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1.
Chinese Journal of Biotechnology ; (12): 561-579, 2021.
Article in Chinese | WPRIM | ID: wpr-878582

ABSTRACT

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.


Subject(s)
Binding Sites , Catalytic Domain , Endopeptidases , Peptide Hydrolases/genetics , Protease Inhibitors , Proteins
2.
Chinese Journal of Biotechnology ; (12): 415-424, 2019.
Article in Chinese | WPRIM | ID: wpr-771365

ABSTRACT

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.


Subject(s)
Aspergillus oryzae , Cloning, Molecular , Endopeptidases , Hydrogen-Ion Concentration , Pichia , Recombinant Proteins , Temperature
3.
Article in Chinese | WPRIM | ID: wpr-772684

ABSTRACT

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.


Subject(s)
Cell Line, Tumor , Cell Movement , Cell Proliferation , Endopeptidases , Metabolism , Humans , Neoplasm Invasiveness , RNA Interference , RNA, Small Interfering , Tongue Neoplasms , Metabolism , Therapeutics , Transfection
4.
Protein & Cell ; (12): 365-379, 2018.
Article in English | WPRIM | ID: wpr-756937

ABSTRACT

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.


Subject(s)
DNA Damage , DNA Repair , Genetics , DNA Replication , Genetics , DNA-Binding Proteins , Genetics , DNA-Directed DNA Polymerase , Genetics , Endopeptidases , Genetics , Gene Knockout Techniques , Humans , Hydrogen Peroxide , Toxicity , NEDD8 Protein , Genetics , Oxidative Stress , Genetics , Proliferating Cell Nuclear Antigen , Genetics , Ubiquitin-Conjugating Enzymes , Genetics , Ubiquitin-Protein Ligases , Genetics , Ubiquitination , Genetics , Ultraviolet Rays
5.
Article in English | WPRIM | ID: wpr-690628

ABSTRACT

<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>


Subject(s)
Cell Movement , Physiology , Cell Proliferation , Cells, Cultured , Endopeptidases , Genetics , Metabolism , Humans , Keratinocytes , Physiology , Signal Transduction , Physiology , Smad Proteins , Genetics , Metabolism , Thiolester Hydrolases , Genetics , Metabolism , Transforming Growth Factor beta3 , Genetics , Metabolism
6.
Article in English | WPRIM | ID: wpr-327203

ABSTRACT

<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>


Subject(s)
Amyloid Precursor Protein Secretases , Metabolism , Amyloid beta-Protein Precursor , Metabolism , Animals , Cyclic AMP Response Element-Binding Protein , Genetics , Metabolism , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Endopeptidases , Genetics , Metabolism , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation , Hippocampus , Metabolism , Pathology , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Immunohistochemistry , Learning , Male , Memory Disorders , Drug Therapy , Genetics , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-1 , Metabolism , Presenilin-2 , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Signal Transduction
7.
Chinese Journal of Biotechnology ; (12): 669-682, 2016.
Article in Chinese | WPRIM | ID: wpr-337432

ABSTRACT

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.


Subject(s)
Antiviral Agents , Chemistry , Carrier Proteins , Chemistry , Drug Resistance, Viral , Endopeptidases , Hepacivirus , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Oligopeptides , Chemistry , Protease Inhibitors , Chemistry , Serine Proteases , Thiazoles , Chemistry , Viral Nonstructural Proteins , Chemistry
8.
Genomics & Informatics ; : 12-19, 2016.
Article in English | WPRIM | ID: wpr-193409

ABSTRACT

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.


Subject(s)
Circadian Rhythm , Depression , Endopeptidases , Mass Spectrometry , Models, Animal , Nervous System , Neuropeptides , Neurotransmitter Agents , Physiological Phenomena , Vital Signs
9.
Chinese Medical Journal ; (24): 2102-2108, 2016.
Article in English | WPRIM | ID: wpr-307460

ABSTRACT

<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>


Subject(s)
Adrenocorticotropic Hormone , Metabolism , Animals , Apoptosis , Cell Proliferation , Physiology , Cell Survival , Physiology , Endopeptidases , Metabolism , Endosomal Sorting Complexes Required for Transport , Metabolism , Enzyme Inhibitors , Pharmacology , Humans , Indenes , Pharmacology , Mice , Pyrazines , Pharmacology , ErbB Receptors , Metabolism , Ubiquitin Thiolesterase , Metabolism
10.
Einstein (Säo Paulo) ; 13(1): 79-88, Jan-Mar/2015. graf
Article in English | LILACS | ID: lil-745885

ABSTRACT

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. .


Subject(s)
Animals , Female , Mice , Disease Models, Animal , Endopeptidases/genetics , Endopeptidases/metabolism , Kidney/metabolism , Kidney/pathology , Mutation/physiology , Superoxides/metabolism , Apoptosis/genetics , Apoptosis/physiology , Collagen/analysis , Creatinine/blood , Erythropoietin/analysis , Fibrosis/genetics , Fibrosis/metabolism , Matrix Metalloproteinase 9/analysis , Oxidative Stress/genetics , Oxidative Stress/physiology , Real-Time Polymerase Chain Reaction , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/metabolism , Superoxide Dismutase/analysis , Superoxides/analysis , Transforming Growth Factor beta/analysis
11.
Rev. bras. ginecol. obstet ; 37(1): 42-51, 01/2015. tab
Article in English | LILACS | ID: lil-732870

ABSTRACT

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.


Subject(s)
Animals , Male , Rats , Cathepsins/analysis , Cystatins/analysis , Cysteine Proteinase Inhibitors/metabolism , Endopeptidases , Leucine/analogs & derivatives , Osteoclasts/chemistry , Osteoclasts/enzymology , Salivary Proteins and Peptides/analysis , Bone Matrix/chemistry , Bone Matrix/enzymology , Cathepsin L , Cysteine Endopeptidases , Cathepsins/antagonists & inhibitors , Cathepsins/metabolism , Cystatins/metabolism , Cysteine Proteinase Inhibitors/toxicity , Leucine/metabolism , Leucine/toxicity , Lysosomes/enzymology , Microscopy, Immunoelectron , Osteoclasts/drug effects , Osteoclasts/ultrastructure , Rats, Wistar , Salivary Cystatins
12.
Article in Chinese | WPRIM | ID: wpr-350529

ABSTRACT

<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>


Subject(s)
Biomarkers , Cathepsin G , Metabolism , Cytokines , Blood , Endopeptidases , Blood , Enzyme-Linked Immunosorbent Assay , Granzymes , Metabolism , Humans , Interferon-beta , Metabolism , Lung Neoplasms , Silicosis , Tuberculosis
13.
Article in Chinese | WPRIM | ID: wpr-239489

ABSTRACT

<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>


Subject(s)
Adult , Asian Continental Ancestry Group , Ethnology , Genetics , Azoospermia , Genetics , Base Sequence , Case-Control Studies , China , Ethnology , Endopeptidases , Genetics , Endosomal Sorting Complexes Required for Transport , Genetics , Genetic Predisposition to Disease , Ethnology , Genotype , Humans , Infertility, Male , Ethnology , Genetics , Male , Molecular Sequence Data , Polymorphism, Single Nucleotide , Ubiquitin Thiolesterase , Genetics
14.
Acta Pharmaceutica Sinica ; (12): 1192-1196, 2015.
Article in Chinese | WPRIM | ID: wpr-257007

ABSTRACT

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.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Metabolism , Cysteine Endopeptidases , Endopeptidases , Metabolism , Gene Expression , Hep G2 Cells , Humans , Peroxisome-Targeting Signal 1 Receptor , RNA, Messenger , Receptors, Cytoplasmic and Nuclear , Metabolism , Transcriptional Activation
15.
Hist. ciênc. saúde-Manguinhos ; 21(4): 1131-1149, Oct-Dec/2014.
Article in Portuguese | LILACS | ID: lil-732507

ABSTRACT

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.


Subject(s)
Animals , Female , Mice , Carcinoma, Lewis Lung/secondary , Cathepsin B/antagonists & inhibitors , Cathepsins/antagonists & inhibitors , Endopeptidases , Leucine/analogs & derivatives , Liver Neoplasms, Experimental/prevention & control , Liver Neoplasms, Experimental/secondary , Neoplasm Invasiveness/prevention & control , Cathepsin L , Collagen , Cysteine Endopeptidases , Carcinoma, Lewis Lung/metabolism , Drug Combinations , Drug Screening Assays, Antitumor , Laminin , Leucine/pharmacokinetics , Leucine/pharmacology , Liver Neoplasms, Experimental/enzymology , Proteoglycans , Tumor Cells, Cultured
16.
Braz. j. microbiol ; 45(3): 903-910, July-Sept. 2014. ilus, graf
Article in English | LILACS | ID: lil-727019

ABSTRACT

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseedoil as an inducer forprotease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.


Subject(s)
Amylases/metabolism , Bacillus/enzymology , Bacillus/metabolism , Bacterial Proteins/metabolism , Detergents/metabolism , Endopeptidases/metabolism , Enzyme Inhibitors/metabolism , Lipase/metabolism , Bacillus/growth & development , Bacillus/isolation & purification , Carbon/metabolism , Culture Media/chemistry , Fermentation , Hydrogen-Ion Concentration , Nitrogen/metabolism , Soil Microbiology , Temperature , Time Factors
17.
Mem. Inst. Oswaldo Cruz ; 109(1): 1-8, 02/2014. tab, graf
Article in English | LILACS | ID: lil-703649

ABSTRACT

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.


Subject(s)
Animals , Endopeptidases/genetics , Schistosoma mansoni/enzymology , Ubiquitin Thiolesterase/genetics , Cercaria/enzymology , Cercaria/genetics , Conserved Sequence/genetics , Evolution, Molecular , Gene Expression , Genome, Helminth/genetics , Genome/genetics , Life Cycle Stages/genetics , Mice, Inbred BALB C , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Alignment , Schistosoma mansoni/genetics , Schistosoma mansoni/growth & development , Transcriptome/physiology , Transcytosis/physiology , Ubiquitin Thiolesterase/classification , Ubiquitin-Specific Proteases/genetics , Ubiquitination/physiology
18.
Iranian Journal of Veterinary Research. 2014; 15 (4): 320-325
in English | IMEMR | ID: emr-166497

ABSTRACT

In the present study, the therapeutic potential of bacteriophages virulent to Staphylococcus aureus associated with goat mastitis were isolated, identified and assessed. Staphylococcus aureus [host or indicator bacterium] was isolated from a goat suffering from clinical mastitis. Based on cultural, morphological, biochemical tests and amplification of S. aureus specific thermonuclease gene in PCR, the identity of the organism was confirmed as S. aureus. Bacteriophages were isolated from soil and faecal samples [n=42] collected from different parts of the Mathura district in Uttar Pradesh [India], and their identity was confirmed by amplification of the bacteriophage-specific endolysin gene fragment in PCR. The thermal tolerance study revealed that all phage isolates were stable at 30 and 40°C with 100% lytic efficacy and their activities reduced to 62-80% at 50°C declining sharply at 60°C with less than 5% efficacy. Likewise, at pH = 6.5 and 7.5, the survivability of all isolates was 100% which reduced to 70-79% and 84-91% at pH = 5.5 and 8.5, respectively. All isolates were stable up to 3 months at 37°C, and for 16 months at 4°C but the stability of their respective endolysins only lasted for 12-23 days at 37°C and 6 months at 4°C. Three of the bacteriophage isolates, S. aureus phage/CIRG/1, S. aureus phage/CIRG/4 and S. aureus phage/CIRG/5 exhibited lytic activity against over 80% of the staphylococcal isolates. The results of the present study provide insight for the use of lytic bacteriophages for therapeutic interventions against multi-drug-resistant S. aureus inducing mastitis in goats


Subject(s)
Animals , Staphylococcus aureus , Mastitis , Goats , Endopeptidases
19.
Chinese Medical Journal ; (24): 4060-4065, 2014.
Article in English | WPRIM | ID: wpr-268423

ABSTRACT

<p><b>BACKGROUND</b>Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors. Despite the advances in therapy over the years, its mortality remains high. The aim of this study was to evaluate the expression of small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) in NSCLC tissues and its role in the regulation of vascular endothelial growth factor (VEGF) expression. We also investigated the association between the expression level of SENP1 and the clinicopathological features and survival of the patients.</p><p><b>METHODS</b>A SENP1 small interfering RNA (siRNA) was constructed and transfected into the NSCLC cells. VEGF gene expression was analyzed by real-time polymerase chain reaction (RT-PCR). Immunohistochemistry staining was used to assess the expression of SENP1 in 100 NSCLC patients and its association with the clinicopathological features and survival was analyzed.</p><p><b>RESULTS</b>VEGF expression was significantly higher in NSCLC tissues than in normal lung tissues. Inhibition of SENP1 by siRNA was associated with decreased VEGF expression. SENP1 was over-expressed in 55 of the 100 NSCLC samples (55%) and was associated with a moderate and low histological tumor grade (3.6%, 38.2%, and 58.2% in high, moderate and low differentiated tumors, respectively, P = 0.046), higher T stage (10.9% in T1, and 89.1% in T2 and T3 tumor samples, P < 0.001) and TNM stage (10.9% in stage I, and 89.1% in stages II and III tumor samples, P < 0.001). The rate of lymph node metastasis was significantly higher in the SENP1 over-expression group (76.4%) than that in the SENP1 low expression group (33.3%, P < 0.001). Sixty three patients received postoperative chemotherapy, including 34 with SENP1 over-expression and 29 with SENP1 low expression. Among the 34 patients with SENP1 over-expression, 22 (64.7%) patients developed recurrence or metastasis, significantly higher than those in the low expression group 27.6% (8/29) (P = 0.005). Multivariate Cox regression analysis showed that lymph node metastasis (P = 0.015), TNM stage (P = 0.001), and SENP1 expression level (P = 0.002) were independent prognostic factors for the survival of NSCLC patients.</p><p><b>CONCLUSIONS</b>SENP1 may be a promising predictor of survival, a predictive factor of chemo-sensitivity for NSCLC patients, and potentially a desirable drug target for lung carcinoma target therapy.</p>


Subject(s)
Antineoplastic Agents , Therapeutic Uses , Blotting, Western , Carcinoma, Non-Small-Cell Lung , Drug Therapy , Genetics , Metabolism , Cell Line, Tumor , Cysteine Endopeptidases , Endopeptidases , Genetics , Metabolism , Female , Humans , Immunohistochemistry , In Vitro Techniques , Lung Neoplasms , Drug Therapy , Genetics , Metabolism , Male , Reverse Transcriptase Polymerase Chain Reaction
20.
Chinese Journal of Cancer ; (12): 285-294, 2014.
Article in English | WPRIM | ID: wpr-320525

ABSTRACT

Ovarian cancer is the leading cause of death in women worldwide. Cisplatin is the core of first-line chemotherapy for patients with advanced ovarian cancer. Many patients eventually become resistant to cisplatin, diminishing its therapeutic effect. MicroRNAs (miRNAs) have critical functions in diverse biological processes. Using miRNA profiling and polymerase chain reaction validation, we identified a panel of differentially expressed miRNAs and their potential targets in cisplatin-resistant SKOV3/DDP ovarian cancer cells relative to cisplatin-sensitive SKOV3 parental cells. More specifically, our results revealed significant changes in the expression of 13 of 663 miRNAs analyzed, including 11 that were up-regulated and 2 that were down-regulated in SKOV3/DDP cells with or without cisplatin treatment compared with SKOV3 cells with or without cisplatin treatment. miRNA array and mRNA array data were further analyzed using Ingenuity Pathway Analysis software. Bioinformatics analysis suggests that the genes ANKRD17, SMC1A, SUMO1, GTF2H1, and TP73, which are involved in DNA damage signaling pathways, are potential targets of miRNAs in promoting cisplatin resistance. This study highlights candidate miRNA-mRNA interactions that may contribute to cisplatin resistance in ovarian cancer.


Subject(s)
Cell Cycle Proteins , Cell Line, Tumor , Chromosomal Proteins, Non-Histone , Cisplatin , Cysteine Endopeptidases , DNA-Binding Proteins , Down-Regulation , Drug Resistance, Neoplasm , Endopeptidases , Female , Humans , MicroRNAs , Nuclear Proteins , Ovarian Neoplasms , Phosphoproteins , RNA, Messenger , Signal Transduction , Transcription Factors, TFII , Tumor Protein p73 , Tumor Suppressor Proteins , Up-Regulation
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