RESUMO
Introducción. Las proteasas y las fosfolipasas son factores de virulencia de Candida spp. que cumplen un papel importante en la invasión de los tejidos. Entre los factores relacionados con el huésped, se encuentran algunos asociados con las características ambientales y otros con la colonización. Objetivo. Determinar la actividad de fosfolipasas y proteasas en aislamientos de especies colonizadoras y patógenas de Candida spp., aisladas de mujeres gestantes de Cartagena de Indias. Materiales y métodos. Se determinó la actividad de fosfolipasas y proteasas en 56 aislamientos mediante degradación del sustrato y cálculo del coeficiente de actividad enzimática. Se compararon las actividades de fosfolipasas y proteasas, entre los aislamientos colonizadores y los patógenos. Resultados. La actividad de la fosfolipasa fue "muy alta" (< 0,69) en 34 aislamientos e, igualmente, la de la proteasa en 14. No hubo diferencias significativas al comparar las actividades de las fosfolipasas y de las de las proteasas, entre los aislamientos colonizadores y los patógenos. Conclusiones. La actividad de las fosfolipasas predominó como factor de virulencia en los aislamientos estudiados. No obstante, no se encontró una diferencia significativa entre los grupos de aislamientos colonizadores y los patógenos, en cuanto a las actividades de fosfolipasas y proteasas.
Introduction. Proteases and phospholipases are virulence factors of Candida spp. that play an important role in tissue invasion. Among the factors related to the host some are associated with environmental characteristics and others with Candida colonization. Objectives. To determine phospholipase and protease activities in colonizing and pathogenic strains, isolated from pregnant women in Cartagena de Indias. Materials and methods. Phospholipase and protease activity was determined in 56 isolates, evaluating substrate degradation and calculating the enzyme activity coefficient. Phospholipase and protease activities were compared between colonizing and pathogenic strains. Results. "Very high" (<0.69) phospholipase and protease activity was found in 34 and 14 isolates, respectively. There was no significant difference when comparing phospholipase and protease activities between colonizing and pathogenic isolates. Conclusions. Phospholipase activity predominated as a virulence factor in the studied strains, but no significant difference was found between colonizing and pathogenic strains for phospholipase and protease activities.
Assuntos
Candidíase Vulvovaginal , Endopeptidases , Fosfolipases , Candida , Fatores de Virulência , MicrobiotaRESUMO
SUMMARY We present the unique case of an adult Brazilian woman with severe short stature due to growth hormone deficiency with a heterozygous G to T substitution in the donor splice site of intron 3 of the growth hormone 1 (GH1) gene (c.291+1G>T). In this autosomal dominant form of growth hormone deficiency (type II), exon 3 skipping results in expression of the 17.5 kDa isoform of growth hormone, which has a dominant negative effect over the bioactive isoform, is retained in the endoplasmic reticulum, disrupts the Golgi apparatus, and impairs the secretion of other pituitary hormones in addition to growth hormone deficiency. This mechanism led to the progression of central hypothyroidism in the same patient. After 5 years of growth and thyroid hormone replacement, at the age of 33, laboratory evaluation for increased weight gain revealed high serum and urine cortisol concentrations, which could not be suppressed with dexamethasone. Magnetic resonance imaging of the sella turcica detected a pituitary macroadenoma, which was surgically removed. Histological examination confirmed an adrenocorticotropic hormone (ACTH)-secreting pituitary macroadenoma. A ubiquitin-specific peptidase 8 (USP8) somatic pathogenic variant (c.2159C>G/p.Pro720Arg) was found in the tumor. In conclusion, we report progression of isolated growth hormone deficiency due to a germline GH1 variant to combined pituitary hormone deficiency followed by hypercortisolism due to an ACTH-secreting macroadenoma with a somatic variant in USP8 in the same patient. Genetic studies allowed etiologic diagnosis and prognosis of this unique case.
Assuntos
Humanos , Feminino , Adulto , Hormônio do Crescimento Humano , Hipersecreção Hipofisária de ACTH , Nanismo Hipofisário/genética , Endopeptidases/genética , Ubiquitina Tiolesterase/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células Germinativas , MutaçãoRESUMO
In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.
Assuntos
Bacillus amyloliquefaciens/genética , Proteínas de Bactérias , Contagem de Células , Endopeptidases/genética , N-Acetil-Muramil-L-Alanina Amidase/genéticaRESUMO
Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.
Assuntos
Sítios de Ligação , Domínio Catalítico , Endopeptidases , Peptídeo Hidrolases/genética , Inibidores de Proteases , ProteínasRESUMO
Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn²⁺ and Cu²⁺ enhanced the activity of PepA, whereas Fe³⁺, Fe²⁺ and Ca² had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.
Assuntos
Aspergillus oryzae , Clonagem Molecular , Endopeptidases , Concentração de Íons de Hidrogênio , Pichia , Proteínas Recombinantes , TemperaturaRESUMO
OBJECTIVE@#This study aimed to explore the influence of Rce1 on invasion and migration of tongue squamous cell carcinoma cells by silencing the Rce1 gene with RNA interference.@*METHODS@#The tongue squamous cell carcinoma Cal-27 and SCC-4 cells were cultured in vitro. The small interfering RNA (siRNA) of the Rce1 gene was designed, and the Rcel gene expression was silenced vialiposome transfection. According to the siRNA transfected by liposome, the experimental group was divided into three groups, namely, Rce1-siRNA-1, Rce1-siRNA-2, and Rce1-siRNA-3 groups. Negative control group was transfected by siCON, and the blank control group was untransfected by siRNA. The Rce1, RhoA, and K-Ras gene expression levels in each group were analyzed by real-time quantitative polymerase chain reaction. The Rce1, RhoA, K-Ras, MMP-2, and MMP-9 protein expression levels were analyzed by Western blot. The invasiveness of tongue cancer cell Cal-27 and SCC-4 were determined by Transwell invasion assay, and cell migration assay was performed by cell scratch assay.@*RESULTS@#Real-time quantitative polymerase chain reaction and Western blot results showed that compared with the negative and blank control groups, the Rce1 gene and protein expression levels in three experimental groups decreased (P0.05). Meanwhile, the MMP-2 and MMP-9 expression levels decreased (P<0.05). Transwell invasion assay results showed that the total number of cells in the PET film of the experimental groups was significantly decreased compared with the control group (P<0.05). The cell scratch test showed that the cell closure time of the scratch in the interference group was significantly longer than those in the control and blank groups (P<0.05).@*CONCLUSIONS@#Silencing Rce1 in vitro can effectively downregulate its expression in tongue squamous cell carcinoma cells Cal-27 and SCC-4 and reduce the migration and invasion abilities of these cells.
Assuntos
Humanos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Endopeptidases , Metabolismo , Invasividade Neoplásica , Interferência de RNA , RNA Interferente Pequeno , Neoplasias da Língua , Metabolismo , Terapêutica , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the effects of YOD1 overexpression on the proliferation and migration of human oral keratinocytes (HOKs), and to clarify whether the mechanisms involve transforming growth factor-β (TGF-β) signaling.</p><p><b>METHODS</b>HOKs were transfected with the plasmid pEGFP-N3-YOD1 containing YOD1. The mRNA levels of YOD1 and TGF-β were determined by qPCR. The protein expressions of YOD1, TGF-β, Smad2/3, Smad4, and phospho-Smad2/3 were determined by western blotting. Cell proliferation and migration were evaluated by Cell Counting Kit-8 assay and wound healing assay, respectively.</p><p><b>RESULTS</b>The mRNA and protein levels of YOD1 were higher in HOKs transfected with YOD1. YOD1 overexpression significantly enhanced the migration of HOKs. The mRNA and protein levels of TGF-β3 were increased by YOD1 overexpression. HOKs transfected with YOD1 exhibited increased phospho-Smad2/3 levels.</p><p><b>CONCLUSION</b>YOD1 overexpression enhances cell migration by promoting TGF-β3 signaling which may play an important role in lip and palate formation. YOD1 mutation may contribute to aberrant TGF-β3 signaling associated with decreased cell migration resulting in NSCLP.</p>
Assuntos
Humanos , Movimento Celular , Fisiologia , Proliferação de Células , Células Cultivadas , Endopeptidases , Genética , Metabolismo , Queratinócitos , Fisiologia , Transdução de Sinais , Fisiologia , Proteínas Smad , Genética , Metabolismo , Tioléster Hidrolases , Genética , Metabolismo , Fator de Crescimento Transformador beta3 , Genética , MetabolismoRESUMO
NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
Assuntos
Humanos , Dano ao DNA , Reparo do DNA , Genética , Replicação do DNA , Genética , Proteínas de Ligação a DNA , Genética , DNA Polimerase Dirigida por DNA , Genética , Endopeptidases , Genética , Técnicas de Inativação de Genes , Peróxido de Hidrogênio , Toxicidade , Proteína NEDD8 , Genética , Estresse Oxidativo , Genética , Antígeno Nuclear de Célula em Proliferação , Genética , Enzimas de Conjugação de Ubiquitina , Genética , Ubiquitina-Proteína Ligases , Genética , Ubiquitinação , Genética , Raios UltravioletaRESUMO
The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Assuntos
Animais , Bactérias/genética , Proteínas de Bactérias/química , Biofilmes , Temperatura Baixa , Laticínios , Endopeptidases/química , Estabilidade Enzimática , Microbiologia de Alimentos , Temperatura Alta , Lipase/química , Leite/microbiologia , Peptídeo Hidrolases/química , Fosfolipases/química , RNA Ribossômico 16S/genética , Alimentos Crus/microbiologia , beta-Galactosidase/químicaRESUMO
<p><b>OBJECTIVE</b>To observe the effects of Huannao Yicong Formula (, HYF) on learning and memory and it's regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer's disease.</p><p><b>METHODS</b>Sixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), Aβand Aβlevels in hippocampus were detected by enzyme-linked immunosorbent assay, γ-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1α (HIF-1α), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction.</p><p><b>RESULTS</b>HYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P<0.01, P<0.05). HYF can decrease the levels of APP, Aβ, Aβand the activity of γ-secretase in hippocampus of Alzheimer's disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA.</p><p><b>CONCLUSION</b>HYF can improve the learning and memory ability by inhibiting the activity of γ-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer's disease.</p>
Assuntos
Animais , Feminino , Masculino , Secretases da Proteína Precursora do Amiloide , Metabolismo , Precursor de Proteína beta-Amiloide , Metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Genética , Metabolismo , Medicamentos de Ervas Chinesas , Farmacologia , Usos Terapêuticos , Endopeptidases , Genética , Metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Hipocampo , Metabolismo , Patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Genética , Metabolismo , Imuno-Histoquímica , Aprendizagem , Transtornos da Memória , Tratamento Farmacológico , Genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1 , Metabolismo , Presenilina-2 , Genética , Metabolismo , RNA Mensageiro , Genética , Metabolismo , Transdução de SinaisRESUMO
Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.
Assuntos
Antivirais , Química , Proteínas de Transporte , Química , Farmacorresistência Viral , Endopeptidases , Hepacivirus , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos , Química , Inibidores de Proteases , Química , Serina Proteases , Tiazóis , Química , Proteínas não Estruturais Virais , QuímicaRESUMO
Neuropeptides produced from prohormones by selective action of endopeptidases are vital signaling molecules, playing a critical role in a variety of physiological processes, such as addiction, depression, pain, and circadian rhythms. Neuropeptides bind to post-synaptic receptors and elicit cellular effects like classical neurotransmitters. While each neuropeptide could have its own biological function, mass spectrometry (MS) allows for the identification of the precise molecular forms of each peptide without a priori knowledge of the peptide identity and for the quantitation of neuropeptides in different conditions of the samples. MS-based neuropeptidomics approaches have been applied to various animal models and conditions to characterize and quantify novel neuropeptides, as well as known neuropeptides, advancing our understanding of nervous system function over the past decade. Here, we will present an overview of neuropeptides and MS-based neuropeptidomic strategies for the identification and quantitation of neuropeptides.
Assuntos
Ritmo Circadiano , Depressão , Endopeptidases , Espectrometria de Massas , Modelos Animais , Sistema Nervoso , Neuropeptídeos , Neurotransmissores , Fenômenos Fisiológicos , Sinais VitaisRESUMO
<p><b>BACKGROUND</b>Two recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).</p><p><b>METHODS</b>The anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.</p><p><b>RESULTS</b>Inhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.</p><p><b>CONCLUSIONS</b>Inhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.</p>
Assuntos
Animais , Humanos , Camundongos , Hormônio Adrenocorticotrópico , Metabolismo , Apoptose , Proliferação de Células , Fisiologia , Sobrevivência Celular , Fisiologia , Endopeptidases , Metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Metabolismo , Inibidores Enzimáticos , Farmacologia , Indenos , Farmacologia , Pirazinas , Farmacologia , Receptores ErbB , Metabolismo , Ubiquitina Tiolesterase , MetabolismoRESUMO
Background Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0-20%), showing maximal growth and enzyme production at 0-5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.
Assuntos
Endopeptidases/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Temperatura , Bacillus/isolamento & purificação , Cloreto de Sódio , Lagos , Álcalis , Tolerância ao Sal , Fermentação , Concentração de Íons de HidrogênioRESUMO
Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. .
Objetivo Estabelecer se a mutação no gene Immp2L induz à fibrose renal e se o envelhecimento exacerba a morfologia renal em camundongos. Métodos Foram usadas fêmeas de camundongos mutantes para proteína semelhante à peptidase 2 da camada interna da mitocôndria, com 3 e 18 meses de idade. Para analisar a fibrose renal, foram usados o escore clássico de fibrose, a coloração com tricrômio de Masson, e a análise de marcadores profibróticos, por meio da reação em cadeia de polimerase em tempo real (superóxido dismutase 1, metalonoproteinase-9, eritropoietina e fator transformador de crescimento beta), e a imunocoloração (fibroblastos e colágeno IV). Marcadores de estresse oxidativo foram determinados por imuno-histoquímica. O número de células apoptóticas renais foi analisado. A função renal foi estimada por creatinina sérica. Resultados Camundongos mutantes jovens apresentaram glomeruloesclerose em quantidade significativamente maior que animais da mesma idade (p=0,034). Os mutantes mostraram maior formação de cilindros tubulares (p=0,025), deposição de colágeno (p=0,019) e maior expressão de colágeno do tipo IV (p<0,001). A expressão de superóxido dismutase 1 foi maior em mutantes jovens (p=0,038). Mutantes idosas exibiram maior expressão dos marcadores de fibroblastos e macrófagos (p=0,007 e p=0,012, respectivamente). As reações da cadeia de polimerase em tempo real da metalanoproteinase-9 e da eritropoietina estavam aumentadas em 2,5 e 6 vezes, respectivamente, em mutantes idosas. A creatinina sérica foi significantemente maior em animais idosos mutantes (p<0,001). Conclusão Essa mutação alterou a arquitetura renal pelo aumento da deposição de matriz extracelular, estresse oxidativo e inflamação, sugerindo papel de proteção de Immp2L contra a fibrose renal. .
Assuntos
Animais , Feminino , Camundongos , Modelos Animais de Doenças , Endopeptidases/genética , Endopeptidases/metabolismo , Rim/metabolismo , Rim/patologia , Mutação/fisiologia , Superóxidos/metabolismo , Apoptose/genética , Apoptose/fisiologia , Colágeno/análise , Creatinina/sangue , Eritropoetina/análise , Fibrose/genética , Fibrose/metabolismo , Metaloproteinase 9 da Matriz/análise , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Superóxido Dismutase/análise , Superóxidos/análise , Fator de Crescimento Transformador beta/análiseRESUMO
Gestational trophoblastic neoplasia (GTN) is the term to describe a set of malignant placental diseases, including invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor. Both invasive mole and choriocarcinoma respond well to chemotherapy, and cure rates are greater than 90%. Since the advent of chemotherapy, low-risk GTN has been treated with a single agent, usually methotrexate or actinomycin D. Cases of high-risk GTN, however, should be treated with multiagent chemotherapy, and the regimen usually selected is EMA-CO, which combines etoposide, methotrexate, actinomycin D, cyclophosphamide and vincristine. This study reviews the literature about GTN to discuss current knowledge about its diagnosis and treatment.
Neoplasia trofoblástica gestacional (NTG) é o termo que descreve o conjunto de anomalias malignas da placenta, incluindo a mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelióide. Ambos a mola invasora e o coriocarcinoma respondem bem à quimioterapia, com taxas de cura superiores a 90%. Desde o advento da quimioterapia, NTG de baixo risco tem sido tratada com monoquimioterapia, pelo geral methotrexate ou actinomicina-D. Casos de NTG de alto risco, contudo, devem ser tratados com poliquimioterapia, e o regime usualmente escolhido é o EMA-CO que combina etoposide, methotrexate, actinomicina-D, ciclofosfamida e vincristina. Esse estudo revê a literatura sobre NTG a fim de discutir os conhecimentos atuais sobre seu diagnóstico e tratamento.
Assuntos
Animais , Masculino , Ratos , Catepsinas/análise , Cistatinas/análise , Inibidores de Cisteína Proteinase/metabolismo , Endopeptidases , Leucina/análogos & derivados , Osteoclastos/química , Osteoclastos/enzimologia , Proteínas e Peptídeos Salivares/análise , Matriz Óssea/química , Matriz Óssea/enzimologia , Catepsina L , Cisteína Endopeptidases , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/toxicidade , Leucina/metabolismo , Leucina/toxicidade , Lisossomos/enzimologia , Microscopia Imunoeletrônica , Osteoclastos/efeitos dos fármacos , Osteoclastos/ultraestrutura , Ratos Wistar , Cistatinas SalivaresRESUMO
<p><b>OBJECTIVE</b>To investigate the changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer.</p><p><b>METHODS</b>Serum samples of patients with silicosis, tuberculosis, and lung cancer were collected. The variation trends of the expression of granzyme A, cathepsin G, apolipoprotein A, and interferon-β (IFN-β) were analyzed using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The concentration of apolipoprotein A of the silicosis group was 200 µg/ml, significantly higher than those of the tuberculosis and lung cancer groups (P < 0.05), and the lung cancer group had a significantly higher concentration of apolipoprotein A compared with the tuberculosis group (P < 0.05). The silicosis group had significantly higher expression of cathepsin G compared with the tuberculosis and lung cancer groups (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in the concentration of cathepsin G (P > 0.05). The tuberculosis group had a significantly higher concentration of granzyme A than the silicosis and lung cancer groups (P < 0.05), and the silicosis group and lung cancer group had similar protein concentration trends (P > 0.05). The tuberculosis group and lung cancer group had significantly higher concentration of IFN-β compared with the silicosis group (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in IFN-β concentration (P > 0.05).</p><p><b>CONCLUSION</b>This study may offer diagnostic markers for the clinical diagnosis of silicosis, tuberculosis, and lung cancer, and could provide a basis for the research, as well as potential molecular targets for the diagnosis and treatment of these diseases.</p>
Assuntos
Humanos , Biomarcadores , Catepsina G , Metabolismo , Citocinas , Sangue , Endopeptidases , Sangue , Ensaio de Imunoadsorção Enzimática , Granzimas , Metabolismo , Interferon beta , Metabolismo , Neoplasias Pulmonares , Silicose , TuberculoseRESUMO
The study aimed to investigate the effects of small ubiquitin-related modifier (SUMO) specific protease 1 (SENP1) on human PXR-mediated MDR1 transcriptional activity and mRNA expression. Empty vector and expression plasmids, including PXR, SENP1 and SENP1 mutant (SENP1m) were transiently transfected into HepG2 and LS174T cells using Lipo2000. Transcriptional activity was detected by dual luciferase reporter gene assay, and mRNA level was measured using real-time polymerase chain reaction. The results showed that SENP1 could remarkably reduce the rifampicin (RIF)-induced MDR1 reporter activity and mRNA level in hPXR over expressed HepG2 and LS174T cells (P < 0.05), whereas adding SENP1m restored the RIF-induced increases (P < 0.05). These results indicated that SENP1 could repress the RIF-induced hPXR-mediated MDR1 transcriptional activity and mRNA expression.
Assuntos
Humanos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Metabolismo , Cisteína Endopeptidases , Endopeptidases , Metabolismo , Expressão Gênica , Células Hep G2 , Receptor 1 de Sinal de Orientação para Peroxissomos , RNA Mensageiro , Receptores Citoplasmáticos e Nucleares , Metabolismo , Ativação TranscricionalRESUMO
<p><b>OBJECTIVE</b>To assess the association of single nucleotide polymorphisms (SNPs) of ubiquitin-specific protease 8 gene (USP8) with male infertility among ethnic Han Chinese from Sichuan.</p><p><b>METHODS</b>A total of 316 infertile males were recruited (case group), which included 72 severe oligozoospermic (SO) cases and 244 non-obstructive azoospermic (NOA) cases. The control group consisted of 149 fertile males. The genotypes of 4 SNPs (rs2241769, rs11857513, rs7174015 and rs3743044) were determined with a Sequenom MassArray technique. The frequencies of genotype, allele and haploptye were analyzed.</p><p><b>RESULTS</b>No significant difference was detected in the allelic or genotypic frequencies of the 4 SNPs between the two groups (P>0.05). Based on linkage disequilibrium analysis and haplotype construction, the frequency distribution of haplotype CAAG showed a significant difference between non-obstructive azoospermic patients and the controls (P=0.021).</p><p><b>CONCLUSION</b>The 4 SNPs (rs2241769, rs11857513, rs7174015 and rs3743044) of USP8 gene may not be associated with male infertility in ethnic Hans from Sichuan. While the haplotype CAAG may be a down-regulating factor for the risk of NOA.</p>
Assuntos
Adulto , Humanos , Masculino , Povo Asiático , Etnologia , Genética , Azoospermia , Genética , Sequência de Bases , Estudos de Casos e Controles , China , Etnologia , Endopeptidases , Genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Genética , Predisposição Genética para Doença , Etnologia , Genótipo , Infertilidade Masculina , Etnologia , Genética , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Ubiquitina Tiolesterase , GenéticaRESUMO
Associadas a projetos de construção da ideia de nação, no Brasil monárquico foram encaminhadas, pelo governo imperial, algumas iniciativas no sentido de materializar propostas de educação física. O objetivo deste artigo é investigar os sentidos e significados atribuídos ao tema na legislação e nos relatórios anuais do Ministério dos Negócios do Império (1831-1889), com especial interesse pelo que se refere ao Rio de Janeiro. A abordagem do assunto nas fontes pesquisadas evidencia que as visões sobre a educação física se deram a partir de uma matriz que articulava concepções de moral, saúde e civilização, tendo que lidar com as condições concretas de um país recém-independente, periférico e com uma burocracia ainda em formação.
In association with its nation building projects, the imperial government in Brazil under monarchic rule took some concrete actions based on proposals for physical education. The aim of this article is to investigate the meanings and significations attributed to this subject in the legislation and the annual reports issued by the Ministry of Business of the Empire (1831-1889), giving special attention to Rio de Janeiro. The approach to the subject in the sources researched demonstrates that the views of physical education took shape through a web of ideas that associated moral, health and civilization conceptions, in a bid to deal with the concrete circumstances of a newly independent peripheral nation with a bureaucratic structure in the process of formation.