RESUMEN
The intestine is a complex organ formed of different types of cell distributed in different layers of tissue. To minimize animal experiments, for decades, researchers have been trying to develop in vitro/ex vivo systems able to mimic the cellular diversity naturally found in the gut. Such models not only help our understanding of the gut physiology but also of intestinal toxicity. This review describes the different systems used to evaluate the effects of drugs/contaminants on intestinal functions and compares their advantages and limitations. The comparison showed that the organotypic model is the best available model to perform intestinal toxicity studies, including on human tissues.
Asunto(s)
Intestinos , Animales , HumanosRESUMEN
Trefoil factors (TFFs) are bioactive peptides expressed by several epithelia, including the intestine, where they regulate key functions such as tissue regeneration, barrier function and inflammation. Although food-associated mycotoxins, including deoxynivalenol (DON), are known to impact many intestinal functions, modulation of TFFs during mycotoxicosis has never been investigated. Here, we analyzed the effect of DON on TFFs expression using both human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that very low doses of DON (nanomolar range) inhibit the secretion of TFFs by human goblet cells (IC50 of 361, 387 and 243 nM for TFF1, 2 and 3, respectively) and prevent wound healing. RT-qPCR analysis demonstrated that the inhibitory effect of DON is related to a suppression of TFFs mRNA expression. Experiments conducted on porcine intestinal explants confirmed the results obtained on cells. Finally, the use of specific inhibitors of signal pathways demonstrated that DON-mediated suppression of TFFs expression mainly involved Protein Kinase R and the MAP kinases (MAPK) p38 and ERK1/2. Taken together, our results show for the first time that at very low doses, DON suppresses the expression and production of intestinal TFFs and alters wound healing. Given the critical role of TFFs in tissue repair, our results suggest that DON-mediated suppression of TFFs contributes to the alterations of intestinal integrity the caused by this toxin.
Asunto(s)
Expresión Génica/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Yeyuno/efectos de los fármacos , Factor Trefoil-3/genética , Tricotecenos/toxicidad , Animales , Células CACO-2 , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Caliciformes/inmunología , Células Caliciformes/metabolismo , Células HT29 , Humanos , Yeyuno/inmunología , Yeyuno/metabolismo , Porcinos , Factor Trefoil-3/metabolismoRESUMEN
Many studies have shown that flavan-3-ols inhibit mammalian alpha-amylases but the published IC50 and Ki values vary up to a thousand times. We therefore tested the effects of 6 pure flavan-3-ols-abundant in green tea-on the activity of pure porcine pancreatic alpha-amylase (PPA) under steady-state kinetic conditions. We used both amylose and maltopentaose as substrates, along with spectrophotometry and chromatography as analytical tools, respectively. A Docking approach was also used to probe the interaction between PPA and each flavan-3-ol. The results showed that the 6 flavan-3-ols inhibit amylose hydrolysis with Ki comprised between 7 and 34 µM, according to a mixed inhibition profile for gallocatechin gallate, and a competitive inhibition profile for the 5 other flavanols. Only the galloyl-containing flavan-3-ols inhibited the maltopentaose hydrolysis with a Ki of about 30 µM according to a noncompetitive profile. We conclude that dietary flavan-3-ols could inhibit starch digestion nonnegligibly. The results of the docking trials were concordant with the kinetic data and have noticeably revealed that the cis-flavan-3-ols epigallocatechin gallate and epicatechin gallate bind similarly to PPA, involving π-stacking with Trp59.
Asunto(s)
Flavonoides/farmacología , Almidón/metabolismo , Té/química , alfa-Amilasas/metabolismo , Amilosa/metabolismo , Animales , Catequina/análogos & derivados , Catequina/farmacología , Digestión , Hidrólisis , Concentración 50 Inhibidora , Modelos Moleculares , Oligosacáridos/metabolismo , Porcinos , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Staphylococcus aureus is one of the most infectious agents among staphylococcal bacteria. Currently many strains of S. aureus have developed resistance against available antibiotics. Therefore, the treatment of infections caused by them is a major challenge. During current study, desogestrel (1), a contraceptive drug, was found to be a potent growth inhibitor of drug resistant strains of S. aureus. Therefore, in search of new and effective agents against multi-drug resistant S. aureus strains, whole-cell bio-catalytic conversion of desogestrel (1) by Cunninghamella blakesleeana ATCC 8688A at pH 7.0 and 25⯰C was carried out, yielding three new metabolites, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6ß,15ß,17ß-triol (2), 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3ß,6ß,17ß-triol (3), and 13-ethyl-11-methylene-18,19-dinor-17α-pregn-20-yn-3α,5α,6ß,17ß-tetraol (4), along with a known metabolite, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6ß,17ß-dihydroxy-3-one (5). Among them, compounds 1-2 showed a potent activity against S. aureus EMRSA-17, S. aureus NCTC 13277 (MRSA-252), and S. aureus NCTC 13143, and clinically isolated Pakistani strain of S. aureus in an in vitro Microplate Alamar Blue Assay (MABA). Vancomycin was used as the standard drug in this assay. In addition, compound 1 also showed a significant activity against vancomycin-resistant S. aureus (VRSA) ATCC 700699. Compounds 1-5 were also evaluated against 3T3 normal cell line (mouse fibroblast) where they all were identified as non-cytotoxic. The present study thus provides new leads for the development of anti-bacterial drugs against MDR S. aureus.
Asunto(s)
Antibacterianos/farmacocinética , Anticonceptivos/farmacocinética , Cunninghamella/metabolismo , Desogestrel/farmacocinética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Biotransformación , Anticonceptivos/química , Anticonceptivos/metabolismo , Desogestrel/química , Desogestrel/metabolismo , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans.
Asunto(s)
Intestino Delgado/enzimología , Hígado/enzimología , Tricotecenos/metabolismo , Acetilación , Adulto , Anciano , Bacterias/enzimología , Células CACO-2 , Carboxilesterasa/metabolismo , Colon/enzimología , Colon/microbiología , Heces/microbiología , Femenino , Microbioma Gastrointestinal , Células Hep G2 , Humanos , Hidrólisis , Intestino Delgado/microbiología , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
The activity of breast milk BSDL was assayed with or without phospholipids as extra-intestinal effector candidates. Phosphatidic acid, lysophosphatidic acid and platelet activating factor but not phosphatidylcholine and lysophosphatidylcholine stimulated BSDL activity at least as efficiently as taurocholate. The apparent dissociation constants of PA and LPA at saturating concentrations of three different substrates were between 0.1 and 13.4 µM and that of PAF was below or equal to 200 pM. Kinetic data suggested the existence of at least one binding site for each of these effectors. PA, LPA and PAF are likely extra-intestinal modulators of BSDL activity.
Asunto(s)
Lipasa/química , Lisofosfolípidos/química , Leche Humana/enzimología , Factor de Activación Plaquetaria/química , Sitios de Unión , Activación Enzimática , Pruebas de Enzimas , Femenino , Humanos , Cinética , Lisofosfatidilcolinas/química , Leche Humana/química , Ácidos Fosfatidicos/química , Fosfatidilcolinas/química , Unión Proteica , Ácido Taurocólico/químicaRESUMEN
1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.
Asunto(s)
Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos Cíclicos/química , Bicarbonato de Sodio/química , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Aminoácidos Cíclicos/metabolismo , Aminoácidos Cíclicos/farmacología , Ácido Ascórbico/química , Ácido Ascórbico/farmacología , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Enlace de Hidrógeno , Cinética , Solanum lycopersicum/enzimología , Modelos Moleculares , Estructura Molecular , Ácidos Fosforosos/química , Ácidos Fosforosos/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bicarbonato de Sodio/metabolismo , Bicarbonato de Sodio/farmacología , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
The digestion of polysaccharides from the wheat cultivars Caphorn and Isengrain was investigated, and the efficiency of an enzyme preparation was tested using the TNO gastrointestinal model (TIM-1). The apparent digestibility (AD) of carbohydrates was determined based on the measurement of organic matter (OM), total monosaccharides, reducing ends (RE) and end products (EP: glucose, maltose and xylobiose). The AD of the OM from Caphorn and Isengrain measured using caecectomised cockerels did not differ from that measured using TIM-1: 72.0 (SD 2.6) v. 70.6 (SD 0.6) % for Caphorn (P = 0.580) and 73.0 (SD 2.3) v. 71.1 (SD 1.9) % for Isengrain (P = 0.252). After the 6 h TIM-1 digestion, 41.4-58.9 % of the OM, RE and EP were recovered from the jejunal compartment and 18.3-27.1 % from the ileal compartment, while ileal deliveries and digestive residues constituted the remainder. A commercial enzyme cocktail tested at 0.2 µl/g of wheat improved TIM-1 digestibility of Caphorn and Isengrain polysaccharides: 3.9 % (P = 0.0203) and 3.4 % (P = 0.0058) based on the OM; 9.7 % (P < 0.0001) and 3.1 % (P = 0.031) based on the total glucose; 47.2 % (P < 0.0001) and 14.2 % (P = 0.0004) based on the RE, respectively. The enzyme cocktail improved the release of the EP for Caphorn (3.8 %, P = 0.008) but not for Isengrain ( − 0.8 %, P = 0.561). The higher efficiency of the enzyme supplementation on the digestion of Caphorn polysaccharides compared with Isengrain seems to be linked to the higher soluble carbohydrate contents and/or less ramified arabinoxylan of Caphorn.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales/efectos de los fármacos , Dieta , Suplementos Dietéticos , Digestión/efectos de los fármacos , Enzimas/farmacología , Polisacáridos/metabolismo , Triticum/química , Alimentación Animal , Animales , Pollos , Glucosa/metabolismo , Íleon/metabolismo , Yeyuno/metabolismo , Masculino , Modelos Biológicos , Especificidad de la Especie , Triticum/clasificaciónRESUMEN
BACKGROUND: The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. RESULTS: High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. CONCLUSION: Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.
Asunto(s)
Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Oligosacáridos/metabolismo , Penicillium/enzimología , Xilanos/metabolismo , Secuencia de Aminoácidos , Endo-1,4-beta Xilanasas/genética , Estabilidad de Enzimas , Cinética , Datos de Secuencia Molecular , Peso Molecular , Penicillium/química , Penicillium/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (â¼3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 µM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.
Asunto(s)
Esterol Esterasa/metabolismo , Ácido Taurocólico/metabolismo , Ácidos y Sales Biliares/metabolismo , Catálisis , Activación Enzimática , Ésteres/química , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Micelas , Leche Humana/enzimología , Nitrofenoles/química , Especificidad por SustratoRESUMEN
Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.
Asunto(s)
Células CACO-2 , Micelas , Vitamina E/química , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/química , Absorción , Células CACO-2/química , Células CACO-2/metabolismo , Enterocitos/química , Enterocitos/metabolismo , Humanos , Hidrólisis , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Microscopía Confocal , Modelos Biológicos , Solubilidad , Esterol Esterasa/química , Esterol Esterasa/metabolismo , Ácido Taurocólico/química , Ácido Taurocólico/metabolismo , Factores de Tiempo , Tocoferoles , Vitamina E/metabolismo , alfa-Tocoferol/metabolismoRESUMEN
In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.
Asunto(s)
Membrana Celular/química , Microdominios de Membrana/química , Obesidad/metabolismo , Páncreas Exocrino/química , Proteómica , Animales , Membrana Celular/ultraestructura , Dieta , Masculino , Microdominios de Membrana/metabolismo , Páncreas Exocrino/metabolismo , Proteínas/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Two genes encoding family 11 endo-(1,4)-beta-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.
Asunto(s)
Endo-1,4-beta Xilanasas/fisiología , Glicósido Hidrolasas/fisiología , Penicillium/enzimología , Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Endo-1,4-beta Xilanasas/biosíntesis , Endo-1,4-beta Xilanasas/genética , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Cinética , Especificidad por Sustrato/fisiologíaRESUMEN
Although data on the behavior of tocopheryl acetate (Tac) in the gut is rare, some studies show that this ester is hydrolyzed in the intestine and the released tocopherol (Tol) is absorbed. An HPLC method is published for the simultaneous analysis of Tol and Tac in mixed micelles. This study shows how this method is used for the measurement of esterase activity on Tac using commercial cholesterol esterase. The rate of hydrolysis is determined in less than 20 min on the basis of both Tac disappearance and Tol appearance. The present method is useful for investigating esterase activities on Tac in model systems that mimick the absorption environment or in any biological or nonbiological medium.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Esterol Esterasa/metabolismo , alfa-Tocoferol/análogos & derivados , Absorción Intestinal , Tocoferoles/análisis , alfa-Tocoferol/análisis , alfa-Tocoferol/metabolismoRESUMEN
This report improves analytical procedures to investigate the behaviour of the two Vitamin E forms, alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac), in model systems mimicking the intestinal medium. We describe how to prepare mixed micelles as vehicle for Tac and Tol and the HPLC method for their quantification in the micelles. Tac and Tol were extracted using ethanol-hexane-drying procedure, whereas the separation and detection were performed in methanol and by UV method, respectively. Both compounds were eluted in less than 4 min. In the range between 1.7 microM and 54 microM of Tac or Tol in the micelles, their recovery were 89% and 81%, respectively, with correlation coefficient over 0.99 and R.S.D. of less than 7.2% in all cases. Limits of detection and quantification for Tac and Tol in mixed micelles ranged between 1 microM and 2 microM and between 3 microM and 5 microM, respectively. The behaviours of Tac and Tol were quite different during the extraction procedure and both were influenced by the vitamin concentration and the relative volume of organic solvents.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Micelas , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/análisis , Estabilidad de Medicamentos , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta , Tocoferoles , alfa-Tocoferol/aislamiento & purificaciónRESUMEN
Pig pancreatic alpha-amylase (PPA), an enzyme belonging to the alpha-amylase family, is involved in the degradation of starch. Like some other members of this family, PPA requires chloride to reach maximum activity levels. To further explain the mechanism of chloride activation, a crystal of wild-type PPA soaked with maltopentaose using a chloride-free buffer was analyzed by X-ray crystallography. A conspicuous reorientation of the acid/base catalyst Glu233 residue was found to occur. The structural results, along with kinetic data, show that the acid/base catalyst is maintained in the active site, in an optimum position, pointing toward the scissile bond-atom, due to the presence of chloride ions. The present study therefore explains the mechanism of PPA activation by chloride ions.