Impact of gastric pH profiles on the proteolytic digestion of mixed ßlg-Xanthan biopolymer gels.

The understanding of how foods are digested and metabolised is essential to enable the design/selection of foods as part of a balanced diet. Essential to this endeavour is the development of appropriate biorelevant in vitro digestion tools. In this work, the influence of gastric pH profile on the in vitro digestion of mixtures of ß-lactoglobulin (ßlg) and xanthan gum prior to and after heat induced gelation was investigated. A conventional highly acidic (pH 1.9) gastric pH profile was compared to two dynamic gastric pH profiles (initial pH of 6.0 vs. 5.2 and H(+) secretion rates of 60 vs. 36 mmol h(-1)) designed to mimic the changes in gastric pH observed during clinical trials with high protein meals. In moving away from the pH 1.9 model, to a pH profile reflecting in vivo conditions, the initial rate and degree of protein digestion halved during the first 45 minutes. After 90 minutes of gastric digestion, all three pH profiles caused similar extents of protein digestion. Given that 50% gastric emptying times of (test) meals are in range of 30-90 min, it would seem highly relevant to use a dynamic pH gastric model rather than a pH 1.9 (USP) or pH 3 model (INFOGEST) in assessing the impact of food structuring approaches on protein digestion. The impact that heat induced gelation had on the degree of gel digestion by pepsin was also investigated. Surprisingly, it was found that heat induced gelation of ßlg-xanthan mixtures at 70-90 °C for 20 minutes lead to a considerable decrease in the rate of proteolysis, which contrasts many studies of dispersed aggregates and gels of ßlg alone whose heating accelerates pepsin activity due to unfolding. In the present case, the formation of a dense protein network created a fine pore structure which restricted pepsin access into the gel thereby slowing proteolysis. This work not only has implications for the in vitro assessment of protein digestion, but also highlights how protein digestion might be slowed, learnings that might have an influence on the design of foods as part of a satisfying balanced diet.

The nutritional status and factors contributing to malnutrition in children with chronic pancreatitis.

BACKGROUND/OBJECTIVES: The present study was undertaken to determine the prevalence of malnutrition among children with chronic pancreatitis (CP). Furthermore, we aimed to evaluate the relationship between etiological factors of CP, its clinical characteristics, and the severity of malnutrition. METHODS: The study included 208 children with CP (113 girls and 95 boys; mean age: 10.8 years, range: 1.6-18 years), hospitalized at our center between 1988 and 2012. The severity of malnutrition was graded on the basis of Cole's ratios, and its prevalence was analyzed according to the etiological factors of pancreatitis. Moreover, the analysis of discrimination was performed to identify the factors contributing to malnutrition among the following variables: age at CP onset, duration of CP, number of CP exacerbations, the number of ERCPs performed, the grade of pancreatic damage documented on imaging, co-occurrence of diabetes, and the results of 72-h fecal fat quantification. RESULTS: We documented features of malnutrition in 52 (25%) children with CP, including 36 (17.3%) patients with moderate malnutrition, and 2 (0.96%) with severe malnutrition. There was no significant difference in the prevalence of malnutrition between groups of patients with various etiological factors of chronic pancreatitis. The age at CP onset showed the best discrimination ability of malnourished patients: the mean age at disease onset in a subgroup of malnourished children was significantly higher than in children with Cole's index >85%. CONCLUSIONS: A considerable percentage of children with CP can suffer from clinically significant malnutrition. Later age at CP onset predisposes to development of malnutrition.

Impact of crema on the aroma release and the in-mouth sensory perception of espresso coffee.

A set of six espresso coffees with different foam characteristics and similar above cup and in-mouth flavour sensory profiles was produced by combination of two varying parameters, the extraction pressure and the filtration of the coffee beverage. The coffees were subsequently evaluated in a comparative manner by a set of analytical (headspace, nose-space) and sensory (Temporal Dominance of Sensations) techniques. The presence of espresso crema in its standard quantity was demonstrated to be associated with the optimum release of pleasant high volatiles, both in the above cup headspace and in-mouth. On the other hand, the TDS study demonstrated that increasing amount of crema was associated with increasing roasted dominance along coffee consumption. Furthermore, a parallel was established between the roasted sensory dominance and the dominant release of 2-methylfuran in the nose-space. This was, however, an indirect link as 2-methylfuran was indeed a chemical marker of roasting but does not contribute to the roasted aroma. Lowering the standard amount of crema by filtration clearly decreased the release of pleasant high volatiles and the in-mouth roasted sensory dominance. On the other hand, increasing the usual crema volume by increasing the extraction pressure did not bring any added value concerning the above cup and in-mouth release of pleasant high volatiles.

The acacia gum arabinogalactan fraction is a thin oblate ellipsoid: a new model based on small-angle neutron scattering and ab initio calculation.

Acacia gum is a branched complex polysaccharide whose main chain consists of 1,3-linked beta-D-galactopyranosyl units. Acacia gum is defined as a heteropolysaccharide since it contains approximately 2% of a polypeptide. The major molecular fraction (F1) accounting for approximately 88% of the total acacia gum mass is an arabinogalactan peptide with a weight-average molecular weight of 2.86 x 10(5) g/mol. The molecular structure of F1 is actually unknown. From small angle neutron scattering experiments in charge screening conditions, F1 appeared to be a dispersion of two-dimensional structures with a radius of gyration of approximately 6.5 nm and an inner dense branched structure. Inverse Fourier transform of F1 scattering form factor revealed a disk-like morphology with a diameter of approximately 20 nm and a thickness below 2 nm. Ab initio calculations on the pair distance distribution function produced a porous oblate ellipsoid particle with a central intricated "network". Both transmission electron microscopy and atomic force microscopy confirm the thin disk model and structural dimensions. The model proposed is a breakthrough in the field of arabinogalactan-protein-type macromolecules. In particular, concerning the site of biosynthesis of these macromolecules, the structural dimensions found in this study would be in agreement with a phloem-mediated long-distance transport. In addition, the structure of F1 could also explain the low viscosity of acacia gum solutions, and its ability to self-assemble and to interact with proteins.

Expression of organ-specific structures and functions in long-term cultures of aggregates from adult rat liver cells.

Freshly isolated hepatocytes, endothelial cells, Kupffer and fat-storing cells from adult rats were found to be able to form spheroidal aggregates within 24 hr when cultured under serum-free and rotatory conditions. The ultrastructure, including intracellular organization and extracellular matrix deposition was investigated in 7-day aggregates by scanning and electron microscopy. The different cell types expressed a histotypic organization and reconstructed their extracellular matrix (fibronectin and laminin) as well as junctional complexes (desmosomes, tight junctions and gap junctions). The aggregates preserved, over a period of at least 7 days, high albumin expression (mRNA) and secretion as well as high secretion of the acute phase protein T-kininogen. Dexamethasone (10(-7) M) increased the tyrosine aminotransferase activity fourfold after a 24-hr exposure. Aggregates cultured in the presence of 10(-7) M dexamethasone showed an increased expression and secretion of albumin concomitantly with a decrease in T-kininogen secretion. Induction of 7-ethoxycoumarin O-deethylase (ECOD) was effective after exposure to 3-methylcholanthrene or Aroclor for 48 hr. Up to a 13-fold increase in (ECOD) activity was found. The results demonstrate that spheroidal cultures of liver cell aggregates obtained under rotatory conditions provide a useful model for studies on the combined cellular interactions between non-parenchymal liver cells and hepatocytes from adult rats.

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.

A simple rapid method to slice biological specimens: an application for non-embedded and embedded Golgi-stained tissue.

A rotary wire saw is faster and simplifies markedly the slicing of soft and hard tissues without apparent loss of quality. Two applications for the Golgi-Cox method (using the rotary wire saw) are described: one eliminates specimen freezing and embedding while the other uses LR-White instead of celloidin, reducing preparation time. Sections of 20 microns to several mm thick can be obtained.

Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocytelike differentiated HT-29 cells: a basis for host-pathogen interactions in the gut.

Enterotoxigenic Escherichia coli are the most common cause of travelers' and infant diarrhea in less-developed countries. In the present work, among several metabolically labeled human diarrheagenic E. coli strains, enterotoxigenic strains expressing colonization factor antigen II were shown to bind to HT-29 intestinal cell monolayers when these cells were grown in conditions promoting their enterocytic differentiation. Indirect immunofluorescence with fimbrial antisera revealed that pathogen attachment was associated with the production of a specific bacterial adhesin, the E. coli surface antigen CS3. Scanning and transmission electron micrographs showed an apical pattern of colonization, characteristic of enterotoxigenic E. coli infections. The above data were consistent with all observations previously made with human enterocytes obtained from intestinal biopsies. The lectin-carbohydrate nature of this cell-cell recognition mechanism was also established. Bacterial binding to differentiated HT-29 cells was inhibited by a mixture of newborn meconium glycopeptides. By coating the cell layers with the plant agglutinin from Evonymus europaea, pathogen attachment was also prevented. Binding of 125I-labeled CS3 adhesin and E. europaea agglutinin to brush border membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose revealed three bands of about 30, 20, and 13 kilodaltons, which acted as receptors for both bacterial and plant lectins. These data suggest that the sugar units to which the bacterial colonization factor CS3 binds are synthesized as carbohydrate chains of three brush border membrane glycoproteins in HT-29 cells by a differentiation-specific pathway.

Transmission acoustic microscopy of tissue sections (1 GHz). Histoacoustics and acoustic staining.

The acoustic microstructure of mouse small intestine has been studied with a transmission acoustic microscope working at 1 GHz and the influence of the histologic processing on the microacoustic pattern has been tested. Unstained thin sections provide pictures rich in details and highly contrasted. Gelatin has been used as hydrosoluble embedding medium and has been compared to paraffin. The former embedding procedure retained the viscoelastic properties of the specimen far more and provided the most detailed pictures. Osmiun tetroxide has been used to demonstrate acoustic staining.

Multiple immunolabeling in histology: a new method using thermo-inactivation of immunoglobulins.

We describe a new method for successive immunofluorescence detection of multiple peptides in single paraffin tissue sections. Immunoglobulins were inactivated at 130 degrees C after each labeling step while tissues were protected with a mixture of phosphate-buffered saline and glycerin. The feasibility of the method is demonstrated by double-staining of rat pituitary corticotrophs with anti-ACTH[1-24] and anti-ACTH[17-39] antiserum. The method is applicable to brain tissue, since single neurons of ovine paraventricular hypothalamus could be triple stained with anti-vasopressin, anti-neurophysins I and II, and anti-CRF antiserum. The usual absorption controls and crossreactivity tests, as well as peptide staining, can be performed on the same tissue section. This new labeling procedure should prove useful in endocrinology, clinical pathology, and the neurosciences.

Corticoliberin-immunoreactive cell bodies localised in two distinct areas of the sheep hypothalamus.

The localisation of corticoliberin producing neurones in the sheep hypothalamus was attempted with an antiserum directed against synthetic ovine CRF by the indirect immunofluorescence procedure. Synthetic ovine corticoliberin-immunoreactive fibres were detected, in order of decreasing importance, in the external median eminence, in the caudal neural lobe around capillaries, at the boundary of the neural and intermediate lobe, around the anterior commissure, in the paraventricular nuclei and in the posterior hypothalamus and midbrain, suggesting that synthetic ovine corticoliberin-related substances act not only on anterior pituitary tissue, but also on the intermediate lobe, on central neurones and on peripheral target organs. Two groups of cell bodies reacted to the anti-synthetic ovine corticoliberin antiserum. The first group was located in the paraventricular nuclei and consisted of 15-20 microns diameter cell bodies with a granular cytoplasm. The second group was located mainly in the dorsolateral caudal hypothalamus, and the cell bodies were smaller (10-15 microns) and had a smooth cytoplasm. No cell bodies were detected in the basal hypothalamus). Synthetic ovine corticoliberin-immunoreactive structures did not contain immunoreactive neurophysin. The synthetic ovine corticoliberin-immunoreaction in the paraventricular neurones was abolished by preincubating the antiserum with synthetic ovine corticoliberin but not with sauvagine or several other peptides. The immunoreaction in the posterior hypothalamic groups was abolished by preincubating the synthetic ovine corticoliberin antiserum with both synthetic ovine corticoliberin and sauvagine, but not with other peptides. The results suggest that the immunoreaction was specific for synthetic ovine corticoliberin in the paraventricular but not posterior hypothalamic region. The relative contribution of both areas to synthetic ovine corticoliberin-like peptides containing nerve terminals of the median eminence remains to be established.