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1.
PLoS One ; 15(7): e0234792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614850

RESUMO

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.


Assuntos
Proteínas Angiogênicas/biossíntese , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Substituição de Aminoácidos , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Encéfalo/citologia , Proteínas de Transporte/análise , Linhagem da Célula , Epitopos/imunologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Desenvolvimento Muscular , Proteína MyoD/análise , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Coelhos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Sequências Repetitivas de Aminoácidos , Pele/citologia , Especificidade da Espécie , Tatuagem , Adulto Jovem
2.
J Agric Food Chem ; 68(20): 5763-5775, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32374605

RESUMO

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.


Assuntos
Glutens/genética , Hordeum/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Proteoma/metabolismo , Antígenos de Plantas/análise , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Técnicas de Inativação de Genes , Globulinas/análise , Globulinas/genética , Globulinas/metabolismo , Glutens/química , Glutens/metabolismo , Hordeum/genética , Hordeum/metabolismo , Espectrometria de Massas , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteoma/química , Proteoma/genética , Proteômica
3.
Biochem Biophys Res Commun ; 524(2): 346-353, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32000999

RESUMO

Recent developments in tissue clearing methods such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) have allowed for the three-dimensional analysis of biological structures in whole, intact tissue, providing greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, preventing the wide application of these techniques to fragile tissues such as developing embryos. Here, we present optimized passive clearing techniques, mPACT-A, that improve tissue rigidity without the expense of optical transparency. We also present a further modified mPACT-A protocol that is specifically optimized for handling mouse embryos, which are small and fragile, such that they easily dismantle when processed via established tissue clearing methods. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PRDM proteins, PRDM7 and PRDM12, in intact cleared mouse embryos at various stages of development. We observed strong PRDM7 and PRDM12 expression in the developing mouse nervous system, suggestive of potential roles in neural development that will be tested in future functional studies.


Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos/embriologia , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Transporte/análise , Desenvolvimento Embrionário , Feminino , Imageamento Tridimensional , Imuno-Histoquímica , Hibridização In Situ , Camundongos/genética , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/análise
4.
Anal Chem ; 92(4): 3417-3425, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31970977

RESUMO

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.


Assuntos
Proteínas de Transporte/análise , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Esfingolipídeos/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Bicamadas Lipídicas/química , Esfingolipídeos/química
5.
Anal Chem ; 92(2): 1963-1971, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31854989

RESUMO

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.


Assuntos
Proteínas de Transporte/análise , Proteínas de Escherichia coli/análise , Glicoproteínas/análise , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Actinobacillus pleuropneumoniae/enzimologia , Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Escherichia coli/química , Proteínas de Escherichia coli/síntese química , Proteínas de Escherichia coli/genética , Glicoproteínas/síntese química , Glicoproteínas/genética , Glicosilação , Glicosiltransferases/química , Mutação , Biblioteca de Peptídeos , Estudo de Prova de Conceito , Proteínas Recombinantes/análise , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética
6.
Mol Carcinog ; 59(1): 126-135, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31713931

RESUMO

Fibrous sheath interacting protein 1 (FSIP1) is frequently activated in a variety of tumors including breast cancer. However, the clinical significance of FSIP1 in hormone receptor (HR)-positive breast cancer is unclear. We analyzed the expression and clinical significance of FSIP1 in human breast cancer databases. A comprehensive analysis of 1094 gene expression profiles of breast cancer in The Cancer Genome Atlas revealed that FSIP1 overexpression correlated with decreased overall survival in HR-positive breast cancer patients. We also showed that knockdown of FSIP1 in T47D and BT474 cell lines resulted in decreased cell proliferation and migration in vitro. Furthermore, we retrospectively examined the expression and prognostic value of FSIP1 in 129 breast cancer patients to examine the expression of FSIP1 by the immunohistochemical method and got the similar results that high expression of FSIP1 predicts poor prognosis. Therefore, FSIP1 has a crucial role in HR-positive breast cancer and represents an attractive therapeutic target for HR-positive breast cancer.


Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Receptores Estrogênicos/análise , Proteínas de Plasma Seminal/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Proteínas de Transporte/análise , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Prognóstico , Proteínas de Plasma Seminal/análise , Transcriptoma , Regulação para Cima
7.
Vet Immunol Immunopathol ; 219: 109972, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733501

RESUMO

This study aimed to determine if intrauterine-infused lipopolysaccharides (LPS) can be translocated to the mammary glands and induce an inflammatory response. Thirty-seven goats were divided into two experiments. Nineteen goats (control group, n = 9; LPS group, n = 10) were subjected to intravenous injection of LPS, and eighteen goats (control group, n = 8; LPS group, n = 10) were subjected to intrauterine infusion of LPS. Milk and blood samples were collected before and after the LPS challenge, to measure the blood leukocyte count (BLC), plasma LPS-binding protein (LBP), milk yield, milk somatic cell count (SCC), lactoferrin (LF), milk lactoperoxidase (LPO) activity, and pro- and anti-inflammatory cytokines in plasma and milk. Mammary gland tissues were collected from the parenchyma before and after the LPS challenge, for immunohistochemistry of LPS. In the intravenous injection experiment, the BLC (P < 0.001) and milk yield (P = 0.009) were lower, whereas the LF concentration (P < 0.001) and milk LPO activity (P < 0.001) were higher in the LPS group compared to that in the control group. LPS was detected in the mammary gland 3 and 24 h after intravenous injection of LPS. In the intrauterine infusion experiment, the mean concentrations of IL-1ß and IL-6 in milk were higher in the LPS group compared to that in the control group (P = 0.004 and P = 0.017, respectively), whereas there were no changes in milk yield or SCC. LPS was detected in the connective tissues and interepithelial spaces of the alveoli of the mammary glands 24 h after intrauterine infusion of LPS. We conclude that intrauterine-infused LPS can be translocated to the mammary glands from the uterus, however, the amount of translocated LPS might not be enough to induce symptoms of clinical or subclinical mastitis.


Assuntos
Lipopolissacarídeos/administração & dosagem , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Mastite/veterinária , Leite/química , Útero/efeitos dos fármacos , Proteínas da Fase Aguda/análise , Administração Intravenosa , Animais , Proteínas de Transporte/análise , Feminino , Cabras/imunologia , Lactação/efeitos dos fármacos , Lactoferrina/análise , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/análise , Leite/citologia
8.
Cell Physiol Biochem ; 53(1): 200-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31287628

RESUMO

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.


Assuntos
Proteínas de Transporte/metabolismo , Glutamina/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Ciclo Celular , Linhagem Celular , Cromatografia Líquida , Fatores de Iniciação em Eucariotos , Glutamina/análise , Insulina/análise , Camundongos , Fibras Musculares Esqueléticas/química , Fosfoproteínas/análise , Fosforilação , Proteínas Proto-Oncogênicas c-akt/análise , Proteína S6 Ribossômica/análise , Espectrometria de Massas em Tandem
9.
World J Gastroenterol ; 25(22): 2699-2705, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31235993

RESUMO

The exocrine structure is significantly affected by diabetes because of endocrine structure-function disorder within the pancreas. Exocrine pancreatic dysfunction (EPD) is the general name of the malabsorption process resulting from inadequate production, release, decreased activation, and/or insufficient degradation of enzymes required for digestion from pancreatic acinar cells. It is important to diagnose patients early and correctly, since there may be both macro- and micro-nutrient deficiency in EPD. In this paper, EPD, the diabetes-EPD relationship, and the predictive, effective factors affecting the emergence of EPD are briefly explained and summarized with contemporary literature and our experienced based on clinical, lab, and radiological findings.


Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Insuficiência Pancreática Exócrina/etiologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Terapia de Reposição de Enzimas , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/terapia , Fezes/enzimologia , Humanos , Pâncreas Exócrino/enzimologia , Pâncreas Exócrino/fisiopatologia , Elastase Pancreática/análise , Elastase Pancreática/metabolismo , Inibidores da Bomba de Prótons/uso terapêutico
10.
APMIS ; 127(8): 588-593, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31233243

RESUMO

Microfibrillar-associated protein 4 (MFAP4) is a non-structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde-fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3-39.1) and 25.5 U/L (18.1-43.3) vs 17.7 U/L (13.7-21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.


Assuntos
Artrite Reumatoide/imunologia , Proteínas de Transporte/análise , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Osteoartrite/imunologia , Membrana Sinovial/imunologia , Idoso , Anticorpos Monoclonais/imunologia , Biomarcadores/análise , Proteínas de Transporte/imunologia , Estudos de Casos e Controles , Proteínas da Matriz Extracelular/imunologia , Feminino , Glicoproteínas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/química , Membrana Sinovial/patologia
11.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096561

RESUMO

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.


Assuntos
Alérgenos/imunologia , Anacardium/química , Imunoglobulina E/imunologia , Pólen/efeitos adversos , Pólen/química , Rinite Alérgica Sazonal/induzido quimicamente , Adolescente , Adulto , Idoso , Alérgenos/química , Animais , Antígenos de Plantas/efeitos adversos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Betula/metabolismo , Brasil , Proteínas de Transporte/análise , Proteínas de Transporte/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Feminino , Humanos , Masculino , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/genética , Proteômica , Rinite Alérgica Sazonal/imunologia , Testes Cutâneos
12.
Int J Mol Med ; 43(6): 2440-2450, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017263

RESUMO

Inflammatory response serves an important role in diabetic nephropathy (DN); however, the mechanism of inflammatory response results in renal damage is not yet clear. The aim of the present study was to investigate the role of the thioredoxin interacting protein (TXNIP)/NOD­like receptor protein 3 (NLRP3) axis­mediated activation of NLRP3 inflammasome in DN. A diabetic rat model was induced by streptozotocin injection. Hematoxylin and eosin (H&E) staining and streptavidin­peroxidase staining were then used to examine the kidney tissue morphology, and TXNIP and NLRP3 protein expression levels, respectively. Furthermore, RNA interference technology was applied to silence the TXNIP gene. TXNIP and NLRP3 inflammasome activation­associated proteins and mRNAs were detected by western blot analysis and RT­qPCR, respectively. Enzyme­linked immunosorbent assay was also used to examine interleukin (IL)­1 and IL­18 expression, while flow cytometry was performed to detect reactive oxygen species production. The data revealed that TXNIP and NLRP3 were overexpressed in kidney tissue of DN rats, and the level of antioxidant genes was downregulated. It was also observed that glucose promoted TXNIP expression and activation of NLRP3 inflammasome in a time­dependent and dose­dependent manner, therefore promoting inflammatory responses. Silencing of TXNIP gene resulted in the downregulation of NLRP3 inflammasome activation, and inhibited the expression levels of IL­1 and IL­18 in a high­glucose environment. Furthermore, low expression of TXNIP promoted the levels of antioxidant genes and reduced the ROS levels. Taken together, the high­glucose environment was able to upregulated the level of inflammatory factors by promoting the expression of TXNIP and the activation of NLRP3 inflammasome, consequently participating in DN.


Assuntos
Proteínas de Transporte/imunologia , Diabetes Mellitus Experimental/imunologia , Nefropatias Diabéticas/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Proteínas de Transporte/análise , Proteínas de Ciclo Celular , Linhagem Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Glucose/imunologia , Humanos , Inflamassomos/análise , Inflamação/imunologia , Inflamação/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/análise , Estresse Oxidativo , Ratos Sprague-Dawley
13.
Anticancer Res ; 39(4): 1869-1874, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30952727

RESUMO

BACKGROUND/AIM: Chronic inflammation in end-stage kidney is associated with the development of pre-neoplastic lesions and renal cell tumors. The aim of this study was to clarify the role of the inflammatory microenvironment in this process. MATERIALS AND METHODS: We used representative microscopic slides from 11 end stage-kidneys containing pre-neoplastic lesions and tumors and applied immunohistochemistry to detect IL-6, SAA1 and LBP expression. We also applied array-based comparative genomic hybridization (CGH) analysis to detect genomic changes in tumor cells. RESULTS: We identified strong expression of IL6, LBP and SAA1 in activated stromal fibroblasts, in proliferating epithelial and tumor cells. Array CGH detected unusual genomic changes in tumor cells. CONCLUSION: Our data indicate that expression of IL6, acute phase protein SAA1 and LBP maintain a long-lasting inflammatory microenvironment that leads to remodeling of end-stage kidneys and the development of unique types of renal cell tumors.


Assuntos
Mediadores da Inflamação/análise , Interleucina-6/análise , Falência Renal Crônica/metabolismo , Neoplasias Renais/química , Rim/química , Lesões Pré-Cancerosas/metabolismo , Microambiente Tumoral , Proteínas da Fase Aguda/análise , Adulto , Idoso , Proteínas de Transporte/análise , Proliferação de Células , Hibridização Genômica Comparativa , Células Epiteliais/química , Células Epiteliais/patologia , Feminino , Fibroblastos/química , Fibroblastos/patologia , Humanos , Imuno-Histoquímica , Rim/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteína Amiloide A Sérica/análise , Células Estromais/química , Células Estromais/patologia
14.
Curr Microbiol ; 76(6): 698-705, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955044

RESUMO

Group A streptococcus (GAS) is an important human pathogen whose clinical isolates differ in their ability to produce hydrogen peroxide (H2O2). H2O2 is primarily produced by the enzyme lactate oxidase (LctO), an in depth in silico research revealed that all genome-sequenced GAS possess the required gene lctO. The importance of lctO for GAS is underlined by its highly conserved catabolite control element (cre box) as well as its perfect promotor sequence in comparison to the known consensus sequences of the Gram-positive model organism Bacillus subtilis. In this study, we provide further insight in the function and regulation of lactate oxidase by analyzing a large group of clinical GAS isolates. We found that H2O2 production increased over time in the late stationary phase; after 4 days of incubation, 5.4% of the isolates showed a positive result at 37 °C, while the rate increased to 16.4% at 20 °C. This correlation between H2O2 production and low temperatures suggests additional regulatory mechanisms for lctO besides catabolite control protein A (CcpA) and indicates that lctO might play a role for GAS energy metabolism at sub-body temperatures. Furthermore, we could identify that H2O2 production was different among clinical isolates; we could correlate H2O2 production to emm-types, indicating that emm-types 6 and 75 had the highest rate of H2O2 production. The emm-type- and temperature-dependent H2O2 production of clinical GAS isolates might contribute to their different survival strategies.


Assuntos
Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Transporte/análise , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxidantes/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/efeitos da radiação , Proteínas de Bactérias/metabolismo , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Temperatura
15.
Rev. esp. enferm. dig ; 111(4): 294-300, abr. 2019. tab
Artigo em Espanhol | IBECS | ID: ibc-189926

RESUMO

Antecedentes: se ha propuesto que el sobrecrecimiento bacteriano del intestino delgado (SBID) y la traslocación bacteriana a través de la pared intestinal se relacionan con el hígado graso no alcohólico (HGNA). El objetivo del presente estudio ha sido estudiar dicha relación en obesos mórbidos. Pacientes y métodos: se incluyeron consecutivamente pacientes con obesidad mórbida previo a su intervención de cirugía bariátrica. Los criterios de exclusión fueron: biopsia hepática normal, otras causas de enfermedad hepática o atrofia de la mucosa duodenal. Se realizó una gastroscopia para cultivo del aspirado duodenal, biopsias duodenales y extracción de sangre venosa periférica para estudio de lipopolisacárido (LPS) y proteína de unión del LPS (LBP). La biopsia hepática se realizó durante la intervención quirúrgica. Resultados: se incluyeron 71 pacientes; 26 fueron excluidos por biopsia hepática normal. Cuarenta y cinco tenían HGNA. Dieciocho eran varones, con edad media de 45,8 años (22-69) e índice de masa corporal (IMC) de 47,8 kg/m2 (37-58); el 25% tuvo SBID en el cultivo del aspirado duodenal. Existió significación estadística entre niveles de LBP y SBID con el grado de esteatosis (p < 0,05 y p = 0,077, respectivamente). No existió relación estadística con el índice de esteatohepatitis no alcohólica (EHNA), aunque sí hubo una tendencia a su asociación. Los niveles de LPS no guardaron relación con el grado de esteatosis o el índice de EHNA. Conclusiones: en pacientes con obesidad mórbida e HGNA se observan mayores niveles circulantes de LBP y mayor frecuencia de SBID cuanto mayor es el grado de esteatosis hepática


Background: small intestinal bacterial overgrowth (SIBO) and bacterial translocation across the intestinal wall have been allegedly associated with non-alcoholic fatty liver (NAFL). Our goal was to study such alleged association in morbidly obese patients. Patients and methods: patients with morbid obesity were consecutively included prior to bariatric surgery. Exclusion criteria included normal liver biopsy, other causes of liver disease, and duodenal mucosal atrophy. A gastroscopy was performed for duodenal aspirate culture and duodenal biopsy, and peripheral venous blood was drawn to assess lipopolysaccharide (LPS) and LPS-binding protein (LBP) levels. A liver biopsy was carried out during surgery. Results: seventy-one patients were included; 26 were excluded because of normal liver biopsy. Forty-five had NAFL. Eighteen were male, mean age was 45.8 years (22-69), and BMI was 47.8 kg/m2 (37-58). A total of 25% had SIBO in their duodenal aspirate culture. There was statistical significance for the association of LBP levels and SIBO with steatosis grade (p < 0.05 and p = 0.077, respectively). There was no statistical association with non-alcoholic steatohepatitis (NASH) index, but a trend towards association was found. LPS levels were not associated with steatosis grade or NASH index. Conclusions: the higher the grade of liver steatosis, the higher were the circulating LBP levels and SIBO rates seen in patients with morbid obesity and NAFL


Assuntos
Humanos , Masculino , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Fígado Gorduroso/microbiologia , Hepatopatia Gordurosa não Alcoólica/microbiologia , Obesidade Mórbida/microbiologia , Translocação Bacteriana/fisiologia , Proteínas da Fase Aguda/análise , Biomarcadores/análise , Estudos Transversais , Proteínas de Transporte/análise , Lipopolissacarídeos/análise , Estudos Prospectivos , Síndrome Metabólica/fisiopatologia
16.
BMC Cancer ; 19(1): 289, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925904

RESUMO

BACKGROUND: The prognostic value of PKM2 and its correlation with tumour cell PD-L1 in lung adenocarcinoma (LUAD) is unclear. METHODS: A total of 506 lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA) dataset and 173 LUAD tumour tissues from Jiangxi Cancer Hospital were used to analyse the correlation between PKM2 and PD-L1 expression. We further established a stable LUAD cell line with PKM2 knockdown and confirmed the association via Western blotting and flow cytometry analysis. Moreover, the prognostic values of PKM2 and PD-L1 were evaluated by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: Based on the above two large cohorts, we found that PKM2 was significantly positively associated with PD-L1 expression (r = 0.132, P = 0.003 and r = 0.287, P < 0.001, respectively). Subsequently, we found that PKM2 knockdown substantially inhibited PD-L1 expression in the A549 LUAD cell line. Moreover, survival analysis showed that higher expression of PKM2 was correlated with significantly shorter overall survival (OS) and disease-free survival (DFS) in lung adenocarcinoma patients (P < 0.001 and P = 0.050, respectively). Subgroup analysis showed that lung adenocarcinoma patients who expressed high PKM2 and PD-L1 levels experienced the poorest OS and DFS. Additionally, multivariate analysis suggested that high PKM2 and PD-L1 expression was an independent prognostic indicator for worse OS and DFS (HR = 1.462, P < 0.001 and HR = 1.436, P = 0.004, respectively). CONCLUSIONS: Our results demonstrated that PKM2 regulated PD-L1 expression and was associated with poor outcomes in lung adenocarcinoma patients.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Antígeno B7-H1/genética , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Hormônios Tireóideos/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adulto , Idoso , Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Hormônios Tireóideos/análise , Hormônios Tireóideos/genética
17.
Biosci Rep ; 39(4)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30902879

RESUMO

There are several phytosanitary problems that have been causing serious damage to the Capsicum crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from Capsicum annuum fruits infected with Colletotrichum gloeosporioides The fungus was inoculated into Capsicum fruits by the deposition of a spore suspension (106 conidia ml-1), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and ß-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of C. gloeosporioides fungi 'in vitro' There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Capsicum/genética , Colletotrichum/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Peptídeos Catiônicos Antimicrobianos/análise , Capsicum/microbiologia , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Defensinas/análise , Defensinas/genética , Frutas/genética , Frutas/microbiologia , Interações Hospedeiro-Patógeno , Doenças das Plantas/genética , Proteínas de Plantas/análise
18.
Phys Chem Chem Phys ; 21(20): 10217-10227, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-30860214

RESUMO

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.


Assuntos
Técnicas de Química Analítica/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Gadolínio/química , Proteínas/análise , Marcadores de Spin , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Mutação , Proteínas/química , Proteínas/genética , Ubiquitina/análise , Ubiquitina/genética
19.
Neuroreport ; 30(6): 446-451, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30817682

RESUMO

Glioblastoma (GBM) is a highly lethal brain tumor, refractory to current therapies. Sperm-associated antigen 4 (SPAG4) is a novel cancer marker with unclear roles in GBM progression. This study aimed to explore the specific effects of SPAG4 on the pathogenesis of GBM. We first investigated the expression level and prognostic power of SPAG4 in patients with GBM using The Cancer Genome Atlas cohort, and then SPAG4 knockdown by RNA interference was performed to reveal the effects of SPAG4 on GBM cells. mRNA and protein expression levels were determined by real-time PCR and western blot. MTT assay was used to examine cell proliferation, and a wound healing assay was performed to detect cell migration. SPAG4 was significantly overexpressed in patients with GBM, and high expression of SPAG4 was associated with a poor prognosis. Silencing of SPAG4 significantly suppressed the proliferation and migration of GBM cells. Meanwhile, decreased expression and phosphorylation of MEK and ERK were identified after SPAG4 knockdown, suggesting that SPAG4 might regulate GBM progression by activating MEK/ERK signaling pathway. Our study revealed that SPAG4 was identified as a cancer biomarker for GBM and might be a promising target for clinical diagnosis and intervention of GBM.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Proteínas de Transporte/metabolismo , Glioblastoma/patologia , Neoplasias Encefálicas/mortalidade , Proteínas de Transporte/análise , Progressão da Doença , Glioblastoma/mortalidade , Humanos , Prognóstico
20.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 31(3): 336-340, 2019 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-30914096

RESUMO

OBJECTIVE: To explore the predictive value of heparin-binding protein (HBP) combined with sequential organ failure assessment (SOFA) score in patients with septic shock. METHODS: Seventy-eight patients with sepsis admitted to intensive care unit (ICU) of Henan Provincial People's Hospital from December 2016 to May 2017 were enrolled. Thirty healthy persons were enrolled as controls. The patient's gender, age, length of ICU stay, and blood culture results, white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), blood lactate (Lac), HBP, SOFA score, acute physiology and chronic health evaluation II (APACHE II) score, organ failure and vasoactive agents usage within 24 hours of admission were recorded. The differences in the above indicators between the groups were compared, and the receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of HBP, SOFA score and their combination in patients with septic shock. RESULTS: All patients were enrolled in the final analysis, including 64 with sepsis and 14 with septic shock. Compared with the sepsis group, the proportion of patients with septic shock who were positive for blood culture, organ failure, and vasoactive agents was higher [57.1% (8/14) vs. 7.8% (5/64), 100.0% (14/14) vs. 65.6% (42/64), 100.0% (14/14) vs. 18.8% (12/64), all P < 0.01], SOFA and APACHE II scores were also higher (SOFA: 8.93±4.16 vs. 5.89±2.68, APACHE II: 22.29±4.89 vs. 15.28±5.14, both P < 0.01); however, there was no significant difference in gender, age or length of ICU stay between the two groups. Compared with the healthy control group, HBP, PCT, CRP and Lac levels were significantly increased in the sepsis group and the septic shock group. HBP in the septic shock group was significantly higher than that in the sepsis group (µg/L: 120.33±43.49 vs. 68.95±54.15, P < 0.05), but there was no significant difference in PCT, CRP or Lac between septic shock group and sepsis group [PCT (µg/L): 1.42 (0.47, 46.00) vs. 0.71 (0.19, 4.50), CRP (mg/L): 102.90±78.12 vs. 102.07±72.15, Lac (mmol/L): 1.81 (1.14, 3.65) vs. 1.59 (1.17, 2.24), all P > 0.05]. It was shown by ROC curve analysis that the area under the ROC curve (AUC) of SOFA score for predicting septic shock was 0.715 [95% confidence interval (95%CI) = 0.540-0.890, P = 0.012], and when the optimal cut-off value was 7.5, the sensitivity was 64.3%, the specificity was 76.6%. The AUC of HBP was 0.814 (95%CI = 0.714-0.913, P < 0.001), and when the optimal cut-off value was 89.43 µg/L, the sensitivity was 78.6%, the specificity was 76.6%; when the two were combined, the AUC was 0.829 (95%CI = 0.724-0.935, P < 0.001), the sensitivity was 92.9%, and the specificity was 61.9%. CONCLUSIONS: HBP can be used as a biological indicator for predicting septic shock, and the accuracy of predicting septic shock can be improved with the combination of SOFA score.


Assuntos
Peptídeos Catiônicos Antimicrobianos/análise , Proteínas Sanguíneas/análise , Proteínas de Transporte/análise , Escores de Disfunção Orgânica , Choque Séptico/diagnóstico , Feminino , Humanos , Masculino , Valor Preditivo dos Testes
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