and inhibited TAK1 phosphorylation, which then negatively regulated K63-linked polyubiquitination of TAK1 and downstream signal transduction. Taken together, these results suggest that EHEC Tir negatively regulates proinflammatory responses by inhibiting the activation of TAK1, which is essential for immune evasion and could be a potential target for the treatment of bacterial infection.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , MAP Quinase Quinase Quinases/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Fatores de Virulência/metabolismo , Animais , Infecções por Escherichia coli/microbiologia , Células HEK293 , Humanos , Macrófagos Peritoneais , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Células RAW 264.7
19.
Vet Res Commun ; 43(2): 115-122, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30989431

RESUMO

Dendritic cells (DC) are important antigen-presenting cells and are among the least characterized immune cells in the chicken. In order to obtain chicken DC, current protocols require isolation of bone marrow myeloid progenitor cells and induction of DC differentiation with supplemental cytokines or negative selection of splenic cell preparations. Chicken peritoneal exudate cells (PEC) have traditionally been a source of various immune cells for ex vivo studies, primarily to investigate heterophils and macrophages. In this study, we observe the presence of CD205+ PEC populations, a marker of DC, as an additional resource to isolate and study chicken primary DCs. A panel of monoclonal antibodies was developed against the chicken CD205 DC marker and used to isolate CD205+ DC from the PEC population using magnetic bead cell sorting. This study reports the development of new anti-CD205 monoclonal antibodies as a reagent for chicken DC research, as well as PEC as a potential source of CD205+ DC for ex vivo studies in the chicken.


Assuntos
Antígenos CD/metabolismo , Separação Celular/veterinária , Galinhas/imunologia , Células Dendríticas/citologia , Lectinas Tipo C/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Células Dendríticas/imunologia , Sefarose/imunologia
20.
Pediatr Rheumatol Online J ; 17(1): 13, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943984

RESUMO

BACKGROUND: Kawasaki disease (KD) is the most common acute coronary vasculitis disease to occur in children. Its incidence has been attributed to the combined effects of infection, genetics, and immunity. Although the etiopathogenesis of KD remains unknown, we have performed a survey of global genetic DNA methylation status and transcripts expression in KD patients in order to determine their contribution to the pathogenesis of KD. METHODS: We recruited 148 participants for this case-control study. The chip studies consisted of 18 KD patients that were analyzed both before undergoing intravenous immunoglobulin (IVIG) treatment and at least 3 weeks afterward, as well as 36 non-KD control subjects, using Illumina HumanMethylation450 BeadChip and Affymetrix GeneChip® Human Transcriptome Array 2.0. We then carried out real-time quantitative PCR on a separate cohort of 94 subjects for validation. RESULTS: According to our microarray study, CD177, a neutrophil surface molecule, appeared to be significantly upregulated in KD patients when compared to controls with epigenetic hypomethylation. After patients received IVIG treatment, CD177 mRNA levels decreased significantly. PCR validation indicated that the CD177 expression is consistent with the Transcriptome Array 2.0 results. Furthermore, the area under the curve values of CD177 between KD patients and controls is 0.937. We also observed significantly higher CD177 levels in typical KD than in incomplete presentation or KD with IVIG resistance. CONCLUSION: In this study, we have demonstrated the epigenetic hypomethylation and increased expression of CD177 during the acute stage of KD. Furthermore, a higher expression of CD177 in KD patients with typical presentation was associated with IVIG resistance.


Assuntos
Isoantígenos/metabolismo , Síndrome de Linfonodos Mucocutâneos/metabolismo , Receptores de Superfície Celular/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Metilação de DNA , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Isoantígenos/sangue , Masculino , Síndrome de Linfonodos Mucocutâneos/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/sangue , Sensibilidade e Especificidade , Transcriptoma/genética
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