RESUMEN
Myrosinase is a cytosolic plant enzyme present in daikon ( Raphanus sativus, Japanese white radish) roots that hydrolyzes 4-methylthio-3-butenyl glucosinolate (MTBGLS) into the natural pungent agent 4-methylthio-3-butenyl isothiocyanate (MTBITC), which possesses antimicrobial, antimutagenic, and anticarcinogenic properties. The concentration of MTBGLS, myrosinase activity, and production of MTBITC in seven daikon varieties (one conventional and six heirlooms) were determined to rank the activity of the glucosinolate-myrosinase system and identify critical factors influencing the production of MTBITC. The six heirloom varieties produced 2.0-11.5 times higher levels of MTBITC as compared to the conventional variety, Aokubi, which is consumed by the present Japanese population. The myrosinase was located exclusively in the outer epidermal layer in Aokubi, and MTBGLS was widely distributed throughout the root tissue. Although the skin is a potentially rich source of myrosinase in Aokubi, the skin is usually peeled off in the current practice of preparing daikon for cooking. New practices are therefore proposed for the preparation of daikon tubers that eliminate the peeling of the skin to avoid removing the enzyme needed to convert MTBGLS to the health-beneficial MTBITC. It is also concluded that the consumption of heirloom daikon varieties in addition to changes in food preparation will optimize the health benefits of daikon.
Asunto(s)
Glucosinolatos/metabolismo , Glicósido Hidrolasas/metabolismo , Raphanus/enzimología , Glucosinolatos/análisis , Glicósido Hidrolasas/genética , Isotiocianatos/metabolismo , Raíces de Plantas/química , Raíces de Plantas/enzimología , ARN Mensajero/análisis , Raphanus/químicaRESUMEN
Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.
Asunto(s)
Ácidos y Sales Biliares/química , Heces/química , Glycine max/química , Proteínas de Plantas/química , Animales , Masculino , Péptidos/aislamiento & purificación , Proteínas de Plantas/farmacología , Ratas , Ratas Endogámicas F344RESUMEN
In order to determine pyroglutamic acid levels in plasma, we developed a method based on precolumn derivatization of the carboxyl group of pyroglutamic acid with 2-nitrophenylhydrazine. Eight-week-old male SD strain rats were administered 200 mg of an acidic peptide fraction obtained from a commercial wheat gluten hydrolysate containing 0.63 mmol/g pyroglutamyl peptide. After administration, significant amounts of free pyroglutamic acid were observed in the ethanol-soluble fraction of the plasma from the portal vein. In addition, pyroglutamate aminopeptidase digestion of the ethanol-soluble fraction liberated significant amounts of pyroglutamic acid, which indicated the presence of the pyroglutamyl peptide. The presence of the pyroglutamyl peptide in the plasma was further confirmed by size exclusion chromatography. The levels of free and peptide forms of pyroglutamic acid increased significantly and reached a maximum (approximately 40 nmol/mL) at 15 and 30 min after administration, respectively.
Asunto(s)
Glútenes/administración & dosificación , Glútenes/química , Péptidos/administración & dosificación , Ácido Pirrolidona Carboxílico/administración & dosificación , Ácido Pirrolidona Carboxílico/sangre , Triticum/química , Animales , Cromatografía en Gel , Hidrólisis , Masculino , Péptidos/química , Péptidos/metabolismo , Vena Porta , Piroglutamil-Peptidasa I/metabolismo , Ácido Pirrolidona Carboxílico/química , RatasRESUMEN
For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteínas en la Dieta/sangre , Alimentos , Péptidos/sangre , Tiocianatos , Cromatografía en Gel , Indicadores y Reactivos , IsotiocianatosRESUMEN
An apparatus for continuous fractionation of peptides on the basis of amphoteric nature of sample peptides was developed. A tank (66.5 cm x 8 cm x 8 cm, L x W x H) was divided into 12 compartments by a thin agarose gel layer. A drain tube (5.5 cm in length and 0.7 cm in i.d.) was fixed through the bottom of each compartment to give a height of 4 cm from the bottom. The tank with 12 compartments and electrodes was referred to as an autofocusing unit. The peptide solution or water was delivered to the sample compartments of the first unit. The solutions drained from the first unit were successively delivered to the second and third units. To the electrodes of three units, a direct electric current was applied. By using the present apparatus, peptides in casein hydrolysate can be continuously fractionated at least for 5 h. Better resolution was obtained in the second and third units.
Asunto(s)
Caseínas/química , Caseínas/metabolismo , Focalización Isoeléctrica/instrumentación , Péptidos/análisis , Concentración de Iones de Hidrógeno , Hidrólisis , Focalización Isoeléctrica/métodosRESUMEN
In the present study, we identified several food-derived collagen peptides in human blood after oral ingestion of some gelatin hydrolysates. Healthy human volunteers ingested the gelatin hydrolysates (9.4-23 g) from porcine skin, chicken feet, and cartilage after 12 h of fasting. Negligible amounts of the peptide form of hydroxyproline (Hyp) were observed in human blood before the ingestion. After the oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20-60 nmol/mL of plasma) after 1-2 h and then decreased to half of the maximum level at 4 h after the ingestion. Major constituents of food-derived collagen peptides in human serum and plasma were identified as Pro-Hyp. In addition, small but significant amounts of Ala-Hyp, Ala-Hyp-Gly, Pro-Hyp-Gly, Leu-Hyp, Ile-Hyp, and Phe-Hyp were contained.
Asunto(s)
Colágeno/sangre , Proteínas en la Dieta/administración & dosificación , Gelatina/administración & dosificación , Péptidos/sangre , Hidrolisados de Proteína/administración & dosificación , Adulto , Animales , Pollos , Cromatografía Líquida de Alta Presión , Gelatina/farmacocinética , Humanos , Hidrolisados de Proteína/farmacocinética , PorcinosRESUMEN
The objective of this study is to clarify the difference in susceptibility to protease digestion by kiwifruit juice between collagen domains under different conditions. In addition, the effect of pre-treatment with kiwifruit juice on collagen in meat during cooking processes was examined. Kiwifruit juice can degrade denatured collagen, but it can not cleave the triple helical domain of collagen. Thus, kiwifruit juice does not have collagenase activity. On the other hand, the cross-linked subunits of acid-soluble collagen were converted to monomeric subunits by kiwifruit juice treatment at acidic pH, suggesting that the globular domains, in which cross-links preferentially occur, can be degraded by kiwifruit juice. The pre-treatment with kiwifruit juice significantly decreased the shear force of connective tissue in comparison with other pre-treatments without protease activity, but inversely increased the liberation of collagen-related peptides in the outer solution by heating processes at 50 and 70 degrees C or by a shorter heating time at 100 degrees C. This can be explained by the protease-mediated degradation of globular domains. However, this effect was not observed with a prolonged heating period at 100 degrees C, and the liberation of collagen-related peptides by pre-treatment with kiwifruit juice at 100 degrees C was less than that at 70 degrees C for all heating periods. Thus, it can be suggested that the pre-treatment with kiwifruit juice might be useful in meat softening under vacuum-cooking and grilling, but not under stewing.
Asunto(s)
Actinidia/enzimología , Colágeno/metabolismo , Frutas/enzimología , Carne/análisis , Péptido Hidrolasas/metabolismo , Animales , Bebidas , Bovinos , Colágeno/química , Tejido Conectivo/metabolismo , Culinaria , Calor , Desnaturalización Proteica , Estructura Secundaria de Proteína , SolubilidadRESUMEN
It has been demonstrated that peptides in enzymatic hydrolysates of proteins can be fractionated on the basis of the amphoteric nature of the sample peptides, by a laboratory-scale isoelectric focusing apparatus, without adding a chemically synthesized carrier ampholyte. This approach is referred to as autofocusing. In the present study, a large-scale (up to 50 L) autofocusing apparatus was developed and tested. A tank (125 cm x 25 cm x 20 cm) was divided into 12 compartments by 11 plates, each with a window covered in a thin agarose gel layer supported by a nylon screen (100 mesh). The compartments at both ends were filled with 0.1 N phosphoric acid (anode) and 0.1 N NaOH (cathode), respectively, functioning as electrode compartments. The remaining compartments were used for sample compartments. Autofocusing was carried out at constant voltage according to two different methods. In method 1, all sample compartments were filled with a 1% water solution of casein or milk whey protein hydrolysates. In method 2, two compartments located in the center of the tank were filled with 5% sample solution and the others were filled with deionized water. Compositional and sequence analyses of the autofocusing fractions revealed that peptides in the two hydrolysates can be fractionated within 24 h by the present apparatus. Better fractionation was obtained by method 2, whereas enrichment of some peptides occurred by using method 1.
Asunto(s)
Proteínas en la Dieta/análisis , Proteínas en la Dieta/metabolismo , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Péptidos/análisis , Aminoácidos/análisis , Caseínas/química , Caseínas/metabolismo , Fraccionamiento Químico , Hidrólisis , Proteínas de la Leche/química , Proteínas de la Leche/metabolismo , Proteína de Suero de LecheRESUMEN
We purified compounds from the husks of psyllium seeds (Plantago ovata Forsk; desert Indian wheat), beginning with an ethanol extraction then followed by HP-20 and silica gel chromatography, which restored gap junctional intercellular communication (GJIC) in v-Ha-ras transfected rat liver epithelial WB-F344 cell line (WB-Ha-ras). GJIC was assessed by a scrape loading dye transfer assay. The active compound was identified as beta-sitosterol based on gas chromatography retention times and electron ionization mass spectroscopy (EI-MS) spectrum of authentic beta-sitosterol. Authentic beta-sitosterol restored GJIC in the tumorigenic WB-Ha-ras GJIC-deficient cells at a dose of 2.4 microM. In addition, a similar phytosterol, stigmasterol, also restored GJIC, albeit at a lower activity. beta-sitosterol and stigmasterol increased the level of connexin43 protein (Cx43) and restored phosphorylation of Cx43 to levels similar to the parental nontransfected cell line. We concluded that the restoration of intercellular communication in the GJIC-deficient, tumorigenic WB-Ha-ras cell line by the ethanol soluble fraction of psyllium seed husks is largely due to the presence of the phytosterol, beta-sitosterol. We discuss implications for dietary modulation of cancer by beta-sitosterol.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Genes ras/fisiología , Hepatocitos/efectos de los fármacos , Psyllium , Sitoesteroles/farmacología , Animales , Western Blotting/métodos , Células Cultivadas/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Hepatocitos/patología , Hipolipemiantes/química , Hipolipemiantes/farmacología , Técnicas In Vitro , Masculino , Ratas , Semillas/química , Sitoesteroles/químicaRESUMEN
Kaempferol induces differentiation in partially differentiated colon cancer cells which express low levels of connexin43 protein and connexin43 mRNA (KNC cells). Differentiation was observed as changes in cell morphology and the activity of alkaline phosphatase. Increased differentiation in kaempferol-treated KNC cells correlated with restoration of gap junctional intercellular communication (GJIC), increased levels of connexin43 protein and its phosphorylation status. Phosphorylation (activation) of Stat3 and Erk was also reduced by kaempferol. An inhibitor of Stat3 phosphorylation also induced morphological changes in KNC cells similar to those in kaempferol-treated cells, suggesting that kaempferol-induced differentiation may be mediated by inhibition of Stat3 phosphorylation. These effects were not observed in HCT116 cells, a poorly differentiated colon cancer cell line deficient in expression of connexin43 mRNA and connexin43 protein. In conclusion, kaempferol might function as an anticancer agent by re-establishing GJIC through enhancement of the expression and phosphorylation of connexin43 protein in a tumorigenic colon cancer cell line that already expresses connexin43 mRNA via a Stat3-dependent mechanism. In contrast, kaempferol had no effect in a tumorigenic colon cancer cell line that did not express connexin43 mRNA and was deficient in GJIC.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Uniones Comunicantes/efectos de los fármacos , Quempferoles/farmacología , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Línea Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/metabolismo , Conexina 43/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Tirfostinos/farmacologíaRESUMEN
Aberrant transcription of the fragile histidine triad (FHIT) gene was investigated in intrahepatic cholangiocellular carcinomas (ICCs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female Syrian golden hamsters. The animals received 70 mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and administration of 20 mg/kg BOP. A total of 14 ICCs were obtained 10 weeks after the beginning of the experiment and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription-polymerase chain reaction analysis. Aberrant transcripts were detected in four out of 14 ICCs (28.6%), as absence in the regions of nucleotides (nt) -75 to 279, nt -75 to 348 and nt -75 to 447. These results suggest that alteration of the FHIT gene may play a role in a small fraction of ICCs induced by BOP in the hamster.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos/patología , Carcinógenos/toxicidad , Colangiocarcinoma/genética , Proteínas de Neoplasias/genética , Nitrosaminas/toxicidad , Transcripción Genética/efectos de los fármacos , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Neoplasias de los Conductos Biliares/inducido químicamente , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/inducido químicamente , Colangiocarcinoma/patología , Cricetinae , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Mesocricetus , Proteínas de Neoplasias/metabolismo , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [alpha1(V)](2)alpha2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.
Asunto(s)
Cromatografía DEAE-Celulosa/métodos , Cromatografía Líquida de Alta Presión/métodos , Colágeno Tipo V/aislamiento & purificación , Tejido Conectivo/metabolismo , Mucosa Intestinal/metabolismo , Pepsina A/metabolismo , Animales , PorcinosRESUMEN
An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.