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At least 127 records · Page 7Linked to original sources
PubMed2004

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation.

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml x 200 mg (-1) feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml x 200 mg (-1) feed DM) for 24 h: y = 0.82 (+/- 0.07)x + 9.87 (+/-3.83), r2 = 0.82, n = 32, P < 0.0001. Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g x kg (-1) DM) and the uCP after incubation for 24 h with rumen fluid (y, g x kg(-1) DM): y = 0.95 (+/-0.10)x - 4.90 (+/-26.70), r2 = 0.75, n = 32, Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.

Animal Feed↗
PubMed2006

Development of an esophagus acellular matrix tissue scaffold.

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.

Animals↗
PubMed1993

Citrus flavonoids stimulate secretion by human colonic T84 cells.

Flavonoids, compounds containing a 2-phenylbenzo(gamma)pyrane nucleus, are universally distributed among vascular plants. Even though flavonoids are ingested in a normal diet in average quantities of 1 g daily, their effects on the digestive system have only been recently investigated. This study used an in vitro model of colonic secretion, monolayers of T84 colonic adenocarcinoma cells mounted in Ussing chambers, to demonstrate that 100 mumol/L of either tangeritin or nobiletin, polymethoxylated flavonoids contained in citrus fruits, stimulated sustained electrogenic chloride secretion with a maximal short-circuit current of 3.3 microA/cm2. In contrast, naringin and hesperidin, glycosylated citrus flavonoids, stimulated minimal secretion, suggesting that carbohydrate substitutions inhibited their secretory potential. The secretion stimulated by tangeritin and nobiletin was synergistic with carbachol but not with vasoactive intestinal peptide and was inhibited by barium chloride, bumetanide, H-89, and Cl- depletion. These properties suggest that tangeritin and nobiletin stimulated Cl- secretion via the cAMP pathway; however, these flavonoids did not stimulate cAMP production to the extent seen with vasoactive intestinal peptide. These flavonoids did not autooxidize, suggesting that reactive oxygen species did not mediate this secretion. These observations suggest that dietary citrus flavonoids may modulate colonic secretion, possibly through direct interaction with intracellular secretory pathways.

Adenocarcinoma↗
PubMedDate not supplied

The Protein Digestibility-Corrected Amino Acid Score (PDCAAS)--a concept for describing protein quality in foods and food ingredients: a critical review.

Protein Digestibility-Corrected Amino Score (PDCAAS) is discussed. PDCAAS is now widely used as a routine assay for protein quality evaluation, replacing the more traditional biological methods [e.g., measurement of the Protein Efficiency Ratio (PER) in rats]. PDCAAS is based on comparison of the essential amino acid content of a test protein with that of a reference essential amino acid pattern and a correction for differences in protein digestibility as determined using a rat assay. Although PDCAAS is a rapid and useful method, it often shows discrepancies when compared to PER values. These discrepancies relate to the following issues: uncertainty about the validity of reference patterns, invalidity of correction for fecal (versus ileal) digestibility, truncation of PDCAAS values to 100%, failure to obtain full biological response after supplementation of the limiting essential amino acid, discrepancies between protein and amino acid digestibility, effects of processing on protein quality, and effects of the presence of antinutritional factors in the matrix containing the protein. Part of the discrepancy between PDCAAS and PER can be overcome by modifications of PDCAAS. This article describes some proposed modifications and puts forward the suggestion that the rat protein fecal digestibility assay be replaced by an in vitro ileal amino acid digestibility assay based on a computer-controlled gastrointestinal model.

Amino Acids↗
PubMed1994

Dynamic construction of a neural network from multiple pattern generators in the lobster stomatogastric nervous system.

In the stomatogastric nervous system (STNS) of the lobster Homarus gammarus, the rhythmic discharge of a pair of identified modulatory neurons (PS cells) is able to construct de novo a functional network from neurons otherwise belonging to other functional networks. The PS interneurons are electrically coupled and possess endogenous oscillatory properties that can be activated synaptically by stimulation of an identified sensory pathway. PS neurons themselves project synaptically onto the three major neural networks (esophageal, gastric mill, and pyloric) of the STNS. When a PS is rhythmically active in vitro, either spontaneously (rarely) or in response to direct stimulation, it dramatically restructures the otherwise independent activity patterns of all three target networks. This functional reconfiguration elicited by a single cell does not rely on changes in neuronal allegiance to pre-existing circuits, or on a simple merger of these different circuits. Rather, PS is responsible for the creation of an entirely new motor rhythm in that, via its widespread synaptic connections, the interneuron is able to subjugate the ongoing activity of the three STNS circuits and selectively appropriate individual elements to its own intrinsic rhythm. In addition, PS excites motor neurons that innervate dilator muscles of a valve situated between the esophagus and the stomach. The reorganization of the regional foregut motor rhythms by the interneuron is therefore coordinated to the opening of this valve, which itself carries sensory receptors that have been found to activate bursting in PS. Our data suggest that the role of PS in massively restructuring stomatogastric output is to generate a unique motor pattern appropriate for swallowing-like behavior. In a wider context, moreover, the results demonstrate that a neural network may not exist as a predefined entity within the CNS, but may be dynamically assembled according to changing behavioral circumstances.

Animals↗
PubMed2000

Reduction of Campylobacter jejuni in a simulated chicken digestive tract by Lactobacilli cultures.

Studies were conducted to investigate the impact of a selected lactobacilli mixed culture on Campylobacter jejuni in simulated chicken digestive tract models. Veronal buffer solutions corresponding to the pH of successive segments of the chicken digestive tract were prepared. The lactobacilli mixtures were prepared by mixing four fresh lactobacilli cultures, including Lactobacillus acidophilus, Lactobacillus fermenentum, Lactobacillus crispatus, and Lactobacillus brevis. The C. jejuni and lactobacilli mixture were mixed with sterile poultry feed, and the previously prepared veronal buffer solutions were then added separately. The mixture was incubated at 41.1 degrees C for various lengths of time with periodic agitation. The feed passage time for five segments of the digestive tract were adopted: crop (pH 4.5), 30 min; proventriculus (pH 4.4), 15 min; gizzard (pH 2.6), 90 min; small intestine (pH 6.2), 90 min; and large intestine (pH 6.3), 15 min. The Campylobacter and lactobacilli were enumerated. An antagonistic effect on C. jejuni by the tested lactobacilli spp. was found in individual sections and the complete simulated digestive tract models. In the simulated complete chicken digestion system, no C. jejuni were found during the final incubation period when a lactobacilli mixture was present. The results of this in vitro study indicate the potential value of future in vivo studies.

Animals↗
PubMed1998

Structural aspects of the gastric H,K ATPase: the M5/M6 domain and alpha beta association.

This review summarizes some of the structural information that has been obtained on the gastric H,K ATPase. Methods such as tryptic digestion, site specific labeling and in vitro translation combine to provide a ten membrane segment model with however reservations as to the full transmembrane nature of M5 or M6. Labeling this region with the thiophilic luminal face reagent omeprazole provided cogent evidence that cys 813 but not cys 822 was labeled. On the other hand, cysteine mutagenesis provided evidence that removal of cys 813 did not affect inhibition of Rb transport by omeprazole whereas removal of cys 822 although not affecting ATPase activity abolished omeprazole inhibition of transport. A model to reconcile these data is presented where M5 and M6 although intramembranal are not transmembrane hairpin structures. Analysis of the region of alpha beta interaction by tryptic digestion and WGA chromatography to define those fragments of alpha that remain beta associated shows that leu 853 to arg 922 in the TM7-loop are a major region of association with the beta subunit. Yeast two hybrid analysis, when combined with these data and those from a chimeric construct, indicates that the sequence Q 907 to R 922 is the important element of interaction in the alpha subunit and no other extracytoplasmic domain was found to interact. Two regions of the beta subunit interact with this region of the alpha subunit between Q64 and N130 as well as A156 and R188. Apparently the beta subunit is folded around a small region of the large extracytoplasmic loop between TM7 and TM8, closer to TM8.

Amino Acid Sequence↗
PubMed1990

Intestinal hydrolysis of pyridoxal 5'-phosphate in vitro and in vivo in the rat. Effect of amino acids and oligopeptides.

The first step in the intestinal absorption of phosphorylated forms of vitamin B6 is intraluminal hydrolysis mediated by alkaline phosphatase. The present studies were performed to evaluate the effect of amino acids and oligopeptides, the products of protein digestion, on the hydrolysis of pyridoxal 5'-phosphate. Models utilized rats and included a cell-free in vitro system and the in vivo, single-pass, perfused jejunal segment. In vitro all amino acids and oligopeptides tested significantly inhibited pyridoxal 5'-phosphate decay (hydrolysis). The degree of inhibition of decay was dependent on the particular compound used, the concentration of that compound, and the pH of the medium. Similar effects for L-lysine concentration and perfusate pH were demonstrated in perfused segments in vivo; by contrast, L-lysine had no effect on pyridoxine uptake. These studies demonstrate that intraluminal hydrolysis of phosphorylated vitamin B6 may be modulated by yet other intraluminal constituents and conditions.

Alkaline Phosphatase↗
PubMed2003

Folic acid and 5-methyltetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model.

Dairy products are a potential matrix for folate fortification to enhance folate consumption in the Western world. Milk folate-binding proteins (FBP) are especially interesting because they seem to be involved in folate bioavailability. In this study, folate bioaccessibility was investigated using a dynamic computer-controlled gastrointestinal model [TNO gastrointestinal model (TIM)]. We used both ultrahigh temperature (UHT)-processed milk and pasteurized milk, differing in endogenous FBP concentrations and fortified with folic acid or 5-methyltetrahydrofolate (5-CH(3)-H(4)folate). To study FBP stability during gastrointestinal passage and the effect of additional FBP on folate bioaccessibility, FBP-fortified UHT and pasteurized milk products were also tested. Folate bioaccessibility and FBP stability were measured by taking samples along the compartments of the gastrointestinal model and measuring their folate and FBP concentrations. Folate bioaccessibility from folic acid-fortified milk products without additional FBP was 58-61%. This was lower (P < 0.05) than that of the 5-CH(3)-H(4)folate-fortified milk products (71%). Addition of FBP reduced (P < 0.05) folate bioaccessibility from folic acid-fortified milk (44-51%) but not from 5-CH(3)-H(4)folate-fortified milk products (72%). The residual FBP levels in the folic acid- and 5-CH(3)-H(4)folate-fortified milk products after gastrointestinal passage were 13-16% and 0-1%, respectively, of the starting amounts subjected to TIM. In conclusion, milk seems to be a suitable carrier for folate, because both folic acid and 5-CH(3)-H(4)folate are easily released from the matrix and available for absorption. However, our results suggest that folic acid remains partly bound to FBP during passage through the small intestine, which reduces the bioaccessibility of folic acid from milk in this model.

Animals↗
PubMed2004

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization.

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 x 10(6) cells per 35-mm plate and grown for 17 days in BGJ(b) medium supplemented with 10% fetal bovine serum, 50 microg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive (40)Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.

Alkaline Phosphatase↗
PubMed2003

An in vitro model system to predict the bioaccessibility of heterocyclic amines from a cooked meat matrix.

To understand the impact of variation in digestion parameters on the release of heterocyclic amines naturally formed during cooking, we developed and characterized a model system to assess the effect of amylase, pepsin, and pancreatin on digestion of well-done chicken. The amounts of MeIQx, DiMeIQx, IFP, and PhIP in the liquid portion of the digestate were compared to levels in the undigested meat to determine the percentage released (accessible fraction). Incubating the meat with amylase and pepsin did not change the accessibility of HAs when compared to incubation with water alone. In contrast, increasing amounts of pancreatin increased the accessibility up to 6.4-fold. Comparing the amounts of the HAs in the liquid to the solid fraction showed that there was more MeIQx, DiMeIQx, and IFP in the liquid fraction. In contrast, PhIP was equally divided between the solid and liquid fractions. For all four compounds, increasing the doneness of the meat decreased the amount of the compound accessible from the meat matrix. Our data suggest that bioaccessability of HAs may vary according to the polarity of the individual HAs and also may depend upon the doneness of the meat. These results may have important ramifications for human feeding studies, which assume that the total amount of each HA in the meat matrix is equally bioavailable.

Animals↗
PubMed2000

Investigation of an in vitro model for predicting the effect of food components on calcium availability from meals.

The availability of calcium from semi-synthetic meals and model food systems was studied by an in vitro method using equilibrium dialysis after simulating gastric digestion. Twelve different food components (phytate, oxalate, wheat fibre- and barley fibre-extract, D-sorbitol, xylitol, galactitol, casein, three different casein phosphopeptide preparations, and lactose) were added to the semi-synthetic meal and their effect on the relative index of availability of 45Ca was studied. Phytate, oxalate, wheat fibre-extract and barley fibre-extract, and, surprisingly, casein had a negative effect on availability of calcium. The most pronounced effect was observed for the addition of phytate. The other dietary factors had no effect on calcium availability. Therefore, this rapid and inexpensive in vitro model could be a useful screening method for factors affecting calcium absorption, especially inhibitory factors, in future human calcium bioavailability studies.

Biological Availability↗
PubMedDate not supplied

Cleaving proteins for the immune system.

Proteasomes are enzymes in eukaryotic cells which cut proteins marked for degradation into fragments. In mammals some of these fragments are used by the immune system to detect proteins of foreign, e.g. viral, origin. Hence reproducing, predicting and possibly understanding the cleaving patterns of proteasomes is an interesting theoretical problem and its solution would be beneficial for vaccine design. The equations connecting cut probabilities, fragment frequencies and so-called cut strengths are derived. A simple model for the time course of protein digestion is used to explain the problem of fragment competition and the possible deviation of in vitro fragment frequencies from those that can be expected in vivo. A family of neural network proteasome models for the reproduction and prediction of cleavage patterns is described in detail together with the webtool PAProC. The first model is based on the experimentally observed cleavage pattern, an intermediate model on the distinction between weak and strong cuts, and the most elaborate model uses quantitative data, i.e., fragment frequencies.

Amino Acid Sequence↗
PubMed1983

5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation.

Most eukaryotic mRNAs are characterized by the presence of a 5'-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNAs in the presence of cap analogues (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNAs. One way of explaining the importance of the 5'-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure, and thus allow the 40S ribosomal subunit to bind to the mRNA [Sonenberg, N., Guertin, D., Cleveland, D., & Trachsel, H. (1981) Cell (Cambridge, Mass.) 27, 563-572]. Our data and those of others indicate that the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogues or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5'-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases has been performed, and the experimental data have been compared with computer-generated models of AMV-4 secondary structure. In addition, the in vitro translatability of AMV-4 has been examined as a function of increasing potassium concentration, conditions that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5'-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5'-terminus can form weak base pairs with internal regions of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1985

Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea urchin eggs.

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of Strongylocentrotus purpuratus increased the free Ca2+ concentration required to elicit exocytosis. Although a small increase in the Ca2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca2+ (0.5 mM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). In this report we demonstrate that NEM inhibits by increasing the Ca2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.

Animals↗