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1.
J Nanosci Nanotechnol ; 20(2): 752-759, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31383070

RESUMO

An Al3+-based metal-organic framework (MOF), CAU-11-COOH, with a V-shaped ligand, DPSDA (3,3'-4,4'-diphenylsulfonetetracarboxylic dianhydride), was prepared using the solvothermal method, and was characterized using powder X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, elemental analysis, thermogravimetric analysis, Brunauer-Emmett-Teller analysis, and CO2 adsorption. The catalytic efficiency of CAU-11-COOH was investigated in the solvent-free cycloaddition of carbon dioxide with epoxides, which yielded five-membered cyclic carbonates under mild reaction conditions. CAU-11-COOH with a co-catalyst, tetrabutylammonium bromide (TBAB), gave higher than 98% yield of epichlorohydrin carbonate at 80 °C without a solvent. A plausible reaction mechanism in which the Lewis acidic metal center, an uncoordinated carboxyl group, and a nucleophilic bromide anion operate synergistically is proposed. The CAU-11-COOH catalysts were found to exhibit high thermal stability and could be reused more than four times without any significant reduction in activity.

3.
Methods Mol Biol ; 1957: 309-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919362

RESUMO

Information contained in the structure of extracellular ligands is transmitted across the cell membrane through allosterically induced changes in G protein-coupled receptor (GPCR) conformation that occur upon ligand binding. These changes, in turn, are imprinted upon intracellular effectors like arrestins and help determine which of its many functions are performed. Intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET), in which both the fluorescence donor and acceptor are contained within the same protein, can be used to report on activation-induced changes in protein conformation. Here, we describe a method using a series of Rluc-arrestin3-FlAsH-BRET biosensors to measure stimulus-induced changes in arrestin conformation in live cells. Each Rluc-arrestin3-FlAsH-BRET construct contains an N-terminal Renilla luciferase fluorescence donor that excites a fluorescent arsenical targeted to a different position within the protein by mutational insertion of a tetracysteine tag motif. Changes in net BRET upon GPCR stimulation can thus be viewed from multiple vantage points within the protein and used to develop an arrestin3 "conformational signature" that is receptor- and ligand-specific. This method can be used to determine how differences in GPCR and ligand structure influence information transfer across the plasma membrane and to classify GPCRs and/or ligands based on their capacity to induce different arrestin3 activation modes.


Assuntos
Arrestina/metabolismo , Arsênico/química , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Fluoresceína/química , Análise de Dados , Células HEK293 , Humanos , Reprodutibilidade dos Testes
5.
Sci Signal ; 11(549)2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254056

RESUMO

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. ß-Arrestins (ßArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete ßArr1/2 and G proteins have cast doubt on the role of ß-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of ßArr1/2 and reconstitution with ßArr1/2 in three different parental and CRISPR-derived ßArr1/2 knockout HEK293 cell pairs to assess the effect of ßArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for ßArr2 or ßArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For ß2 adrenergic receptors (ß2ARs) and ß1ARs, ßArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with ßArr1/2. Loss of desensitization and receptor internalization in CRISPR ßArr1/2 knockout cells caused ß2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing ßArr1/2. These data suggest that ßArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from ßArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

7.
J Lipid Res ; 58(2): 325-338, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27881715

RESUMO

HDL normally transports about 50-70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.


Assuntos
Lipoproteínas HDL/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Receptores Depuradores Classe B/metabolismo , Esfingosina/análogos & derivados , Animais , Aorta/metabolismo , Transporte Biológico/genética , Cálcio/metabolismo , Células HEK293 , Humanos , Lipoproteínas HDL/genética , Técnicas de Cultura de Órgãos , Ratos , Receptores de Lisoesfingolipídeo/genética , Receptores Depuradores Classe B/genética , Transdução de Sinais , Esfingosina/metabolismo
10.
Int J Mol Sci ; 17(9)2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27598132

RESUMO

Polydeoxyribonucleotide (PDRN), a deoxyribonucleotide polymer, is popularly used for faster healing of cutaneous wounds and boosting of neocollagenesis of photoaged skin among current dermatologic practitioners. Some patients receiving PDRN injection treatment also reported improvement of photoaging-associated mottled pigmentation (PMP). To investigate the effect of PDRN on cutaneous melanogenesis, we examined the effect of PDRN and an available product (Placentex(®)) containing PDRN on melanogenesis using human melanocytes-keratinocytes cocultures and mouse melanocytes. Melanin content, tyrosinase activity, and levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein (TRP-1) were determined. Intracellular signaling pathways were assessed by Western blotting. PDRN and Placentex(®) led to decreases in melanin content, tyrosinase activity, and MITF and TRP-1 expression with concomitant increases in phosphorylated forms of extracellular signal-regulated protein kinase (ERK) and AKT in mouse melanocytes. More importantly, both PDRN and Placentex(®) significantly suppressed the melanin content in human melanocyte-keratinocyte cocultures. Clinical evaluation of six female patients with facial hyperpigmentation after three sessions of intradermal PDRN injections using a 5-point scale revealed that PDRN led to more than noticeable improvements in hyperpigmented lesions. This is the first study to demonstrate that PDRN, which is known for its wound-healing properties, may have novel anti-melanogenesis and potential skin whitening properties.


Assuntos
Melanócitos/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Preparações Clareadoras de Pele/farmacologia , Pigmentação da Pele/efeitos dos fármacos , Adulto , Idoso , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Melaninas/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Polidesoxirribonucleotídeos/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Preparações Clareadoras de Pele/administração & dosagem , Tripsina/genética , Tripsina/metabolismo
11.
Lasers Surg Med ; 48(9): 878-886, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27551954

RESUMO

BACKGROUND: Non-invasive devices for fat reduction involving high-intensity focused ultrasound (HIFU) are attracting attention. HIFU can deliver energy to the desired depth and can ablate subcutaneous adipose tissue (SAT), but purpura and pain may still limit its use. OBJECTIVES: The aim of this study was to investigate the effects of a novel HIFU device for fat destruction with a contact cooling system compared to HIFU without contact cooling. METHODS: A group of three pigs were administered a series of four HIFU treatments with or without contact cooling over a period of 12 weeks. Energy fluence parameters ranged from 60 to 300 J/cm2 . Immediately after the treatment and at 1, 4, and 12 weeks, the tissue was studied by hematoxylin and eosin (H&E), Masson-trichrome, toluidine blue, CD68 staining, and transmission electron microscopy. Three human volunteers also received treatment with this HIFU device with cooling and were evaluated subjectively and objectively by computed tomography (CT). RESULTS: HIFU treatment with a contact cooling decreased the skin surface temperature and prevented epidermal damage. Ecchymosis was observed on the non-cooled area immediately after HIFU treatment, but not on the cooled area. Histological analyses on both areas (cooled and non-cooled) revealed disrupted adipocytes in the treatment area immediately, at 1 and 4 weeks following treatment. Lipophagic histiocytic fat necrosis was evident at 4 weeks. Finally, at 12 weeks all inflammation subsided, and the lobules were markedly atrophied with reduced SAT thickness. The human volunteers experienced reduction of a few centimeter-range reduction in waist circumference after 4 weeks and pain was tolerable without bruising. CONCLUSIONS: HIFU treatment with a cooling system efficiently destroyed adipocytes. This novel HIFU device with an added contact cooling system may provide an effective, safe and less painful treatment as a non-invasive device for fat reduction. Lasers Surg. Med. 48:878-886, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Crioterapia , Ablação por Ultrassom Focalizado de Alta Intensidade/instrumentação , Lipectomia/instrumentação , Gordura Subcutânea/cirurgia , Adulto , Animais , Feminino , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Humanos , Lipectomia/métodos , Gordura Subcutânea/patologia , Suínos
12.
Sci Rep ; 6: 30217, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27456065

RESUMO

Disruption of the TGF-ß pathway is associated with liver fibrosis and suppression of liver tumorigenesis, conditions associated with low Vitamin D (VD) levels. However, potential contributions of VD to liver tumor progression in the context of TGF-ß signaling remain unexplored. Our analyses of VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3(+/-) mice, with a three-fold increase in TLR7 expression compared to controls. ChIP and transcriptional assays confirm Smad3 binding at two TLR7 promoter SBE sites. Molecular interactions between TGF-ß pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-ß pathway member expression levels and ß-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-ß member expression with lower ß-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-ß pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and ß-catenin activation. VD could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling.


Assuntos
Neoplasias Hepáticas Experimentais/patologia , Glicoproteínas de Membrana/metabolismo , Proteína Smad3/genética , Receptor 7 Toll-Like/metabolismo , Fator de Crescimento Transformador beta/genética , Deficiência de Vitamina D/complicações , Proteínas Wnt/metabolismo , Animais , Humanos , Neoplasias Hepáticas Experimentais/complicações , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Vitamina D/administração & dosagem
13.
J Cosmet Laser Ther ; 18(7): 381-386, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27249461

RESUMO

BACKGROUND: Laser toning using low-fluence 1064-nm Q-switched neodymium-doped yttrium aluminum laser (QSNY) has gained popularity in the treatment of photoaging-associated mottled pigmentation (PMP). However, hypopigmentation or lack of efficacy has been reported depending on the fluences used. OBJECTIVE: To compare a novel fractional 1064-nm QSNY with conventional 1064-nm QSNY for the treatment of photoaging-associated mottled pigmentary lesions except epidermal lesions of lentigines and freckles through a randomized, split-face, double-blind study. MATERIALS AND METHODS: Thirteen Asian women were treated every week for 6 weeks with fractional 1064-nm QSNY on one side of the face and conventional 1064-nm QSNY on the other side. We evaluated the pigmentation area and severity index (PSI), melanin index, erythema index, and the patient's global assessment of improvement. RESULTS: At three months post-treatment, the PSI score improved compared with baseline, by 14.48% on the conventional 1064-nm QSNY side and 21.81% on the fractional 1064-nm QSNY side. Both groups showed improvements in the melanin index. CONCLUSION: Both fractional 1064-nm QSNY and strictly low-fluence conventional 1064-nm QSNY are moderately effective against PMP and other photoaging signs. Fractional laser toning shows better subjective outcomes than conventional toning.


Assuntos
Epidermólise Bolhosa Simples/terapia , Terapia a Laser/métodos , Lasers de Estado Sólido/uso terapêutico , Terapia com Luz de Baixa Intensidade/métodos , Luz Solar/efeitos adversos , Adulto , Grupo com Ancestrais do Continente Asiático , Terapia Combinada , Método Duplo-Cego , Feminino , Humanos , Satisfação do Paciente , Estudos Prospectivos , Resultado do Tratamento
14.
Int J Mol Sci ; 17(6)2016 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-27314341

RESUMO

Solar lentigo (SL) is a representative photoaging skin disorder. Alteration of the main epidermal constituent cells-keratinocytes and melanocytes-in relation to the photoaged dermal environment or chemokine/cytokine network is suggested as its pathogenesis. Among these, we focused on monocyte chemoattractant protein-1 (MCP-1), as it is known to be associated with tissue aging. For the first time, we report that the MCP-1 receptor, CCR2, is expressed in normal human melanocytes. In SL tissue, there was an increase of CCR2+Melan A+ melanocytes with positivity to Rb protein compared to peri-lesional normal skin. MCP-1 induced the proliferation of normal human melanocytes without a significant change in the melanin content. MCP-1 treatment in normal human keratinocytes showed an increase in senescence-associated ß-galactosidase staining and p53 and p21 protein expressions. In summary, MCP-1 may participate in the development of SL by affecting epidermal constituent cells, for example, by inducing melanocyte proliferation and keratinocyte senescence.


Assuntos
Células Epidérmicas , Epiderme/metabolismo , Lentigo/etiologia , Lentigo/metabolismo , Luz Solar/efeitos adversos , Idoso , Biomarcadores , Biópsia , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacologia , Epiderme/efeitos dos fármacos , Epiderme/efeitos da radiação , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Lentigo/tratamento farmacológico , Lentigo/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Melanócitos/efeitos da radiação , Pessoa de Meia-Idade , beta-Galactosidase/metabolismo
15.
Ann Dermatol ; 28(2): 255-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27081280
16.
Nature ; 531(7596): 665-8, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27007854

RESUMO

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in ß-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of ß-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in ß-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average ß-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.


Assuntos
Arrestinas/química , Arrestinas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Animais , Ativação Enzimática , Células HEK293 , Humanos , Ligantes , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Conformação Proteica , Transporte Proteico , Ratos , Receptores Acoplados a Proteínas-G/química , beta-Arrestinas
17.
J Cutan Pathol ; 43(4): 324-33, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26695102

RESUMO

BACKGROUND: Few studies have evaluated the histopathologic features of cutaneous extranodal natural killer (NK)/T-cell lymphoma (ENKTL), and the histopathologic spectrum of this disease according to its clinical morphology remains unclear. OBJECTIVE: This study investigated the differences in pathologic findings of cutaneous ENKTL depending on clinical morphology. METHODS: A total of 41 cases of cutaneous ENKTL were included. Skin lesions were classified according to clinical morphology as: (i) nodular lesions, (ii) cellulitis or abscess-like swellings and (iii) erythematous to purpuric patches. Histopathologic variables were compared between groups. RESULTS: Perivascular infiltration of tumor cells and vasculopathy in the dermis and subcutaneous layer were common microscopic findings irrespective of clinical morphology. Erythematous to purpuric patches were mainly composed of small-sized tumor cells, whereas medium- to large-tumor cells were predominant in lesions of other clinical morphologies. The density of tumor cell infiltration was significantly higher in cellulitis or abscess-like lesions or nodular lesions compared with erythematous to purpuric patches. A panniculitis-like pattern and angiocentricity were less common in patch lesions than in cellulitis-like swelling and nodular lesions. CONCLUSION: There is a histopathologic spectrum of cutaneous ENKTL that is dependent on the clinical morphology.


Assuntos
Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Feminino , Humanos , Masculino , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/patologia , Estudos Retrospectivos
18.
J Dermatol ; 43(5): 526-31, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26507367

RESUMO

Rosacea has a wide spectrum of clinical features, which include persistent facial redness, flushing, telangiectasia, inflammatory papules/pustules, hypertrophy and/or ocular features. The prognosis of rosacea according to clinical subtype has not been evaluated. We analyzed the prognosis of rosacea in 234 patients, which included 120 patients with mixed subtype, 75 with the erythematotelangiectatic rosacea subtype and 39 with the papulopustular rosacea (PPR) subtype. The prognosis of rosacea was classified as: (i) no improvement; (ii) partial remission; and (iii) complete remission. The frequencies of complete remission, time to complete remission and 1-year complete remission rate were compared between subtypes. Follow-up periods ranged 2-72 months (median follow-up, 17.5). Aggravation of the disease was found in 50.4% of patients during follow up. Partial or complete remission was noted in 61.5% and 20.9% of patients, respectively. The median time to complete remission was 56.0 months. The prognosis of disease was more favorable for patients with the PPR subtype than for patients with other subtypes with respect to the frequency of complete remission, median time to complete remission and the 2-year complete remission rate. In conclusion, papulopustular rosacea without remarkable centrofacial erythema showed a more favorable prognosis than other subtypes. Erythematotelangiectatic lesions in rosacea patients present a challenge for the treatment of rosacea.


Assuntos
Eritema/diagnóstico , Rosácea/classificação , Rosácea/diagnóstico , Índice de Gravidade de Doença , Adolescente , Adulto , Idoso , Criança , Face , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Adulto Jovem
20.
Ann Dermatol ; 27(4): 439-41, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26273162

RESUMO

Cutaneous metastasis from gastric adenocarcinoma is uncommon, and the eyelid is a rare metastatic site. Three patterns of clinical presentation of eyelid metastasis have been described: nodular, infiltrative, and ulcerated. The infiltrative pattern, also known as an inflammatory diffuse pattern or mask-like metastasis, can be easily misdiagnosed as cellulitis or contact dermatitis. Here, we report a case of gastric adenocarcinoma in a 75-year-old man who presented with a localized erythematous plaque on his eyelid that developed four months earlier. The patient had been treated with an antimicrobial agent owing to suspicion of preseptal cellulitis. Gastric adenocarcinoma metastasis was diagnosed on the basis of histopathological examination and immunophenotyping (i.e., cytoplasmic epithelial membrane antigen, cytokeratin- 7, cytokeratin-20, and carcinoembryonic antigen). For patients with malignant neoplasms, persistent skin lesions similar to cellulitis or contact dermatitis should be suspected of metastasis derived from an internal malignancy, even for very rare sites of metastasis.

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