Small Intestinal Explants Research Articles

Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex.

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i.e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.

Organ specificity of epithelial cells grown in tissue culture from explants obtained from various levels of the rat gut

Monolayer cultures of epithelial cells grown from explants of fetal rat small intestine can differentiate into columnar as well as goblet cells in tissue culture (Kondo et al., Exp cell res 153 (1984) 12) [9]. In this study we have cultured tissue from various levels of the fetal rat gastrointestinal tract in order to characterize those cell types that can be cultured from these tissues and to determine the growth potential of these cells using this culture system. Explants of esophagus and forestomach tissue yielded monolayer outgrowths of squamous epithelial cells which grew as closely apposed polygonal cells capable of developing cornified envelopes. Explants of the glandular portion of the stomach yielded outgrowths of densely packed cells which were more pleiomorphic and which did not desquamate or form cornified envelopes. Explants of small intestine and colon yielded outgrowths of epithelial cells, some of which differentiated into goblet cells, while others developed a 'columnar' cell morphology. The following differences between squamous- and non-squamous cultures were observed. Squamous epithelium showed self-sustained growth, while growth of non-squamous epithelial cells became self-limiting. Cytochalasin B (CB) (5 micrograms/ml for 1 h) induced contraction of the whole cell sheet of non-squamous cells, but of only individual squamous cells. Squamous cell outgrowth was observed from tissues derived from fetuses of all ages (13-day fetuses through to 3-week-old rats); whereas non-squamous epithelial outgrowth was poor when rats older than the 21st gestational day were studied. These results indicate that cultures established in this manner developed the general characteristics of the cells of the organ from which they were derived, even when undifferentiated fetal tissue was used. The growth conditions needed for columnar epithelium are more stringent than those needed for squamous tissues. This technique opens the way for further characterization of growth requirements of gastrointestinal columnar epithelium.

In vivo and in vitro metabolism of 2,4-dinitrotoluene in strain a mice

The elimination of metabolism of a single dose (100 mg/kg) of 2,4-dinitrotoluene (2,4-DNT) in A/J mice were examined. After intraperitoneal administration, elimination was rapid, with 70% of the dose appearing in the urine within 4 hr. Four hours after oral administration, only 28.5% of the dose was excreted in the urine, which increased to 66% after 8 hr. Elimination via the feces was minimal (<2.1% of the dose) in both cases. From 0.5 to 4 hr after intraperitoneal administration, 3.6 to 8.8% of the urinary metabolites was unconjugated while 2.4 to 8.8% was present in the glucuronide fraction. After oral administration these amounts were 5.5 to 6.8% and 20.5 to 28.2% respectively. After both intraperitoneal and oral administration, no unchanged 2,4-DNT could be detected in the urine, and 2,4-dinitrobenzyl alcohol (2,4-DNBAlc) represented the most abundant identifiable neural metabolite. Small amounts of 2,4-diaminotoluene, 2-amino-4-nitroobenzyl alcohol, 2-( N-acethyl)amino-4-nitrotoluene, 4-amino-2-nitrotoluene (4A2NT), and 2-amino-4-nitrotoluene (2A4NT) were also present. In almost all cases the largest proportion of metabolites represented unknowns, some of which exhibited the chromatographic properties of carboxylic acid metabolites. Metabolism of 2,4-DNT by liver and lung microsomes yielded mainly 2,4-DNBAlc with lower amounts of 4A2NT and 2A4NT, and their formation was dependent on the presence of oxygen and NADPH. Pretreatment of the animals with 2,3,7,8-tetrachlorodibenzo- p-dioxin resulted in increased yields of all three metabolites. Aerobic metabolism of 2,4-DNT by explants of small intestine, large intestine, or by cecal contents yielded 2,4-DNBAlc, 2A4NT, 4A2NT and 4-( N-acetyl)amino-2-nitrotoluene (4Ac2NT). The proportion of reduced metabolites (2A4NT, 4A2NT, and 4Ac2NT) was much higher in these systems than with liver or lung microsomes and their formation by small intestine and cecal contents was enhanced several-fold under anaerobic conditions, while that of 2,4-DNBAlc was abolished. It is concluded that 2,4-DNT metabolism in the A/J mouse is rapid and complete and that the major neutral urinary metabolite is 2,4-DNBAlc. Minor amounts of reduced or partially reduced products appear to be formed mainly in the intestine, with a major role by its microflora.

230 CELL PROLIFERATION IN HUMAN FETAL SMALL INTESTINE

Cellular kinetic data are not available for human fetal intestinal tissue. Therefore an investigation was undertaken to establish the tritiated thymidine (3H-TdR) incorporation into DNA and the labelling indices (LI) in the epithelium (E), mesenchymal (ME) and the muscular (M) layers between 8 and 15 weeks gestation. Explants of small intestine were cultured in serum-free Leibovitz L-15 medium at 37°C in 95% air-5% CO2. The LI were established on radioautographs of explants cultured for 2 h in presence of 2 μCi of 3H-TdR per ml of culture medium. The incorporation of 3H-TdR into DNA was evaluated after a 6 h period and results expressed as DPM/μg DNA. Between 8 and 10 weeks' gestation, the LI were 28, 14 and 9% for respectively the E, ME and M layers. Between 10 and 12 weeks there was a decrease of the LI which remained more or less constant between 12 and 15 weeks (15, 9 and 6%). Labelled epithelial cells were noted at all levels of the epithelium prior to or at the beginning of villus formation and later on were observed only in the intervillous area. The addition of hydrocortisone (50 ng/ml) to the culture medium during 5 days induced a significant increase (98%) of 3H-TdR incorporation into DNA. This overall increase of DNA synthesis was reflected at the epithelial cell level where the LI increased by 57%. These experiments provide some basic data on cell proliferation in fetal intestine and indicate that explant culture technique should be useful for the study of the regulators of cell proliferation during the development of human intestine.

Biosynthesis of intestinal microvillar proteins: Rapid expression of cytoskeletal components in microvilli of pig small intestinal mucosal explants

Using alkaline extraction to separate cytoskeletal and membrane proteins of intestinal microvilli, the kinetics of assembly of these two microvillar protein compartments was studied by pulse-chase labelling of pig small intestinal mucosal explants, kept in organ culture. Following a 10 min pulse of [ 35S]methionine, the membrane proteins did not appear in the microvillar fraction until after 40–60 min of chase. In contrast, the cytoskeletal components, of which the 110-kDa protein and villin were immunologically identified, were expressed in the microvillar fraction immediately after the 10 min pulse. These different kinetics of appearance indicate that the two microvillar protein compartments have separate mechanisms of biosynthesis and microvillar expression.

Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules.

The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestinal explants. On the ultrastructural level, monensin (1 microM) caused an increasingly extensive dilation and vacuolization of the Golgi complex during 4h exposure of the explants. On the molecular level, the effect of monensin was twofold. (1) The processing from the initial high-mannose-glycosylated form to the mature complex-glycosylated form was arrested. For some of the enzymes studied, intermediate stages between the high-mannose and complex forms could be seen, probably corresponding to 'trimmed' or partially complex-glycosylated polypeptides. (2) Labelled microvillar enzymes failed to reach their final destination. These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes. The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes in the microvillar membrane during a 3 h labelling period. Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the microvillar membrane.

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV.

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase.

The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-beta-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a 'high-mannose' oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20--40 min or 60--90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60--90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes.

Explants of pig small intestine were maintained at 37 degrees C in organ culture for periods up to 24 h in a system using Trowell T-8 medium supplemented with 10% foetal-calf serum. The mucosal morphology was well preserved during culture, as judged by light and electron microscopy. The explant contents of protein and two brush-border enzymes, microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.5), were not significantly modified during culture compared with controls, but a moderate, continuous release of both protein and enzyme activities into the medium was observed. Continuous labelling with [35S]methionine resulted in an even incorporation of radioactivity in the protein components, and the rate of labelling only moderately decreased over the 24 h period. The polypeptide compositions of sucrase (EC 3.2.1.48)--isomaltase (EC 3.2.1.10), maltase--glucoamylase (EC 3.2.1.20) lactase (EC 3.2.1.23)--phlorizin hydrolase (EC 3.2.1.62), microvillus aminopeptidase and aspartate aminopeptidase (EC 3.4.11.7) synthesized during culture were studied, and some were found to be similar to those of the pro-forms of the enzymes isolated from animals that had had their pancreatic duct disconnected 3 days before being killed. These results confirmed earlier findings of the existence of pro-forms of some of the microvillar enzymes and thus indicate a low activity of pancreatic proteinases in the culture system.

Comparative tissue explant requirements of Columbia-SK, C(M) and M.M. viruses, and of Theiler's encephalomyelitis virus.

SummaryIn a fluid medium consisting of 3 cc of Simms' solution and 1.25 cc of oxserum ultrafiltrate plus 0.25 cc of selected minced tissue in glucosol solution, the Col. SK, C(M) and M.M. viruses were found to grow freely on tissue explants of brain, lung, small intestine, heart muscle, and tail from embryo mice, but not on explants of tongue skeletal muscle, kidney, liver, or spleen from the same embryos. Growth of all 3 viruses was also obtained on explants of minced 5-day chick embryos, but not to an equal degree.The GD VII strain of Theiler's encephalomyelitis virus was carried through 15 culture passages on explants of brain and tail tissue, but failed to show well defined or continued growth on explants of other tissues and organs from embryo mice. Unlike the GD VII strain, the FA strain of Theiler's virus failed to grow on explants of tail tissue. Both strains grew on explants of chick embryo tissue, but the FA strain grew less freely than the GD VII strain, even on explants of mouse brain tissue, ...