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1.
Life Sci Alliance ; 6(4)2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36669792

RESUMO

Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreção Tipo IV , Plasmídeos/genética , DNA , Esterases/genética
2.
Bioconjug Chem ; 34(1): 248-256, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36621834

RESUMO

Enzyme-responsive drug delivery systems have drawn much attention in the field of cancer theranostics due to their high sensitivity and substrate specificity under mild conditions. In this study, an amphiphilic polymer T1 is reported, which contains a tetraphenylethene unit and a poly(ethylene glycol) chain linked by an esterase-responsive phenolic ester bond. In aqueous solution, T1 formed stable micelles via self-assembly, which showed an aggregation-induced emission enhancement of 32-fold at 532 nm and a critical micelle concentration of 0.53 µM as well as esterase-responsive activity. The hydrophobic drug doxorubicin (DOX) was efficiently encapsulated into the micelles with a drug loading of 21%. In the presence of the esterase, the selective decomposition of drug-loaded T1 micelles was observed, and DOX was subsequently released with a half-life of 5 h. In vitro antitumor studies showed that T1@DOX micelles exhibited good therapeutic effects on HeLa cells, while normal cells remained mostly intact. In vivo anticancer experiments revealed that T1@DOX micelles indeed suppressed tumor growth and had reduced side effects compared to DOX·HCl. The present work showed the potential clinical application of esterase-responsive drug delivery in cancer therapy.


Assuntos
Micelas , Polietilenoglicóis , Humanos , Polietilenoglicóis/química , Células HeLa , Esterases , Portadores de Fármacos/química , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Polímeros/química , Sistemas de Liberação de Medicamentos , Concentração de Íons de Hidrogênio
3.
Food Chem ; 407: 135154, 2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-36502727

RESUMO

Pichia kudriavzevii was one of the important aroma-producing fungi in the solid-state fermentation of Baijiu, and immobilization was an effective strategy for improving microbial performance. Herein, P. kudriavzevii cells were immobilized in a gel network that crosslinked by chitosan and sodium alginate to form sodium alginate/chitosan-P. kudriavzevii microspheres (SA/CS-PMs). Their structural characteristics and formation processes were characterized by SEM and FT-IR. The effect of synthesis conditions on the performance of microspheres were determined by single-factor experiments. Under the optimal conditions, the SA/CS-PMs could increase the amylase activity of the fermentation broth by 57.18%, the esterase activity by 66.13%, the content of ester by 67.04%, and could be reused at least three times. Further research results indicated that the content of ester could be increased significantly in Baijiu solid-state fermentation with the SA/CS-PMs. In conclusion, the SA/CS-PMs could improve the ester production ability of P. kudriavzevii by increasing the esterase activity, which was a valuable exploration of directional biosynthesis and a feasible strategy to improve solid-state fermentation quality.


Assuntos
Quitosana , Fermentação , Alginatos , Ésteres , Microesferas , Espectroscopia de Infravermelho com Transformada de Fourier , Pichia , Esterases
4.
Pestic Biochem Physiol ; 188: 105229, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36464331

RESUMO

Culex tritaeniorhynchus is a major Japanese encephalitis virus vector distributed in Southeast Asia and surrounding countries. The aim of the present study is to investigate insecticide resistance status among 10 Cx. tritaeniorhynchus populations of the Mediterranean region of Turkey. Bioassay results indicated that all of the populations were resistant or at least possibly resistant to 1,1'-(2,2,2-Trichloroethane-1,1-diyl) bis (4-chlorobenzene) (DDT) (4%), [(dimethoxyphosphorothioyl) sulfanyl] butanedioate, Diethyl (malathion) (5%), and 2-[(Propan-2-yl) oxy] phenyl methylcarbamate (propoxur) (0,1%). Whereas, some of the populations were still susceptible to 3-Phenoxybenzyl (1RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate (permethrin) (0,75%) and (S)-Cyano (3-phenoxy phenyl) methyl (1R,3R)-3-(2,2-dibromoethen-1-yl)-2,2-dimethylcyclopropane-1-carboxylate (deltamethrin) (0,05%). Biochemical analysis results showed altered alpha esterase, beta esterase, para-nitrophenyl acetate (PNPA), and glutathione-s-transferase (GST) levels in some populations while all of the populations had increased oxidase levels except for the Yumurtalik population. Additionally, all of the populations had sensitive acetylcholinesterase (AChE) levels similar to the control group except for the Erzin population. Correlation analysis showed a significant correlation between mortality rates for deltamethrin and alpha esterase, beta esterase, PNPA, and GST levels while mortality rates for permethrin were significantly correlated with GST levels. An allele-specific polymerase chain reaction (AS-PCR) detected high L1014F allele frequency in the populations. Overall results indicate the urgent need for monitoring and mapping of insecticide resistance in Cx. tritaeniorhynchus populations of the study area for effective vector control management.


Assuntos
Culex , Animais , Culex/genética , Permetrina , Acetilcolinesterase/genética , Mosquitos Vetores , Mutação , Esterases , Glutationa Transferase
5.
Pestic Biochem Physiol ; 188: 105271, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36464376

RESUMO

The acute toxicity of chlorpyrifos and chlorpyrifos-oxon (organophosphorothioate insecticides) was examined alone and in combination with atrazine (triazine herbicide) and alachlor (chloroacetanilide herbicide) to honey bees (Apis mellifera). Atrazine and alachlor were observed to not be acutely toxic to bees at doses up to 10 and 4 µg per bee, respectively. However, atrazine significantly increased chlorpyrifos toxicity by 3-fold while reducing chlorpyrifos-oxon toxicity by 1.8-fold. These changes in toxicity are correlated with significant 1.3- and 1.2-fold inhibition of acetylcholinesterase (AChE) activity in bees exposed to chlorpyrifos and chlorpyrifos-oxon, respectively. Atrazine significantly increased cytochrome P450, general esterase, and glutathione S-transferase (GST) activities by 1.5-, 1.2-, and 1.2- fold respectively, in bees compared to untreated individuals. Alachlor increased chlorpyrifos toxicity by 2.5-fold but did not affect the toxicity of chlorpyrifos-oxon. Exposure to alachlor and chlorpyrifos did not affect AChE compared to chlorpyrifos alone. However, exposure to chlorpyrifos-oxon and alachlor significantly increased acetylcholinesterase (AChE) activity by 1.4-fold. GST activity, but not P450 or general esterases, was significantly increased in bees exposed to alachlor. These data provide evidence that triazine and chloroacetanilide herbicide exposure alters detoxification enzyme activity and, in turn, alters the sensitivity of bees to organophosphorothioate insecticides. Importantly, these data can be used to guide future studies aiming to test safety profiles for pollinators and expand regulatory framework required for pesticide registration.


Assuntos
Atrazina , Clorpirifos , Inseticidas , Abelhas , Animais , Atrazina/toxicidade , Clorpirifos/toxicidade , Acetilcolinesterase , Inseticidas/toxicidade , Triazinas , Esterases
6.
Viruses ; 14(12)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36560743

RESUMO

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: 112NVENLEEL119, 117EELRSLFS124, and 170PIQDAQ175. Epitope 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection.


Assuntos
Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Humanos , Animais , Camundongos , Epitopos , Vírus da Influenza A Subtipo H9N2/genética , Hemaglutininas , Esterases , Anticorpos Monoclonais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Anticorpos Antivirais , Galinhas
7.
Anal Chem ; 94(51): 17922-17929, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36515388

RESUMO

Chymotrypsin, an extensively known proteolytic enzyme, plays a substantial role in maintaining physiological functions, including protein digestion, immune response, and tissue repair. To date, intense attention has been focused on the invention of efficient and sensitive chemical tools for chymotrypsin activity measurement. Among them, the "nonpeptide"-based chymotrypsin probe design strategy utilizing the esterase activity of chymotrypsin has been well-developed due to its low cost and high atom-economy feature. However, the ester-bond-based nature of these probes make them possibly vulnerable to esterases and active chemicals. These defects strictly restricted the application of the previously reported probes, especially for imaging in living systems. Therefore, to acquire fluorogenic probes with sufficient stability and specificity for chymotrypsin sensing in a complicated biological environment, a more stable skeleton for nonpeptide-based chymotrypsin probe construction is urgently needed. Herein, a novel nonpeptide-based fluorogenic probe for specific chymotrypsin activity sensing was designed and synthesized by the substitution of an ester-based linker with a heptafluorobutylamide moiety. The acquired probe, named TMBIHF, showed high selectivity toward various enzymes and reactive chemicals, while it retained high sensitivity and catalytic efficiency toward chymotrypsin. Moreover, TMBIHF was successfully applied for monitoring chymotrypsin activity and pancreas development in live zebrafish, specific sensing of exogenous and endogenous chymotrypsin in nude mice, and visualizing chymotrypsin-like activity-dependent cellular apoptosis, thus providing an alternative and reliable way for chymotrypsin-targeted biosensor or prodrug construction.


Assuntos
Quimotripsina , Peixe-Zebra , Animais , Camundongos , Quimotripsina/metabolismo , Camundongos Nus , Peixe-Zebra/metabolismo , Esterases/metabolismo , Corantes Fluorescentes
8.
Biopharm Drug Dispos ; 43(6): 247-254, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36519186

RESUMO

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04 ± 1.99 µM, 434.60 ± 12.46 nM/min/mg protein, and 21.69 ± 0.28 ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24 ± 0.04 mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.


Assuntos
Hidrolases , Proteína de Ligação a Fosfatidiletanolamina , Humanos , Cloridrato de Prasugrel/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Clopidogrel , Hidrolases/metabolismo , Esterases/metabolismo , Intestinos
9.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499468

RESUMO

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity.


Assuntos
Aminoácidos , Lipase , Filogenia , Lipase/metabolismo , Esterases/metabolismo , Transferases
10.
Int J Mol Sci ; 23(23)2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36499718

RESUMO

Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth's biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes' quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.


Assuntos
Esterases , Lipase , Esterases/metabolismo , Lipase/metabolismo , Temperatura Baixa
11.
Chem Commun (Camb) ; 58(96): 13329-13332, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36373630

RESUMO

A tumor-targeting therapy strategy is urgently needed to increase the accumulation of drugs in tumors and reduce the side effects in normal tissues. Herein, we developed an esterase-activatable curcumin prodrug Cur-RGD for tumor-targeting therapy. Armed with the tumor-targeting RGD peptide and in situ esterase-triggered drug release, this prodrug Cur-RGD can efficiently improve the therapeutic effect of curcumin in tumors.


Assuntos
Antineoplásicos , Curcumina , Nanopartículas , Neoplasias , Pró-Fármacos , Humanos , Curcumina/farmacologia , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Esterases , Oligopeptídeos , Neoplasias/tratamento farmacológico , Portadores de Fármacos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral
12.
Dent Mater ; 38(12): 2041-2051, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36428113

RESUMO

OBJECTIVE: To investigate the effects of salivary esterases on biostability of collagen treated by galloylated polyphenols. METHODS: Human dentin was microtomed into 6-µm-thick films, which were demineralized and treated for 60 s using solutions containing 0.6% and 2% of one of the crosslinkers: tannic acid (TAC), epigallocatechin gallate (EGCG), epigallocatechin (EGC), and N-[3-dimethylaminopropyl]-N'-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and for 1 h using EDC/NHS. Half of the treated and untreated (control) films were subjected to human saliva incubation. Collagen biostability was assessed via exogenous protease biodegradation by weight loss and hydroxyproline release, and endogenous MMPs by in situ zymography. The degradation products of galloylated polyphenols (TAC and EGCG) by saliva were monitored using proton nuclear magnetic resonance (1H NMR) and gel permeation chromatography (GPC). The esterase activity of saliva induced by the crosslinkers was also assessed. RESULTS: Collagen films treated with TAC and EGCG exhibited significantly improved biostability (p < 0.05); however, the enhanced biostability was severely reduced after saliva incubation (p < 0.001). For EDC/NHS treated collagen, saliva incubation showed negligible effect on the biostability. 1H NMR studies confirmed the esterase-catalyzed hydrolysis of the galloyl. GPC measurements showed decreased molecular weight of TAC in saliva indicating its chemical degradation. Both TAC and EGCG showed much higher esterase activity than other treatment groups. SIGNIFICANCE: The galloyl group plays important role in collagen crosslinking, inducing higher biostability. However, galloylated polyphenols crosslinked on collagen are highly susceptible to metabolism of human saliva by salivary esterase, dramatically compromising the enhanced biostability.


Assuntos
Colágeno , Polifenóis , Humanos , Polifenóis/farmacologia , Peso Molecular , Esterases , Dentina
13.
Int J Mol Sci ; 23(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36362119

RESUMO

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1-15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH1AB1C, efficiently hydrolysed (azo)casein at pH 6.5-8.0 and 60-70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express.


Assuntos
Esterases , Peptídeo Hidrolases , Esterases/metabolismo , Peptídeo Hidrolases/metabolismo , Endopeptidases/metabolismo , Domínio Catalítico/genética , Ésteres/metabolismo , Concentração de Íons de Hidrogênio
14.
BMC Complement Med Ther ; 22(1): 259, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195907

RESUMO

BACKGROUND: The leaf of Ceylon cinnamon (true cinnamon) is traditionally claimed for a variety of health benefits. However, reported scientific information is scanty and needs urgent attention for value addition. METHODS: Ethanolic (95%) and Dichloromethane:Methanol (DM, 1:1 v/v) leaf extracts of Ceylon cinnamon were evaluated for a range of medically important bioactivities namely anti-inflammatory [nitric oxide scavenging activity (NOSA), superoxide scavenging activity (SCA), COX1 and COX2 inhibition], growth inhibition & cytotoxicity against MCF7, HePG2 and AN3CA carcinoma cell lines, glutathionase-S-transferase (GST) inhibition and antilipidemic (anti-HMG-CoA reductase, anti-lipase, anti-cholesterol esterase, and cholesterol micellization inhibition) properties in vitro (n = 3). Further, a range of bioactive compounds in both leaf extracts was also quantified (n = 3). RESULTS: Both leaf extracts had all the investigated bioactive compounds and possessed moderately potent bioactivities compared to the reference drugs used in the study. Ethanolic leaf extract (ELE) exhibited the highest activities (IC50: µg/mL) for NOSA (40.26 ± 0.52), SCA (696.24 ± 40.02), cholesterol esterase inhibition (110.19 ± 1.55), cholesterol micellization inhibition (616.69 ± 7.09), GST inhibition (403.78 ± 2.70) and growth inhibition (GI50: 144.84 ± 1.59-269.00 ± 0.51) & cytotoxicity (LC50: 355.44 ± 9.38-717.71 ± 23.69) against studied cancer cell lines. In contrast, COX1 & COX2 (IC50: 6.62 ± 0.85 and 44.91 ± 3.06 µg/mL) and HMG-CoA reductase & lipase inhibitory activities (36.72 ± 4.74 and 19.71 ± 0.97% inhibition at 200 and 600 µg/mL) were highest in DM extract. ELE also showed the highest quantities (0.81 ± 0.06-104.38 ± 1.79) of tested compounds (mg/g extract) where eugenol was the highest and gallic acid was the lowest among quantified. CONCLUSION: Both leaf extracts of Ceylon cinnamon had all the tested bioactive compounds and possess all the investigated bioactivities. This is the 1st study to report all the investigated bioactivities of the leaf of Ceylon Cinnamon.


Assuntos
Cinnamomum zeylanicum , Óleos Voláteis , Anti-Inflamatórios/farmacologia , Coenzima A , Ciclo-Oxigenase 2 , Esterases , Eugenol , Ácido Gálico , Metanol , Cloreto de Metileno , Óxido Nítrico , Oxirredutases , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Superóxidos , Transferases
15.
Biomater Sci ; 10(22): 6517-6524, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36190132

RESUMO

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear etiology and insufficient therapeutic efficacy. The development of specific, effective and safe IBD treatment drugs is of great clinical significance. Curcumin (Cur) is a good candidate to prevent and manage inflammatory diseases (such as IBD) due to its antioxidant and anti-inflammatory effects with safety profile. However, its poor aqueous solubility and instability under physiological conditions greatly limit its therapeutic efficacy. Herein, we exploited a Cur precursor Cur-FFEYp to locally deliver and slowly release Cur at inflamed regions for treatment of IBD by a sequential self-assembly and disassembly strategy. The much higher catalytic efficiency of alkaline phosphatase (ALP) than esterase towards Cur-FFEYp validated the sequential ALP-induced self-assembly with the formation of Cur hydrogel and esterase-guided disassembly with the slow release of Cur. In cell and animal experiments, Cur-FFEYp can effectively enhance the anti-inflammatory effect of Cur on inflammatory macrophages and significantly alleviate two types of IBD. We envision that by using other biomarkers to conduct the sequential self-assembly and disassembly processes and replacing other drugs, our smart strategy could be easily adjusted for the treatment of more diseases or cancers.


Assuntos
Curcumina , Doenças Inflamatórias Intestinais , Animais , Curcumina/farmacologia , Curcumina/uso terapêutico , Hidrogéis , Doenças Inflamatórias Intestinais/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Esterases
16.
Int J Biochem Cell Biol ; 153: 106316, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36280040

RESUMO

Notum, which belongs to the α/ß hydrolase family, is a deacylated extracellular protein that regulates the Wnt signaling pathway. Studies have found that Notum participates in the progression of colorectal cancer and hepatocellular carcinoma, but its role in oral squamous cell carcinoma (OSCC) is currently unclear. This study aimed to explore the role of Notum in regulating OSCC and further reveal the underlying mechanisms. Various approaches including bioinformatics analysis, enzyme-linked immunosorbent assay and immunohistochemical staining were used to detect the expression of Notum in OSCC cells and tissues. Cell counting kit-8 assay, clone formation assay, wound healing assay, transwell assay and in-gel zymography assay were explored to evaluate the regulation of Notum in OSCC proliferation and migration. Hoechst 33342/PI assay, cell immunofluorescence, flow cytometry and in vivo tumorigenesis experiment were applied for OSCC apoptosis. Real-time quantitative polymerase chain reaction analysis was performed for mRNA level while western blotting was conducted to detect protein expression. The results showed that Notum was highly expressed in OSCC tissues and cells, and Notum promoted the proliferation and migration of OSCC cells while it inhibited their apoptosis. Furthermore, signaling pathway analysis showed that Notum led to potential pro-survival of OSCC through crosstalk between sonic hedgehog (Shh) and Wnt/ß-catenin signaling via phosphorylation of glycogen synthase kinase-3 beta. These results will help to elucidate the mechanism and also provide new ideas for targeted treatment of OSCC.


Assuntos
Esterases , Neoplasias Bucais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Esterases/genética , Esterases/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Via de Sinalização Wnt/genética
17.
Sci Rep ; 12(1): 17712, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271284

RESUMO

Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity-response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae.


Assuntos
Clorpirifos , Praguicidas , Animais , Azinfos-Metil , Clorpirifos/metabolismo , Praguicidas/farmacologia , Larva , Transcriptoma , Compostos Organofosforados/farmacologia , Antioxidantes/metabolismo , Bufo arenarum/metabolismo , Esterases/metabolismo , Poliaminas/metabolismo , Fatores de Transcrição/metabolismo
18.
Chem Biol Interact ; 368: 110228, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36283465

RESUMO

Beclomethasone dipropionate (BDP) is an inhaled glucocorticoid used for maintenance treatment of asthma in adults and children. BDP is a prodrug activated in lung when hydrolyzed to its major active metabolite beclomethasone-17-monopropionate (17-BMP), which can be further deactivated to beclomethasone (BOH). The specific hydrolases contributing to these processes have not been identified which warrants an investigation to enable a better assessment of the drug-drug interaction (DDI) liability and a better management of drug efficacy and systemic toxicity. In the present study, the pulmonary metabolism of BDP was investigated using both human lung S9 (HLuS9) and recombinant carboxylesterase 1 (CES1) S9. By employing the relative activity approach, we tested the hypothesis of CES1 being the major enzyme involved. Assessment of other hydrolases were conducted in an assay with selective esterase inhibitors. In addition, the DDI potentials between BDP and Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were evaluated due to the increasing use of inhaled cannabis both recreationally and medically. The mechanism of DDI was conducted in an in vitro time-dependent inhibition assay, and further interpreted utilizing a proposed model. In HLuS9, BDP was efficiently metabolized almost completely to 17-BMP, which was then converted to BOH at a much lower rate. CES1 was found as a minor contributor accounting for only 1.4% of BDP metabolism in HLuS9, while arylacetamide deacetylase might be the main enzyme involved. Both THC and CBD inhibited the HLuS9 mediated BDP hydrolysis in a reversible manner, with reported IC50 values estimated as 8.98 and 36.8 µM, respectively. Our proposed model suggested a moderately decreased 17-BMP exposure in lung by concomitant THC from a cannabis cigarette, while the effects from orally administered CBD was expected to be of no clinical relevance.


Assuntos
Beclometasona , Canabidiol , Cannabis , Fumar Maconha , Adulto , Criança , Humanos , Administração por Inalação , Beclometasona/administração & dosagem , Beclometasona/uso terapêutico , Cannabis/efeitos adversos , Dronabinol , Esterases , Glucocorticoides , Fumar Maconha/efeitos adversos
19.
Mikrochim Acta ; 189(11): 413, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216987

RESUMO

A self-enhanced electrochemical luminescence (ECL) composite material g-C3N4-CdTe QDs was prepared. The combination of g-C3N4 and CdTe QDs can amplify the ECL signal and improve the stability. Based on this discovery, g-C3N4-CdTe QDs and acetylcholine esterase (AChE) were used to construct an ECL sensor for organophosphorus pesticides (OP) detection. The sensor showed a strong initial ECL signal in PBS containing S2O82-. It is because that g-C3N4 not only acts as a co-reaction promoter to amplify the ECL signal of the CdTe QDs/S2O82- system but also acts as a carrier with large specific surface area to adsorb more CdTe QDs and improve the sensitivity of the sensor. The reaction of AChE and acetylthiocholine (ATCl) was hindered by organophosphorus pesticides (OPs). The ECL signal was enhanced by the addition of OPs, and a linear relationship was displayed between the increasing value and the concentration of malathion. A good linear range from 2.52 × 10-13 to 2.52 × 10-8 mol L-1 was obtained and the limit of detection was 8.4 × 10-14 mol L-1 under optimized experimental conditions. The results indicated that the sensor had promising applications for the detection of OPs in vegetable samples.


Assuntos
Técnicas Biossensoriais , Compostos de Cádmio , Nanoestruturas , Praguicidas , Pontos Quânticos , Acetilcolina , Acetiltiocolina , Técnicas Biossensoriais/métodos , Esterases , Malation , Compostos Organofosforados , Telúrio
20.
J Agric Food Chem ; 70(41): 13349-13357, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36205442

RESUMO

Corn bran is an abundant coprocessing stream of corn-starch processing, rich in highly substituted, diferuloyl-cross-linked glucurono-arabinoxylan. The diferuloyl cross-links make the glucurono-arabinoxylan recalcitrant to enzymatic conversion and constitute a hindrance for designing selective enzymatic upgrading of corn glucurono-arabinoxylan. Here, we show that two bacterial feruloyl esterases, wtsFae1A and wtsFae1B, each having a carbohydrate-binding module of family 48, are capable of cleaving the ester bonds of the cross-linkages and releasing 5-5', 8-5', 8-5' benzofuran, and 8-O-4' diferulate from soluble and insoluble corn bran glucurono-arabinoxylan. All four diferulic acids were released at similar efficiency, indicating nondiscriminatory enzymatic selectivity for the esterified dimer linkages, the only exception being that wtsFae1B had a surprisingly high propensity for releasing the dimers, especially 8-5' benzofuran diferulate, indicating a potential, unique catalytic selectivity. The data provide evidence of direct enzymatic release of diferulic acids from corn bran by newly discovered feruloyl esterases, i.e., a new enzyme activity. The findings yield new insight and create new opportunities for enzymatic opening of diferuloyl cross-linkages to pave the way for upgrading of recalcitrant arabinoxylans.


Assuntos
Benzofuranos , Zea mays , Zea mays/química , Hidrolases de Éster Carboxílico/química , Xilanos/química , Ácidos Cumáricos/química , Fibras na Dieta , Ésteres , Amido , Esterases
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