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1.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576130

RESUMO

Several studies have sought new therapies for obesity and liver diseases. This study investigated the effect of the trypsin inhibitor isolated from tamarind seeds (TTI), nanoencapsulated in chitosan and whey protein isolate (ECW), on the liver health status of the Wistar rats fed with a high glycemic index (HGLI) diet. The nanoformulations without TTI (CW) and ECW were obtained by nanoprecipitation technique, physically and chemically characterized, and then administered to the animals. The adult male Wistar rats (n = 20) were allocated to four groups: HGLI diet + water; standard diet + water; HGLI diet + ECW (12.5 mg/kg); and HGLI diet + CW (10.0 mg/kg), 1 mL per gagave, for ten days. They were evaluated using biochemical and hematological parameters, Fibrosis-4 Index for Liver Fibrosis (FIB-4), AST to Platelet Ratio Index (APRI) scores, and liver morphology. Both nanoparticles presented spherical shape, smooth surface, and nanometric size [120.7 nm (ECW) and 136.4 nm (CW)]. In animals, ECW reduced (p < 0.05) blood glucose (17%), glutamic oxalacetic transaminase (39%), and alkaline phosphatase (24%). Besides, ECW reduced (p < 0.05) APRI and FIB-4 scores and presented a better aspect of hepatic morphology. ECW promoted benefits over a liver injury caused by the HGLI diet.


Assuntos
Quitosana/química , Dieta , Índice Glicêmico , Fígado/lesões , Nanopartículas/química , Tamarindus/química , Inibidores da Tripsina/farmacologia , Proteínas do Soro do Leite/química , Animais , Glicemia/metabolismo , Jejum/sangue , Homeostase , Insulina/sangue , Resistência à Insulina , Rim/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Masculino , Nanopartículas/ultraestrutura , Ratos Wistar , Valores de Referência
2.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443447

RESUMO

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.


Assuntos
Antifúngicos/farmacologia , Enzimas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Polissacarídeos/metabolismo , Liofilização , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Peso Molecular , Proteólise/efeitos dos fármacos , Alimentos de Soja , Espectrofotometria Ultravioleta , Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
3.
In Vivo ; 35(5): 2551-2558, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410942

RESUMO

BACKGROUND/AIM: We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. MATERIALS AND METHODS: The action of TI (1-10 µg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. RESULTS: The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. CONCLUSION: Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.


Assuntos
Amilases , Inibidores da Tripsina , Animais , Insulina/metabolismo , Secreção de Insulina , Masculino , Pâncreas/metabolismo , Ratos , Ratos Wistar , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
4.
Sci Rep ; 11(1): 15849, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349162

RESUMO

Extracorporeal membrane oxygenation (ECMO) is a life-saving intervention for patients suffering from respiratory or cardiac failure. The ECMO-associated morbidity and mortality depends to a large extent on the underlying disease and is often related to systemic inflammation, consecutive immune paralysis and sepsis. Here we tested the hypothesis that human α1-antitrypsin (SERPINA1) due to its anti-protease and anti-inflammatory functions may attenuate ECMO-induced inflammation. We specifically aimed to test whether intravenous treatment with α1-antitrypsin reduces the release of cytokines in response to 2 h of experimental ECMO. Adult rats were intravenously infused with α1-antitrypsin immediately before starting veno-arterial ECMO. We measured selected pro- and anti-inflammatory cytokines and found, that systemic levels of tumor necrosis factor-α, interleukin-6 and interleukin-10 increase during experimental ECMO. As tachycardia and hypertension developed in response to α1-antitrypsin, a single additional bolus of fentanyl and midazolam was given. Treatment with α1-antitrypsin and higher sedative doses reduced all cytokine levels investigated. We suggest that α1-antitrypsin might have the potential to protect against both ECMO-induced systemic inflammation and immune paralysis. More studies are needed to corroborate our findings, to clarify the mechanisms by which α1-antitrypsin inhibits cytokine release in vivo and to explore the potential application of α1-antitrypsin in clinical ECMO.


Assuntos
Débito Cardíaco/efeitos dos fármacos , Citocinas/metabolismo , Oxigenação por Membrana Extracorpórea/métodos , Hemodinâmica , Inibidores da Tripsina/farmacologia , alfa 1-Antitripsina/farmacologia , Animais , Masculino , Ratos , Ratos Endogâmicos Lew
5.
J Basic Microbiol ; 61(8): 709-720, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34228389

RESUMO

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.


Assuntos
Ascomicetos/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Tripsina/metabolismo , Antioxidantes , Endófitos/metabolismo , Fermentação , Loranthaceae , Simulação de Acoplamento Molecular , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
7.
Food Chem ; 358: 129891, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33940290

RESUMO

Quercetin is a well-studied natural product with multiple pharmacological properties. In this study, we demonstrated that quercetin suppressed protein digestion in the intestinal fluid by inhibiting trypsin, a key digestive enzyme. However, we also observed a previously unknown property of quercetin: promoting the intestinal absorption of proteins. In addition, the promoted protein absorption was mediated by internalization of digested oligopeptides in the intestinal epithelia rather than increasing the intestinal paracellular permeability. Notably, four other flavonoids also achieved such enhanced intestinal absorption, suggesting that this effect was associated with the aglycone flavonol backbone, but not related to their inhibitory potencies against trypsin. This study demonstrates that quercetin exhibits dual effects on protein digestion and absorption: 1) suppressing protein digestion by inhibiting trypsin in the intestinal fluid; 2) promoting the intestinal absorption of oligopeptides in the intestinal villi cells.


Assuntos
Proteínas na Dieta/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Quercetina/farmacologia , Animais , Líquidos Corporais/efeitos dos fármacos , Líquidos Corporais/metabolismo , Proteínas na Dieta/metabolismo , Digestão/efeitos dos fármacos , Cães , Flavonoides/farmacologia , Absorção Intestinal/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Células Madin Darby de Rim Canino , Masculino , Permeabilidade , Proteólise , Ratos Sprague-Dawley , Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
8.
Food Funct ; 12(8): 3527-3538, 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33900335

RESUMO

This study explored the effects of lotus seedpod oligomeric procyanidins (LSOPC) and their main monomer catechin (CC) on the formation of advanced glycation end products (AGEs) and Caco-2 cytotoxicity during gastrointestinal digestion. Studies have found that LSOPC and CC inhibited the AGEs formation effectively in simulated gastrointestinal digestion and protected Caco-2 cells from AGEs attack. The effect of CC on the inhibition of AGEs formation was significantly better than that of LSOPC. Further, they could effectively inhibit the digestive enzyme activity, reactive oxygen species, RAGE-p38MAPK-NF-κB signaling pathway, inflammatory factors (tumor necrosis factor alpha, interleukin 6), and adhesion factors (intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1) to protect Caco-2 cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Trato Gastrointestinal/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Nelumbo/química , Proantocianidinas/farmacologia , Sementes/química , Apoptose/efeitos dos fármacos , Células CACO-2 , Catequina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Digestão , Trato Gastrointestinal/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Pepsina A/antagonistas & inibidores , Espécies Reativas de Oxigênio/análise , Tocoferóis/análise , Inibidores da Tripsina/farmacologia
9.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805525

RESUMO

Food proteins and peptides are able to exert a variety of well-known bioactivities, some of which are related to well-being and disease prevention in humans and animals. Currently, an active trend in research focuses on chronic inflammation and oxidative stress, delineating their major pathogenetic role in age-related diseases and in some forms of cancer. The present study aims to investigate the potential effects of pseudocereal proteins and their derived peptides on chronic inflammation and oxidative stress. After purification and attribution to protein classes according to classic Osborne's classification, the immune-modulating, antioxidant, and trypsin inhibitor activities of proteins from quinoa (Chenopodium quinoa Willd.), amaranth (Amaranthus retroflexus L.), and buckwheat (Fagopyrum esculentum Moench) seeds have been assessed in vitro. The peptides generated by simulated gastro-intestinal digestion of each fraction have been also investigated for the selected bioactivities. None of the proteins or peptides elicited inflammation in Caco-2 cells; furthermore, all protein fractions showed different degrees of protection of cells from IL-1ß-induced inflammation. Immune-modulating and antioxidant activities were, in general, higher for the albumin fraction. Overall, seed proteins can express these bioactivities mainly after hydrolysis. On the contrary, higher trypsin inhibitor activity was expressed by globulins in their intact form. These findings lay the foundations for the exploitation of these pseudocereal seeds as source of anti-inflammatory molecules.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fatores Imunológicos/farmacologia , Proteínas de Vegetais Comestíveis/isolamento & purificação , Proteínas de Vegetais Comestíveis/farmacologia , Sementes/química , Amaranthus/química , Anti-Inflamatórios não Esteroides/química , Antioxidantes/farmacologia , Células CACO-2 , Fracionamento Químico , Chenopodium quinoa/química , Fagopyrum/química , Humanos , Fatores Imunológicos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Vegetais Comestíveis/farmacocinética , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia
10.
Bioorg Med Chem Lett ; 40: 127939, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33713780

RESUMO

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.


Assuntos
Benzoatos/farmacologia , Enteropeptidase/antagonistas & inibidores , Guanidinas/farmacologia , Inibidores da Tripsina/farmacologia , Animais , Benzoatos/síntese química , Benzoatos/farmacocinética , Células CHO , Bovinos , Cricetulus , Dieta Hiperlipídica , Fezes/química , Guanidinas/síntese química , Guanidinas/farmacocinética , Humanos , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/enzimologia , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Proteínas/metabolismo , Relação Estrutura-Atividade , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/farmacocinética
11.
J Enzyme Inhib Med Chem ; 36(1): 480-490, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33491503

RESUMO

Trypsin inhibitors from tamarind seed have been studied in vitro and in preclinical studies for the treatment of obesity, its complications and associated comorbidities. It is still necessary to fully understand the structure and behaviour of these molecules. We purifed this inhibitor, sequenced de novo by MALDI-TOF/TOF, performed its homology modelling, and assessed the interaction with the trypsin enzyme through molecular dynamics (MD) simulation under physiological conditions. We identified additional 75 amino acid residues, reaching approximately 72% of total coverage. The four best conformations of the best homology modelling were submitted to the MD. The conformation n°287 was selected considering the RMSD analysis and interaction energy (-301.0128 kcal.mol-1). Residues Ile (54), Pro (57), Arg (59), Arg (63), and Glu (78) of pTTI presented the highest interactions with trypsin, and arginine residues were mainly involved in its binding mechanism. The results favour bioprospecting of this protein for pharmaceutical health applications.


Assuntos
Simulação de Dinâmica Molecular , Extratos Vegetais/farmacologia , Tamarindus/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Relação Dose-Resposta a Droga , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Sementes/química , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação
12.
Chem Biol Drug Des ; 97(3): 607-627, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32946175

RESUMO

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.


Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Antígenos CD2/química , Antígenos CD58/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Estabilidade Proteica , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Inibidores da Tripsina/farmacologia
13.
Protein Pept Lett ; 28(6): 665-674, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33191881

RESUMO

BACKGROUND: Protease inhibitors have been isolated from plants and present several biological activities, including immunomodulatory action. OBJECTIVE: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. METHODS: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15-240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). RESULTS: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15-30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as Δψm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. CONCLUSION: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.


Assuntos
Moringa oleifera/enzimologia , Proteínas de Plantas , Inibidores da Tripsina , Animais , Comportamento Animal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Flores/química , Hemólise/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Plantas/toxicidade , Baço/citologia , Testes de Toxicidade Aguda , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/toxicidade
14.
PLoS One ; 15(12): e0244031, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33378351

RESUMO

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.


Assuntos
Campylobacter rectus/metabolismo , Campylobacter/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Periplásmicas/genética , Inibidores de Serino Proteinase/metabolismo , Inibidores da Tripsina/metabolismo , Animais , Campylobacter/genética , Campylobacter rectus/genética , Células Cultivadas , Galinhas , Humanos , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/antagonistas & inibidores , Homologia de Sequência , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/farmacologia , Inibidores da Tripsina/farmacologia
15.
Poult Sci ; 99(10): 5007-5017, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988537

RESUMO

Trypsin inhibitors (TI) resident in soybeans affects protein utilization. While heat treatment influences residual TI, it simultaneously affects the structure and solubility of the soybean proteins and confounds any response to exogenous proteases. Using purified TI, the effect of exogenous protease to TI can be dissociated from changes in the soybean protein. Thus, the current study was designed to evaluate the growth performance and protein utilization responses of broiler chickens to purified TI and exogenous protease. Soybean meal (SBM) was preanalyzed for basal TI (2,996 TIU/g SBM), formulated into nutritionally adequate experimental diets to contain 1,033 TIU/g diet, and purified TI was added at 9,000 TIU/g diet. A total of 320 Cobb-500 broiler chicks were allocated to 4 diets, each with 8 replicate cages and 10 birds per replicate. The experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). On day 7, 14, and 21 posthatching, protease supplementation improved the BW gain (P < 0.01) and gain to feed ratio (P < 0.05) of birds. On day 14 and 21 posthatching, the relative weight of pancreas increased (P < 0.05) with added TI but was reduced (P < 0.001) with protease supplementation. Apparent ileal digestibility of all amino acids, except methionine, decreased (P < 0.001) with added TI but increased (P < 0.05) with protease supplementation. Jejunal MUC-2 was downregulated (P < 0.01) and SCL7A-2 was upregulated (P < 0.05) by protease supplementation. Duodenal trypsin and chymotrypsin activities reduced (P < 0.05) with added TI but increased (P < 0.01) with protease supplementation. Exogenous protease produced longer villi (P < 0.05) and deeper crypts (P < 0.01) in the jejunal tissue. In conclusion, dietary addition of purified TI negatively affects nutrient utilization by broiler chickens. Furthermore, the study showed that the efficacy of the exogenous protease might be independent of dietary TI concentration.


Assuntos
Aminoácidos , Galinhas , Dieta , Suplementos Nutricionais , Peptídeo Hidrolases , Soja , Inibidores da Tripsina , Aminoácidos/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Digestão/efeitos dos fármacos , Peptídeo Hidrolases/farmacologia , Inibidores da Tripsina/farmacologia
16.
Biomolecules ; 10(9)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872343

RESUMO

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman-Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman-Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2' site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the ß subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman-Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.


Assuntos
Antineoplásicos/isolamento & purificação , Ranidae/metabolismo , Inibidores da Tripsina/isolamento & purificação , Motivos de Aminoácidos , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Quimotripsina/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Hemolíticos/química , Hemolíticos/isolamento & purificação , Hemolíticos/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/farmacologia
17.
Int J Mol Sci ; 21(17)2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32878020

RESUMO

Wheat amylase trypsin inhibitors (ATIs) represent a common dietary protein component of gluten-containing cereals (wheat, rye, and barley). They act as toll-like receptor 4 ligands, and are largely resistant to intestinal proteases, eliciting a mild inflammatory response within the intestine after oral ingestion. Importantly, nutritional ATIs exacerbated inflammatory bowel disease and features of fatty liver disease and the metabolic syndrome in mice. For Alzheimer's disease (AD), both inflammation and altered insulin resistance are major contributing factors, impacting onset as well as progression of this devastating brain disorder in patients. In this study, we evaluated the impact of dietary ATIs on a well-known rodent model of AD (5xFAD). We assessed metabolic, behavioral, inflammatory, and microbial changes in mice consuming different dietary regimes with and without ATIs, consumed ad libitum for eight weeks. We demonstrate that ATIs, with or without a gluten matrix, had an impact on the metabolism and gut microbiota of 5xFAD mice, aggravating pathological hallmarks of AD. If these findings can be translated to patients, an ATI-depleted diet might offer an alternative therapeutic option for AD and warrants clinical intervention studies.


Assuntos
Doença de Alzheimer/patologia , Comportamento Animal , Microbioma Gastrointestinal , Inflamação/patologia , Placa Amiloide/patologia , Triticum/enzimologia , Inibidores da Tripsina/farmacologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Amilases/química , Animais , Dieta/efeitos adversos , Modelos Animais de Doenças , Feminino , Imunidade Inata , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Amiloide/metabolismo , Tripsina/química
18.
J Nat Prod ; 83(10): 3030-3040, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-32997497

RESUMO

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin-Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to favor a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladderlike connectivity of their disulfide bonds. Interhelical interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the antiparallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function; however, their diverse structural features despite a common fold suggest that additional bioactivities yet to be revealed are likely.


Assuntos
Dobramento de Proteína , Proteínas de Armazenamento de Sementes/química , Sequência de Aminoácidos , Dissulfetos/química , Sequências Hélice-Alça-Hélice , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica em alfa-Hélice , Proteínas de Armazenamento de Sementes/síntese química , Proteínas de Armazenamento de Sementes/farmacologia , Inibidores da Tripsina/farmacologia
19.
J Physiol Biochem ; 76(4): 573-586, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32794154

RESUMO

Hepatic encephalopathy (HE) is a devastating neuropsychiatric presentation of the advanced hepatic insufficiency. It is associated with high morbidity and mortality. Aquaporin-4 (AQP4), the principal astrocyte water channel, is primarily involved in brain edema development. Ulinastatin (ULI) is a potent protease inhibitor, extracted from fresh human urine. We hypothesized that ULI could be neuroprotective in acute HE through molecular targeting of brain AQP4, which is known to be upregulated in HE. To induce acute liver failure (ALF), the rats were acutely intoxicated with thioacetamide (TAA). Animals were randomized into HE- and ULI-treated HE groups, with control normal group. Total bilirubin, albumin, serum aminotransferases, and serum/brain ammonia/proinflammatory cytokines, blood-brain barrier (BBB) integrity/tight junction proteins, brain water content, and neurological scores were assessed. Additionally, brain AQP4 and α-Syntrophin mRNA expression and protein levels were evaluated by quantitative real-time PCR and enzyme-linked immunosorbent assay, respectively. Brain and liver tissues were stripped and processed for further microscopic and histological analyses. ULI exerted potent dual neuro/hepato protective potential, improved neurological score, animals' survival, ameliorated brain edema, probably via anti-inflammatory activity, preserved BBB integrity, down-regulated AQP4 expression, and membrane polarization by decreased α-syntrophin level, with rescued brain bioenergetics. ULI could be tooled as a possible therapeutic option in HE in ALF.Graphical abstract The possible ULI mediated protection in TAA-induced HE rat model.


Assuntos
Aquaporina 4/metabolismo , Glicoproteínas , Encefalopatia Hepática/tratamento farmacológico , Inibidores da Tripsina , Animais , Glicoproteínas/administração & dosagem , Glicoproteínas/farmacologia , Masculino , Terapia de Alvo Molecular , Ratos , Inibidores da Tripsina/administração & dosagem , Inibidores da Tripsina/farmacologia
20.
Cancer Lett ; 491: 108-120, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32841713

RESUMO

Breast cancer is the most common malignant tumor among women worldwide, and triple-negative breast cancer is the most aggressive type of breast cancer, which does not respond to hormonal therapies. The protease inhibitor, EcTI, extracted from seeds of Enterolobium contortisiliquum, acts on the main signaling pathways of the MDA-MB-231 triple-negative breast cancer cells. This inhibitor, when bound to collagen I of the extracellular matrix, triggers a series of pathways capable of decreasing the viability, adhesion, migration, and invasion of these cells. This inhibitor can interfere in the cell cycle process through the main signaling pathways such as the adhesion, Integrin/FAK/SRC, Akt, ERK, and the cell death pathway BAX and BCL-2. It also acts by reducing the main inflammatory cytokines such as TGF-α, IL-6, IL-8, and MCP-1, besides NFκB, a transcription factor, responsible for the aggressive and metastatic characteristics of this type of tumor. Thus, the inhibitor was able to reduce the main processes of carcinogenesis of this type of cancer.


Assuntos
Citocinas/antagonistas & inibidores , Fabaceae/química , Glicosaminoglicanos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Inibidores da Tripsina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citocinas/biossíntese , Feminino , Humanos , Metaloproteinases da Matriz/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Inibidores da Tripsina/uso terapêutico
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