RESUMO
The aim of this work is to evaluate the antitumor effect mediated by the proteasome inhibitors of Inula viscosa extracts on skin carcinogenesis. Female Swiss albino mice were divided into five groups depending on the combination of skin cancer-inducing 7,12-dimethylbenz(a)anthracene (DMBA) and extract of Inula viscosa treatments. Histology of the affected skin and measurement of proteasome activity were performed to demonstrate the effect of Inula viscosa on mice. The identification of the molecules responsible for this inhibitory activity was carried out through the docking studies. The results showed that Inula viscosa extracts inhibit the development of papilloma in mice. Therefore, the best chemopreventive action of Inula viscosa was observed on mice in which extract treatment was performed before and after the induction of skin carcinogenesis. It was revealed that the ingestion of extracts Inula viscosa delays the formation of skin papillomas in animals and simultaneously decreases the size and number of papillomas, which is also reflected on the skin histology of the mice treated. Structure-activity relationship information obtained from component of Inula viscosa particularly tomentosin, inuviscolide, and isocosticacid demonstrated that distinct bonding modes in ß 1, ß 2, and ß 5 subunits determine its selectivity and potent inhibition for ß 5 subunit.
Assuntos
Antineoplásicos/uso terapêutico , Inula/química , Extratos Vegetais/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , 9,10-Dimetil-1,2-benzantraceno , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Células CACO-2 , Quimotripsina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Simulação de Acoplamento Molecular , Papiloma/tratamento farmacológico , Papiloma/patologia , Extratos Vegetais/análise , Extratos Vegetais/farmacocinética , Extratos Vegetais/farmacologia , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Testes de ToxicidadeRESUMO
Lucilia sericata, one of the most common species of the Calliphoridae family, is found in large numbers around droppings, garbage and carcasses. This fly species is important in medicine, forensics and veterinary medicine. The larvae of the parasite are important both in veterinary medicine and in combating of the animal diseases, as they cause significant losses in animal production. Since they are one of the first fly colonies to settle on corpses, they can also be used in determining the time of death in the field of forensic medicine. L.sericata larvae used in Maggot debridement treatment (MDT) which is a treatment method with fly larvae, help wound healing by destroying necrotic tissues and infectious agents in wounds. While the larvae protect themselves from polymicrobial flora with the proteins they secrete; at the same time, they make an interesting contribution to wound healing with these molecules secreted. One of the most important molecules discovered in recent years is lucimycin which has an antifungal effect. In addition, lucifensin and chymotrypsin secretions have gained importance in recent years due to their antibacterial effects and especially their effects on resistant gram-negative and positive bacteria. There is a need for the discovery of the molecules that can be alternative in the treatment of non-healing wounds or that can be applied together with existing antibiotics. It is necessary to investigate the antimicrobial characterization of the compounds involved in maggot therapy and their mechanisms. The aim of this study was to clone, molecular characterization and analysis of the antigenic structures of lucifensin and chymotrypsin genes, which are important defensin molecules secreted by L.sericata larvae used in MDT. Primarily, the cultivation of L.sericata colonies to be used in molecular studies were performed. Later, RNA isolation and cDNA synthesis from larvae were carried out. Lucifensin and chymotrypsin genes were individually inserted into the pJet1.2 plasmid by cloning reactions. The presence of the recombinant plasmid was confirmed by PCR screening and DNA sequence analysis methods in all steps. Nucleotide and amino acid based molecular characterizations of these two genes, which are important larval components in wound treatment, have been made. Antigenic regions and three-dimensional structures of the proteins were obtained. The isolate numbered MT495795 of the L.sericata lucifensin gene and the isolate numbered MT495794 of the chymotrypsin gene were registered to GenBank. This data reported for the first time in the Republic of Turkey will contribute to the literature. From the beginning of the 20th century until the discovery of the antibiotics, MDT was applied especially on soldiers but did not find much application area after the discovery of the antibiotics. Drug resistance, which is the most important problem encountered in the treatment of the wounds today, has led to the recall of MDT and its mechanism of action. In this study the data, obtained will constitute a source for the multidisciplinary studies of the scientists from different fields on the discovery and applicability of the important moleculesin the treatment of the wounds.
Assuntos
Quimotripsina , Defensinas , Dípteros , Animais , Quimotripsina/genética , Quimotripsina/metabolismo , Desbridamento , Defensinas/genética , Defensinas/metabolismo , Dípteros/genética , Humanos , Larva , TurquiaRESUMO
This study was aimed to explore the comparative acidifying properties of 2-hydroxy-4-(methylthio) butanoic acid (HMTBA) and a combination of DL-methionine (DLM) and acidifier in male broiler production. A total of 480 1-day-old broiler chicks were randomly divided into four treatments: A (low HMTBA, 0.057% HMTBA); B (low acidifier, 0.05% DLM + 0.057% acidifier); C (high HMTBA, 0.284% HMTBA); and D (high acidifier, 0.25% DLM + 0.284% acidifier). At 21 d, growth performance, chyme pH, digestive enzyme activities, and intestinal microflora were measured. The pH of crop, gizzard, and ileum contents was higher in the HMTBA treatment group than in DLM + acidifier treatment group. Furthermore, acidifier supplementation promoted growth of butyrate-producing bacteria such as Faecalibacterium, whereas high HMTBA (0.284%) inhibited the proliferation of acid-producing bacteria including Roseburia and Collinsella. The chymotrypsin activity was lower in the HMTBA group than in the DLM + acidifier group. In contrast, high-level HMTBA group showed higher average daily gain and average daily feed intake than the DLM + acidifier group. These results suggested that HMTBA work through different pathways with DLM plus acidifier.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Galinhas/crescimento & desenvolvimento , Galinhas/microbiologia , Galinhas/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Metionina/análogos & derivados , Metionina/farmacologia , Animais , Quimotripsina/metabolismo , Papo das Aves , Ingestão de Alimentos/efeitos dos fármacos , Conteúdo Gastrointestinal , Moela das Aves , Concentração de Íons de Hidrogênio , Íleo , Masculino , Aumento de Peso/efeitos dos fármacosRESUMO
Whey, the main by-product of the dairy industry, is frequently disposed of in the environment without any treatment due to the high cost of this process. Alternatively, whey can be used as a medium to culture lactic acid bacteria and produce value-added products such as bacteriocins. In this work, we attempted to improve bacteriocin production by Lactobacillus plantarum ST16Pa in a whey powder formulation supplemented with additional sources of carbon, nitrogen, and vitamin B12 at different levels and varying the agitation intensity according to a Plackett-Burman experimental design. Only the addition of tryptone positively influenced the production of this bacteriocin. The results allowed us to identify a supplemented whey formulation, comprising 150 g/L of whey total solids plus 10 g/L of tryptone and soybean extract, whose fermentation by Lb. plantarum ST16Pa in shake flasks under agitation at 150 rpm led to a cell-free supernatant with an antimicrobial activity against Listeria innocua 6a CLIST 2865 (inhibition zone of 13.23 mm) close to that previously obtained in de Man, Rogosa and Sharpe medium by other authors. These results are significant considering that the same strain cultured in cheese whey did not previously display any antimicrobial activity.
Assuntos
Bacteriocinas/biossíntese , Lactobacillus plantarum/metabolismo , Soro do Leite/metabolismo , Animais , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Reatores Biológicos/normas , Queijo/microbiologia , Quimotripsina/metabolismo , Fermentação , Ácido Láctico/análise , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/crescimento & desenvolvimento , Lactose/análise , Listeria/metabolismo , Pós , Pronase/metabolismo , Tripsina/metabolismo , Soro do Leite/química , Proteínas do Soro do Leite/metabolismoRESUMO
We tested the amyloid fibril formation inhibitory effect of seven teas diluted in 55% ethanol at pH 7.0 at a protein concentration of 0.15 mg/ml α-chymotrypsin. In the experiments we investigated the formation and inhibition of amyloid fibrils by turbidity measurements, aggregation kinetics experiments and Congo red binding assay. The results suggest that the different teas effectively inhibit the formation of amyloidlike fibrils. The two most potent inhibitors were peppermint and melilot, extracts which almost completely inhibited the formation of aggregates in 5-fold dilution. The inhibitory effect on the aggregation formation of melilot and peppermint extracts was concentration dependant. The extent of inhibition was found to be proportional with the total concentration of phenolic compounds.
Assuntos
Amiloide/efeitos dos fármacos , Quimotripsina/efeitos dos fármacos , Extratos Vegetais/farmacologia , Achillea , Amiloide/metabolismo , Camomila , Quimotripsina/metabolismo , Datura stramonium , Humanos , Técnicas In Vitro , Melissa , Mentha piperita , Folhas de Planta , Agregação Patológica de Proteínas/metabolismo , Salvia , UrticaceaeRESUMO
Enzyme hydrolysates (trypsin, papain, pepsin, α-chymotrypsin, and pepsin-pancreatin) of Tinospora cordifolia stem proteins were analyzed for antioxidant efficacy by measuring (1) 1,1-diphenyl-2-picrylhydrazyl (DPPHâ¢) radical scavenging activity, (2) 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical scavenging capacity, and (3) Fe2+ chelation. Trypsin hydrolysate showed the strongest DPPH⢠scavenging, while α-chymotrypsin hydrolysate exhibited the highest ABTS+ scavenging and Fe2+ chelation. Undigested protein strongly inhibited the gastrointestinal enzymes, trypsin (50% inhibition at enzyme/substrate ratio = 1:6.9) and α-chymotrypsin (50% inhibition at enzyme/substrate ratio = 1:1.82), indicating the prolonged antioxidant effect after ingestion. Furthermore, gel filtration purified peptide fractions of papain hydrolysates exhibited a significantly higher ABTS+ and superoxide radical scavenging as compared to non-purified digests. Active fraction 9 showing the highest radical scavenging ability was further purified and confirmed by MALDI-TOF MS followed by MS/MS with probable dominant peptide sequences identified are VLYSTPVKMWEPGR, VITVVATAGSETMR, and HIGININSR. The obtained results revealed that free radical scavenging capacity of papain hydrolysates might be related to its consistently low molecular weight hydrophobic peptides.
Assuntos
Sequestradores de Radicais Livres/análise , Peptídeos/análise , Tinospora/química , Antioxidantes , Quimotripsina/metabolismo , Sequestradores de Radicais Livres/química , Hidrólise , Papaína/metabolismo , Pepsina A/metabolismo , Peptídeos/química , Extratos Vegetais/análise , Extratos Vegetais/química , Tripsina/metabolismoRESUMO
A novel chymotrypsin inhibitor, which detected in the seed of wild emmer wheat (Triticum dicoccoides), was purified by ion-exchange chromatography, affinity chromatography and Ultracentrifugation. On the basis of its specificity, this inhibitor was named WeCI (wild emmer chymotrypsin inhibitor). SDS-PAGE analysis displayed that the purified WeCI is a single chain polypeptide with a molecular weight of approximately 13kDa. The inhibition constants (Ki) for amylase and bovine pancreatic chymotrypsin were 1.12×10-9M and 2.41×10-9M, respectively. Automated sequencing and mass spectrometry analyses revealed that WeCI is a neutral monomeric protein consisting of 119 residues. In vitro, WeCI strongly suppressed bovine pancreatic chymotrypsin as well as chymotrypsin-like activities separated from the midgut of the beet armyworm Spodoptera exigua. No inhibitory activities were found against bovine pancreatic trypsin, bacterial subtilisin, or porcine pancreatic elastase. The primary structure of WeCI was markedly similar (46-95%) to those of several proteins belonging to the wheat crop chymotrypsin/α-amylase inhibitor superfamily and displayed the typical sequence motif of the α-amylase inhibitor-seed storage protein group. WeCI significantly inhibited the growth and development of Spodoptera exigua, dependent on inhibitor concentration. WeCI significantly increased the mortality rate of Spodoptera exigua and caused a significant decrease in its fertility.
Assuntos
Quimotripsina/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Spodoptera/efeitos dos fármacos , Triticum/química , Animais , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Israel , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/química , Extratos Vegetais/farmacologia , Sementes/química , Spodoptera/química , Spodoptera/enzimologia , Spodoptera/crescimento & desenvolvimento , SuínosRESUMO
The objective of the present study was to identify the proteome pattern, isolate and study the functions of selective proteins from Ferula asafoetida root exudate using chromatographic techniques. The root exudate proteins were fractionated using ion-exchange and gel filtration chromatography. A range of bioactive protein fractions were then separated in sufficient quantity which is the focus of this study. Based on studies, here we report three main proteins with molecular weights 14kDa, 27kDa, and 39kDa. The biological and pharmacological activities of both purified and unpurified proteins obtained were extensively studied to understand their significance. The study revelaed that 27kDa protein interestingly stabilized trypsin activity in 24h of time and retained about 64% of the enzyme activity. Analyses confirmed 40°C and pH 8.0 are the optimum temperature and pH respectively. The 39kDa protein remarkably increased the activity of chymotrypsin and the 14kDa protein showed anti-bacterial activity against Pseudomonas aeruginosa. Invariably all of the three purified proteins showed enhanced anti-oxidant activity. In conclusion, results here obtained suggested that the primary metabolites (proteins) in asafoetida are mainly responsible for its versatile biological and pharmacological activities.
Assuntos
Fracionamento Químico/métodos , Ferula/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Quimotripsina/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Peso Molecular , Proteínas de Plantas/química , Raízes de Plantas/química , Tripsina/metabolismoRESUMO
This experiment was conducted to investigate the effects of protected essential oils and organic acids mixture on poultry feeding. A total of 450 1-day-old Cobb 500 chicks were randomly allotted into three treatments with six replicates. Birds were offered a basal diet (C), basal diet with 0.15 g/kg enramycin premix (A) and basal diet with 0.30 g/kg protected essential oils and organic acids mixture product (P). The results showed that protected essential oils and organic acids mixture supplementation reduced average daily feed intake and ratio of feed to gain (F/G) at 22-42 days of age, and F/G during 1-42 days of age also declined (P < 0.05). Product supplementation improved spleen index, villus height and crypt depth of the jejunum at 42 days when compared with the control (P < 0.05). In addition, secretory immunoglobulin A level of ileal mucosa and trypsin and chymotrypsin activities of intestinal tract were higher in the P treatment. Bacterial sequence analysis of the intestinal tract revealed that protected essential oils and organic acids mixture supplementation changed gut microflora mainly in Lactobacillus. These data suggested that dietary mixture of organic acids and essential oils addition could be used in the poultry industry as an antibiotic growth promoter alternative.
Assuntos
Ácidos , Ração Animal , Galinhas/crescimento & desenvolvimento , Galinhas/microbiologia , Dieta/veterinária , Suplementos Nutricionais , Microbioma Gastrointestinal , Óleos Voláteis , Animais , Quimotripsina/metabolismo , Ingestão de Alimentos/fisiologia , Imunoglobulina A Secretora/metabolismo , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/metabolismo , Lactobacillus , Tripsina/metabolismo , Aumento de PesoRESUMO
BACKGROUND: Spirulina platensis is an excellent source of proteins (>60%) that can be hydrolyzed into bioactive peptides. RESULTS: In this study, whole proteins of Spirulina platensis were extracted and hydrolyzed using three gastrointestinal endopeptidases (pepsin, trypsin and chymotrypsin). Subsequently, gel filtration chromatography was employed to separate hydrolysates, and four fractions (Tr1-Tr4) were obtained. Among them, Tr2 showed the strongest anti-proliferation activities on three cancer cells (MCF-7, HepG-2 and SGC-7901), with IC50 values of <31.25, 36.42 and 48.25 µg mL-1 , respectively. Furthermore, a new peptide, HVLSRAPR, was identified from fraction Tr1. This peptide exhibited strong inhibition on HT-29 cancer cells with an IC50 value of 99.88 µg mL-1 . CONCLUSION: Taken together, these peptides possessed anti-proliferation activities on cancer cells and low cytotoxicity on normal cells, suggesting that they might serve as a natural anticancer agent for nutraceutical and pharmaceutical industries. © 2016 Society of Chemical Industry.
Assuntos
Proteínas de Algas/isolamento & purificação , Anticarcinógenos/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Descoberta de Drogas , Hepatócitos/efeitos dos fármacos , Neoplasias/prevenção & controle , Spirulina/química , Proteínas de Algas/efeitos adversos , Proteínas de Algas/química , Proteínas de Algas/farmacologia , Sequência de Aminoácidos , Anticarcinógenos/efeitos adversos , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Proteínas de Bactérias/efeitos adversos , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , China , Quimotripsina/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Suplementos Nutricionais/efeitos adversos , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Peso Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Oligopeptídeos/efeitos adversos , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Pepsina A/metabolismo , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Hidrolisados de Proteína/química , Tripsina/metabolismoAssuntos
Espectrometria de Massas/métodos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Quimotripsina/química , Quimotripsina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Inibidores de Proteases/farmacologia , Reprodutibilidade dos Testes , Tripsina/química , Tripsina/metabolismoRESUMO
An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.
Assuntos
Quimases/genética , Clonagem Molecular , DNA Complementar , Cifozoários/enzimologia , Cifozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Quimases/antagonistas & inibidores , Quimases/química , Quimases/metabolismo , Quimotripsina/metabolismo , Evolução Molecular , Cinética , Fluoreto de Fenilmetilsulfonil/farmacologia , Filogenia , Ligação Proteica , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 µM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the ß5/ß6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (Ki = 0.42 µM). The solution structure of 9 and 38 was solved by (1)H NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 µM) is slightly more potent than 9 (IC50 = 34.3 µM) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors.
Assuntos
Quimotripsina/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Conformação Proteica , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato , Interface Usuário-ComputadorRESUMO
Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonahm) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonahm reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.
Assuntos
Coagulação Sanguínea , Quimotripsina/metabolismo , Desbridamento/métodos , Larva/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Quimotripsina/química , Quimotripsina/genética , Drosophila melanogaster/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The objective of this study was to evaluate effects of dietary crude protein (CP) intake on ileal amino acid digestibilities and expression of genes for digestive enzymes in growing and finishing pigs. In Experiment 1, 18 growing pigs (average initial BW = 36.5 kg) were assigned randomly into one of three treatments (n = 6/treatment group) representing normal (18 % CP), low (15 % CP), and very low (12 % CP) protein intake. In Experiment 2, 18 finishing pigs (average initial BW = 62.3 kg) were allotted randomly into one of three treatments (n = 6/treatment group), representing normal (16 % CP), low (13 % CP) and very low (10 % CP) protein intake. In both experiments, diets with low and very low CP were supplemented with crystalline amino acids to achieve equal content of standardized ileal digestible Lys, Met, Thr, and Trp, and were provided to pigs ad libitum. Daily feed intake, BW, and feed/gain ratios were determined. At the end of each experiment, all pigs were slaughtered to collect pancreas, small-intestine samples, and terminal ileal chymes. Samples were used for determining expression of genes for digestive enzymes and ileal amino acid digestibilities. Growing pigs fed the 12 % CP and 15 % CP diets had lower final body weight (P < 0.01) and ADG (P < 0.0001) when compared with pigs fed the 18 % dietary CP diet. Growing pigs fed with the 12 % CP diet showed higher digestibilities for CP (P < 0.05), DM (P < 0.05), Lys (P < 0.0001), Met (P < 0.01), Cys (P < 0.01), Thr (P < 0.01), Trp (P < 0.05), Val (P < 0.05), Phe (P < 0.05), Ala (P < 0.05), Cys (P < 0.01), and Gly (P < 0.05) than those fed the 18 % CP diet. Finishing pigs fed the 16 % CP diet had a higher (P < 0.01) final body weight than those fed the 10 % CP diet. mRNA levels for digestive enzymes (trypsinogen, chymotrypsin B, and dipeptidases-II and III) differed among the three groups of pigs (P < 0.05), and no difference was noted in the genes expression between control group and lower CP group. These results indicated that a reduction of dietary CP by a six-percentage value limited the growth performance of growing-finishing pigs and that a low-protein diet supplemented with deficient amino acids could reduce the excretion of nitrogen into the environment without affecting weight gain.
Assuntos
Aminoácidos/metabolismo , Ração Animal/análise , Proteínas Alimentares/metabolismo , Íleo/metabolismo , Suínos/genética , Animais , Peso Corporal , Quimotripsina/genética , Quimotripsina/metabolismo , Dieta com Restrição de Proteínas , Proteínas Alimentares/análise , Digestão , Feminino , Íleo/enzimologia , Masculino , Suínos/crescimento & desenvolvimento , Suínos/metabolismo , Tripsinogênio/genética , Tripsinogênio/metabolismoRESUMO
The whey protein ß-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified ß-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified ß-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to ß-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.
Assuntos
Compostos Alílicos/química , Dissulfetos/química , Lactoglobulinas/química , Ácidos Sulfínicos/química , Quimotripsina/metabolismo , Dicroísmo Circular , Alho , Conformação Molecular , Pepsina A/metabolismo , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Compostos de Sulfidrila/química , Tripsina/metabolismoRESUMO
Linseed (Linun usitatissimum L.) is an important oilseed whose nutritional value can be impaired due to presence of antinutritional factors and low protein digestibility. Protein fractions from raw linseed meal were extracted, isolated and analyzed in vitro and in vivo. Globulins, the major protein fraction of linseed, showed low in vitro susceptibility to trypsin and chymotrypsin, but its in vivo digestibility was 93.2 %. Albumin fraction had high trypsin inhibition activity (5250 Inhibition Units g(-1)) and presented low molecular mass protein bands, similar to known trypsin inhibitors. Raw linseed consumption caused negative effects on rat growth and reduction of intestinal villi. Results indicate that raw linseed meal must not be used as an exclusive source of protein regardless of the major proteins have high digestibility; digestive enzymes inhibitors in raw linseed probably reduces the protein utilization.
Assuntos
Proteínas Alimentares/farmacologia , Digestão , Linho/química , Crescimento/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Inibidores da Tripsina/efeitos adversos , Tripsina/metabolismo , Animais , Disponibilidade Biológica , Quimotripsina/metabolismo , Proteínas Alimentares/metabolismo , Humanos , Intestinos/efeitos dos fármacos , Masculino , Peso Molecular , Valor Nutritivo , Extratos Vegetais , Proteínas de Plantas/metabolismo , Ratos Wistar , Sementes/químicaRESUMO
Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious peptides to enteric lactobacilli including L. gasseri after peptic digestion in the gastrointestinal tract. This is the first report showing peptide requirement of L. gasseri and efficacy of pepsinolysis on the growth of L. gasseri and its relatives in milk. This study would contribute to increasing usability of L. gasseri and its relatives as probiotics in dairy foods.
Assuntos
Lactobacillus/crescimento & desenvolvimento , Leite/microbiologia , Peptídeos/metabolismo , Aminoácidos/metabolismo , Animais , Caseínas/metabolismo , Bovinos , Quimotripsina/metabolismo , Trato Gastrointestinal/microbiologia , Leite/química , Nitrogênio/química , Pepsina A/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Probióticos/metabolismoRESUMO
A library of structurally distinct enaminones was synthesized using sonication or Ru(II) catalysis to couple primary, secondary, and tertiary thioamides with α-halocarbonyls or α-diazocarbonyls. Screening the library for proteasome inhibition using a luciferase-based assay identified seven structurally diverse compounds. Two of these molecules targeted luciferase, while the remaining five exhibited varying potency and specificity for the trypsin-like, chymotrypsin-like, or caspase-like protease activities of the proteasome. Physiological relevance was confirmed by showing these molecules inhibited proteasomal degradation of the full-length protein substrate p21cip1 expressed in tissue culture cells. A cell viability analysis revealed that the proteasome inhibitors differentially affected cell survival. Results indicate a subset of enaminones and precursor molecules identified in this study are good candidates for further development into novel proteasome inhibitors with potential therapeutic value.
Assuntos
Peptidomiméticos/química , Peptidomiméticos/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Animais , Quimotripsina/antagonistas & inibidores , Quimotripsina/química , Quimotripsina/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Cetonas/síntese química , Cetonas/química , Cetonas/farmacologia , Camundongos , Células NIH 3T3 , Peptidomiméticos/síntese química , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tripsina/química , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Seeds of cereals (Gramineae) are a rich source of serine proteinase inhibitors of most of the several inhibitor families. In the present study, trypsin and chymotrypsin inhibitory activities was detected in the seed flour extracts of three varieties of maize (Zea maize) and six varieties of sorghum (Sorghum bicolor). The maize variety, Hi Teck 2031 and the sorghum variety, Giza 10 were found to have higher trypsin and chymotrypsin inhibitory potentials compared to other tested varieties for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Maize and sorghum purified proteins showed a single band on SDS-PAGE corresponding to molecular mass of 20.0 and 15.2 kDa for maize and sorghum PIs respectively. The purified inhibitors were stable at temperature below 60 °C and were active at wide range of pH from 2 to 12 pH. The kinetic analysis revealed non-competitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation and mean pupal weight of S.littoralis where maize PI was more effective than sorghum PI. It may be concluded that maize and sorghum protease inhibitor gene(s) could be potential targets for future studies in developing insect resistant transgenic plants.