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1.
Rev. argent. microbiol ; 51(1): 12-17, mar. 2019. ilus, tab
Article in Spanish | LILACS | ID: biblio-1003276

ABSTRACT

Phytophtora capsici es un patógeno que incide sobre cultivos de la familia de las solanáceas causando pérdidas económicas en cultivos de pimientos, tomates, berenjenas y cur-cubitáceas. En este trabajo evaluamos el efecto del quitosano de bajo grado de polimerización (QBP) sobre el crecimiento de P. capsici y sobre la regulación génica de este fitopatógeno a nivel transcripcional. A una concentración de 0,4mg/l de QBP se obtuvo un 88% de inhibición en el crecimiento; concentraciones superiores a 1,6 mg/l inhibieron el crecimiento en un 100%. Mediante ensayos de cambio en la movilidad electroforética de ácidos nucleicos se comprobó que el quitosano interactúa con el ADN y el ARN del hongo frente a concentraciones entre 2 y 4 mg/l de ADN y entre 0,5 y 3 mg/l de ARN. Además, se efectuó un análisis de despliegue diferencial de los productos de amplificación por RT-PCR de los ARN mensajeros de P. capsici obtenidos en presencia o ausencia de QBP; este mostró cambios en el perfil de expresión inducidos por el tratamiento con quitosano. El análisis bioinformático de las secuencias de los transcritos expresados diferencialmente sugiere que el QBP afectó la regulación génica de elementos involucrados en la síntesis de quitina y de proteínas de unión a hidratos de carbono.


Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4 mg/l for DNA and 0,5 to 3 mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.


Subject(s)
Phytophthora/genetics , Gene Expression/drug effects , Gene Expression Regulation/drug effects , Chitosan/administration & dosage , Phytophthora/drug effects , Electrophoretic Mobility Shift Assay/methods , Chitosan/therapeutic use , Polymerization
2.
Yonsei Medical Journal ; : 1045-1053, 2019.
Article in English | WPRIM | ID: wpr-762055

ABSTRACT

PURPOSE: To explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis. MATERIALS AND METHODS: The mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice. RESULTS: The mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects. CONCLUSION: Our findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.


Subject(s)
Animals , Bile Ducts , Blotting, Western , Carcinoma, Hepatocellular , Cholestasis , Electrophoretic Mobility Shift Assay , Hep G2 Cells , Humans , Lentivirus , Ligation , Liver , Male , Mice , Multidrug Resistance-Associated Proteins , Phosphorylation , Real-Time Polymerase Chain Reaction , RNA, Messenger , RNA, Small Interfering , Tumor Necrosis Factor-alpha , Up-Regulation
3.
Article in English | WPRIM | ID: wpr-764310

ABSTRACT

BACKGROUND: Abnormal upregulation of prostaglandin E₂ (PGE₂) is considered to be a key oncogenic event in the development and progression of inflammation-associated human colon cancer. It has been reported that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme catabolizing PGE₂, is ubiquitously downregulated in human colon cancer. 15-Deoxy-Δ(12,14)-prostaglandin J₂ (15d-PGJ₂), a peroxisome proliferator-activated receptor γ ligand, has been shown to have anticarcinogenic activities. In this study, we investigate the effect of 15d-PGJ₂ on expression of 15-PGDH in human colon cancer HCT116 cells. METHODS: HCT116 cells were treated with 15d-PGJ₂ analysis. The expression of 15-PGDH in the treated cells was measured by Western blot analysis and RT-PCR. In addition, the cells were subjected to a 15-PGDH activity assay. To determine which transcription factor(s) and signaling pathway(s) are involved in 15d-PGJ₂-induced 15-PGDH expression, we performed a cDNA microarray analysis of 15d-PGJ₂-treated cells. The DNA binding activity of AP-1 was measured by an electrophoretic mobility shift assay. To determine whether the AP-1 plays an important role in the 15d-PGJ₂-induced 15-PGDH expression, the cells were transfected with siRNA of c-Jun, a major subunit of AP-1. To elucidate the upstream signaling pathways involved in AP-1 activation by 15d-PGJ₂, we examined its effect on phosphorylation of Akt by Western blot analysis in the presence or absence of kinase inhibitor. RESULTS: 15d-PGJ₂ (10 μM) significantly upregulated 15-PGDH expression at the mRNA and protein levels in HCT-116 cells. 15-PGDH activity was also elevated by 15d-PGJ₂. We observed that genes encoding C/EBP delta, FOS-like antigen 1, c-Jun, and heme oxygenase-1 (HO-1) were most highly induced in the HCT116 cells following 15d-PGJ₂ treatment. 15d-PGJ₂ increased the DNA binding activity of AP-1. Moreover, transfection with specific siRNA against c-Jun significantly reduced 15-PGDH expression induced by 15d-PGJ₂. 15d-PGJ₂ activates Akt and a pharmacological inhibitor of Akt, LY294002, abrogated 15d-PGJ₂-induced 15-PGDH expression. We also observed that an inhibitor of HO-1, zinc protoporphyrin IX, also abrogated upregulation of 15-PGDH and down-regulation of cyclooxygenase-2 expression induced by 15d-PGJ₂. CONCLUSIONS: These finding suggest that 15d-PGJ₂ upregulates the expression of 15-PGDH through AP-1 activation in colon cancer HCT116 cells.


Subject(s)
Blotting, Western , Colon , Colonic Neoplasms , Cyclooxygenase 2 , DNA , Down-Regulation , Electrophoretic Mobility Shift Assay , HCT116 Cells , Heme Oxygenase-1 , Heme , Humans , Oligonucleotide Array Sequence Analysis , Oxidoreductases , Peroxisomes , Phosphorylation , Phosphotransferases , RNA, Messenger , RNA, Small Interfering , Transcription Factor AP-1 , Transfection , Up-Regulation , Zinc
4.
Pesqui. vet. bras ; 38(3): 430-435, mar. 2018. tab
Article in Portuguese | LILACS, VETINDEX | ID: biblio-964371

ABSTRACT

O objetivo desse estudo foi o de avaliar as frações proteicas em secreções colostrais de vacas acometidas por mastite clínica imediatamente após o parto. Para tanto, foram utilizadas 30 vacas da raça Holandesa distribuídas em três grupos, a saber: Grupo I (GI)- 10 vacas pluríparas sadias, Grupo II (GII) 10 vacas pluríparas que pariram com mastite assintomática e Grupo III (GIII) 10 vacas pluríparas que pariram com mastite clínica. Foram avaliadas as concentrações de imunoglobulina a (IgA), lactoferrina (LF), albumina, imunoglobulina G (IgG), ß-lactoglobulina (ß-Lg) e α-lactoalbumina (α-La) por meio da eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE).Observou-se que a IgG, LF e a albumina variaram entre as glândulas com mastite assintomática e clínica quando comparadas às glândulas sadias, e que a presença de um único microrganismo é capaz de promover alterações no proteinograma, com ou sem manifestações clínicas na glândula mamária.(AU)


The aim of this study was to evaluate the protein fractions in colostral secretions of cows affected by mastitis immediately after calving. Therefore, 30 Holstein cows were divided into three groups: Group I (GI) composed of ten multiparous cows calving without mastitis; Group II (GII) composed of ten multiparous cows calving with subclinical mastitis, and Group III (GIII) composed of ten multiparous cows calving with mastitis. The concentration of immunoglobulin A (IgA), lactoferrin (LF), albumin, immunoglobulin G (IgG), ß-lactoglobulin (ß-Lg) and α-lactoalbumin (α-La) was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the IgG, LF and albumin vary among glands of subclinical and clinical mastitis and healthy and that the presence of a bacteria in the mammary gland was the key role for changing of the pattern of serum protein source.(AU)


Subject(s)
Animals , Female , Cattle , Colostrum/enzymology , Electrophoretic Mobility Shift Assay/classification , Mastitis, Bovine/classification
5.
Protein & Cell ; (12): 848-866, 2018.
Article in English | WPRIM | ID: wpr-758025

ABSTRACT

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.


Subject(s)
Animals , Cells, Cultured , DNA-Binding Proteins , Metabolism , Electrophoretic Mobility Shift Assay , Humans , Immunoprecipitation , Mice , MicroRNAs , Genetics , Metabolism , Neoplasms , Genetics , Metabolism
6.
Article in English | WPRIM | ID: wpr-740091

ABSTRACT

BACKGROUND: Oxidative stress occurs in white adipose tissue and dysregulates the expression of adipokines secreted from adipocytes. Since adipokines influence inflammation, supplementation with antioxidants might be beneficial for preventing oxidative stress-mediated inflammation in adipocytes and inflammation-associated complications. β-Carotene is the most prominent antioxidant carotenoid and scavenges reactive oxygen species in various tissues. The purpose of this study was to determine whether β-carotene regulates the expression of adipokines, such as adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in 3T3-L1 adipocytes treated with glucose/glucose oxidase (G/GO). METHODS: 3T3-L1 adipocytes were cultured with or without β-carotene and treated with G/GO, which produces H2O2. mRNA and protein levels in the medium were determined by a real-time PCR and an ELISA. DNA binding activities of transcription factors were assessed using an electrophoretic mobility shift assay. RESULTS: G/GO treatment increased DNA binding affinities of redox-sensitive transcription factors, such as NF-κB, activator protein-1 (AP-1), and STAT3. G/GO treatment reduced the expression of adiponectin and increased the expression of MCP-1 and RANTES. G/GO-induced activations of NF-κB, AP-1, and STAT3 were inhibited by β-carotene. G/GO-induced dysregulation of adiponectin, MCP-1, and RANTES were significantly recovered by treatment with β-carotene. CONCLUSIONS: β-Carotene inhibits oxidative stress-induced inflammation by suppressing pro-inflammatory adipokines MCP-1 and RANTES, and by enhancing adiponectin in adipocytes. β-Carotene may be beneficial for preventing oxidative stress-mediated inflammation, which is related to adipokine dysfunction.


Subject(s)
Adipocytes , Adipokines , Adiponectin , Adipose Tissue, White , Antioxidants , beta Carotene , Chemokine CCL2 , Chemokine CCL5 , DNA , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Inflammation , Oxidative Stress , Oxidoreductases , Reactive Oxygen Species , Real-Time Polymerase Chain Reaction , RNA, Messenger , Transcription Factor AP-1 , Transcription Factors
7.
Article in English | WPRIM | ID: wpr-713700

ABSTRACT

PURPOSE: Peroxisome proliferator-activated receptor γ (PPARγ) is involved in the pathology of numerous diseases including atherosclerosis, diabetes, obesity, and cancer. Matrix metalloproteinases (MMPs) play a significant role in tissue remodeling related to various processes such as morphogenesis, angiogenesis, tissue repair, invasion, and metastasis. We investigated the effects of PPARγ on MMP expression and invasion in breast cancer cells. METHODS: MCF-7 cells were cultured and then cell viability was monitored in an MTT assay. Western blotting, gelatin zymography, real-time polymerase chain reaction, and luciferase assays were performed to investigate the effect of the synthetic PPARγ ligand troglitazone on MMP expression. Transcription factor DNA binding was analyzed by electrophoretic mobility shift assay. A Matrigel invasion assay was used to assess the effects of troglitazone on MCF-7 cells. RESULTS: Troglitazone did not affect MCF-7 cell viability. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced MMP-9 expression and invasion in MCF-7 cell. However, these effects were decreased by troglitazone. TPA increased nuclear factor κB and activator protein-1 DNA binding, while troglitazone inhibited these effects. The selective PPARγ antagonist GW9662 reversed MMP-9 inhibition by troglitazone in TPA-treated MCF-7 cells. CONCLUSION: Troglitazone inhibited nuclear factor κB and activator protein-1-mediated MMP-9 expression and invasion of MCF-7 cells through a PPARγ-dependent mechanism.


Subject(s)
Atherosclerosis , Blotting, Western , Breast Neoplasms , Breast , Cell Survival , DNA , Electrophoretic Mobility Shift Assay , Gelatin , Luciferases , Matrix Metalloproteinase 9 , Matrix Metalloproteinases , MCF-7 Cells , Morphogenesis , Neoplasm Metastasis , NF-kappa B , Obesity , Pathology , Peroxisomes , PPAR gamma , Real-Time Polymerase Chain Reaction , Transcription Factor AP-1 , Transcription Factors
8.
Article in English | WPRIM | ID: wpr-717803

ABSTRACT

Loss of response to anti-tumor necrosis factor (anti-TNF) agents in the treatment of inflammatory bowel disease (IBD) is a major consideration to maintain sustained response. Reversal of immunogenicity can re-establish response and increase the durability of these agents. Strategies to reverse immunogenicity include dose-intensification and/or the addition of an immunomodulator. However, there is a relative paucity of data on the efficacy of such interventions in pediatric IBD patients. Available reports have not strictly utilized homogenous mobility shift assay, which reports on anti-drug antibodies even in the presence of detectable drug, whereas prior studies have been confounded by the use of drug sensitive assays. We report four pediatric inflammatory bowel disease patients with successful reversal of immunogenicity on an anti-TNF agent using dose intensification and/or addition of an immunomodulator.


Subject(s)
Antibodies , Biological Products , Child , Electrophoretic Mobility Shift Assay , Humans , Inflammatory Bowel Diseases , Necrosis
9.
Yonsei Medical Journal ; : 1222-1231, 2018.
Article in English | WPRIM | ID: wpr-719241

ABSTRACT

PURPOSE: Cockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292). MATERIALS AND METHODS: mRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting. RESULTS: GCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration. CONCLUSION: GCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.


Subject(s)
Airway Obstruction , Asthma , Blattellidae , Blotting, Western , Cockroaches , Collagenases , DNA , Electric Impedance , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Epithelium , Extracellular Matrix , Fluorescent Antibody Technique , Humans , Matrix Metalloproteinase 1 , Matrix Metalloproteinases , Phosphorylation , Phosphotransferases , Protein Kinases , Real-Time Polymerase Chain Reaction , RNA, Messenger , RNA, Small Interfering , Tight Junction Proteins , Tight Junctions , Transcription Factors , Transfection
10.
Article in English | WPRIM | ID: wpr-742218

ABSTRACT

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.


Subject(s)
Antibodies , Blotting, Western , Calcium-Binding Proteins , Cations, Divalent , Dyneins , EF Hand Motifs , Electrophoretic Mobility Shift Assay , Fasciola hepatica , Humans , Molecular Weight , Opisthorchiasis , Opisthorchis , Parasites , Protein Isoforms
11.
Article in English | WPRIM | ID: wpr-159621

ABSTRACT

Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.


Subject(s)
Astrocytes , Central Nervous System , Culture Media, Serum-Free , Cytokines , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-8 , Interleukins , Toll-Like Receptors , Tumor Necrosis Factor-alpha
12.
Braz. j. med. biol. res ; 49(9): e5388, 2016. tab, graf
Article in English | LILACS | ID: biblio-951696

ABSTRACT

Galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGF-βRI), is the only known TGF-β pathway inhibitor. In the present study, we investigated the effect of galunisertib on taurocholate (TAC)-induced acute pancreatitis (AP) in rats, and the role of TGF-β and NF-κB signaling pathways. AP was induced by infusion of TAC into the pancreatic duct of Sprague-Dawley male rats (n=30). The rats (220±50 g) were administered galunisertib intragastrically [75 mg·kg-1·day-1 for 2 days (0 and 24 h)]. Serum IL-1β, IL-6, TNF-α, amylase (AMY), lipase (LIP), and myeloperoxidase (MPO) levels were measured by ELISA. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); NF-κBp65 and TGF-β1 proteins, as well as TGF-βRI and p-Smad2/3 proteins, were detected by western blot assay. Cell apoptosis was detected by TUNEL assay. H&E staining was used to evaluate the histopathological alterations of the pancreas. Galunisertib treatment attenuated the severity of AP and decreased the pancreatic histological score. In addition, number of apoptotic cells were significantly increased in the galunisertib-treated group (16.38±2.26) than in the AP group (8.14±1.27) and sham-operated group (1.82±0.73; P<0.05 and P<0.01, respectively). Galunisertib decreased the expression levels of TGF-βRI and p-Smad2/3 and inhibited NF-κB activation and p65 translocation compared with the sham-operated group. Furthermore, serum IL-1β, IL-6, TNF-α, AMY and LIP levels and tissue MPO activity were significantly decreased in the galunisertib-treated group. Our data demonstrate that galunisertib attenuates the severity of TAC-induced experimental AP in rats by inducing apoptosis in the pancreas, inhibiting the activation of TGF-β signals and NF-κB as well as the secretion of pro-inflammatory cytokines.


Subject(s)
Animals , Male , Pancreatitis/drug therapy , Pyrazoles/therapeutic use , Quinolines/therapeutic use , Protein-Serine-Threonine Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Pancreas/drug effects , Pancreas/pathology , Pancreatitis/pathology , Enzyme-Linked Immunosorbent Assay , Blotting, Western , Acute Disease , Interleukin-6/blood , Treatment Outcome , Apoptosis , Peroxidase/analysis , In Situ Nick-End Labeling , Electrophoretic Mobility Shift Assay , Interleukin-1beta/blood , Amylases/blood , Lipase/blood
13.
Article in English | WPRIM | ID: wpr-159297

ABSTRACT

BACKGROUND: Hyperoside, a flavonoid which is mainly found in Hypericum perforatum L., has many biological effects. One of the most important effects is to prevent the oxidative stress induced by reactive oxygen species. However, the molecular mechanisms underlying its effect are not fully understood. Oxidative stress is implicated in the occurrence of various physical diseases. A wide array of enzymatic antioxidant defense systems include NADH: quinone oxidoreductase 1, superoxide dismutase, and heme oxygenase-1 (HO-1). In the present study, the protective effects of hyperoside against hydrogen peroxide-induced oxidative stress in human lens epithelial cells, HLE-B3, were investigated in terms of HO-1 induction. METHODS: The protein and mRNA expressions of HO-1 were examined by Western blotting and reverse transcriptase-PCR assays, respectively. To evaluate the ability of hyperoside to activate nuclear factor erythroid 2-related factor 2 (Nrf2), Western blotting and electrophoretic mobility shift assay were performed with nuclear extracts prepared from HLE-B3 cells treated with hyperoside. The activation of extracellular signal-regulated kinase (ERK), the upstream kinase of Nrf2 signaling, was monitored by Western blot analysis. The protective effect of hyperoside in HLE-B3 cells against hydrogen peroxide was performed by MTT assay. RESULTS: Hyperoside increased both the mRNA and protein expression of HO-1 in a time- and dose-dependent manner. In addition, hyperoside elevated the level of of Nrf2 and its antioxidant response element-binding activity, which was modulated by upstream of ERK. Moreover, it activated ERK and restored cell viability which was decreased by hydrogen peroxide. CONCLUSIONS: Hyperoside is an effective compound to protect cells against oxidative stress via HO-1 induction.


Subject(s)
Antioxidants , Blotting, Western , Cell Survival , Electrophoretic Mobility Shift Assay , Epithelial Cells , Heme Oxygenase-1 , Humans , Hydrogen , Hydrogen Peroxide , Hypericum , NAD , Oxidative Stress , Phosphotransferases , Reactive Oxygen Species , RNA, Messenger , Superoxide Dismutase
14.
Article in English | WPRIM | ID: wpr-121858

ABSTRACT

BACKGROUND: Isoflavones are biologically active compounds that occur naturally in a variety of plants, with relatively high levels in soybean. Tectorigenin, an isoflavone, protects against hydrogen peroxide (H2O2)-induced cell damage. However, the underlying mechanism is unknown. METHODS: The MTT assay was performed to determine cell viability. Catalase activity was assessed by determining the amount of enzyme required to degrade 1 μM H2O2. Protein expression of catalase, phospho-extracellular signal-regulated kinase (ERK), IκB-α, and NF-κB were evaluated by Western blot analysis. A mobility shift assay was performed to assess the DNA-binding ability of NF-κB. Transient transfection and a NF-κB luciferase assay were performed to assess transcriptional activity. RESULTS: Tectorigenin reduced H2O2-induced death of Chinese hamster lung fibroblasts (V79-4). In addition, tectorigenin increased the activity and protein expression of catalase. Blockade of catalase activity attenuated the protective effect of tectorigenin against oxidative stress. Furthermore, tectorigenin enhanced phosphorylation of ERK and nuclear expression of NF-κB, while inhibition of ERK and NF-κB attenuated the protective effect of tectorigenin against oxidative stress. CONCLUSIONS: Tectorigenin protects cells against oxidative damage by activating catalase and modulating the ERK and NF-κB signaling pathway.


Subject(s)
Animals , Blotting, Western , Catalase , Cell Death , Cell Survival , Cricetinae , Cricetulus , Electrophoretic Mobility Shift Assay , Extracellular Signal-Regulated MAP Kinases , Fibroblasts , Hydrogen Peroxide , Isoflavones , Luciferases , Lung , NF-kappa B , Oxidative Stress , Phosphorylation , Phosphotransferases , Soybeans , Transfection
15.
Article in English | WPRIM | ID: wpr-125901

ABSTRACT

BACKGROUND AND PURPOSE: The wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD). METHODS: Immunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay. RESULTS: Immunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements. CONCLUSIONS: Our findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis.


Subject(s)
Blotting, Western , Cytoplasm , Dermatomyositis , Electrophoretic Mobility Shift Assay , Embryonic Development , Female , Fluorescent Antibody Technique , Genes, Reporter , Homeostasis , Humans , Luciferases , Lung Diseases, Interstitial , Muscular Dystrophy, Duchenne , Polymyositis , Pregnancy , Pulmonary Fibrosis , Regeneration , Wound Healing
16.
J. pediatr. (Rio J.) ; 91(2): 175-182, Mar-Apr/2015. tab
Article in English | LILACS | ID: lil-745947

ABSTRACT

OBJECTIVE: To analyze the prevalence of excess weight and low height, and identify associated factors among children younger than five years. METHODS: Cross-census study. A total of 1,640 children from two municipalities in Piauí, Brazil were included. RESULTS: The prevalence of low height was 10.9% (95% CI: 9.3 to 12.4), inversely associated with mother's younger age and low level of education, lower socioeconomic status, mothers who had fewer than six prenatal consultations, and households that had more than one child younger than 5 years. Excess weight prevalence was 19.1% (95% CI: 17.2 to 21.0), and remained inversely associated with lower maternal age, low maternal education, and cesarean delivery. Stunting was greater in children aged between 12 and 23 months, while excess weight decreased with age. CONCLUSIONS: It is noteworthy that the stunting rate, although decreasing, is still high, while the prevalence of excess weight, even in this very poor area, already exceeds the expected percentage for a population with better socioeconomic level. .


OBJETIVO: Analisar a prevalência de excesso de peso e déficit de altura e identificar fatores associados entre menores de cinco anos. MÉTODOS: Estudo censitário transversal. Foram incluídas 1.640 crianças de dois municípios do Piauí, Brasil. RESULTADOS: A prevalência de déficit de altura foi 10,9% (IC95%: 9,3-12,4), inversamente associado com menor idade e escolaridade materna, menor condição socioeconômica, mães que fizeram menos de seis consultas pré-natal e se nessas casas havia mais de uma criança menor de cinco anos. O excesso de peso teve prevalência de 19,1% (IC95%: 17,2-21,0) e manteve-se inversamente associado com menor idade da mãe, baixa escolaridade materna e parto cesáreo. O déficit de altura foi maior para crianças entre 12 e 23 meses, enquanto o excesso de peso diminuiu com a idade. CONCLUSÕES: Destaca-se que o déficit de altura, embora esteja diminuindo, ainda é elevado, enquanto a prevalência de excesso de peso, mesmo nessa área muito pobre, já supera o percentual esperado para uma população com melhores condições socioeconômicas. .


Subject(s)
Adult , Child , Child, Preschool , Female , Humans , Male , Bone Density , DNA Methylation , Promoter Regions, Genetic , Retinoid X Receptor alpha/genetics , CpG Islands , Electrophoretic Mobility Shift Assay , Prospective Studies , Vitamin D/analogs & derivatives , Vitamin D/blood
17.
Article in Korean | WPRIM | ID: wpr-172595

ABSTRACT

OBJECTIVE: Resveratrol is well-known for its anti-inflammatory, anti-oxidant effects on several diseases. We investigated whether dietary supplementation with resveratrol may suppress joint inflammation and destruction in a mouse model of collagen-induced arthritis (CIA). METHODS: Mice were randomly divided into two groups; CIA mice with normal diet-fed and CIA mice fed a 0.05% resveratrol diet. The effect of resveratrol on arthritis was assessed by clinical scoring system. The plain radiographs of paws were obtained to evaluate the effects on preventing bone destruction. Joint inflammation, cartilage damage, and osteoclastic bone resorption were checked by staining with H&E, Safranin-O, and tartrate resistant acid phosphatase (TRAP). Levels of pro-inflammatory cytokines were checked by enzyme-linked immunosorbent assay. The level of expression of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay (EMSA). RESULTS: Dietary supplementation with resveratrol led to mitigated severity of arthritis compared to the normal diet group (6.7+/-0.8 vs. 2.7+/-0.6, p<0.01). Resveratrol-fed mice showed decreased bone destruction on radiograph (3.4+/-0.3 vs. 2.0+/-0.2, p<0.01), and showed significantly inhibited pathological changes (inflammation 2.0+/-0.3 vs.3.2+/-0.2, p<0.01; cartilage damage 1.5+/-0.3 vs. 3.2+/-0.2, p<0.01; pannus formation 1.4+/-0.3 vs. 3.0+/-0.3, p<0.01; erosion; 1.4+/-0.2 vs. 3.3+/-0.3, p<0.01). Generation of TRAP-positive osteoclasts was inhibited in the resveratrol-fed mice (55.3+/-12.7 vs. 3.27+/-0.8, p<0.01). Resveratrol-fed mice showed decreased levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6,monocyte chemoattractant protein 1, and the soluble receptor activator of NF-kappaB ligand in joint tissues and sera. Expression of NF-kappaB, measured by EMSA, was decreased in resveratrol-fed mice. CONCLUSION: Dietary supplementation with resveratrol mitigates inflammation and bone destruction in CIA mice.


Subject(s)
Acid Phosphatase , Animals , Antioxidants , Arthritis , Arthritis, Experimental , Arthritis, Rheumatoid , Bone Resorption , Cartilage , Cytokines , Diet , Dietary Supplements , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Inflammation , Interleukins , Joints , Mice , NF-kappa B , Osteoclasts , Receptor Activator of Nuclear Factor-kappa B , Tumor Necrosis Factor-alpha
18.
Article in English | WPRIM | ID: wpr-264548

ABSTRACT

Epithelial-mesenchymal transition (EMT) plays an important role in fibrotic diseases. We have previously showed that silica induces EMT in human bronchial epithelial cells (BECs); however, the underlying mechanism of silica-induced EMT is poorly understood. In the present study, we investigated the role of Snail in silica-induced EMT in human BECs in vitro. Human BECs were treated with silica at various concentrations and incubation times. Then MTT assay, western blot, electrophoretic mobility shift assay (EMSA), and small interfering RNA (siRNA) transfection were performed. We found that silica increased the expression and DNA binding activity of Snail in human BECs. SNAI siRNA inhibited the silica-induced expression of Snail. Moreover, SNAI siRNA upregulated the expression of epithelial marker E-cadherin, but attenuated the expression of mesenchymal marker α-smooth muscle actin and vimentin in silica-stimulated cells. These results suggest that Snail mediates the silica-induced EMT in human BECs.


Subject(s)
Actins , Metabolism , Blotting, Western , Bronchi , Cell Biology , Metabolism , Cadherins , Metabolism , Cell Culture Techniques , Cell Line , Cell Survival , Electrophoretic Mobility Shift Assay , Epithelial Cells , Cell Biology , Metabolism , Epithelial-Mesenchymal Transition , Humans , Particle Size , RNA, Small Interfering , Genetics , Silicon Dioxide , Toxicity , Snail Family Transcription Factors , Transcription Factors , Genetics , Metabolism
19.
Mem. Inst. Oswaldo Cruz ; 109(3): 356-361, 06/2014. tab, graf
Article in English | LILACS | ID: lil-711732

ABSTRACT

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.


Subject(s)
Humans , Electrophoretic Mobility Shift Assay , Mycobacterium tuberculosis/isolation & purification , Nontuberculous Mycobacteria/isolation & purification , /genetics , Bacterial Typing Techniques , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium Infections/microbiology , Mycobacterium tuberculosis/classification , Nontuberculous Mycobacteria/classification , Polymerase Chain Reaction
20.
Genomics & Informatics ; : 240-246, 2014.
Article in English | WPRIM | ID: wpr-61836

ABSTRACT

Mutation in HNF1B, the hepatocyte nuclear factor-1beta (HNF-1beta) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-1beta in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic beta-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in beta-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.


Subject(s)
Animals , Codon , COS Cells , Diabetes Mellitus, Type 2 , DNA , Electrophoretic Mobility Shift Assay , Gene Expression , Glucose , Glucose Transporter Type 2 , Hepatocyte Nuclear Factor 1-beta , Humans , Insulin , Luciferases , Metabolism , Point Mutation , RNA, Messenger
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