Apoptotic process in the monkey small intestinal epithelium: I. Association with glutathione level and its efflux

The apoptotic process in the normal gastrointestinal mucosa is of interest due to its possible role in physiological cell renewal. The aim of this study was to identify the apoptotic process in the monkey small intestine and the association of glutathione level and its efflux in this process. Monkey small intestinal epithelial cells were separated in to different fractions consisting of villus, middle and crypt cells. Apoptosis was identified by DNA ladder pattern and Hoechst staining. The level of glutathione, its efflux and the enzymes involved in its metabolism were quantitated in these fractions. Apoptotic cells were identified predominantly in the villus tip cell fractions by both DNA ladder pattern and Hoechst dye staining. Glutathione level was 7 fold higher in the crypt cells as compared to villus tip cells the middle cells showing a gradual decrease. A similar pattern was seen in mitochondrial content of glutathione. As the cells mature from crypt to villus, there is increased efflux of GSH, which may be responsible for the decreased level of GSH in apoptotic villus cells. In the monkey small intestine, apoptotic cells are seen in the villus tip fractions and the glutathione level and its efflux may play a role in this process.     

In vitro polarographic studies of rat brain oxygen uptake in simulated uraemia and hepatic coma

The villi and crypts of the small intestine of the albino rat (Rattus norvigicus) have been enumerated and measured at different ages from 2 weeks to 8 months. The number of villi increased up to the third month of age and the number of crypts up to the eighth month. In the proximal intestine, the mean length of the villus base increased up to the fifth month as ridge-shaped villi were formed. Villus height was greater proximally than distally and this, and the crypt depth, remained constant from the end of the first month of age. The total villus circumference per unit area of intestine, and the villus circumference per crypt was the same proximally and distally, and was relatively constant after the first month of age. The total circumference of the crypt mouths per square millimeter of intestine was the same proximally and distally and, at all ages, was greater than the total villus circumference. The villus surface area per square millimeter of intestine, or per crypt, remained relatively constant after the first month and was greater proximally than distally, due only to the taller villi proximally. The change from finger-shaped to ridge-shaped villi did not affect the villus mucosal surface area. The changes in villus shape are probably not determined by differences in the rate of crypt cell production.     

Apoptotic process in the monkey small intestinal epithelium: 2. Possible role of oxidative stress

Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis and this type of cell death can be inhibited by various antioxidants. In our accompanying paper, we have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. The aim of the present study was to evaluate the possible relationship between oxidative stress, antioxidant levels and the apoptotic process in the monkey small intestinal epithelium. Monkey small intestinal epithelial cells were isolated into different fractions consisting of villus, middle and crypt cells. Mitochondrial function was assessed by the reduction of the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), with and without succinate. The extent of lipid peroxidation was assessed by measuring the formation of conjugated diene, depletion of polyunsaturated fatty acids and alpha-tocopherol. Level of antioxidant enzymes like, superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase were also quantitated in various cell fractions. MTT reduction was significantly decreased in villus cells as compared to the cells from other fractions and this was evident even in presence of the respiratory substrate, succinate. Increased formation of conjugated diene and depletion of polyunsaturated fatty acids were seen in villus and crypt cells as compared to middle fraction cells. The alpha-tocopherol level was decreased in both villus and crypt cells as compared to cells from middle region. Significant decrease of SOD activity was seen in the villus tip cells and a slight decrease was seen in the crypt fractions. Glutathione dependent enzymes like GST, GPx and GSH reductase showed higher activity in the villus fractions. A similar observation was also seen in the catalase activity. This study has shown that although oxidative stress is seen in both villus and crypt cells, decreased mitochondrial function was seen in villus tip cells which may be responsible for apoptotic process in the intestinal epithelium.     

Intraepithelial lymphocytes from villus tip and crypt portions of the murine small intestine show distinct characteristics

BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) are located between epithelial cells that are thought to display unique features and functions at the small intestinal villus tip and crypt levels. We have addressed whether the spatial differences in the intestinal epithelium extend to IELs and subsequent cross-talk between IELs and epithelial cells. METHODS: IELs were isolated from villus tip and crypt portions of mouse small intestine and then compared for spontaneous cytokine production and responsiveness to interleukin (IL)-2 and/or IL-7. RESULTS: No difference was observed between number of beta IELs in villus tips and crypts, whereas a trend toward increased frequencies of IELs bearing the gamma delta form of T-cell receptor was noted in villus tips. Interestingly, the number of beta IELs producing interferon gamma and IL-5 was significantly reduced in the cells from crypts compared with villus tips. Furthermore, villus tip beta IELs exhibited higher responses to stimulation signals provided by IL-2 and/or IL-7 than their crypt counterpart. Such functional differences were not observed with gamma delta IELs from the two intestinal sites. CONCLUSIONS: Distinct molecular cross-talk between IELs and epithelial cells occurs in intestinal villus tips and crypts.     

A comparative study of the glycolipids of human, bird and fish testes and of human sperm

The glycolipids of human testis and sperm have been compared. Both adult testis and the sperm exhibited remarkably complex, but generally similar, patterns of glycolipids. In particular, both contained appreciable amounts of the sulfogalactosylmonoalkylmonoacylglycerol, recently shown to be the principal glycolipid of the testis and sperm of a number of animals. In contrast, immature (prebuteral) human testis did not contain this compound. To extend knowledge on the possible distribution of sulfogalactosylmonoalkylmonoacylglycerol in the testes of other chordates, we have also analysed the glycolipids of the testes of a number of birds and fish. None of the testes from these species contained the above compound. Instead, sulfogalactosylceramide was found to be a major glycolipid of the testis of mature fowl, duck and skate-fish and sulfogalactosylglucosylceramide of the testis of mature salmon and trout. Immature duck testis contained only a trace of sulfogalactosylceramide. These studies reveal intriguing differences between the sulfatides of various chordates, lend support to the concept that sulfatides increase markedly in testis at a specific stage of spermatogenesis and suggest an important role for sulfatides in testicular and spermatozoal function.     

Aglycone modulation of glycolipid receptor function

The aglycone has been largely ignored in consideration of glycoconjugate function. Evidence is reviewed which suggests that the role of the lipid in glycolipid carbohydrate function may be particularly significant. The lipid moiety can promote or reduce carbohydrate exposure of membrane glycolipids. Theoretical calculation has indicated that the plane of the plasma membrane can restrict the permitted conformations of a given glycolipid oligosaccharide. Thus the lipid moiety may influence the relative conformation of such carbohydrate sequences. Evidence of ceramide regulation of glycolipid function can be found in studies of enzyme substrate specificity, antiglycolipid recognition and bacterial/host cell interactions. Studies of verotoxin binding to its glycolipid receptor globotriaosyl ceramide indicate that modulation of receptor function by glycolipid fatty acid content plays an important role in in vitro binding assays, cell cytotoxicity and intracellular routing.     

The role of the facilitating cell in the establishment of donor chimerism and transplantation tolerance

Guanylin, structurally related to the heat-stable enterotoxin of E. coli, is a 15-amino-acid peptide isolated from rat small intestine. We investigated the morphological effects of an intravenous injection of rat and human guanylin upon the rat intestine. Various doses of rat guanylin were injected intravenously in anesthetized rats. After 5, 10 and 30 min, rats were killed by intracardiac perfusion with aldehyde fixative, and specimens of the intestine were then prepared for light and electron microscopy. Intravenously injected rat guanylin rapidly induced mucus secretion from crypt goblet cells in the duodenum. About half of the crypt goblet cells secreted mucous granules by compound exocytosis within 5 min. The villus goblet cells, in contrast, were not sensitive to guanylin. Goblet cells in the jejunum were less responsive than those in the duodenum. This secretory response was rare in the ileum and colon. Human guanylin produced similar results. The mucus secretion induced by guanylin was inhibited by a prior-injection of atropine, but not hexamethonium. Moreover, guanylin induced intense edema in the mucosa and submucosa of the small intestine 5 min after the injection, which disappeared after 30 min. A prior-injection of atropine did not block the appearance of edema. In conclusion, the intravenous injection of guanylin induces two phenomena related to water movement: (1) compound exocytosis of mucous granules from crypt goblet cells in the rat duodenum and jejunum; (2) perineural, inter-epithelial and intra-epithelial edema in the rat small intestine.     

Human brush border myosin-I and myosin-Ic expression in human intestine and Caco-2BBe cells

The human intestinal cell line, Caco-2BBe, has been established as an excellent model system for analysis of the enterocyte cytoskeleton including that of the actin rich apical brush border. To facilitate its use for functional analysis of a major component of the brush border, brush border myosin-I, human cDNAs encoding the heavy chain of this class I myosin were isolated and sequenced. The identity of this myosin as human brush border myosin-I was verified based on similarity with other vertebrate sequences, as well as its expression profile at both the RNA and protein levels. Localization of the protein in human intestine along the crypt-villus axis closely resembles that previously determined for brush border myosin-I in chicken, and is quite distinct from that of myosin-Ic, another myosin-I expressed in human intestine and Caco-2BBe cells. In immature cells of the crypt, brush border myosin-I staining is low, and there is significant cytosolic and basolateral localization, while villus cells stain much more intensely, and the protein is primarily localized to the brush border. Localization of myosin-Ic is essentially the inverse of brush border myosin-I in that crypt cells exhibit higher levels of staining, while villus cells have very low levels of myosin-Ic. The expression of both myosins-I was also examined during cell-contact induced differentiation of Caco-2BBe cells where expression and changes in localization closely resemble those that accompany differentiation of enterocyte in vivo.     

The ultrastructure of the cell center in the enterocytes of mouse embryos and newborn mice

The centrosome structure was studied in differentiating small intestine enterocytes of mouse embryos (day 16 to 17 of gestation) and newborn mice. It is shown that fine structure of the centrosome in embryonic enterocytes differs from that found in cells of the adult mice. The centrosome of the crypt cells is more active: a greater number of cytoplasmic microtubules terminates there; mother centriole has two types of satellites; both centrioles are surrounded by fine fibrillar material. In 20% of crypt cells, replication of centrioles is observed. In the upper part of the villus of embryonic intestine, there still are two centrioles per cell, but they are located 3-7 um apart and devoid of any cytoplasmic microtubules attached to or directed toward them. In newborn mice, centrosomes of cells located at the bottom of the crypt are less active, as compared with centrosomes of embryonic cells. There are 1-3 satellites on mother centriole and few cytoplasmic microtubules closing to the centrosome. In 20% of cells from the bottom of the crypt centrioles are replicating. In the lateral part of the crypt, centrosome is inactive: centrioles are located 1-3 um apart and do not replicate. In the villus, centrioles undergo changes similar to those observed in the villus of adult mice. Centrioles loose portions of microtubule triplets. Near the top of the villus, only one centriole was found in two of 25 studied cells. In the enterocytes of the murine small intestine, centrioles are always located far from nuclei and close to the apical cell surface (1-3 um from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane.     

The effect of transposition to jejunum on epithelial cell kinetics in an ileal segment

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4--7 days to reach values normal for jejunum after 14--30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with 3H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after 3H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.     

Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine, parasitic nematode, Ascaris suum

The acidic glycolipid fraction (AF) of the porcine, parasitic nematode, Ascaris suum , consisted of two subfractions. The major component AF II reacted with orcinol-sulfuric acid and molybdate, while the minor component AF I gave a positive reaction with azure-A, a cationic dye specific for sulfatides. Sugar constituent analysis, methanolysis, methylation analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary-ion mass spectrometry, and gas-liquid chromatography/mass spectrometry specified AF II to be an unusual phosphoinositolglycosphingolipid (Galalpha1-Ins-P-1ceramide) and the minor component AF I to be a 3-sulfogalactosylcerebroside (HSO3-3Galss1-1ceramide). The ceramide moiety of both components consisted of lignoceric (C24:0) and cerebronic (C24h:0) acids and mainly C17 iso-branched sphingosine. Immunohistochemical localization studies of the glycolipid-bound antigenic determinants with a polyclonal antiserum against AF II and an anti-sulfatide monoclonal antibody against AF I revealed the presence of the AF II-epitope in the intestine, whereas the AF I-epitope was found in the hypodermis, contractile zone of somatic muscle cells and the external musculature of the uterus. To our knowledge, this is the first report of the presence of a sulfatide in an invertebrate.     

Predictive models of carpal tunnel syndrome causation among VDT operators

Gpx2 mRNA, encoding a selenium-dependent glutathione peroxidase (GPX-GI), has been found to be highly expressed in the gastrointestinal tract (GI) mucosal epithelium. In this study, we show that GPX-GI is produced in the mucosal epithelium of the adult rat GI tract and that the activity levels are comparable to that from GPX-1. Post-mitochondrial supernatant GPX activity from the mucosal epithelium of the complete length of the small intestine was partially purified. A sample enriched for putative GPX-GI was fractionated by SDS-polyacrylamide gel electrophoresis. Polypeptides of 21 kDa and 22 kDa were digested with trypsin. After resolving the tryptic peptides by high pressure liquid chromatography (HPLC), the major peaks were analyzed for their amino acid sequence by Microflow-HPLC-Tandem Mass Spectrometry and automated Edman degradation sequencing. Both methods revealed that the 21-kDa sample contained rat GPX-GI determined by the sequence homology with the deduced mouse GPX-GI polypeptide sequence. Rat GPX-1 was also detected in the samples. AntiGPX-GI and antiGPX-1 antibodies were used to determine the distribution of the respective isoenzyme activities along the length of the intestine and with respect to the crypt to villus axis in rats. GPX-GI and GPX-1 activities were uniformly distributed in the middle and lower GI tract and with respect to the crypt to villus axis. GPX-GI activity accounted nearly the same percentage of the total GPX activity as GPX-1 in all of the these compartments. Studies on the distal ileum segment of wildtype and Gpx1 gene knockout mice showed that GPX-GI activity was also at parity with GPX-1 in the mucosal epithelium of this segment.     

Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Cell proliferation and crypt fission are controlled independently

Crypt fission is now established as an important mechanism of intestinal growth and regeneration. It has been proposed that increased crypt size is the stimulus for crypt fission, because crypts preparing for fission are generally larger. Consequently, we investigated the effects of epidermal growth factor (EGF) and dimethylhydrazine, which are both known to stimulate crypt cell proliferation, on crypt fission in the rat intestine. We also examined whether the effects of EGF on both proliferation and crypt fission are modified by the pretreatment with dimethylhydrazine for 16 weeks, dimethylhydrazine was then discontinued for 8 weeks, followed by intravenous infusion of EGF for 1 week. There were four groups: vehicle alone, EGF alone, dimethylhydrazine alone, and dimethylhydrazine followed by EGF infusion. The rats were killed at 25 weeks and rates of intestinal crypt cell production, crypt size, and crypt fission were determined. Intravenously infused EGF significantly increased crypt cell production rate, but the magnitude of the effect decreased from the proximal to the distal colon. EGF caused an increase in crypt area, possibly reflecting an increase in crypt size. Importantly dimethylhydrazine had no significant effect on crypt cell production rate nor on crypt area in the distal colon, but it did cause an increase in crypt area in the mid-colon. The crypt fission index was significantly decreased by EGF and increased by dimethylhydrazine. There was no qualitative interaction between EGF and dimethylhydrazine. These results demonstrate the marked proliferative effect of intravenously infused EGF in the colon of orally fed rats, with significant site effects (P = 0.0007); the effect was greatest in the proximal colon and disappeared in the distal colon. The observation that EGF reduced crypt fission indicates that increased cell proliferation, per se, is not a stimulus for crypt fission. This is further supported by the observation that dimethylhydrazine increases crypt fission in crypts of normal size in the distal colon without significantly increasing cell proliferation. These results suggest that increasing crypt cellularity by proliferation is not sufficient to induce crypt fission, and factors other than increased crypt size by proliferation can control crypt fission. It is also probable that cell proliferation and crypt fission are independently regulated. Crypt fission appears to play a considerable role in the intestinal response to carcinogens.     

Ultrastructural changes in the cell center during enterocyte differentiation in the mouse

Centrioles in the enterocytes of murine small intestine are located far away from the nuclei and close to the apical surface of cells (1-3 microns from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane. Centrosomes (cell center) in enterocytes undergo subsequent involution in respect to the position of cell in the crypt-villus system. Five zones were studied separately: the bottom of the crypt (approx. 1/3 of the appendix), the upper part of the crypt (before its transition into the villus), the basal part, the middle and top of the villus. A centrosome in the crypt contains two centrioles (maternal and daughter) located close to each other. In 10 of 27 cells studied centrioles were replicating. A maternal centriole has 2-3 pericentriolar satellites and appendages. Several cytoplasmic microtubules run towards mother centrioles. Centrioles at the upper part of the crypt split at a distance up to 1.5 microns from each other. The average number of cytoplasmic microtubules and pericentriolar satellites decreases. At the basal part of the villus, centrioles split a distance up to 5 microns from each other; seldom microtubules may be found in a radius of 1 micron around centrioles and none of them is attached to centrioles or satellites. Starting from the middle of the villus, centrioles sometimes may have incomplete triplets of microtubules at the distal and (or) the proximal end. At the upper part of the villus there is only one centriole per cell, that was found in 10 of 50 cells studied on a complete series of 0.2 micron thick sections.     

The impact of primary and modular nursing delivery systems on perceptions of caring behavior

BACKGROUND: The somatostatin analogue octreotide impairs intestinal regeneration and the adaptive response to intestinal resection by inhibition of enterocyte migration and proliferation and increased apoptosis. Epidermal growth factor (EGF) stimulates regeneration and adaptation by increasing proliferation and reducing apoptosis. The aim of this study was to determine the effect of EGF on octreotide-induced enterocyte apoptosis. METHODS: Twenty-four rabbits underwent patch enteroplasty in the distal ileum to stimulate the mucosa. There were four study groups: octreotide 250 microgram/kg/day, EGF 40 microgram/kg/day, EGF plus octreotide, and control. Normal ileal mucosa adjacent to the patch was evaluated at 7 days for villus height, crypt depth, crypt cell production rate (CCPR), and in situ end labeling of DNA fragmentation. RESULTS: Octreotide alone increased apoptosis compared with controls at the villus tip (40 +/- 7% vs 18 +/- 7%, P < 0.05), lateral villus (9 +/- 2% vs 3 +/- 2%, P < 0.05), and crypt (15 +/- 3% vs 10 +/- 3%, P < 0. 05). EGF decreased apoptosis in the crypt (2 +/- 1%) and villus (6 +/- 1% villus tip and 1 +/- 1% lateral villus, P < 0.05) compartments. EGF inhibited octreotide-induced apoptosis in the crypt (5 +/- 2%) but not the villus (31 +/- 5% villus tip and 6 +/- 2% lateral villus, P < 0.05). Mean DNA fragmentation was significantly greater in octreotide-treated animals (P < 0.05). The octreotide-treated animals had reduced crypt depth and villus height but normal CCPR compared with controls. EGF increased CCPR and crypt depth compared with controls. Combining EGF and octreotide resulted in crypt depth and CCPR similar to those of controls but reduced villus height. CONCLUSIONS: EGF inhibits octreotide-induced apoptosis. This effect is greater in crypt than in villus enterocytes. Octreotide appears to have both direct and indirect effects on enterocyte apoptosis.     

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.     

The effects of exogenous sphingosine on Neuro2a cells are strictly related to the overall capacity of cells to metabolize sphingosine

Neuro2a cells were exposed to different doses (1-40 nmol/10(6) cells) of [C3-3H]sphingosine and the relationship between metabolism and biological effects of sphingosine was investigated. Sphingosine appeared to be rapidly taken up and metabolized. The incorporation of sphingosine was not merely dependent on its concentration but primarily on the dose per cell of administered sphingosine. At low doses, [3H]sphingosine represented a minor portion of the cellular radioactivity, and N-acylated metabolites, particularly ceramide, largely prevailed over degradation products. Concomitantly with ceramide increase, Neuro2a differentiation took place. With increasing exogenous sphingosine/doses, the acylation process reached saturation. From this point on, [3H]sphingosine started accumulating and eventually cell toxicity occurred. In conclusion, the biological effects exerted by exogenous sphingosine on Neuro2a cells are not merely dependent on the long-chain base concentration in the culture medium, but are strictly related to the cellular dose of exogenous sphingosine and to the capacity of cells to metabolize sphingosine.     

Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum

In hereditary hemochromatosis (HH), intestinal absorption of dietary iron is increased, leading to excessive iron accumulation in tissues and resultant organ damage. The HFE protein, which is defective in HH, normally is expressed in crypt enterocytes of the duodenum where it has a unique, predominantly intracellular localization. In placenta, the HFE protein colocalizes with and forms a stable association with the transferrin receptor (TfR), providing a link between the HFE protein and iron transport. In the present study, we examined the relationship of the HFE protein to the TfR in enterocytes of the human duodenum and measured the uptake of transferrin-bound iron and ionic iron by isolated crypt and villus enterocytes. Immunocytochemistry showed that the HFE protein and TfR both are expressed in the crypt enterocytes. Western blots showed that, as was the case in human placenta, the HFE protein in crypt enterocytes is physically associated with the TfR and with beta2-microglobulin. The crypt cell fraction exhibited dramatically higher transferrin-bound iron uptake than villus cells. On the other hand, the villus cells showed 2-3 times higher uptake of ionic iron than crypt cells. We propose that the HFE protein modulates the uptake of transferrin-bound iron from plasma by crypt enterocytes and participates in the mechanism by which the crypt enterocytes sense the level of body iron stores. Impairment of this function caused by HFE gene mutations in HH could provide a paradoxical signal in crypt enterocytes that programs the differentiating enterocytes to absorb more dietary iron when they mature into villus enterocytes.