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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.
Mem. Inst. Oswaldo Cruz ; 116: e200584, 2021. graf
Article in English | LILACS | ID: biblio-1250360

ABSTRACT

In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.


Subject(s)
Humans , Plasmodium vivax/genetics , Malaria, Vivax , Genetic Variation/genetics , Brazil , Protozoan Proteins/genetics , Catalytic Domain
3.
Chinese Journal of Biotechnology ; (12): 1021-1030, 2020.
Article in Chinese | WPRIM | ID: wpr-826874

ABSTRACT

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.


Subject(s)
Carboxylic Ester Hydrolases , Chemistry , Metabolism , Catalytic Domain , Crystallography , Pectins , Metabolism , Protein Structure, Tertiary , Substrate Specificity
4.
Article in Chinese | WPRIM | ID: wpr-826365

ABSTRACT

To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.


Subject(s)
Animals , Catalytic Domain , Down-Regulation , HEK293 Cells , Humans , Mitochondria , Protein Phosphatase 2 , Rats , tau Proteins
5.
Braz. arch. biol. technol ; 63: e20180573, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132185

ABSTRACT

Abstract This work reports the study of the potential application of Zn/TiO2 catalysts, obtained by the sol-gel method, in processes of environmental decontamination through the reactions of photodegradation of textile dye, followed by electrospray mass spectrometry. The catalysts synthesis was performed according to a 2² factorial design with repetition at the central point. The characterization techniques used were: N2 adsorption measurements (BET method), scanning electron microscopy with energy dispersive X-ray (MEV/EDS), X-ray diffraction and point of zero charge (PZC). The photocatalytic tests were performed in batch in the presence of sunlight, and to evaluate the degradation kinetics study, a rapid direct injection electrospray mass spectrometry (DI-ESI-MS) method has been developed. By the photocatalytic tests, the calcination temperature of 400 °C has shown the best results of discoloration for the reactive Orange-122 dye (99.76%) in a reaction time of 2h. The discoloration kinetics were a pseudo-first order, and a statistical analysis was performed to investigate the effects of the variables and to optimize the conditions of discoloration to the dye. After the reactional time of 2 h, an ion of m/z 441.5 was detected by ESI-MS, indicating that the photocatalytic process was effective for the degradation of the dye to secondary compounds.


Subject(s)
Azo Compounds/toxicity , Biodegradation, Environmental , Decontamination/methods , Tandem Mass Spectrometry/methods , Environmental Restoration and Remediation/methods , Waste Water , Photochemistry , Textiles/toxicity , Microscopy, Electron, Scanning , Catalysis , Catalytic Domain , Spectrometry, Mass, Electrospray Ionization , Coloring Agents , Photobioreactors , Models, Theoretical
6.
Article in English | WPRIM | ID: wpr-765110

ABSTRACT

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser¹¹⁷⁹ (p-eNOS-Ser¹¹⁷⁹). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser¹¹⁷⁹ levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser¹¹⁷⁹ levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser¹¹⁷⁹ levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser¹¹⁷⁹ dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.


Subject(s)
Acetylcholine , Animals , Aorta , Blotting, Western , Catalytic Domain , Endothelial Cells , In Vitro Techniques , Losartan , Mice , Mice, Obese , Nitric Oxide Synthase Type III , Nitric Oxide , Okadaic Acid , Peroxisomes , Phosphorylation , Protein Phosphatase 2 , Rats , Receptor, Angiotensin, Type 1 , Relaxation , Renin-Angiotensin System , Vascular Diseases
7.
Article in English | WPRIM | ID: wpr-763184

ABSTRACT

PURPOSE: Amplified mesenchymal-epithelial transition factor, MET, is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). Although multiple MET tyrosine kinase inhibitors (TKIs) are being actively developed for MET-driven NSCLC, the mechanisms of acquired resistance to MET-TKIs have not been well elucidated. To understand the mechanisms of resistance and establish therapeutic strategies, we developed an in vitro model using the MET-amplified NSCLC cell line EBC-1. MATERIALS AND METHODS: We established capmatinib-resistant NSCLC cell lines and identified alternative signaling pathways using 3′ mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative polymerase chain reaction and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the resistant cell lines. RESULTS: We found that EBC-CR1 showed an epidermal growth factor receptor (EGFR)‒dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells that had overexpression of EGFR-MET heterodimer dramatically responded to combined capmatinib with afatinib. In addition, EBC-CR3 cells derived from EBC-CR1 cells that activated EGFR with amplified phosphoinositide-3 kinase catalytic subunit α (PIK3CA) were sensitive to combined afatinib with BYL719, a phosphoinositide 3-kinase α (PI3Kα) inhibitor. CONCLUSION: Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib-resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3Kα inhibitors may be effective therapeutic strategies in capmatinib-resistant NSCLC patients.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Catalytic Domain , Cell Line , Cell Proliferation , Humans , In Vitro Techniques , Parents , Phosphotransferases , Polymerase Chain Reaction , Protein-Tyrosine Kinases , ErbB Receptors , RNA, Messenger
8.
Article in Chinese | WPRIM | ID: wpr-775139

ABSTRACT

OBJECTIVE@#To study the effect of hyperoxic exposure on the dynamic expression of heme oxygenase-1 (HO-1) and glutamate-L-cysteine ligase catalytic subunit (GCLC) in the lung tissue of preterm neonatal rats.@*METHODS@#Cesarean section was performed for rats on day 21 of gestation to obtain 80 preterm rats, which were randomly divided into air group and hyperoxia group after one day of feeding. The rats in the air group were housed in room air under atmospheric pressure, and those in the hyperoxia group were placed in an atmospheric oxygen tank (oxygen concentration 85%-95%) in the same room. Eight rats each were selected from each group on days 1, 4, 7, 10, and 14, and lung tissue samples were collected. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissue at different time points after air or hyperoxic exposure. Western blot and RT-qPCR were used to measure the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats at different time points after air or hyperoxic exposure.@*RESULTS@#Compared with the air group, the hyperoxia group had a significant reduction in the body weight (P<0.05). Compared with the air group, the hyperoxia group had structural disorder, widening of alveolar septa, a reduction in the number of alveoli, and simplification of the alveoli on the pathological section of lung tissue. Compared with the air group, the hyperoxia group had significantly lower relative mRNA expression of HO-1 in the lung tissue on day 7 and significantly higher expression on days 10 and 14 (P<0.05). Compared with the air group, the hyperoxia group had significantly lower mRNA expression of GCLC in the lung tissue on days 1, 4, and 7 and significantly higher expression on day 10 (P<0.05). Compared with the air group, the hyperoxia group had significantly higher protein expression of HO-1 in the lung tissue on all days, and the protein expression of GCLC had same results as HO-1, except on day 1 (P<0.05).@*CONCLUSIONS@#Hyperoxia exposure may lead to growth retardation and lung developmental retardation in preterm rats. Changes in the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats may be associated with the pathogenesis of hyperoxia-induced lung injury in preterm rats.


Subject(s)
Animals , Animals, Newborn , Catalytic Domain , Cesarean Section , Cysteine , Female , Glutamates , Heme Oxygenase-1 , Humans , Hyperoxia , Infant, Newborn , Lung , Pregnancy , Rats , Rats, Sprague-Dawley
9.
Article in English | WPRIM | ID: wpr-739224

ABSTRACT

The development of next generation sequencing (NGS) has led to marked advancement of our understanding of genetic events mediating the initiation and progression of thyroid cancers. The NGS studies have confirmed the previously reported high frequency of mutually-exclusive oncogenic alterations affecting BRAF and RAS proto-oncogenes in all stages of thyroid cancer. Initially identified by traditional sequencing approaches, the NGS studies also confirmed the acquisition of alterations that inactivate tumor protein p53 (TP53) and activate phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in advanced thyroid cancers. Novel alterations, such as those in telomerase reverse transcriptase (TERT) promoter and mating-type switching/sucrose non-fermenting (SWI/SNF) complex, are also likely to promote progression of the BRAF(V600E)-driven thyroid cancers. A number of genetically engineered mouse models (GEMM) of BRAF(V600E)-driven thyroid cancer have been developed to investigate thyroid tumorigenesis mediated by oncogenic BRAF and to explore the role of genetic alterations identified in the genomic analyses of advanced thyroid cancer to promote tumor progression. This review will discuss the various GEMMs that have been developed to investigate oncogenic BRAF(V600E)-driven thyroid cancers.


Subject(s)
Animals , Carcinogenesis , Catalytic Domain , Mice , Mice, Transgenic , Negotiating , Proto-Oncogene Proteins B-raf , Proto-Oncogenes , Telomerase , Thyroid Gland , Thyroid Neoplasms
10.
Laboratory Animal Research ; : 185-194, 2018.
Article in English | WPRIM | ID: wpr-718850

ABSTRACT

The different polymorphisms of the transcription factor 7-like 2 (TCF7L2) gene promote variances in diabetes susceptibility in humans. We investigated whether these genotypes also promote differences in diabetic susceptibility in commercial pigs. Growing pigs (Landrace, both sex, 50–60 kg) with the C/C (n=4) and T/T (n=5) TCF7L2 genotypes were identified and intravenously injected with streptozotocin (STZ, 40 mg/kg) twice in weekly intervals, then a high-energy diet was offered. Oral glucose tolerance tests, blood analyses and the homeostasis model assessment-insulin resistance (HOMA-IR) index calculations were performed. The animals were sacrificed at the end of 12 weeks of treatment to reveal the pancreas histomorphometry. The results showed that all of the treated pigs grew normally despite exhibiting hyperglycemia at two weeks after the induction. The glycemic level of the fasting or postprandial pigs gradually returned to normal. The fasting insulin concentration was significantly decreased for the T/T carriers but not for the C/C carriers, and the resulting HOMA-IR index was significantly increased for the C/C genotype, indicating that the models of insulin dependence and resistance were respectively developed by T/T and C/C carriers. The histopathological results illustrated a significant reduction in the pancreas mass and insulin active sites, which suggested increased damage. The results obtained here could not be compared with previous studies because the TCF7L2 background has not been reported. Growing pigs may be an excellent model for diabetic in children if the animals are genetically pre-selected.


Subject(s)
Animals , Catalytic Domain , Child , Diabetes Mellitus , Diet , Fasting , Genotype , Glucose Tolerance Test , Homeostasis , Humans , Hyperglycemia , Insulin , Pancreas , Streptozocin , Swine , Transcription Factors
11.
Article in English | WPRIM | ID: wpr-718588

ABSTRACT

BACKGROUND/OBJECTIVES: The honeysuckle berry (HB) contains ascorbic acid and phenolic components, especially anthocyanins, flavonoids, and low-molecular-weight phenolic acids. In order to examine the potential of HB as a hepatoprotective medicinal food, we evaluated the in vitro anti-oxidant and anti-inflammatory activities of Korean HB (HBK) and Chinese HB (HBC). MATERIALS/METHODS: Antioxidant and anti-inflammatory effects of the extracts were examined in HepG2 and RAW 264.7 cells, respectively. The anti-oxidant capacity was determined by DPPH, SOD, CAT, and ARE luciferase activities. The production of nitric oxide (NO) as an inflammatory marker was also evaluated. The Nrf2-mediated mRNA levels of heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase [quinone] 1 (Nqo1), and glutamate-cysteine ligase catalytic subunit (Gclc) were measured. The concentrations of HB extracts used were 3, 10, 30, 100, and 300 µg/mL. RESULTS: The radical scavenging activity of all HB extracts increased in a concentration-dependent manner (P < 0.01 or P < 0.05). SOD (P < 0.05) and CAT (P < 0.01) activities were increased by treatment with 300 µg/mL of each HB extract, when compared to those in the control. NO production was observed in cells pretreated with 100 or 300 µg/mL of HBC and HBK (P < 0.01). Treatment with 300 µg/mL of HBC significantly increased Nqo1 (P < 0.01) and Gclc (P < 0.05) mRNA levels compared to those in the control. Treatment with 300 µg/mL of HBK (P < 0.05) and HBC (P < 0.01) also significantly increased the HO-1 mRNA level compared to that in the control. CONCLUSIONS: Thus, the Korean and Chinese HBs were found to possess favorable in vitro anti-oxidant and anti-inflammatory activities. Nrf2 and its related anti-oxidant genes were associated with both anti-oxidant and anti-inflammatory activities in HB-treated cells. Further studies are needed to confirm these in vivo effects.


Subject(s)
Animals , Anthocyanins , Ascorbic Acid , Asian Continental Ancestry Group , Catalytic Domain , Cats , Flavonoids , Fruit , Glutamate-Cysteine Ligase , Heme Oxygenase-1 , Humans , In Vitro Techniques , Lonicera , Luciferases , Nitric Oxide , Oxidoreductases , Phenol , RNA, Messenger
12.
Mem. Inst. Oswaldo Cruz ; 113(8): e180098, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040602

ABSTRACT

β-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to β-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two β-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against β-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional β-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional β-lactamases are widespread in nature; these findings also raise concern that bifunctional β-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.


Subject(s)
beta-Lactamases/genetics , Catalytic Domain/genetics , Genomics , Environmental Microbiology , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/isolation & purification
13.
Article in English | WPRIM | ID: wpr-727934

ABSTRACT

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.


Subject(s)
Amino Acids , Catalytic Domain , Consensus , Hydrogen-Ion Concentration , Immunoprecipitation , Mutagenesis, Site-Directed , Protein Phosphatase 1 , Signal Transduction
14.
Article in English | WPRIM | ID: wpr-741612

ABSTRACT

The present study was undertaken to investigate the isolated compounds from the stem bark of Garcinia atroviridis as potential cholinesterase inhibitors and the ligand-enzyme interactions of selected bioactive compounds in silico. The in vitro cholinesterase results showed that quercetin (3) was the most active AChE inhibitor (12.65 ± 1.57 µg/ml) while garcinexanthone G (6) was the most active BChE inhibitor (18.86 ± 2.41 µg/ml). It is noteworthy to note that compound 6 was a selective inhibitor with the selectivity index of 11.82. Molecular insight from docking interaction further substantiate that orientation of compound 6 in the catalytic site which enhanced its binding affinity as compared to other xanthones. The nature of protein-ligand interactions of compound 6 is mainly hydrogen bonding, and the hydroxyl group of compound 6 at C-10 is vital in BChE inhibition activity. Therefore, compound 6 is a notable lead for further drug design and development of BChE selective inhibitor.


Subject(s)
Butyrylcholinesterase , Catalytic Domain , Cholinesterase Inhibitors , Cholinesterases , Computer Simulation , Drug Design , Garcinia , Hydrogen Bonding , In Vitro Techniques , Quercetin , Xanthones
15.
Article in English | WPRIM | ID: wpr-648047

ABSTRACT

Dysfunction or loss of blood vessel causes several ischemic diseases. Although endothelial progenitor cells (EPCs) are a promising source for cell-based therapy, ischemia-induced pathophysiological condition limits the recovery rate by causing drastic cell death. To overcome this issue, we attempted to develop a cell-targeted peptide delivery and priming system to enhance EPCbased neovascularization using an engineered M13 bacteriophage harboring nanofibrous tubes displaying ∼ 2700 multiple functional motifs. The M13 nanofiber was modified by displaying RGD, which is an integrin-docking peptide, on the minor coat protein, and bymutilayering SDKPmotifs,which are the key active sites for thymosin b4, on themajor coat protein. The engineered M13 nanofiber dramatically enhanced ischemic neovascularization by activating intracellular and extracellular processes such as proliferation, migration, and tube formation in the EPCs. Furthermore, transplantation of the primed EPCs with the M13 nanofiber harboring RGD and SDKP facilitated functional recovery and neovascularization in a murine hindlimb ischemia model. Overall, this study demonstrates the effectiveness of theM13 nanofiber-based novel peptide deliveryandprimingstrategy inpromotingEPC bioactivity and neovessel regeneration. To our knowledge, this is first report onM13 nanofibers harboring dual functional motifs, the use of which might be a novel strategy for stem and progenitor cell therapy against cardiovascular ischemic diseases.


Subject(s)
Animals , Bacteriophages , Blood Vessels , Catalytic Domain , Cell Death , Endothelial Progenitor Cells , Hindlimb , Ischemia , Nanofibers , Regeneration , Stem Cells , Thymosin
16.
Cell Journal [Yakhteh]. 2017; 19 (Supp. 1): 37-43
in English | IMEMR | ID: emr-189338

ABSTRACT

Objective: There is a positive correlation between higher serum phytoestrogen concentrations and lower risk of breast cancer. The activation of telomerase is crucial for the growth of cancer cells; therefore, the aim of this study was to examine the effects of enterolactone [ENL] and enterodiol [END] on this enzyme


Materials and Methods: In this experimental study, we performed the viability assay to determine the effects of different concentrations of ENL and END on cell viability, and the effective concentrations of these two compounds on cell growth. We used western blot analysis to evaluate human telomerase reverse transcriptase catalytic subunit [hTERT] expression and polymerase chain reaction [PCR]-ELISA based on the telomeric repeat amplification protocol [TRAP] assay for telomerase activity


Results: Both ENL and END, at 100 micro M concentrations, significantly [P<0.05] reduced cell viability. However, only the 100 micro M concentration of ENL significantly [P<0.05] decreased hTERT protein levels and telomerase activity. Lower concentrations of ENL did not have any significant effects on telomerase activity and hTERT protein levels


Conclusion: High concentration of ENL decreased the viability of MCF-7 breast cancer cells and inhibited the expression and activity of telomerase in these cells. Although END could reduce breast cancer cell viability, it did not have any effect on telomerase expression and activity


Subject(s)
Humans , Female , Lignans , Telomerase/drug effects , Catalytic Domain , Breast Neoplasms
17.
Article in English | WPRIM | ID: wpr-147982

ABSTRACT

Previously, we demonstrated that galangin (3,5,7-trihydroxyflavone) protects human keratinocytes against ultraviolet B (UVB)-induced oxidative damage. In this study, we investigated the effect of galangin on induction of antioxidant enzymes involved in synthesis of reduced glutathione (GSH), and investigated the associated upstream signaling cascades. By activating nuclear factor-erythroid 2-related factor (Nrf2), galangin treatment significantly increased expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). This activation of Nrf2 depended on extracellular signal-regulated kinases (ERKs) and protein kinase B (AKT) signaling. Inhibition of GSH in galangin-treated cells attenuated the protective effect of galangin against the deleterious effects of UVB. Our results reveal that galangin protects human keratinocytes by activating ERK/AKT-Nrf2, leading to elevated expression of GSH-synthesizing enzymes.


Subject(s)
Catalytic Domain , Extracellular Signal-Regulated MAP Kinases , Glutamate-Cysteine Ligase , Glutathione Synthase , Glutathione , Humans , Keratinocytes , Proto-Oncogene Proteins c-akt
18.
Article in English | WPRIM | ID: wpr-158424

ABSTRACT

Mice deficient in the toll-like receptor (TLR) or the myeloid differentiation factor 88 (MyD88) are resistant to acute liver failure (ALF) with sudden death of hepatocytes. Chalcone derivatives from medicinal plants protect from hepatic damages including ALF, but their mechanisms remain to be clarified. Here, we focused on molecular basis of piperidylmethyloxychalcone (PMOC) in the treatment of TLR/MyD88-associated ALF. C57BL/6J mice were sensitized with D-galactosamine (GalN) and challenged with Escherichia coli lipopolysaccharide (LPS, TLR4 agonist) or oligodeoxynucleotide containing unmethylated CpG motif (CpG ODN, TLR9 agonist) for induction of ALF. Post treatment with PMOC sequentially ameliorated hepatic inflammation, apoptosis of hepatocytes, severe liver injury and shock-mediated death in ALF-induced mice. As a mechanism, PMOC inhibited the catalytic activity of TGF-β-activated kinase 1 (TAK1) in a competitive manner with respect to ATP, displaced fluorescent ATP probe from the complex with TAK1, and docked at the ATP-binding active site on the crystal structure of TAK1. Moreover, PMOC inhibited TAK1 auto-phosphorylation, which is an axis in the activating pathways of nuclear factor-κB (NF-κB) or activating protein 1 (AP1), in the liver with ALF in vivo or in primary liver cells stimulated with TLR agonists in vitro. PMOC consequently suppressed TAK1-inducible NF-κB or AP1 activity in the inflammatory injury, an early pathogenesis leading to ALF. The results suggested that PMOC could contribute to the treatment of TLR/MyD88-associated ALF with the ATP-binding site of TAK1 as a potential therapeutic target.


Subject(s)
Adenosine Triphosphate , Animals , Apoptosis , Catalytic Domain , Chalcone , Death, Sudden , Escherichia coli , Hepatocytes , In Vitro Techniques , Inflammation , Liver , Liver Failure, Acute , Mice , Myeloid Differentiation Factor 88 , Phosphotransferases , Plants, Medicinal , Toll-Like Receptors
19.
Annals of Dermatology ; : 20-25, 2017.
Article in English | WPRIM | ID: wpr-37418

ABSTRACT

BACKGROUND: Acne vulgaris has been linked to the Western diet. Hyperglycemic diet increases insulin and insulin-like growth factor (IGF)-1. Deeper insights into IGF-1-mediated signal pathway are critical importance to understand the impact of Western diet. OBJECTIVE: We investigated the effect of IGF-1 on the expression of inflammatory biomarkers and sebum production in cultured sebocytes. METHODS: Polymerase chain reaction and enzyme-linked immunosorbent assay were performed to measure changes in the expression of inflammatory biomarkers including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IGF1R, IGFBP2, sterol response element-binding protein (SREBP), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PI3KCA) after the treatment of cultured sebocytes with 10⁻⁷ M or 10⁻⁵ M IGF-1. Sebum production was evaluated after the treatment of cultured sebocytes with 10⁻⁷ M or 10⁻⁵ M IGF-1 using lipid analysis. RESULTS: The expression levels of IL-1β, IL-6, IL-8, and TNF-α in cultured sebocytes after treatment with 10⁻⁷ M or 10⁻⁵ M IGF-1 were increased. Increased gene expression levels of NF-κB in cultured sebocytes were also shown after 10⁻⁷ M or 10⁻⁵ M IGF-1 treatments. Gene expression of these inflammatory biomarkers was decreased after 10⁻⁷ M or 10⁻⁵ M IGF-1 treatment in the presence of 100 nM NF-κB inhibitor. Treatment with 10⁻⁷ M or 10⁻⁵ M IGF-1 increased the gene expression levels of IGF1R, IGFBP2, SREBP and PI3KCA in cultured sebocytes. Sebum production from cultured sebocytes treated with 10⁻⁷ M or 10⁻⁵ M IGF-1 was also increased. CONCLUSION: It is suggestive that IGF-1 might be involved in the pathogenesis of acne by increasing both expression of inflammatory biomarkers and also sebum production in sebocytes.


Subject(s)
Acne Vulgaris , B-Lymphocytes , Biomarkers , Catalytic Domain , Diet , Diet, Western , Enzyme-Linked Immunosorbent Assay , Gene Expression , Insulin , Insulin-Like Growth Factor I , Interleukin-6 , Interleukin-8 , Interleukins , Polymerase Chain Reaction , Sebum , Signal Transduction , Tumor Necrosis Factor-alpha
20.
Article in English | WPRIM | ID: wpr-69358

ABSTRACT

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.


Subject(s)
Amino Acids , Catalytic Domain , DNA, Complementary , Echinostoma , Endoribonucleases , Escherichia coli , Intestine, Small , Oligonucleotides, Antisense , Parasites , Ribonuclease H , Ribonucleases , RNA , Trematoda
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