Short communication: Estimation of ileal digestibility in chickens using single- and dual-tracer methods.

Fractionation of digesta, as occurs during gastrointestinal transit in chickens, complicates accurate measurements of ileal digestibility using tracers. Dual-tracer methods using separate tracers for solid and fluid digesta phases may improve the accuracy of digestibility measurements when assumptions of the single tracer method are violated. The aim of the present study was to compare the apparent ileal digestibility (AID) of nutrients calculated with single- and dual-tracer methods in chickens fed diets varying in particle size, anticipating digesta phase separation in the proximal gastrointestinal tract. A total of 112 Dekalb White (BW: 1.53 ± 0.107 kg) and 112 Bovans Black (BW: 1.79 ± 0.127 kg) 29-week-old laying hens were distributed over 32 pens (seven birds/pen). Within breed, pens were randomly assigned to one of two experimental diets (coarse vs fine oat hulls; n = 8 replicate pens per diet/breed combination). Diets were supplemented with TiO2 (3 g/kg) and Co-EDTA (2 g/kg). On days 34, 35, or 36, birds were euthanised and digesta from the ileum was collected for tracer and nutrient analyses. Apparent ileal digestibility was subsequently calculated by single- and dual-tracer methods. Although coarse oat hulls were hypothesised to increase the fractionation of solid and fluid digesta phases, no breed or diet × method interactions were found. Using a single tracer method based on TiO2, AID of nitrogen (N) was overestimated by 3%-units (P < 0.01) compared with the dual-tracer method, whereas AID estimates of DM, starch, fat, and non-starch polysaccharides did not differ (P > 0.09) and precision of all AID estimates was improved. In conclusion, these results show that although from a conceptual perspective, dual-tracer methods are presumed to better account for the variation in flow behaviour of different digesta phases, AID estimates obtained by the commonly used single tracer method using solid-phase tracer TiO2 were more precise and only marginally differed from estimates obtained by a dual-tracer method using distinct tracers for solid (TiO2) and liquid (Co-EDTA) digesta phases. Considering technical and economical constraints, the single tracer method may thus be the method of choice in many situations. Only when digestibility of proteins or amino acids is of specific interest, single tracer methods using a solid-phase tracer may not suffice. Nevertheless, for both single- and dual-tracer methods, tracer selection is critical, and the choice of tracers should depend on the nutrient(s) of interest.

Polysaccharide source altered ecological network, functional profile, and short-chain fatty acid production in a porcine gut microbiota.

Several validated dynamic in vitro models of the colon have been developed for humans, but there is no dynamic in vitro fermentation model for pigs. This study was conducted to modify the human, dynamic, computer-controlled TNO in vitro model of the colon (TIM-2) for pigs and investigate effects of different starch sources and polysaccharides on swine microbiota structure, ecological network, predictive functional profile, and short-chain fatty acids production. Our study showed that three different types of starch or two polysaccharides greatly impacted microbiota composition. Co-occurrence network analysis indicated that microbiota fed with different sources of starch changed the network topological properties. Functional profiles were predicted to vary significantly among the three starch treatments, and the original pig faecal inoculum was more similar to maize starch treatment. On the other hand, compared with maize starch and arabinoxylans (AX), the microbial composition of the original inoculum was more similar when AX-XG (arabinoxylans and xyloglucan) were added, and the functional profile of the original inoculum also clustered with AX-XG. The cumulative production of acetic, propionic, and butyric acid on maize starch were significantly higher than those on potato starch and wheat starch, while only the amount of acetic acid was significant higher on AX-XG than that on AX. In conclusion, supplementation of maize starch as the starch source together with AX and XG, leads to the bacteria being more stable in the in vitro model and closer to the original inoculum and microbial function compared to potato starch, wheat starch and AX. A maize basal diet may improve energy absorption in the large intestine in growing pigs.

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion.

BACKGROUND: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.

Mutational analysis of the putative devazepide binding site of the CCK(A) receptor.

Recently a molecular model was proposed for the binding site of the antagonist 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl) -1H-indole-2-carboxamide (devazepide) on the cholecystokinin-A (CCK(A)) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129-148). Fifteen amino acids were identified, including hydrophilic ones such as Ser139, Asn349 and Ser379, that might interact with the carboxamide moiety in devazepide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin-(26-33)-peptide amide (CCK-8) and CCK-8-evoked Ca2+ mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity.

Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts 11.

To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number of ts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stage ts11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures.

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10(-3) M were transferred to hormone-free medium containing 10(-2) M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10(-4) M calcium were transferred to hormone-free medium with 10(-3) M calcium. At calcium concentrations between 6·10(-3) and 10(-2) M globular stage somatic embryos were found in cultures supplemented with 2·10(-6) M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.