Interaction of concanavalin a with bacterial lipopolysaccharides in agarose gel.

Binding of fluorescein isothiocyanate-labeled concanavalin A to a series of molecular species of lipopolysaccharide (LPS), purified from pathogenic bacteria, was studied via agarose gel precipitation experiments and the results were compared with available structural data.The LPS species could be divided into ConA-reactive and non-reactive ones. Reactivity resided in the O-specific chain of LPS, and binding to the lipid A or core moieties of LPS could not be demonstrated by the present methods. The α-D-glucose or α-D-mannose residues of the repeating O-specific oligosaccharide units appeared to be recognized by ConA, except when blocked by steric hindrance. Specificity of the reaction was verified by inhibition with 2% D-glucose. Binding by bacterium-specific sugar-residues could not be demonstrated.For precipitation to occur, polyvalency was required both for LPS and ConA, and the resulting precipitation appeared to be promoted by hydrophobic interactions between the lipid A moieties of LPS molecules. The LPS species were differently retained by the agarose gel, which can be explained by differences in their micellar structure in aqueous solution. E. coli O83 LPS did not readily diffused in 1% agarose gel, but its precipitation with ConA could be demonstrated either at elevated temperature or mixing it previously with molten agarose (Mancini's arrangement).

Effect of orally administered plant lectins on intestinal liquor accumulation and amylase activity in rats.

Short-term effects of orally administered plant lectins, with special reference to the Phaseolus vulgaris agglutinin (phytohaemagglutinin, PHA), were studied in growing rats. The orally administered PHA elicited a dose-dependent accumulation of liquor with elevated pH in the proximal small intestine. Although the concentration of alpha-amylase activity did not change, total alpha-amylase activity slightly, but significantly increased in the gut. When a panel of plant lectins with different carbohydrate binding specificities was tested at the dose of 100 mg/kg body weight, most of them stimulated the secretion of liquor, but the total alpha-amylase activity was increased only by PHA, ConA or WGA.

Origin and mediation of secretion induced by oral phytohaemagglutinin (PHA) in rats.

After oral administration several gut-binding lectins induce accumulation of liquor and amylase in the proximal small intestine. Orally administered Phaseolus vulgaris phytohaemagglutinin (PHA) was used to study the mediation of these effects in rats. The regulation of amylase secretion clearly differed from that of the liquor. The amylase activity was of pancreatic origin, in agreement with the known cholecystokinin-releasing effect of PHA. It appears that CCK exerts its effect both directly and by facilitating neural stimulatory pathways. Intestinal secretion was identified as the source of the liquor, without a contribution by other secretions. It was mediated by a local cholinergic reflex with the involvement of both muscarinic and nicotinic acetylcholine receptors. It is speculated that the observed enteric reflex may enable the gut to transport secreted antibacterial peptides or secretory antibodies from the crypts to adherent bacteria on adjacent villi.

Effect of orally and intraperitoneally administered plant lectins on food consumption of rats.

A panel of orally administered lectins (100 mg/kg b.w.) of different binding specificities was tested for suppression of voluntary food consumption in prefasted rats. PHA isolectins (Phaseolus vulgaris) and RPA-I (Robinia pseudoacacia) were found to exert a marked and significant effect, but two other gut-binding lectins, i.e. SBA (Glycine max) and WGA (Triticum vulgar) and several non-binding lectins were ineffective. In cannulated rats PHA infused into the duodenum induced food suppression, i.e. binding of the lectin to the mouth or stomach was unnecessary. Suppression of food consumption lasted through the whole nocturnal feeding period, control (BSA) and experimental (PHA) curves of cumulative food consumption showed a V-like divergence. Suppression by PHA or RPA-I showed very similar time courses, but a long-lasting inhibition of gastric emptying was only observed in the RPA-treated animals. Intraperitoneally administered lectins suppressed food consumption much more effectively than the oral ones, whereas Galanthus nivalis agglutinin (ONA) had little or no effect. It is concluded that lectins can be used as effective tools for the modulation of food consumption and gastric emptying in experimental animals.

Binding of FITC-labelled lectins to the gastrointestinal epithelium of the rat.

Biotechnology uses lectin genes to transfect into crop plants for protection against insects and nematodes. On the other hand, the information is limited on lectin-binding properties of cells in the gastrointestinal tract. Therefore, binding of a panel of FITC-labelled plant lectins to gastrointestinal cells of the rat was studied. In the stomach, cytoplasmic staining of parietal cells by PHA appeared to be due to glycoproteins attached to the tubulovesicles. PNA also stained the parietal cells, but only in the isthmus and neck regions, reacting with desialylated glycoproteins. WGA bound to the mucous neck cells with higher affinity than to the surface and foveolar mucous cells. The mucous cells were also stained by SNA-I, UEA-I and, less intensively, by LCA. Chief cells did not show detectable reaction with any of the applied lectins. Binding of PHA to gastric cells showed differences when compared with the results of in vivostudies. Small intestinal brush border was stained with UEA-I and SNA-I, the latter lectin also strongly stained the surface of small intestinal crypts. Both lectins reacted with the mucus of goblet cells. In the large intestine UEA-I and SNA-I stained the goblet cells at the base and upper part of the crypts, respectively. Accordingly, we provided evidences for the unique lectin-binding phenotype of the various segments of the gastrointestinal tract.

Both free and complexed trypsin inhibitors stimulate pancreatic secretion and change duodenal enzyme levels.

Secretion of pancreatic digestive enzymes was measured in pancreatic cannulated rats after duodenal stimulation with Kunitz or Bowman-Birk protease inhibitors or their complexes with trypsin and/or chymotrypsin. Free and complexed inhibitors were bound by the duodenal epithelium, stimulated the discharge of cholecystokinin, and significantly increased secretion rates of alpha-amylase, trypsinogen, and chymotrypsinogen. Inasmuch as secretion rates returned to basal levels with cholecystokinin-A receptor antagonists, the stimulation was likely to be mediated by cholecystokinin. Soya factors also influenced the duodenal concentration of pancreatic enzymes under simulated feeding conditions. Thus the level of alpha-amylase increased while the trypsin concentration decreased in rats gavaged with free or complexed inhibitors. The same was true for chymotrypsin when the Bowman-Birk inhibitor was used, but the Kunitz inhibitor and its trypsin complex actually raised the luminal concentration of chymotrypsin. Accordingly, because soya inhibitors remained effective in stimulating pancreatic secretion after elimination of their inhibitory activity by complex formation, it is questionable whether the signal for cholecystokinin secretion was solely due to lowering of duodenal protease levels.

Lack of acid-resistant trypsin inhibitor in mare's colostrum: short communication.

Mare's colostrum was collected and examined for the presence of trypsin inhibitors. It was found to contain a low level of trypsin inhibitor which could be denatured by 2.5% trichloroacetic acid and, therefore, it clearly differs from the acid-resistant colostral inhibitor of Artiodactyla and Carnivora. This finding is exceptional for a species that concentrates IgG in the colostrum and whose newborn absorbs colostral proteins non-selectively by the gut. It appears that the presence of colostral trypsin inhibitor is not essential for the transmission of maternal immunity via the colostrum and the gut.

Demonstration of acidity in intestinal vacuoles of the suckling rat and pig.

Fluorescence staining characteristics of "large vacuoles," i.e. vacuoles ranging up to almost cell size, were studied in suckling rats and pigs. In the distal epithelium of the small intestine of suckling rat, yellow autofluorescence and accumulation of orally administered FITC-dextran were observed in the supranuclear vacuole. In both species the weakly basic amino dye acridine orange (AO) stained the nuclei at neutral pH bright yellow-green and the transport and digestive vacuoles bright red or orange. It is concluded that trapping and accumulation of the dye (red shift) were due to the acidity of the vacuolar interior. Assessment of the vacuolar pH in rat enterocytes is in agreement with published data on lysosomal pH values. Acidic buffers, lysosomotropic and destructive agents, or illumination with bright light induced irreversible fading of AO-stained vacuoles; the color of the porcine transport vacuoles was the most labile. This fading was used to differentiate vacuoles from other structures, e.g., vacuolar inclusion bodies and goblet cells. In suckling rat, staining characteristics of the gut epithelium changed on Days 19 and 20 of postnatal age. Detection of acidity in the distal (digestive) vacuoles supports the lysosome-like nature of their function. They appear to constitute an auxiliary, intracellular digestive system for the young animal. However, the function of acidity in the non-digestive transport vacuoles of newborn pig is unclear.

Fate of the antinutritive proteins of soyabean in the ovine gut.

Defatted raw soyabean flour was administered intraruminally at a level of 10 g/kg body weight to a sheep fitted with ruminal, duodenal, and ileal cannulas. Soyabean lectin and trypsin inhibitor appeared in the duodenum within one hour, both reached the terminal ileum and disappeared within 24 hours. The passage of the lectin along the gut progressively lagged behind that of the inhibitor, which indicates that the lectin binds to the intestinal surface. The effect of the inhibitors was neutralized by a marked pancreatic hypersecretion. Contrary to earlier assumptions, the antinutritional proteins were not effectively degraded in the forestomachs.

Observation of duodenal bile shuttle in the sheep.

Relation between duodenal anatomy and function was studied in cannulated sheep. It was concluded that during the interperistaltic periods, gravitational backflow of bile and admixed pancreatic juice towards the pylorus may provide a means for desorption of enterokinase from the mucosal surface of the cranial duodenum. The biliary back and forth ("bile shuttle") may contribute to the utilization of duodenal enterokinase.

Binding and degradation of lectins by components of rumen liquor.

The binding of 15 different plant lectins to feed particles and microbes in rumen liquor, and their degradation were studied in vitro. The rate of degradation assessed from the label released when radioactive iodine-labelled lectins were incubated with rumen liquor conflicted with the rates calculated from measurements of the survival of the antigenic structure (immuno-rocket electrophoresis) or the biological function (haemagglutination) of the lectins. Thus solubilization of the radioactive label indicated that Concanavalin A (Con A), but not the soyabean agglutinin, SBA, or kidney bean phytohaemagglutinin, PHA-E3L, was stable to rumen proteolysis. In contrast, both SBA and PHA-E3L were shown by immuno-rocket electrophoresis or haemagglutination tests to be highly resistant to breakdown, while the degradation of Con A proceeded at a constant slow rate under the same conditions. This was in accord with the previously established general stability of lectins in the gut of single-stomach animals. Of the 15 lectins, SBA, favin (Vicia faba lectin) and Con A were bound by hay and the particle fraction of rumen liquor. This was, in part, specific and reversible in the presence of appropriate sugars. Most pure bacterial strains preferentially bound lectins with specificity for glucose/mannose (favin and Con A), while rumen fungi reacted with SBA. The level of binding was low with other lectins. However, inter-strain differences of lectin-binding were found in Selenomonas ruminantium and Ruminococcus flavefaciens. Clearly, as some lectins were not fully degraded in the rumen, they could be expected to depress the utilization of the diet not only in single-stomach animals but, possibly, also in ruminants.

Tubular reabsorption of protein by porcine kidneys during neonatal alimentary proteinuria.

The ability of renal proximal tubular cells to reabsorb protein early in postnatal life was investigated using goat haemoglobin as tracer. The haemoglobin was intracardially administered to newborn piglets. The kidneys were fixed for light and electron microscopy 4 h later. Piglets killed immediately after birth and those allowed to suck colostrum for 4 h were used as controls. The proximal tubular cells of newborn, unsuckled piglets already contained absorptive vacuoles. Haemoglobin was absorbed in some absorptive vacuoles of the proximal tubules. The tracer was also demonstrable in the urine, mainly in the form of methaemoglobin. Although proximal tubular cells of newborn piglets are able to absorb protein, this absorption is of limited extent and excess protein is voided with the urine.

Development of porcine digestive enzymes with special reference to ribonuclease.

The activity of digestive enzymes was determined in the gastrointestinal tract of newborn, 1-, 3- and 5-week-old piglets and in adult pigs. The pancreas of newborn piglets contained considerable ribonuclease (RNase) activity, which continued to increase with age. After the initial values an opposite tendency was found in the intestinal contents, probably due to the increase of proteolytic degradation with advancing age. Serum RNase showed little age dependence. The time-course of development of pancreatic RNase resembled more that of proteolytic enzymes than tha of amylase. The data indicate that a high pancreatic secretion of RNase commences much before the appearance of RNA in the diet.

Development of ovine digestive enzymes with special reference to ribonuclease.

Twenty Merino lambs of four age groups (1 day, 2, 4 and 7 weeks) and 8 adult Merino wethers were killed. The development of pancreatic and gastrointestinal enzymes was followed by determining RNase, amylase, lipase, trypsin, chymotrypsin and total proteolytic (azocaseinase) activity. Pancreatic protein content, rumen and abomasal pH and abomasal clotting time were also determined. Pancreatic RNase was already present in the newborn lambs and significantly rose in the first 2 weeks of life and before reaching adult values. The increase was more marked and went to higher adult values than in the pig (Baintner and Farkas, 1989). The time-course resembled that of pancreatic amylase and chymotrypsin; pancreatic trypsin and azocaseinase also showed some similarities, but pancreatic lipase had a different time course. Small intestinal RNase also changed differently; it showed a maximum at 4 weeks and had trends opposite to total proteolytic activity, indicating partial digestion of the enzyme by intestinal proteases. Rumen and caecal RNase activities may be indicative of microbial growth and fermentation rate; they showed mostly opposite tendencies in the two localities. In contrast to the pig (Baintner and Farkas, 1989), pancreatic and small intestinal trypsin:chymotrypsin ratios did not show significant increase during development in sheep.

Investigations about influence of the content of plant crude protein in the ration on the utilization of urea in dairy cattle. 6. Digestive enzymes and their interactions in contents of proximal small intestine.

Forty-two samples were taken from the contents of the proximal small intestine of two lactating dairy cows fitted with re-entrant duodenal cannula. Most samples were free of detectable amylase activity. The (chymo)trypsinogen present was only partially activated to (chymo)trypsin. The activation was continued in vitro: slowly at the original pH of the samples (between pH 2.8 and 4.2), and faster after neutralization or a slight alkalinization. The effect of Ca, EDTA and soybean inhibitor on the activation of trypsinogen was also studied. The pancreatic enzymes were inactive in the acid pH range of the samples, but pepsin was markedly active. At pH 3.8 casein was digested rapidly by purified pepsin and slowly by the samples (agar-plate experiments). In model experiments performed with purified enzymes, pepsin digested (chymo)trypsin rapidly at pH 1-2 and slowly at pH 3.8. In the intestinal juice (chymo)trypsin and their zymogens seemed to be unaffected by pepsin under the conditions of the samples. It is concluded that the conditions prevailing in the duodenum/upper jejunum of the experimental cows account for a gastric-type digestion, despite the presence of pancreatic enzymes. In vivo the intestinal contents pass in distal direction. Meanwhile the pH of the chyme gradually increases and gives rise to first an increase of enterokinase activity accounting for a faster activation of the zymogens; second a start of function of activated pancreatic proteases and third a gradual decrease of pepsin activity and finally to its irreversible denaturation. Thus the development of intestinal type digestion is delayed in ruminants.