Occurrence of a new hematoside in the kidney of guinea pig

We have isolated a new hematoside from guinea pig kidney. Like the usual hematoside (II3NeuAc LacCer), isolated from human erythrocytes, this new hematoside contained glucose, galactose, and N-acetylneuraminic acid in an equimolar proportion. By thin-layer chromatography (TLC), however, it migrated faster than the usual hematoside. After mild alkaline hydrolysis the TLC mobility of this ganglioside became identical to that of the usual hematoside. The sialic acid in this ganglioside was susceptible to Clostridial neuraminidase. Based on TLC mobility and the results of periodate oxidation, the sialic acid of the new hematoside was identified as 9-O-acetyl-N-acetylneuraminic acid. Therefore, the structure of this new hematoside is 9-O-Ac-NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GLc beta 1 leads to 1'Cer.     

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly (P less than 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.     

The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

The mucosa of the small intestine: development of the cellular lipid composition during enterocyte differentiation and postnatal maturation

Alterations in lipids linked to intestinal maturation and enterocyte differentiation were reviewed. The 3 main lipid components of cell membranes, ie cholesterol, phospholipids and glycolipids, were examined. Cell phospholipid content increases from the crypts to the mid-villus, which accounts for membrane development and organelle growth in differentiating cells. Changes in the proportion of phospholipid polar head groups occur in brush border membrane during postnatal maturation of the small intestine. The possibility that phospholipid fatty acid composition in differentiating cells might be altered by dietary lipids is discussed. Cholesterol biosynthesis mainly occurs in crypt and lower villus cells whereas its absorption from luminal content and esterification into lipoproteins occur in upper villus mature cells. Cholesterol cell content increases in mature cells in comparison to immature cells on the one hand, and in the distal by comparison with proximal parts of the intestine on the other. Increasing cholesterol content is generally correlated with decreasing membrane fluidity, which in turn could modulate functional properties of the mucosa. Glycosphingolipids are mainly found in the brush border membrane, which contains 20-30% glycolipids by weight of total lipids. These components tend to reinforce the membrane stability and significantly contribute to the surface properties of epithelial cells. The latter undergo noticeable changes during cell differentiation and postnatal maturation. Significant changes in both the glycosidic and lipophilic parts of glycosphingolipid molecules occur in differentiating cells and are of possible importance in the process of mucosal maturation. It is possible that the addition of a terminal sialic acid (sialyltransferase activity) instead of a terminal galactose (galactosyltransferase) to an endogenous acceptor (lactosylceramide) could constitute an important event in the differentiation process, and may account for the increasing content of hematosides along the intestinal villus of rat. Alterations in lipid counterpart mainly consist of hydroxylation of fatty acids in hematosides during postnatal maturation or in glucosylceramides during cell differentiation. Collectively these intestinal lipid changes may contribute in part to the development of mucosal barrier, selective permeability and functional properties of the mature intestinal mucosa.     

Sialoglycoconjugate changes during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat

Previous studies indicated a reproducible pattern of altered glycosphingolipid biosynthesis accompanying late stages of liver tumorigenesis in the rat induced by the carcinogen 2-acetylaminofluorene. The sequence began with a dramatic elevation in CMP-sialic acid:lactosylceramide sialyltransferase and was followed by sequential elevations and eventual depressions in other enzymes catalyzing sugar transfers to glycolipid acceptors. The present study focused on the early events of glycolipid biosynthesis during the first 11 weeks of 2-acetylaminofluorene administration according to the same feeding schedule as used previously. Transient elevations in CMP-sialic acid synthetase and elevations in neutral glycosphingolipid precursors to gangliosides were found to precede the major elevations in CMP-sialic acid:lactosylceramide sialyltransferase (GM3 synthetase) noted earlier. Two cycles of response were observed prior to the initiation of the sustained enhancement of biosynthesis of precursor ganglioside, GM3, and/or a significant increase in total or lipid-soluble sialic acid. In vitro rates of sialyl transfer from CMP-sialic acid to endogenous protein acceptors were not altered. The results suggest that the previous observations of altered ganglioside biosynthesis following 2-acetylaminofluorene administration are not an isolated occurrence but may represent late events in a sequence or 'cascade' of biochemical change involving, as well, biosynthesis of ganglioside precursors, CMP-sialic acid and neutral glycosphingolipids.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.     

Rabbit muscle gangliosides

Four ganglioside fractions were isolated from rabbit muscle: one hematoside and three hexosamine-containing species. They were analyzed for hexoses, hexosamine, sialic acid, fatty acids, and long-chain base content. The molar ratios of sphingosine-hexose-hexosamine-sialic acid were: for hematoside, 1:2:0:1; for the disialogangliosides, 1:3:1:2; and for trisialoganglioside, 1:3:1:3. The carbohydrates were studied by thin-layer and paper chromatography. The hexoses were glucose and galactose; the hexosamine was N-acetylgalactosamine and the sialic acid was N-acetylneuraminic acid. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography. The fatty acid composition was similar in all of the four gangliosides. The most abundant fatty acids were 16:0 and 18:0, but significant amounts of 16:1, 18:1, 20:0, and 22:0 were also found. Hydroxy fatty acids were not detected. In all of the muscle gangliosides the main long-chain bases were C(18)-sphingenine and C(20)-sphingenine. In hematoside there were also measurable amounts of C(18)-sphinganine and C(20)-sphinganine, whereas in the major gangliosides only traces of C(18)-sphinganine were detected.     

Sialic acids. Enzymatic synthesis of Tay-Sachs ganglioside.

A particulate preparation from embryonic chicken brain catalyzed the transfer of N-acetylgalactosamine from uridine diphospho-N-acetylgalactosamine to the ganglioside GM3 (hematoside, sialyllactosylceramide). The kinetic properties of the transferase were determined. The product was isolated and on the basis of chemical analysis and chromatographic behavior was shown to be Tay-Sachs ganglioside (GM2). The particulate preparation also utilized N-acetyl-D-glucosamine and some of its derivatives as acceptors, but partial heat inactivation and substrate competition experiments indicated that the two classes of acceptors, hematoside and N-acetylglucosamine, were substrates for different N-acetylgalactosaminyltransferases. The enzyme that utilized hematoside showed low but detectable activity with analogues such as lactosylceramide and sialyllactose, but no activity with a wide range of other beta-galactosides and glycosphingolipids. These results are in accord with a proposed pathway for the biosynthesis of the gangliosides and for the patterns of these substances in different cell types and tissues.     

Dietary fiber decreases cholesterol and phospholipid synthesis in rat intestine

The effects of fiber ingestion on the incorporation of oleic acid into triglyceride and lecithin, acetate incorporation into cholesterol, and monosaccharide and amino acid transport were determined in rat intestine. Prolonged pectin (10% by weight) ingestion caused a decrease in jejunal and ileal cholesterol synthesis (33% and 52%, respectively). Pectin ingestion reduced cholesterol synthesis by 60% in ileal crypt cells, but did not affect cholesterol synthesis in the jejunal or ileal villus cells or in jejunal crypt cells. Cholesterol synthesis in isolated crypt cells was markedly less than in isolated villus cells. Prolonged ingestion of a fiber-free diet supplemented with either cellulose or pectin (10% and 5% by weight, respectively) decreased jejunal lecithin glucose and leucine absorption but did not affect jejunal triglyceride synthesis.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.     

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.     

Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets.

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterations might be related to the appearance of specific functions.     

Disaccharidase-deficient animals have normal ultrastructure of intestinal brush border membranes

Summary The intestinal disaccharidases, lactase, sucrase-isomaltase complex, and glucoamylase are proteins intimately associated with the brush-border membrane of the epithelial cell. These three enzyme activities are found in the intestine of the adult rat; lactase and glucoamylase activities are primarily associated with the intestine of the infant rat. Only glucoamylase and isomaltase activities are detected in the intestine of the California sea lion, Zalophus californianus. The activities of these enzymes are detected only in villus cells, and not in crypt cells.We have carried out electron microscopic studies of negatively stained brush-border preparations of intestinal crypt and villus cells; from the intestine of the 10-day-old rat and from that of the California sea lion. The density of the knob-like structures protruding from the brush-border membranes was not significantly different in any of these preparations. The diameter of the knobs on the preparations from crypt cells was smaller than the diameters of the knobs found on membranes prepared from the other sources. These data are discussed in terms of the relationship between the presence of knob structures and disaccharidase activities associated with the brush-border membranes.     

Improved isolation of villus and crypt cells from rat small intestinal mucosa.

An enzymic method is described which allows the isolation under comparable conditions of crypt and villus cells from rat jejunum with normal morphologic appearance and high metabolic activity when compared with previous preparations. The method is based on a differential scraping of short lengths of everted small intestine to yield two villus cell fractions and a gut wall residue. The scrapings and the gut tube are incubated for the same length of time in a HEPES-buffered modified Hanks' balanced salt solution containing hyaluronidase, DNase, and soybean trypsin inhibitor. The cells of the crypt region are recovered by a further scraping of the digested gut wall. Cells from all fractions are dispersed by gentle agitation, washed, and harvested by centrifugation. The final crypt and villus cells are 95--99% viable by dye exclusion and exhibit 5--20% cross-contamination on the basis of differential marker enzymes. The isolated crypt and villus cells prepared by the new procedure are suitable for comparative studies of metabolic activity in the absence of chelation-induced structural and metabolic abnormalities.     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Ganglioside composition of normal and leukemic bovine lymphocytes

The ganglioside composition of bovine peripheral lymphocytes was shown to change sharply under lymphoid leukemia. In normal lymph, lymph nodes, spleen and blood lymphocytes the major ganglioside is N-glycolylhematoside, whereas in calf thymus lymphocytes appreciable amounts of more polar components (GM1- and GD1a-like gangliosides) were found. In leukemic lymphocytes isolated from the same tissues the hematoside content is decreased, while the amount of more polar gangliosides is increased. Possible causes of the altered ganglioside pattern in leukemic lymphocytes are discussed.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.