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Digestion Products (digestion + products)
Selected AbstractsHow do enamelysin and kallikrein 4 process the 32-kDa enamelin?EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Yasuo Yamakoshi The activities of two proteases , enamelysin (MMP-20) and kallikrein 4 (KLK4) , are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. [source] Using the polymer partitioning method to probe the thermodynamic activity of poorly water-soluble drugs solubilized in model lipid digestion productsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2003Ben J. Boyd Abstract The thermodynamic activity of solubilized drug is an important determinant of the extent of absorption of lipophilic drugs from the gastrointestinal tract. In this study, the polymer partitioning method was evaluated for its use in the determination of the thermodynamic activity of lipophilic drugs when solubilized in colloidal digestion products, using drug in dilute solution as a reference ideal solution. The lipophilic drugs griseofulvin, diazepam, and danazol partitioned into a polymeric receiver phase from non-micellar solution as a function of drug lipophilicity. The concentration of drug that partitioned into the polymer was linearly proportional to the concentration of free drug in solution, and this allowed the measured partition coefficient to be utilized as an indicator of the drug activity coefficient. The addition of a solubilizing species such as bile salt micelles caused a reduction in drug activity of a similar magnitude to that predicted from micelle equilibrium solubility data in the identical micellar solutions. The addition of micelle swelling lipids such as lecithin and fatty acids resulted in further reductions in activity coefficient. The ability to measure drug activity in model digestive systems has potential for application in the rational development of improved lipid-based formulations of poorly water-soluble drugs for oral administration. © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1262,1271, 2003 [source] Determination of the small cell lung cancer associated biomarker pro-gastrin-releasing peptide (ProGRP) using LC-MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2007Bjørn Winther Abstract Small cell lung cancer is a rapidly growing neoplasm with high mortality. A recently discovered biomarker, pro-gastrin-releasing peptide (ProGRP), is used as a specific diagnostic marker for the disease. The present methods of quantification are based on the immunoassay techniques RIA and ELISA. Our object was to develop an LC-MS method for the detection and quantification of ProGRP using specific tryptic digestion products from the recombinant peptide ProGRP (31,98), a sequence common to three isoforms of ProGRP. The conditions for enzymatic cleavage were optimized and MS compatibility was obtained. Digestion of ProGRP (31,98) yielded an array of peptide products and these were evaluated for further method development. The peptide product NLLGLIEAK proved to be the preferable candidate to monitor ProGRP due to signal intensity, column retention, and peptide specificity. The identity of this product was verified by means of LC-MS/MS and the linearity of the calibration curve evaluated. LOD was calculated to be 13.9 pg on column (O.C.). Plasma samples spiked with ProGRP (31,98) prior to digestion verified the suitability of this digest product for the determination of ProGRP. LC-MS may prove to be a valuable tool for biomarker mediated diagnosis in the future. [source] Optimization of a gastrointestinal model applicable to the evaluation of bioaccessibility in fish feedsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2009Mariam Hamdan Abstract BACKGROUND: Although several types of in vitro digestibility assays have been applied to nutritional evaluation of feeds for aquatic organisms, all of them are based on the use of closed reactors and do not simulate the gastric phase of the digestion. Our objective was to evaluate the suitability of a gastrointestinal model based on the use of a digestion cell provided by a semi-permeable reaction chamber, which allows continuous removal of digestion products as they are produced. We tested the effects of some factors, like the inclusion of a gastric phase, reaction temperature or bile salts on the hydrolysis of feed proteins by fish enzymes. RESULTS: We found that the most suitable operational conditions to simulate the digestion process must include a short acid pre-digestion as well as the use of bile salts in the reaction mixture. Acid pre-digestion resulted in a significant increase in the liberation of amino acids which represented more than twice that measured when using a single phase. The addition of two bile salts (45 µmol L,1 sodium taurocholate + chenodesoxycolate) resulted in almost a threefold increase in the hydrolysis of feed protein. The use of the described open system also allows the evaluation of carbohydrate hydrolysis as well as determination of residual undigested matter, in a similar manner to that carried out in ruminants with the DAISY system. CONCLUSION: Results suggest the system can be a very suitable model for evaluation of bioaccessibility in fish feeds. Copyright © 2009 Society of Chemical Industry [source] Identification of endo- and exo-polygalacturonase activity in Lygus hesperus (Knight) salivary glands,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Maria de la Paz Celorio-Mancera Abstract Polygalacturonase (PG) activity found in the salivary gland apparatus of the western tarnished plant bug (WTPB, Lygus hesperus Knight) has been thought to be the main chemical cause of the damage inflicted by this mirid when feeding on its plant hosts. Early viscosity and thermal stability studies of the PG activity in L. hesperus protein extracts were difficult to interpret. Thus, it has been suggested that one or more PG protein(s) with different hydrolytic modes of action are produced by this mirid. In order to understand the quantitative complexity of the WTPB salivary PG activity, PG purification from a protein extract from salivary glands excised from L. hesperus insects was performed using affinity and ion exchange chromatography. To elucidate the qualitative complexity of the purified PGs, the digestion products generated by the PGs were separated using high performance anion exchange chromatography with pulsed amperometric detection. At least five PG proteins were detected; these differing in terms of their glycosylation, mass-to-charge ratios, and/or molecular mass. The characterization of the products generated by these PGs showed that endo- and exo-acting PGs are produced by WTPB. Although none of the PGs was purified to homogeneity, the present work provides biochemical evidence of a multiplicity of PGs that degrade the pectin component of the plant tissue in different fashions. The implications of these findings affect the understanding of WTPB feeding damage and, potentially, help identify ways to control this important crop pest. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Gastro-duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potentialCLINICAL & EXPERIMENTAL ALLERGY, Issue 10 2006T. Eiwegger Summary Background The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. Methods An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. Results In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments Mr L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P<0,05; Wilxocon's signed rank test). The Th-2 type cytokine polarization of T cells from peanut allergic donors (IFN-,/IL-13 ratio and IFN-,/IL-4 ratio of CFSElow CD4+ T cells) remained unchanged regardless of the level of digestion. Histamine release of basophils from peanut allergic individuals was induced to the same extent by native Ara h 1 and its digestion products. Conclusion Gastro-duodenal digestion fragments of Ara h 1 retain T cell stimulatory and IgE-binding and cross-linking properties of the intact protein. [source] | |||||||||||||