The development of arylsulphatase in the small intestine of the rat

1. Arylsulphatase activity was measured in stomach, proximal and distal third of small intestine, colon, liver and kidney of foetal and neonatal Sprague-Dawley rats and Swiss mice, with nitrocatechol sulphate as substrate. 2. The specific activity in the distal small intestine, but not in the stomach, proximal small intestine or colon, increased about fourfold between 5 and 16 days after birth in both conventional and germ-free rats. 3. No comparable increase occurred in the distal small intestine of the mouse. 4. The specific activity of acid phosphatase in the distal small intestine of the rat rose only slightly when the arylsulphatase activity increased. 5. The pH optimum and Michaelis constant of arylsulphatase activity of the distal small intestine were similar for 1-day-old, 9-day-old and adult rats. 6. When extracts of distal small intestine of 1-day-old and 9-day-old rats were incubated together, the arylsulphatase activities were additive.     

Characterization, distribution and biosynthesis of the major ganglioside of rat intestinal mucosa.

The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine.     

Mucosal inflammation in a genetic model of spontaneous type I diabetes mellitus

The BioBreeding (BB) rat provides a model of spontaneous type I diabetes mellitus that closely resembles the human disease. Diabetes-prone BB rats demonstrate increased intestinal permeability prior to the development of insulinitis. Studies suggest that alterations in intestinal permeability can lead to increased intestinal inflammatory activity. Diabetes-prone (BBdp) and diabetes-resistant (BBdr) BB rats were examined at 45 days and at >70 days of age following the development of clinical disease (BBd). In separate experiments, tissue was assayed for myeloperoxidase (MPO) or fixed for histological assessment and immunohistochemistry. Blood was obtained for leukocyte MPO measurements and morphological assessment of circulating leukocytes. MPO activity was significantly elevated in the distal small intestine of 45-day-old BBdp rats. In contrast, at >70 days of age, MPO activity was significantly increased throughout the small intestine of BBd and non-diabetic BBdp rats. Subsequently, all measurements were performed in >70-day-old rats. An increase in inflammatory infiltrate was noted in the distal small intestine of BBd rats by light microscopy. Infiltrating cells were identified as bands (a maturing cell type of the neutrophil lineage) and mature neutrophils. The findings suggest diabetes susceptibility is associated with an increase in intestinal inflammatory activity.     

Regional differences in the appearance of adult-type glycosphingolipids in the small intestine of inbred rats at weaning time

The small intestine of 15- to 33-day-old rats was cut into four segments: duodenum, proximal jejunum, distal jejunum, and ileum. Neutral glycosphingolipids and gangliosides were purified from each segment and analyzed by thin-layer chromatography in order to study the developmental appearance of adult-type glycolipids at each level of the small intestine. Type 1 A-6 glycolipid was first detected in the ileum at 15 days and subsequently in the jejunum and duodenum at 19 days of age. N-Glycolylneuraminic acid was expressed first in the ileum at 17 days, then in the proximal jejunum at 21 days, but only after 29 days in the duodenum. In each region, 6-8 days were required between first detection and full expression of N-glycolylneuraminic acid. The presence of 2-hydroxylated fatty acids in glucosylceramide was found first in the ileum at 19 days, 2-3 days before appearing in the duodenum and proximal jejunum. A period of 2-3 days was necessary to reach full adult-type level of 2-hydroxylated fatty acids in glucosylceramide. These results show that adult-type glycolipids appear earlier in the distal than in the proximal region of the rat small intestine, and that different glycolipids appear at different times and at different rates. The finding that the biochemical differentiation of the whole small intestine expands over a period of 3 days to 2 weeks, depending on the region and the glycolipid, before being fully completed indicates that, in addition to the time lag observed between the distal and the proximal region, the new cells arising from the crypt of Lieberkhün after 15 days of age are not at once fully differentiated.     

Immunohistochemical observations of immunoglobulin A in the Paneth cells of germ-free and formerly-germ-free rats

Summary The localization of secretory immunoglobulin A (IgA) in Paneth cells was immunohistochemically studied in germ-free (Gf) and ex-Gf rats that had been injected with feces obtained from specific-pathogen-free (SPF) rats. In Gf as well as SPF rats, the secretory granules of Paneth cells and the brush borders of crypt cells exhibited IgA immunoreactivity. At 12 and 24 h after inoculation, it was found that, concomitant with the occurrence of considerable degranulation, the IgA immunoreactivity in Paneth cells disappeared, except of the margin of supranuclear vacuoles. In contrast, the IgA immunoreactivity of the crypt-cell brush borders was unchanged. Four days after inoculation, secretory granules exhibiting IgA immunoreactivity reaccumulated in Paneth cells. The present study suggests that Paneth cells regulate the bacterial milieu in the intestine by releasing secretory granules containing IgA into the crypt lumen.     

Immunohistochemical observations of immunoglobulin A in the Paneth cells of germ-free and formerly-germ-free rats

The localization of secretory immunoglobulin A (IgA) in Paneth cells was immunohistochemically studied in germ-free (Gf) and ex-Gf rats that had been injected with feces obtained from specific-pathogen-free (SPF) rats. In Gf as well as SPF rats, the secretory granules of Paneth cells and the brush borders of crypt cells exhibited IgA immunoreactivity. At 12 and 24 h after inoculation, it was found that, concomitant with the occurrence of considerable degranulation, the IgA immunoreactivity in Paneth cells disappeared, except of the margin of supranuclear vacuoles. In contrast, the IgA immunoreactivity of the crypt-cell brush borders was unchanged. Four days after inoculation, secretory granules exhibiting IgA immunoreactivity reaccumulated in Paneth cells. The present study suggests that Paneth cells regulate the bacterial milieu in the intestine by releasing secretory granules containing IgA into the crypt lumen.     

Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine

To study an enzymatic basis for the postnatal changes in intestinal glycosylation, the activities of sialyl- and fucosyltransferases were determined in the particulate fraction of mucosal cells prepared from rat small intestine of various ages. The results show that sialyltransferase activity was present in increased levels compared to adults during the preweaning period (1-2 weeks) and subsequently declined 5-fold to adult levels after weaning, while fucosyltransferase activity was decreased compared to adults in the first 3 weeks of life, rapidly increased at 4 weeks, and reached adult levels (10-fold) by 5 weeks. The changes in both sialyl- and fucosyltransferase activities were reflected by the membranous content of glycosidic-bound sialic acid and fucose, respectively. Cortisone injection precociously induced a decreased sialyltransferase activity and an increased fucosyltransferase activity in 2-week-old suckling rats. This study indicates that the activities of sialyl- and fucosyltransferases were reciprocally related and modulated by cortisone action in the developing intestine. These enzyme changes may be responsible for the previously noted shift from sialylation to fucosylation of the intestinal mucosa during maturation.     

Postnatal changes in the distribution and elemental composition of Paneth cells in normal and corticosteroid-treated rats

Summary Paneth cells containing zinc-rich granules were found in the small intestine of 6-day-old rats. These cells were more numerous in older animals and were consistently most common in the distal ileum. The zinc content of granules from 10-day-old rats was similar to that found in adults (ca 300 mg atoms/kg dry weight) but no calcium could be detected. An injection of cortisone acetate at 5 days resulted in a premature increase in the numbers of Paneth cells in 10-day-old rats. The cell granules contained normal, adult levels of zinc, a calcium concentration of ca 400 mg atoms/kg dry weight and also an increased concentration of phosphorus.     

Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone-epidermal growth factor

Abstract. There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone-epidermal growth factor (URO-EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population.
The number of mitoses and labelled cells per crypt, and thus the crypt cell production rates, were significantly decreased in the TPN group when compared to the orally fed. URO-EGF significantly increased both proliferative indices and the number of dividing cells per crypt. Crypt cell production in the small intestine was restored to those levels seen in the orally fed rats, moreover, labelling per crypt in the colon was increased to more than twice that of orally fed rats. The location of the mean labelling position and the half maximum labelling position followed the changes in crypt length in the small intestine, but to a lesser extent; thus the growth fraction was significantly increased in the TPN rats in comparison with the orally fed and the URO-EGF treated groups. Similar changes in these positions were seen in the colon, but the growth fraction was much reduced in the URO-EGF treated rats, as a consequence of the large increase in crypt length without a concomitant alteration in label distribution.     

Increase of sialyltransferase activity in the small intestine following thermal injury in rats

The acute phase protein response following inflammation is associated with an increased total protein-bound carbohydrate content in plasma in the form of glycoproteins. Glycosyltransferases in liver may serve as a regulator of this increased glycosylation activity in the plasma and may also serve as a marker for the acute phase response. Sialyltransferase is an example of a glycosyltransferases in which sialic acid is transferred to oligosaccharides of glycopeptides in the Golgi prior to glycopeptide secretion. In this study, sialyltransferase activities were determined in plasma, liver, and intestinal mucosa following a standardized 25% body surface area thermal injury in the rat. A statistically significant increase in sialyltransferase activity was found in liver and small intestine which were maximal at 24 hours after the injury. These increased sialyltransferase activities were accompanied by a statistically significant 2 to 4 fold elevation in plasma sialyltransferase activity at 24 hours. The plasma and liver elevations in these activities were similar to elevations seen in other models of acute inflammation using turpentine injections and bacterial infections. The increased sialyltransferase activity within the rat intestine was comparable to increases in intestinal sialyltransferase activity following colchicine treatment and may represent a similar mechanism(s).     

Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat

Summary A modification of Weiser's (1973) cell isolation method was used in order to study the developmental pattern of various intestinal enzyme activities in villus and crypt cells of normal rats from 5 days after birth until 8 weeks. Alkaline phosphatase and enterokinase activities were always located in the upper villus zone during postnatal development. Enterokinase activity was higher in the upper villus cells during the third week of life than after this period. Aminopeptidase activity was located in the crypt cells during the first week, its maximum activity remained in this area until the third week. At this time, sucrase activity appeared in the crypt cells, then aminopeptidase and sucrase activities rose to the villus zone during the fourth week. Amylase activity was detected along the entire crypt-villus axis 5 days after birth, reaching maximum activity in crypt cells at the end of the first week and in the upper villus cells after the fourth week. In contrast with the other enzymes studied almost all amylase activity was soluble in the youngest animals whereas at weaning most of the activity appeared in a particulate form in the villus cells. But in the crypt cells the ratio between particulate and soluble form remained unchanged until the adult stage. Various hypotheses are advanced to explain the patterns of evolution of the different enzymes.     

Bringing target-matched PI3King from the bench to the clinic

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.     

CONTROL OF INTESTINAL EPITHELIAL REPLACEMENT: LACK OF EVIDENCE FOR A TISSUE-SPECIFIC BLOOD-BORNE FACTOR

The small intestine of rats was cut across in two places, about 14 and 50% of the length of the small intestine from the pylorus, and continuity was re-established by suturing the proximal and distal ends. The resulting sac of small intestine, averaging 36% of the total length of the small intestine, had its upper end closed off, and its lower end anastomosed, either to the intestine-in-continuity (an ‘intestine-sac’), or to the skin of the abdominal wall (a ‘skin-sac’). On the ninth post-operative day, the cell production rate in squashes of micro-dissected whole crypts of Lieberkühn was measured by mitotic blockade with Colcemid. The rate of cell production in unoperated and sham-operated rats was 30 cells/crypt/hr, throughout the length of the small intestine. In the intestine in continuity, the rate increased to an average of 46 cells/crypt/hr above the anastomosis, and to 54 cells/crypt/hr below it. At the lower end of the ‘intestine-sac’, which drained into the intestine-in-continuity, the rate was 39 cells/crypt/hr, while in the lower end of the sac which drained to skin the rate of cell production was only 16 cells/crypt/hr. This significantly lower cell production rate in intestine which was not in contact with ingesta is taken to be evidence of the importance of local, rather than blood-borne factors in the control of epithelial replacement.     

Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine

Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.     

POPULATION DYNAMICS OF INTESTINAL EPITHELIA IN THE RAT TWO MONTHS AFTER PARTIAL RESECTION OF THE ILEUM

Sprague-Dawley rats that had been subjected 2 months previously to partial resection (10 per cent) of the small intestine and an equal number of control rats were injected with tritiated thymidine and sacrificed at intervals during the subsequent 16 hours. Segments of duodenum, jejunum and ileum were prestained by the Feulgen technique and radioautographed. The proportion of crypt cells bearing labeled nuclei, the percentage of labeled crypt cells in mitosis and the appearance of labeled crypt cells on the villi were determined. Comparison of control and resected rats showed that (a) the proportion of intestinal crypt cells incorporating thymidine was considerably greater and uniformly high throughout the shortened intestine, (b) the life cycle of crypt cells was slightly reduced, and was uniform throughout the shortened intestine, and (c) the time during which cells were retained in crypts was markedly reduced. On the basis of persistent, generalized increase in the production of crypt cells, and on prior evidence that the epithelial cells of shortened intestine continue to have a brief life span and evidence of metabolic immaturity, the existence of a humoral factor, tentatively called "intestinal epithelial growth hormone," is postulated.     

Effect of cortisone on the developmental pattern of the neutral and the acid β-galactosidases of the small intestine of the rat

1. The developmental pattern and effect of cortisone on acid beta-galactosidase and neutral beta-galactosidase were studied in postnatal rats by a recently proposed method for their independent determination. 2. After birth the acid beta-galactosidase activity increases in the ileum, whereas it decreases slightly in the jejunum. On day 16 after birth the activity in the ileum decreases and in 20-day-old rats activity in both parts of the intestine decreases to adult values. In suckling animals the activity in the ileum exceeds the jejunal activity severalfold and in adult animals the activity in the jejunum is slightly higher than that in the ileum. 3. Neutral beta-galactosidase activity is high after birth and decreases in both jejunum and ileum after day 20 after birth. In 12-20-day-old rats activity in both parts is essentially the same, but in adult animals jejunal activity exceeds ileal activity four-to five-fold. 4. Cortisone (0.5, 2.0 or 5.0mg/100g body wt. daily for 4 days) does not influence the activity of either enzyme in 60-day-old rats. Acid beta-galactosidase activity is decreased after cortisone treatment in 8-, 12-, 16-and 18-day-old rats, with sensitivity to cortisone increasing with the approach of weaning. No effect of cortisone on acid beta-galactosidase is seen in 8-day-old rats. Neutral beta-galactosidase activity is increased in the ileum of 8-, 12-, 16- and 18-day old rats, but only in the jejunum of 8-and 12-day-old rats.     

Properties of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase in Villous and Crypt Cells of the Rat Small Intestine

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in microsomes of villous and crypt cells from the jejunal and ileal epithelia of rats fed commercial pellet were studied. The optimum pH of the microsomal reductase from villi and crypts was 7.0~7.2 and the Km for HMG-CoA was 41.7 µm. The reductase specifically required dithiothreitol for its activity. The activity was higher in ileal populations than in the jejunum. Responses of the reductase in the villous fraction to feeding cholesterol and taurocholate in combination or cholestyramine resembled those observed in crypt cells. Thus, the properties of microsomal HMG-CoA reductase in villous and crypt cells from the small intestine are similar each other, and they are possibly the same enzyme.     

Multiple forms of mutarotases from the kidney, liver, and small intestine of rats: purification, properties, subcellular localization and developmental changes

Comparative studies of mutarotase [aldose 1-epimerase, EC 5.1.3.3] from the kidney, liver and small intestine of rats were performed placing in the focus on the study of multiple forms. The findings obtained are as follows. Mutarotases from the kidney and liver of adult rats were both separated into four forms (types I-IV) by DEAE-cellulose column chromatography, whereas only two forms (types I and II) were detected in the small intestine. Liver mutarotase type I was further separated into types I1 and I2 by column chromatography on hydroxylapatite. Types I and II from the kidney and type II from the liver were purified to homogeneity as judged by isoelectric focusing on thin layer polyacrylamide gel. Of various physicochemical properties, only the Km for alpha-D-xylose and the isoelectric point were different among the multiple forms. Liver mutarotase was immunohistochemically localized in the nuclei of parenchymal cells and small intestine enzyme in the nuclei of mucosal cells, indicating similarity with the localization of kidney enzyme (in the nuclei of epithelial cells of renal tubules and glomeruli) which was reported in our previous paper [Experientia (1979) 35, 1094-1097]. The kidney mutarotase level increased gradually after birth and reached a maximum near adult level within 20 days. This developmental pattern was essentially the same as that in the liver but clearly different from that in the small intestine, in which the mutarotase activity of suckling rats was several times higher than that of adult rats. Distribution patterns of multiple forms (types I-IV) of the enzyme in the kidney and liver of 10-day-old rats were similar to those in respective tissues of adult rats. On the other hand, the small intestine of 10-day-old rats contained four forms (types I-IV), whereas there were only two forms (types I and II) in adult rats.