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1.
Biochemistry ; 62(2): 535-542, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36598875

RESUMO

Kazal inhibitors hold high potential as scaffolds for therapeutic molecules, taking advantage of the easily exchangeable canonical binding loop. Different Kazal inhibitor backbones have been suggested to be therapeutically useful, but the impact of different Kazal-like scaffolds on binding properties is still largely unknown. Here, we identified trypsin-targeting human serine protease inhibitor Kazal type 1 (SPINK1) homologues in different mammalian species that cluster in two P2-P1 combinations, implying the coevolution of these residues. We generated loop exchange variants of human SPINK1 for comparison with Kazal inhibitors from related species. Using comprehensive biophysical characterization of the inhibitor-enzyme interactions, we found not only affinity but also pH resistance to be highly backbone-dependent. Differences are mostly observed in complex stability, which varies by over one order of magnitude. We provide clear evidence for high backbone dependency within the Kazal family. Hence, when designing Kazal inhibitor-based therapeutic molecules, testing different backbones after optimizing the canonical binding loop can be beneficial and may result in increased affinity, complex stability, specificity, and pH resistance.


Assuntos
Inibidor da Tripsina Pancreática de Kazal , Animais , Humanos , Mamíferos , Tripsina/química , Inibidor da Tripsina Pancreática de Kazal/química
2.
Br J Dermatol ; 188(1): 100-111, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36689511

RESUMO

BACKGROUND: Mendelian disorders of cornification (MeDOC) are a group of heterogeneous genodermatoses with different genetic bases. The pathogenesis of a substantial group of MeDOC remains to be elucidated. OBJECTIVES: To identify a new causative gene and the pathogenesis of a previously undescribed autosomal-dominant cornification disorder. METHODS: Whole-exome sequencing was performed in three families with the novel cornification disorder to identify the disease-causing variants. As the variants were located around the signal peptide (SP) cleavage site of a kallikrein-related peptidase, SP cleavage, subcellular localization and extracellular secretion of the variants were evaluated in eukaryotic overexpression systems by Western blotting or immunocytochemistry. Then the trypsin-like and chymotrypsin-like proteolytic activity of the peptidase and degradation of its catalytic substrate were assayed using the patients' stratum corneum (SC) samples. The morphology of the lamellar bodies and corneodesmosomes (CDs) in the patients' SC was ultrastructurally examined. A mouse model harbouring the equivalent variant was constructed and evaluated histologically. RESULTS: We identified two heterozygous variants affecting Gly50 in kallikrein-related peptidase (KLK)11 in a familial case and two sporadic cases with the new disorder, which is characterized by early-onset ichthyosiform erythroderma or erythrokeratoderma. KLK11 belongs to the family of kallikrein-related peptidases participating in skin desquamation by decomposing CDs, a process essential for shedding of the SC. In vitro experiments demonstrated that the variants perturbed the SP cleavage of KLK11, leading to subcellular mislocalization and impaired extracellular secretion of the KLK11 Gly50Glu variant. Both trypsin-like and chymotrypsin-like proteolytic activities were significantly decreased in the patients' SC samples. Reduced proteolysis of desmoglein 1 and delayed degeneration of CDs were detected in patients' SC, indicating delayed skin desquamation. Consistently, the patients showed a thickened, dense SC, indicating abnormal skin desquamation. Mice harbouring the homozygous c.131G>A (p.Gly44Glu) Klk11 variant, which is equivalent to KLK11 c.149G>A (p.Gly50Glu) in humans, exhibited hyperkeratosis and abnormal desquamation, partially recapitulating the phenotype. CONCLUSIONS: We provide evidence that variants at Gly50 affecting the SP cleavage of KLK11 cause a new autosomal-dominant cornification disorder with abnormal desquamation. Our findings highlight the essential role of KLKs in maintaining homeostasis of skin keratinization and desquamation.


Assuntos
Quimotripsina , Sinais Direcionadores de Proteínas , Humanos , Animais , Camundongos , Tripsina/metabolismo , Quimotripsina/metabolismo , Calicreínas/química , Calicreínas/metabolismo , Pele/metabolismo
3.
Methods Mol Biol ; 2619: 239-248, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662474

RESUMO

This chapter describes a method for the purification of urinary trypsin inhibitor (UTI), a small chondroitin sulfate proteoglycan with Ser-proteinase inhibitory activity, excreted at high levels into urine following an inflammatory condition. The method consists of two fractionation steps: an anion-exchange chromatography and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue G-250 gel staining. Several UTI bands are excised from gel, minced, destained, and dehydrated for extraction with SDS-containing buffer, at 60 °C for 24 h. This allows for obtaining a highly purified UTI sample useful for both structural and functional studies.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Cromatografia , Eletroforese em Gel de Poliacrilamida , Ânions , Tripsina
4.
PLoS One ; 18(1): e0279204, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36652464

RESUMO

BACKGROUND: Previous clinical and in vitro investigations have supported the efficacy of a glycerol throat spray containing cold-adapted cod trypsin (ColdZyme) against respiratory viruses causing the common cold bycreating a protective mucosal barrier shown to deactivate common cold virus in vitro and decrease pharyngeal rhinovirus load. METHODS AND FINDINGS: This was a double-blind, randomized, parallel-group, placebo-controlled study conducted at 10 German sites to evaluate the efficacy of the medical device ColdZyme, a glycerol mouth spray containing cold-adapted cod trypsin for a naturally occurring common cold versus placebo spray. Adults experiencing a minimum of three common colds during the previous year, but otherwise healthy, were enrolled to begin treatment with the mouth spray or placebo six times daily at first sign of a common cold. Jackson's symptom scale and the 9-item Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) quality of life (QoL) domain and a sore throat scale were recorded daily by subjects, as well as any use of allowed rescue treatment. Between January and April 2019, 701 subjects were enrolled and randomly assigned to the ColdZyme group (n = 351) or the placebo group (n = 350). Of the 701 subjects, 438 (62.5%) subjects developed symptoms typical of common cold, and all 438 started study treatment (n = 220 in the ColdZyme group and n = 218 in the placebo group). The demographic profile of the treatment groups were comparable with 68.1% female and almost all subjects being Caucasian (98.4%). The age ranged between 18 and 70 years with a mean age of 41.3 (±14.4) years. There were no differences between the groups in primary and major secondary endpoints, however, the assessment using the WURSS-21 QoL domain and Jackson score suggests a slightly faster recovery with ColdZyme as symptoms and complaints affecting the quality of life were shortened by about 1 day. The beneficial effect of ColdZyme was particularly noticeable on the fifth day of the common cold. A positive difference between treatment groups was also seen for the subjects' assessments of global efficacy of the investigational product A robust safety profile for ColdZyme was demonstrated throughout the study. CONCLUSION: The safety and tolerability of ColdZyme have been confirmed in a large study population and further establishes evidence of a faster recovery from common cold symptoms. Early self-diagnosis and early use of ColdZyme mouth spray is a safe alternative for treatment of naturally occurring colds.


Assuntos
Resfriado Comum , Adulto , Humanos , Feminino , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Idoso , Masculino , Resfriado Comum/diagnóstico , Resfriado Comum/tratamento farmacológico , Resfriado Comum/complicações , Qualidade de Vida , Sprays Orais , Glicerol/uso terapêutico , Tripsina , Método Duplo-Cego , Rhinovirus , Boca
5.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614215

RESUMO

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.


Assuntos
Antineoplásicos , Humanos , Recoverina , Células A549 , Sobrevivência Celular , Tripsina/farmacologia , Antineoplásicos/farmacologia , Receptores Acoplados a Proteínas G/genética , Esferoides Celulares
6.
Sci Rep ; 13(1): 1493, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707546

RESUMO

Protein hydrolysates from dietary sources possess many physiological and biological properties. Artocarpus altilis is an evergreen multipurpose plant with many benefits. Therefore, this study evaluates in vitro antioxidant and anti-inflammatory properties of A. altilis protein hydrolysates. Protein was isolated from A. altilis and hydrolysed with pepsin and trypsin separately using different enzyme: substrate ratios (1:8, 1:16, 1:32). Antioxidant properties investigated included Fe2+-chelating, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide radical scavenging activities. Anti-inflammatory activities were determined using effects on hypotonic solution-induced cell lysis on red blood cell membrane stabilisation and heat-induced protein denaturation. The degree of hydrolysis of trypsin hydrolysate increased with increasing enzyme-substrate ratio, while pepsin hydrolysate decreased as the enzyme-substrate ratio increased. The dominant amino acids in A. altilis protein and hydrolysates were glutamate, aspartate and leucine. Protein hydrolysates obtained from pepsin and trypsin digestion had DPPH scavenging abilities of 43.0 ± 0.01% and 22.2 ± 0.01%, respectively. However, trypsin-hydrolysed protein had a high Fe2+-chelating ability, while pepsin-hydrolysed protein had high hydrogen peroxide scavenging ability. Trypsin-hydrolysed protein showed good membrane stability and inhibition of protein denaturation. The results indicated that A. altilis protein hydrolysates possess significant antioxidant and anti-inflammatory effects and can further lend support to food industries as functional foods.


Assuntos
Artocarpus , Fabaceae , Doença de Parkinson , Antioxidantes/farmacologia , Antioxidantes/química , Artocarpus/química , Hidrolisados de Proteína/farmacologia , Hidrolisados de Proteína/química , Frutas/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismo , Peróxido de Hidrogênio , Fabaceae/metabolismo
7.
Insect Biochem Mol Biol ; 152: 103893, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36513274

RESUMO

Digestion and absorption of old cuticles during insect molting are necessary for new cuticle formation, during which complicated enzyme catalysis is essential. To date, a few carboxypeptidases, aminopeptidases and serine proteases (mostly trypsins) connected with cuticle digestion, zymogen activation and histological differentiation during the ecdysis of lepidopteran, dipteran and hymenopteran insects have been identified. However, little is known about these proteins in hemimetabolous insects. In this study, we identified 33 candidate trypsin and trypsin-like homologs, 14 metallocarboxypeptidase and 32 aminopeptidase genes in the brown planthopper Nilaparvata lugens, a hemipteran rice pest. Among the proteins encoded by these genes, 9 trypsin-like proteases, 3 metallocarboxypeptidases and 1 aminopeptidase were selected as potential procuticle hydrolases by bioinformatics analysis and in vivo validation. RNA interference targeting these genes demonstrated that 3 trypsin-like proteases (NlTrypsin-8, NlTrypsin-29 and NlTrypsin-32) genes and 1 metallocarboxypeptidase (NlCpB) gene were found to be essential for ecdysis in N. lugens; specifically, gene silencing led to incomplete cuticle degradation and arrested ecdysis, causing lethal morphological phenotype acquisition. Spatiotemporal expression profiling by quantitative PCR and western blotting revealed their specific expression in the integument and their periodic expression during each stadium, with a peak before ecdysis and eclosion. Transmission electron microscopy demonstrated corresponding ultrastructural defects after RNAi targeting, with NlCpB-silenced specimens having the most undigested old procuticles. Immunohistochemical staining revealed that NlTrypsin-8, NlTrypsin-29 and NlCpB were predominantly located in the exuvial space. This research further adds to our understanding of proteases and its potential role in insect ecdysis.


Assuntos
Hemípteros , Muda , Animais , Tripsina/metabolismo , Muda/genética , Hemípteros/metabolismo , Serina Proteases/metabolismo , Interferência de RNA , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas de Insetos/metabolismo
8.
Mar Environ Res ; 183: 105849, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36565507

RESUMO

In the marine environment, plastic pollution may occur simultaneously with hypoxia. However, current ecological risk assessments of nanoplastics have rarely considered the impact of additional environmental factors, such as hypoxia. In this study, we investigated the effect of polystyrene nanospheres (PS-NPs) on the digestive performance (antioxidant system and digestive enzymes) of mussels Mytilus galloprovincialis under different patterns of hypoxia (normoxia, constant hypoxia, and fluctuating hypoxia). The result showed that PS-NPs caused oxidative damage in the digestive glands of mussels, while all patterns of hypoxia exacerbated this oxidative damage. Activities of four digestive enzymes (α-amylase, cellulase, trypsin, and lipase) were examined. Among these, the activity of the α-amylase was inhibited by PS-NPs, and the inhibition was aggravated by all the hypoxia patterns. The cellulase activity and trypsin activity was enhanced by PS-NPs, and the increase was further stimulated by hypoxia. Lipase activity was not affected by PS-NPs alone, but significant inhibition was detected after the coexposure to PS-NPs and hypoxia. Conclusively, the combined stress of hypoxia and nanoplastics can significantly affect the digestive performance of mussels and may alter the mussel nutrient uptake strategy. Our work has provided new insight into the ecological risk assessment of plastics under global climate change.


Assuntos
Celulases , Mytilus , Poluentes Químicos da Água , Animais , Antioxidantes , Microplásticos , Tripsina/farmacologia , Proteínas , Plásticos/toxicidade , Poliestirenos/toxicidade , Hipóxia , Lipase/farmacologia , alfa-Amilases/farmacologia , Celulases/farmacologia , Poluentes Químicos da Água/toxicidade
9.
Sci Total Environ ; 864: 161068, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36565887

RESUMO

A large amount of household food waste (HFW) is produced yearly, resulting in environmental problems and financial burdens. Bio-production of lactic acid (LA), a high value-added platform chemical, from HFW by anaerobic fermentation is a promising way of resource recovery. However, the LA production yield from HFW is low. This paper compared several pretreatment methods (hydrothermal pretreatment, chemical pretreatment, and combined hydrothermal and chemical pretreatment) to improve LA production from HFW. The result showed that the combined pretreatment (alkali-thermal pretreatment at pH 10 and 120 °C) significantly increased the LA production than single hydrothermal and chemical pretreatment. The pretreatment process promoted the dissolution of organics, especially the polysaccharides and amino acids, and further influenced the LA production by Lactobacillus rhamnosus ATCC 7469. Among the amino acids, aspartic acid (Asp), threonine (Thr), glutamic acid (Glu), glycine (Gly), alanine (Ala), cystine (Cys), valine (Val), isoleucine (Ile), arginine (Arg), and proline (Pro) significantly correlated with LA concentration.


Assuntos
Aminoácidos , Eliminação de Resíduos , Sequência de Aminoácidos , Alimentos , Solubilidade , Tripsina , Polissacarídeos
10.
Pancreatology ; 23(1): 48-56, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36517351

RESUMO

BACKGROUND: PRSS1 and PRSS2 constitute the only functional copies of a tandemly-arranged five-trypsinogen-gene cluster (i.e., PRSS1, PRSS3P1, PRSS3P2, TRY7 and PRSS2) on chromosome 7q35. Variants in PRSS1 and PRSS2, including missense and copy number variants (CNVs), have been reported to predispose to or protect against chronic pancreatitis (CP). We wondered whether a common trypsinogen pseudogene deletion CNV (that removes two of the three trypsinogen pseudogenes, PRSS3P2 and TRY7) might be associated with CP causation/predisposition. METHODS: We analyzed the common PRSS3P2 and TRY7 deletion CNV in a total of 1536 CP patients and 3506 controls from France, Germany, India and Japan by means of quantitative fluorescent multiplex polymerase chain reaction. RESULTS: We demonstrated that the deletion CNV variant was associated with a protective effect against CP in the French, German and Japanese cohorts whilst a trend toward the same association was noted in the Indian cohort. Meta-analysis under a dominant model yielded a pooled odds ratio (OR) of 0.68 (95% confidence interval (CI) 0.52-0.89; p = 0.005) whereas an allele-based meta-analysis yielded a pooled OR of 0.84 (95% CI 0.77-0.92; p = 0.0001). This protective effect is explicable by reference to the recent finding that the still functional PRSS3P2/TRY7 pseudogene enhancers upregulate pancreatic PRSS2 expression. CONCLUSIONS: The common PRSS3P2 and TRY7 deletion CNV was associated with a reduced risk for CP. This finding provides additional support for the emerging view that dysregulated PRSS2 expression represents a discrete mechanism underlying CP predisposition or protection.


Assuntos
Pancreatite Crônica , Tripsinogênio , Humanos , Alelos , Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença , Genótipo , Mutação , Pancreatite Crônica/genética , Tripsina/genética , Tripsinogênio/genética
11.
Anal Chem ; 95(2): 628-637, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36549687

RESUMO

A newly synthesized proteome reflects perturbations sensitively and maintains homeostasis in cells. To investigate the low abundant newly synthesized proteins (NSPs) from a complex background proteome, an enrichment process with high selectivity and reliability is essential. Here, we have developed a strategy to realize comprehensive analysis of NSPs by integrating tandem orthogonal proteolysis (TOP) with cleavable bioorthogonal tagging (CBOT) called TOP-CBOT. A solid-phase-conjugated probe with a clickable moiety and a protease-cleavable site was designed, which allowed NSPs to be covalently captured along with tandem release by trypsin and orthogonal tobacco etch virus (TEV) protease. Our method has integrated the advantages of protein-level and peptide-level enrichment. Trypsin digests larger number of peptides from the recovered proteins for NSPs identification and quantification, while the specific tag-contained peptides from TEV data set enabled further NSPs confirmation. Integrating information from two complementary data sets, the reliability in NSPs identification and quantitation were remarkably enhanced. A total of 3699 proteins were recovered in the trypsin data set. Additionally, 1931 proteins were confirmed as NSPs with 5019 identified peptides in the TEV data set, over 90% of which were overlapped with the tryptic data set. Our strategy was further applied to profile NSP degradation kinetics during rapamycin-induced macroautophagy. The newly synthesized proteome displayed varied alteration of degradation rates among stimulation and more than half of NSPs showed decreased half-lives during autophagy.


Assuntos
Peptídeos , Proteoma , Proteoma/análise , Tripsina/metabolismo , Proteólise , Reprodutibilidade dos Testes , Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo
12.
ACS Appl Mater Interfaces ; 15(1): 494-510, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36577517

RESUMO

Targeting the limitation of antimicrobial peptides (AMPs) application in vivo, self-assembled AMPs library with specific nanostructures is expected to gradually overtake monomer AMPs libraries in the future. Peptide polymers are fascinating self-assembling nanoscale structures that have great advantage in biomedical applications because of their satisfactory biocompatibility and versatile properties. Herein, we describe a strategy for inducing the self-assembly of T9W into nanostructured antimicrobial micelles with evidently improved pharmacological properties, that is, PEGylation at the C-terminal of T9W (CT9W1000), an antibacterial biomaterial that self-assembles in aqueous media without exogenous excipients, has been developed. Compared with parental molecular, the CT9W1000 is more effective against Pseudomonas aeruginosa, and its antibacterial spectrum had also been broadened. Additionally, CT9W1000 micelles had higher stability under salt ion, serum, and acid-base environments. Importantly, the self-assembled structure is highly resistant to trypsin degradation, probably allowing T9W to be applied in clinical settings in the future. Mechanistically, by acting on membranes and through supplementary bactericidal mechanisms, CT9W1000 micelles contribute to the antibacterial process. Collectively, CT9W1000 micelles exhibited good biocompatibility in vitro and in vivo, resulting in highly effective treatment in a mouse acute lung injury model induced by P. aeruginosa PAO1 without drug resistance. These advances may profoundly accelerate the clinical transformation of T9W and promote the development of a combination of peptide-based antibiotics and PEGylated nanotechnology.


Assuntos
Lesão Pulmonar Aguda , Peptídeos Antimicrobianos , Micelas , Pseudomonas aeruginosa , Animais , Camundongos , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Modelos Animais de Doenças , Testes de Sensibilidade Microbiana , Tripsina/metabolismo , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/microbiologia , Nanoestruturas/química , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Farmacorresistência Bacteriana
13.
Food Chem ; 408: 135063, 2023 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-36535182

RESUMO

Ovomucoid (OVM) is a critical anti-nutritional factor in egg, which may reduce nutrient utilization. In this study, the effects of polyphenols on the trypsin inhibitory activity (TIA) of OVM were investigated by exploring the structural changes and interaction mechanisms. The results found that TIA decreased to 62.34% and 90.41% as epigallocatechin gallate (EGCG) and gallic acid (GA) were added individually. EGCG and GA interacted with OVM via static quenching and hydrophobic interaction. They induced a transition of OVM conformation from disorder to order. Infrared and fluorescence quenching analysis showed that the interaction between EGCG or GA and OVM was spontaneous, and hydrophobic interaction was the predominant force. The mechanism suggested that polyphenols affect the protein conformation by spontaneously binding to OVM in hydrophobic interactions, and lowering the TIA through reduced hydrophobicity. In summary, EGCG may be a promising OVM trypsin activity inactivator, which could also guarantee safety of egg products.


Assuntos
Catequina , Polifenóis , Polifenóis/farmacologia , Ovomucina , Tripsina , Conformação Proteica , Catequina/farmacologia , Ovos , Ácido Gálico/farmacologia
14.
Food Chem ; 408: 135230, 2023 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-36549163

RESUMO

The work aimed to assess the antioxidant ability and obtain a new antioxidant peptide from rice bran protein. Rice bran protein was hydrolyzed by Alcalase, Neutral, Pepsin, Chymotrypsin, and Trypsin, separately. Trypsin hydrolysate (T-RBPH) showed high Fe2+ chelating activity (IC50, 2.271 ± 0.007 mg/mL), DPPH and hydroxyl radical scavenging ability (IC50, 0.191 ± 0.006 and 1.038 ± 0.034 mg/mL). Moreover, T-RBPH could alleviate the H2O2-induced oxidative damage in Caco-2. The T-RBPH was purified and identified by UF, GF, FPLC, and LC-MS/MS. Finally, 9-amino acid peptide-AFDEGPWPK with low molecular weight (1045.48 Da), high antioxidant activity, good safety, and solubility was screened by in silico method and chemical oxidation determination, and its interaction with Keap1 was also demonstrated. The ORAC and DPPH radical scavenging ability of AFDEGPWPK were 44.16 ± 0.79 and 28.38 ± 0.14 µmol TE/mM. Moreover, the Molecular docking and Western blot (WB) results showed that AFDEGPWPK could enter the binding pocket in the Kelch domain and activate Keap1/Nrf2/HO-1 pathway.


Assuntos
Antioxidantes , Oryza , Humanos , Antioxidantes/farmacologia , Antioxidantes/química , Hidrolisados de Proteína/química , Oryza/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Cromatografia Líquida , Tripsina/metabolismo , Simulação de Acoplamento Molecular , Peróxido de Hidrogênio/metabolismo , Células CACO-2 , Espectrometria de Massas em Tandem , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/química
15.
Cell Tissue Bank ; 23(4): 641-652, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34545505

RESUMO

There is no consensus between the protocols used for the isolation, maintenance and cultivation of Adipose-derived stem cells (ADSCs) for therapeutic purposes. Thus, was evaluated the maintenance method of ADSCs submitted to enzymatic disaggregation by trypsin. Was made (1st until 10th passage) immunophenotyping, cell differentiation assays, comet assay, differential cell death, apoptosis, cell viability and membrane integrity by flow cytometry.The results showded that trypsinization,did not induce genomic instability, also did not alter the tail moment. The cell death assay, showed that only on the 10th passage there was a significant reduction and was cofirmed by flow cytometry that is apoptosis. The viability showded significant reduction only in 10th passage, this was related to the loss of integrity of membrane, proven by flow cytometry. The quantities varied along the passages (11 × 105 to 2 × 105). Qualitatively, it can be observed that as the number of cells decreases, there is also a reduction in the juxtaposition of ADSCs and increased of the cell size, it is started in 6th passage. In view of the results, it is suggested for more safety, that ADSCs cultured until the 5th passage being used in human transplantation procedures.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco , Humanos , Tripsina/metabolismo , Células Cultivadas , Instabilidade Genômica
16.
Pestic Biochem Physiol ; 188: 105269, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36464374

RESUMO

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.


Assuntos
Bacillus thuringiensis , Mariposas , Serpinas , Animais , Serpinas/genética , Quimotripsina/genética , Tripsina , Peptídeo Hidrolases , Mariposas/genética
17.
Biofouling ; 38(9): 876-888, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36503292

RESUMO

The biological impact of chemical formulations used in various coating applications is essential in guiding the development of new materials that directly contact living organisms. To illustrate this point, an investigation addressing the impact of chemical compositions of polydimethylsiloxane networks on a common platform for foul-release biofouling management coatings was conducted. The acute toxicity of network components to barnacle larvae, the impacts of aqueous extracts of crosslinker, silicones and organometallic catalyst on trypsin enzymatic activity, and the impact of assembled networks on barnacle adhesion was evaluated. The outcomes of the study indicate that all components used in the formulation of the silicone network alter trypsin enzymatic activity and have a range of acute toxicity to barnacle larvae. Also, the adhesion strength of barnacles attached to PDMS networks correlates to the network formulation protocol. This information can be used to assess action mechanisms and risk-benefit analysis of PDMS networks.


Assuntos
Incrustação Biológica , Thoracica , Animais , Tripsina , Biofilmes , Incrustação Biológica/prevenção & controle , Silicones/química
18.
Int J Mol Sci ; 23(24)2022 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-36555366

RESUMO

Only 3-5% of heavy alcohol users develop acute alcohol pancreatitis (AAP). This suggests that additional triggers are required to initiate the inflammatory process. Genetic susceptibility contributes to the development of AAP, and SPINK1 mutation is a documented risk factor. We investigated the prevalence of the SPINK1(N34S) mutation in patients with AAP compared to heavy alcohol users who had never suffered an episode of pancreatitis. Blood samples for the mutational analysis from patients with first episode (n = 60) and recurrent AAP (n = 43) and from heavy alcohol users without a history of AAP (n = 98) as well as from a control population (n = 1914) were obtained. SPINK1 mutation was found in 8.7% of the patients with AAP. The prevalence was significantly lower in healthy controls (3.4%, OR 2.72; 1.32-5.64) and very low in alcoholics without pancreatitis (1.0%, OR 9.29; 1.15-74.74). In a comparison adjusted for potential cofounders between AAP patients and alcoholics, SPINK1 was found to be an independent marker for AAP. The prevalence of the SPINK1 mutation is overrepresented in AAP patients and very low in alcoholics without pancreatitis. This finding may play a role in understanding the variable susceptibility to AAP found in heavy alcohol users.


Assuntos
Consumo de Bebidas Alcoólicas , Pancreatite , Inibidor da Tripsina Pancreática de Kazal , Humanos , Predisposição Genética para Doença , Mutação , Pancreatite/genética , Fatores de Risco , Tripsina/genética , Inibidor da Tripsina Pancreática de Kazal/genética , Consumo de Bebidas Alcoólicas/efeitos adversos
19.
Nat Commun ; 13(1): 7959, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36575174

RESUMO

The progression of cancer from localized to metastatic disease is the primary cause of morbidity and mortality. The interplay between the tumor and its microenvironment is the key driver in this process of tumor progression. In order for tumors to progress and metastasize they must reprogram the cells that make up the microenvironment to promote tumor growth and suppress endogenous defense systems, such as the immune and inflammatory response. We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression. Here, we identify a novel tumor-mediated mechanism that represses the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a hitherto undescribed activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1. Based on the ability of PRSS2 to reprogram the tumor microenvironment, this discovery could lead to the development of therapeutic agents that are indication agnostic.


Assuntos
Neoplasias , Trombospondina 1 , Humanos , Trombospondina 1/genética , Trombospondina 1/metabolismo , Microambiente Tumoral/genética , Neoplasias/genética , Tripsina , Tripsinogênio
20.
Anal Chem ; 94(51): 18099-18106, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36515251

RESUMO

To regulate nanostructure synthesis is of crucial importance for developing various applications, including catalysis, bioanalysis, and optical devices. Herein, the morphology and peroxidase (POD)-mimicking activity of peptide-templated copper nanoassemblies (Cu NAs) are regulable with peptide types. The Cu NAs templated with peptide containing single cysteine are uniform nanoclusters with strong POD-like activity. However, the Cu NAs templated with peptide containing two cysteines are fusiform-like with very weak POD-like activity. Unexpectedly, the POD-like activity of Cu NAs templated with peptide containing two cysteines with lysine between the cysteines is significantly enhanced when trypsin is incubated, which is unchanged for the Cu NAs templated with peptide containing two cysteines without lysine between the cysteines. The remarkably enhanced POD-mimicking activity originates from trypsin specifically shearing the peptide bond on the lysine, thereby allowing the aggregated Cu NAs to unravel into individual nanoclusters. Therefore, a robust colorimetric sensing platform was constructed for sensitive and selective detection of trypsin, which showed a linear concentration range of 3-1000 nM and a detection limit of 0.82 nM (S/N = 3). More interestingly, featured by trypsin inhibitor restraining trypsin activity, it enabled us to screen trypsin inhibitors as well. Subsequently, the developed assay was applied to detect trypsin in serum samples with good accuracy and reproducibility. Thus, this strategy shows great potential application in the clinic for diagnosis of trypsin-indicating diseases as well as the screening of trypsin inhibitor-based anti-cancer drugs.


Assuntos
Cobre , Nanopartículas Metálicas , Cobre/química , Inibidores da Tripsina/farmacologia , Tripsina/química , Reprodutibilidade dos Testes , Lisina , Nanopartículas Metálicas/química , Peptídeos , Limite de Detecção
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