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1.
Phytochemistry ; 165: 112055, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31261031

RESUMO

Twenty-one known Amaryllidaceae alkaloids of various structural types and one undescribed alkaloid, named narcimatuline, have been isolated from fresh bulbs of Narcissus pseudonarcissus L. cv. Dutch Master. The chemical structures were elucidated by combination of MS, HRMS, 1D and 2D NMR spectroscopic techniques, and by comparison with literature data. Narcimatuline amalgamates two basic scaffolds of Amaryllidaceae alkaloids in its core, namely galanthamine and galanthindole. All isolated compounds were evaluated for their in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyl oligopeptidase (POP), and glycogen synthase kinase-3ß (GSK-3ß) inhibitory activities. The most interesting biological profile was demonstrated by newly isolated alkaloid narcimatuline.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Alcaloides de Amaryllidaceae/farmacologia , Inibidores da Colinesterase/farmacologia , Narcissus/química , Fármacos Neuroprotetores/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/isolamento & purificação , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/isolamento & purificação , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Serina Endopeptidases/metabolismo , Relação Estrutura-Atividade
2.
Artigo em Inglês | MEDLINE | ID: mdl-31161918

RESUMO

Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended.


Assuntos
Cerveja/análise , Glutens/análise , Serina Endopeptidases/metabolismo , Ensaio de Imunoadsorção Enzimática , Fermentação , Glutens/metabolismo , Hidrólise
3.
Cell Mol Life Sci ; 76(16): 3193-3206, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201463

RESUMO

Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-ß (Aß) plaques are one of the pathological hallmarks in AD patient brains. Aß accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. However, so far disease-modifying drugs targeting ß- and γ-secretase pathways seeking a decrease in the production of toxic Aß peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin ß acts as an alternative ß-secretase, capable of generating truncated Aß2-x peptides that have been described to be increased in AD patients. This indicates an important ß-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin ß within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin ß itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin ß. Overall, we highlight the current cellular, molecular and structural understanding of meprin ß as alternative ß-secretase within the complex protease web, regulating APP processing in health and disease.


Assuntos
Proteína ADAM10/metabolismo , Metaloendopeptidases/metabolismo , Proteína ADAM10/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Presenilina-1/metabolismo , Proteólise , Serina Endopeptidases/metabolismo
4.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 753-757, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204927

RESUMO

OBJECTIVE: To analyze the expression level of the serum soluble E cadherin (SE-CAD) and Matriptase and its clinical significance for evaluation of the disease condtions and prognosis in patients with acute myeloid leukemia (AML). METHODS: One hundred and ten patients diagnosed as AML in our hospital were divided into 3 groups: newly diagnosed group (38 cases), remission group (40 cases) and recurrence group (32 cases). The expression levels of serum matriptase were detected by Western blot, and the expression levels of serum SE-CAD were detected by ELISA. The serum levels of serum SE-CAD and matriptase among 3 groups were compared. Followin-up for one year, according to the outcome of patients, all the patients were divided into 2 groups: the survival group and death group. The serum levels of SE-CAD and Matriptase were compared between 2 groups. The correlation of serum levels of SE-CAD and matriptase with the survival of AML patients was analyzed by multivariate Logistic analysis. The evaluation value of the serum levels of SE-CAD and matriptase for the prognosis of the patients with AML were analyzed by receiver operating characteristic curves (ROC). RESULTS: The serum levels of SE-CAD and matriptase were siginificantly different among 3 groups (P<0.05). The serum levels of SE-CAD and matriptase in remission group were lowest (P<0.05), and the serum levels of SE-CAD and matriptase were not different between newly diagnoses and recurrence groups (P>0.05). Multivariate Logistic analysis showed that the serum levels of SE-CAD and matriptase were independent risk factors for the prognosis of AML patients (OR=3.157, P<0.05, OR=2.426, P<0.05). By follow-up for 1 year, the serum expression levels of SE-CAD and Matriptase in survival group were lower than that in death group. ROC curve showed that when the cut-off value of matriptase level was 0.73 and SE-CAD level was 3.42 ng/ml, the AUC of predictions for the poor prognosis in AML patients was 0.849 (P<0.05), the sensitivity was 85.6% (95%CI: 0.810~0.924) and specificity was 89.6% (95%CI: 0.849~0.941). CONCLUSION: The serum levels of SE-CAD and matriptase can perfectly evaluate the condition and short-term prognosis of the patients with AML.


Assuntos
Leucemia Mieloide Aguda , Caderinas , Humanos , Prognóstico , Serina Endopeptidases
5.
Nat Commun ; 10(1): 2853, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253808

RESUMO

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucDF106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Serina Endopeptidases/metabolismo , Arabidopsis/imunologia , Proteínas de Bactérias/genética , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/genética
6.
Subcell Biochem ; 92: 187-219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214988

RESUMO

Signal peptidases are the membrane bound enzymes that cleave off the amino-terminal signal peptide from secretory preproteins . There are two types of bacterial signal peptidases . Type I signal peptidase utilizes a serine/lysine catalytic dyad mechanism and is the major signal peptidase in most bacteria. Type II signal peptidase is an aspartic protease specific for prolipoproteins. This chapter will review what is known about the structure, function and mechanism of these unique enzymes.


Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo
7.
Medicine (Baltimore) ; 98(18): e15164, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31045759

RESUMO

The aim of this study was to evaluate the cytomorphologic maturity and molecular activation of cancer-associated fibroblasts (CAFs) in the intratumoral stroma and invasive front in colorectal cancer and understand how they affect cancer invasion and long-term oncological outcomes.The cytomorphologic maturity of and α-smooth muscle actin (α-SMA), fibroblast activation protein α (FAPα), and fibroblast-specific protein 1 (FSP-1) expression in CAFs in the intratumoral stroma (CAF) and the invasive front (CAF) of colorectal cancer tissues were compared (n = 147). The correlations between CAF maturation, molecular activity markers, and cancer invasion were evaluated by network analysis. Overall survival and systemic recurrence were analyzed to assess the oncological effects of CAF properties.The cytomorphologic maturation rate was comparable between CAF and CAF. The presence of mature CAFs was related to epidermal growth factor receptor overexpression in cancer cells. Expression rates of α-SMA (96.6%-98.0%) and FAPα (18.6%-22.9%) were similar between CAF and CAF. FSP-1 expression was more frequent in CAF than in CAF (66.4% vs 58.2%, P = .038). There was a significant decrease in FSP-1 expression in CAF and CAF in higher stages. The infiltrating growth pattern of the tumor was more frequent in the immature CAF. In colorectal cancer with perineural invasion and lymph node metastasis, FSP-1 expression in CAF was significantly lower. On multivariate analysis using the Cox proportional hazards model, immature CAF was found to be an independent prognostic factor of overall survival. In non-metastatic (stage I-III) colorectal cancer patients, CAF maturity was not a prognostic factor for systemic recurrence.Cytomorphologic maturity and molecular activation markers were similar between CAFs in the intratumoral stroma and invasive front of colorectal cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Colorretais/patologia , Tumores do Estroma Gastrointestinal/patologia , Invasividade Neoplásica/patologia , Actinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Receptores ErbB/metabolismo , Feminino , Tumores do Estroma Gastrointestinal/metabolismo , Gelatinases/metabolismo , Humanos , Metástase Linfática/patologia , Masculino , Proteínas de Membrana/metabolismo , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Serina Endopeptidases/metabolismo , Análise de Sobrevida
8.
Analyst ; 144(11): 3659-3667, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31074478

RESUMO

We report a highly sensitive and rapid electrochemical method for the detection of endotoxin, based on a Limulus amebocyte lysate (LAL) assay using redox cycling at a pair of electrodes in a nanocavity for electrochemical signal amplification. We have previously developed Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as a substrate for the amperometric LAL assay, and in this work, Z-Leu-Gly-Arg-aminomethylferrocene (LGR-AMF) was newly prepared. They were examined as substrates for a LAL-based endotoxin assay using a nanocavity device. During the last step of the endotoxin-induced LAL cascade reaction, pAP or AMF is generated from the substrate, which can be detected electrochemically with efficient signal amplification by redox cycling between the two electrodes in the nanocavity. A device with a 190 nm-high nanocavity was fabricated by photolithography. With the fabricated device in model assay solutions prepared by mixing LGR-pAP and pAP, we demonstrated that pAP could be quantitatively detected from the difference in oxidation potentials between LGR-pAP and pAP. For LGR-AMF and AMF, a difference in the formal potential of 0.1 V was obtained which was considered to be insufficient to distinguish AMF from LGR-AMF. However, we showed for the first time that analytes such as AMF can be detected by differences in diffusion coefficients between the analyte and coexisting molecules (such as LGR-AMF) using a device with high redox-cycling efficiency. Next, the endotoxin assay was performed using the fabricated nanocavity device. Using this method, endotoxin was detected at concentrations as low as 0.2 and 0.5 EU L-1 after LAL reaction times of 1 h and 30 min, respectively, using the LGR-pAP substrate. However, the endotoxin assay using LGR-AMF was not successful because the clotting enzyme did not react with LGR-AMF. This problem might be solved by further design of the substrate. Our nanocavity device represents an effective platform for the simple and rapid detection of endotoxin with high sensitivity.


Assuntos
Endotoxinas/análise , Nanoestruturas/química , Aminofenóis/química , Animais , Proteínas de Artrópodes/química , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Endopeptidases/química , Endotoxinas/química , Precursores Enzimáticos/química , Desenho de Equipamento , Compostos Ferrosos/química , Caranguejos Ferradura/enzimologia , Oligopeptídeos/química , Oxirredução , Platina/química , Serina Endopeptidases/química , Titânio/química
9.
Food Chem Toxicol ; 129: 466-475, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082461

RESUMO

Targeted degrading Aspergillus niger-derived prolyl endopeptidase (AN-PEP) is promising in gluten hydrolysis because it specifically cleaves the proline-rich sites in gluten. The current study aims to understand the safety aspects of AN-PEP hydrolysed low immunoreactive wheat flours by testing immune responses using cell line and animal models. In the AN-PEP hydrolysed wheat flour (AN-PEP HWF) gliadin extract, there was no increase in the levels of zonulin-1 (Zo-1) and pro-inflammatory cytokines (IL-6 and IL-8) but a significant increase was noted in the control wheat flour (CWF) gliadin-treated Caco-2 cells. The Zo-1 localization in Caco-2 cells was significantly noted in the reacted positive fluorescence cells that were treated with the control wheat flour. Further, a safety evaluation of HWF was carried out in gluten-sensitized BALB/c mice. Mouse anti-gliadin (IgG, IgA and IgE) antibodies were significantly generated in the CWF treated animals rather than the AN-PEP HWF groups. The serum pro-inflammatory (IL-1ß, IL-4, IL-6, IL-15, TNF-α and IFN-γ) markers were observed in significant levels in CWF challenged mice and a similar trend was observed in ex-vivo splenocyte cells. A small intestine histopathological sectioning revealed that there are no abnormalities or structural changes in AN-PEP HWF challenged mice.


Assuntos
Doença Celíaca/imunologia , Farinha , Glutens/toxicidade , Serina Endopeptidases/metabolismo , Triticum/metabolismo , Animais , Células CACO-2 , Feminino , Humanos , Hidrólise , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/metabolismo
10.
J Mol Neurosci ; 68(2): 261-274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949956

RESUMO

The level of miR-181a decreases rapidly in N2a cells following oxygen-glucose deprivation/reperfusion, but its role in this process is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-181a. We hypothesized that miR-181a reduces neuronal apoptosis and protects neurons by targeting reelin. Second mitochondria-derived activator of caspases (Smac) is a protein located in mitochondria that regulates apoptosis. The pro-apoptotic effect of Smac is achieved by reversing the effects of apoptosis-inhibiting proteins (IAPs), particularly X-linked inhibitor of apoptosis (XIAP). We also evaluated the effect of miR-181a on the Smac/IAP signaling pathway after oxygen-glucose deprivation and reperfusion in N2a cells. The miR-181a level, apoptosis rate, and the levels of reelin mRNA and protein, Smac, and XIAP were assessed in N2a cells subjected to oxygen-glucose deprivation for 4 h and reperfusion for 0, 4, 12, or 24 h with/without an miR-181a mimic, or mismatched control. Direct targeting of reelin by miR-181a was assessed in vitro by dual luciferase assay and immunoblotting. Pre-treatment with miR-181a mimicked the increase in the miR-181a level in N2a cells after oxygen-glucose deprivation/reperfusion, resulting in a significant decrease in the apoptosis rate. Changes in the miR-181a level in N2a cells were inversely correlated with reelin protein expression. Direct targeting of the reelin 3' untranslated region by miR-181a was verified by dual luciferase assay, which showed that miR-181a significantly inhibited luciferase activity. The Smac level was significantly lower in the miR-181a mimics than the normal control and mimics-cont groups (P < 0.01), whereas the level of XIAP was increased slightly. These findings suggest that miR-181a protects neurons from apoptosis by inhibiting reelin expression and regulating the Smac/IAP signaling pathway after oxygen-glucose deprivation/reperfusion injury.


Assuntos
Apoptose , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glucose/deficiência , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
11.
PLoS Genet ; 15(4): e1007739, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30990817

RESUMO

Sleep disordered breathing (SDB)-related overnight hypoxemia is associated with cardiometabolic disease and other comorbidities. Understanding the genetic bases for variations in nocturnal hypoxemia may help understand mechanisms influencing oxygenation and SDB-related mortality. We conducted genome-wide association tests across 10 cohorts and 4 populations to identify genetic variants associated with three correlated measures of overnight oxyhemoglobin saturation: average and minimum oxyhemoglobin saturation during sleep and the percent of sleep with oxyhemoglobin saturation under 90%. The discovery sample consisted of 8,326 individuals. Variants with p < 1 × 10(-6) were analyzed in a replication group of 14,410 individuals. We identified 3 significantly associated regions, including 2 regions in multi-ethnic analyses (2q12, 10q22). SNPs in the 2q12 region associated with minimum SpO2 (rs78136548 p = 2.70 × 10(-10)). SNPs at 10q22 were associated with all three traits including average SpO2 (rs72805692 p = 4.58 × 10(-8)). SNPs in both regions were associated in over 20,000 individuals and are supported by prior associations or functional evidence. Four additional significant regions were detected in secondary sex-stratified and combined discovery and replication analyses, including a region overlapping Reelin, a known marker of respiratory complex neurons.These are the first genome-wide significant findings reported for oxyhemoglobin saturation during sleep, a phenotype of high clinical interest. Our replicated associations with HK1 and IL18R1 suggest that variants in inflammatory pathways, such as the biologically-plausible NLRP3 inflammasome, may contribute to nocturnal hypoxemia.


Assuntos
Hexoquinase/genética , Subunidade alfa de Receptor de Interleucina-18/genética , Oxiemoglobinas/metabolismo , Sono/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Moléculas de Adesão Celular Neuronais/genética , Biologia Computacional , Proteínas da Matriz Extracelular/genética , Feminino , Redes Reguladoras de Genes , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Hipóxia/sangue , Hipóxia/genética , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas do Tecido Nervoso/genética , Oxigênio/sangue , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Serina Endopeptidases/genética , Síndromes da Apneia do Sono/sangue , Síndromes da Apneia do Sono/genética , Adulto Jovem
12.
Dis Markers ; 2019: 4864370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984307

RESUMO

Background: Influence of TMPRSS6 A736V and HFE (C282Y and H63D) polymorphisms on serum hepcidin-25 levels and iron status parameters in end-stage renal disease (ESRD) patients stratified according to gender has not been previously investigated. In addition, we aimed to evaluate the diagnostic accuracy of the parameters to separate iron-deficiency anemia (IDA) from anemia of chronic disease. Materials and Methods: Iron status parameters and genetic analysis were performed in 126 ESRD patients and in 31 IDA patients as the control group. Results: ESRD patients had significantly higher ferritin and hepcidin-25 (<0.001) relative to IDA patients. Cut-off values with the best diagnostic accuracy were found for hepcidin ≥9.32 ng/mL, ferritin ≥48.2 µg/L, transferrin saturation ≥16.8%, and MCV ≥81 fL. Interaction between gender and HFE haplotypes for the hepcidin-25 and ferritin levels in ESRD patients (p = 0.005, partial eta squared = 0.09; p = 0.027, partial eta squared = 0.06, respectively) was found. Serum transferrin was influenced by the combined effect of gender and TMPRSS6 A736V polymorphism in ESRD patients (p = 0.002, partial eta squared = 0.07). Conclusion: Our findings could contribute to the further investigation of mechanisms involved in the pathophysiology and important gender-related involvement of the TMPRSS6 and HFE polymorphisms on anemia in ESRD patients.


Assuntos
Anemia Ferropriva/genética , Proteína da Hemocromatose/genética , Hepcidinas/sangue , Falência Renal Crônica/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Serina Endopeptidases/genética , Adulto , Idoso , Anemia Ferropriva/sangue , Feminino , Ferritinas/sangue , Humanos , Ferro/sangue , Falência Renal Crônica/sangue , Falência Renal Crônica/patologia , Masculino , Pessoa de Meia-Idade , Transferrina/análise
13.
J Agric Food Chem ; 67(18): 5240-5249, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31008594

RESUMO

Fluoride is a widespread environmental pollutant that can induce low sperm quality and fertilizing ability; however, the underlying mechanism still remains unclear. Hence, we aimed to investigate the influence of fluoride on the sperm fertilizing ability via some key proteins in the epididymis. For this, 40 adult rats were assigned randomly into four groups. The control group was given distilled water, while the other three groups were given 25, 50, and 100 mg of NaF/L via drinking water for 56 days, respectively. After 1 day, epididymides were processed for sperm-egg binding, RNA extraction, western blot, and immunofluorescence analysis. Fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer. A further study revealed that fluoride altered the expression levels of genes and proteins related to acrosome reaction in vivo, including SPAM1, ACR, and PRSS21. However, fluoride only affected the expression of the ACR protein only in the epididymis but not in the testis. Fluoride also affected the expression levels of the membrane proteins CD9 and CD81 of epididymosomes in the epididymis. From the results, it can be concluded that fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer, which could be one of the reasons for decreased fertility ability in males treated with fluoride. These results provide some theoretical guidance and new ideas for treatments of low fertility, infertility, and other reproductive diseases.


Assuntos
Acrosina/metabolismo , Moléculas de Adesão Celular/metabolismo , Epididimo/efeitos dos fármacos , Fluoretos/farmacologia , Hialuronoglucosaminidase/metabolismo , Serina Endopeptidases/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Acrosina/genética , Animais , Moléculas de Adesão Celular/genética , Epididimo/metabolismo , Fertilização/efeitos dos fármacos , Hialuronoglucosaminidase/genética , Masculino , Ratos , Ratos Sprague-Dawley , Serina Endopeptidases/genética , Testículo/efeitos dos fármacos , Testículo/metabolismo
14.
Mol Immunol ; 109: 1-11, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30836204

RESUMO

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Células Epiteliais/imunologia , Pyroglyphidae/imunologia , Receptor PAR-1/imunologia , Receptor PAR-2/imunologia , Mucosa Respiratória/imunologia , Serina Endopeptidases/imunologia , Células A549 , Alérgenos/farmacologia , Animais , Proteínas de Artrópodes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Receptor PAR-1/genética , Receptor PAR-2/genética , Serina Endopeptidases/farmacologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/imunologia , Células Th2/patologia
15.
Clin Biochem ; 67: 12-15, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30890412

RESUMO

BACKGROUND: Corin is a serine protease known to convert B-type natriuretic peptide (BNP) prohormone into BNP and its amino-terminal fragment (NT-proBNP). In mice lacking corin, high blood pressure and proteinuria were found at late gestational stages, with associated delayed trophoblast invasion and impaired spiral artery remodeling in the uterus. We hypothesize that both NT-proBNP and soluble corin elevation predict the presence of preeclampsia in pregnant patients with hypertension. METHODS: We prospectively enrolled 149 pregnant women with a history of chronic hypertension or gestational hypertension presenting at a tertiary-care hospital. We compared plasma NT-proBNP and soluble corin concentrations based on their preeclamptic status. RESULTS: In our study cohort, 62 patients with preeclampsia had lower gestational age than 87 patients without preeclampsia (33.3 ±â€¯3 versus 36.6 ±â€¯3 weeks; P < .001), otherwise the baseline characteristics were similar. We observed higher NT-proBNP concentrations in patients with preeclampsia compared to those without preeclampsia (304.3 [96.34, 570.4] vs. 60.8 [35.61, 136.8] ng/L, P < .001), with no differences between chronic and gestational hypertension. However, the concentration of corin was not statistically different between the two groups (1756 [1214, 2133] vs. 1571 [1171, 1961] ng/L, P = .1087). ROC curve analysis demonstrated stronger predictive value of NT-proBNP compared to soluble corin in predicting the presence of preeclampsia in our study population (AUC 0.7406 vs. 0.5789, P < .0001). CONCLUSION: While corin may contribute to mechanistic underpinnings of the development of preeclampsia in animal models, soluble corin likely has no diagnostic role in human pregnancies for preeclampsia beyond natriuretic peptide levels.


Assuntos
Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Pré-Eclâmpsia/sangue , Serina Endopeptidases/sangue , Adulto , Biomarcadores/sangue , Feminino , Humanos , Gravidez , Estudos Prospectivos , Solubilidade
16.
eNeuro ; 6(1)2019 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30863790

RESUMO

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Técnicas Genéticas , Neurônios/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Cultura Primária de Células , Distribuição Aleatória , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Transcrição Genética , Transcriptoma
17.
J Mol Neurosci ; 68(1): 91-98, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847724

RESUMO

Smad anchor for receptor activation (SARA) is an important regulator of transforming growth factor ß (TGF-ß) signaling by recruiting Smad2/3 to TGF-ß receptors. We recently demonstrated that the expressions of SARA and level of downstream phospho-Smad3 (p-Smad3) were upregulated in the brain in the epileptic rat model, but were never examined in patients with temporal lobe epilepsy (TLE). In this study, we examined the expressions of SARA and level of p-Smad3 in brain tissues of TLE patients using immunohistochemistry and western blot to demonstrate that SARA activation in neurons is sufficient to facilitate TGF- ß pathway in patients to regulate epilepsy. We found that the expressions of SARA and level of p-Smad3 were significantly upregulated in neurons of the temporal cortex of TLE patients compared to controls. Moreover, SARA and p-Smad3 were strongly stained in the cytoplasm in the temporal cortex of TLE patients. Our results indicate that upregulation of SARA and p-Smad3 in cortex neurons might be involved in the development of intractable temporal lobe epilepsy.


Assuntos
Epilepsia do Lobo Temporal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Serina Endopeptidases/genética , Proteína Smad3/genética , Adolescente , Adulto , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Serina Endopeptidases/metabolismo , Proteína Smad3/metabolismo , Lobo Temporal/metabolismo , Regulação para Cima
18.
Chem Commun (Camb) ; 55(35): 5064-5067, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30896004
19.
Artigo em Inglês | MEDLINE | ID: mdl-30885835

RESUMO

Tibetan pigs, indigenous to Tibetan plateau, are well adapted to hypoxia. So far, there have been not any definitively described genes and functional sites responsible for hypoxia adaptation for the Tibetan pig. The whole genome-wide association studies in human suggested that genetic variations in TMPRSS6 was associated with hemoglobin concentration (HGB) and red cell counts (RBC). Here we conducted resequencing of the nearly entire genomic region (40.1 kb) of the candidate gene TMPRSS6 in 40 domestic pigs and 40 wild boars along continuous altitudes and identified 708 SNPs, in addition to an indel (CGTG/----) in the intron 10. We conduct the CGTG indel in 838 domestic pigs, both the CGTG deletion frequency and the pairwise r2 linkage disequilibrium showed an increase with elevated altitudes, suggesting that TMPRSS6 has been under Darwinian positive selection. As the conserved core sequence of hypoxia-response elements (HREs), the deletion of CGTG in Tibetan pigs decreased the expression levels of TMPRSS6 mRNA and protein in the liver revealed by real-time quantitative PCR and western blot, respectively. We compared domestic pigs and Tibetan pigs living continuous altitudes, found that the blood-related traits with the increase of altitude, however, the HGB did not increase with the elevation in Tibetan pigs. Genotype association analysis results dissected a genetic effect on reducing HGB by 13.25 g/L in Gongbo'gyamda Tibetan pigs, decreasing mean corpuscular volume (MCV) by 4.79 fl in Diqing Tibetan pigs. In conclusion, the CGTG deletion of TMPRSS6 resulted in lower HGB and smaller MCV, which could reflect a blunting erythropoiesis and improving blood viscosity as well as erythrocyte deformability. It remains to be determined whether a blunting of erythropoiesis for TMPRSS6 or others genetic effects are the physiological adaptations among Tibetan pigs.


Assuntos
Aclimatação/fisiologia , Viscosidade Sanguínea/fisiologia , Eritropoese/fisiologia , Proteínas de Membrana , Polimorfismo de Nucleotídeo Único , Seleção Genética/fisiologia , Serina Endopeptidases , Animais , Desequilíbrio de Ligação/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Suínos , Tibet
20.
Genetics ; 212(1): 53-63, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30862621

RESUMO

The Q-system is a binary expression system that works well across species. Here, we report the development and demonstrate the applications of a split-QF system that drives strong expression in Drosophila, is repressible by QS, and is inducible by a small nontoxic molecule (quinic acid). The split-QF system is fully compatible with existing split-GAL4 and split-LexA lines, thus greatly expanding the range of possible advanced intersectional experiments and anatomical, physiological, and behavioral assays in Drosophila, and in other organisms.


Assuntos
Drosophila/genética , Expressão Gênica , Transgenes , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Drosophila/genética , Feminino , Técnicas Genéticas , Masculino , Ácido Quínico , Serina Endopeptidases/genética , Fatores de Transcrição/genética
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