Biomedical subjects

J F Beaulieu

Publications and source records attributed to J F Beaulieu.

At least 55 records · Page 3Linked to original sources

PubMed1994

Reciprocal expression of laminin A-chain isoforms along the crypt-villus axis in the human small intestine.

BACKGROUND/AIMS: Intestinal epithelial cell proliferation, migration, and differentiation are susceptible to various influences along the crypt-villus axis including compositional changes in the basement membrane and differential expression of receptors for these components. However, laminin has been consistently detected at the epithelial basement membrane without significant variation in relation to the crypt-villus functional unit. The aim of this study was to examine the distribution of individual laminin chains. METHODS: The patterns of distribution of laminin A, M (an A-chain variant), B1, and B2 chains as well as the integrins alpha 6 beta 1 and alpha 6 beta 4, two laminin receptors, were determined by indirect immunofluorescence in the adult human small intestine. RESULTS: Markedly distinct patterns of laminin chain expression were observed along the crypt-villus axis. The A chain was found predominantly associated with the differentiated epithelial cells on the villus whereas the M chain was restricted to the basement membrane of crypts. However, both B1 and B2 chains were detected all along the intestinal basement membrane. Furthermore, alpha 6 beta 1 and alpha 6 beta 4 integrins were found to be expressed by all enterocytes. CONCLUSIONS: These observations show a reciprocal expression of laminin isoforms along the crypt-villus axis and support the concept that the intestinal epithelial basement membrane is subjected to intricate compositional changes.

Adult↗

PubMed1994

Tenascin may not be required for intestinal villus development.

Tenascin, a large extracellular matrix protein, is subject to complex spatial and temporal patterns of expression in the course of various organogenetic processes including the formation of the small intestinal villus. In the present study, the presence of tenascin was analyzed in human fetal colonic villi, which are transient structures that are programmed to disappear at the time of colonic mucosal remodeling. While the labeling of muscles and peripheral mesenchyme was similar in both segments, surprisingly the colonic villus cores were mostly devoid of tenascin as opposed to those of the small intestine. Western blot analysis revealed that the 220 and 320 kDa forms of tenascin were detected in both segments. However, the 200 kDa form present in small intestinal villi was absent in colonic villi. These data suggest that tenascin under its 200 kDa form is not required for villus formation.

Blotting, Western↗

PubMed1993

Expression of tenascin in the developing human small intestine.

The expression of tenascin (Tn), an extracellular matrix glycoprotein, was analyzed in the human small intestine. Three major oligomeric forms of intestinal Tn (320 kDa, 220 kDa and 200 kDa) were identified in both the fetus and the adult. The intestinal 200 kDa form was not found expressed by intestinal mesenchymal cells in culture. During development, the amounts of the 320 and 220 kDa forms remained relatively constant between 9-20 weeks of gestation as compared to sucrase-isomaltase and a-smooth muscle actin while the 200 kDa immunoreactive component increased substantially during this period of active intestinal morphogenesis. Further experiments by microdissection revealed that the 200 kDa component was the major form of Tn in the intestinal villus. These results suggest that this newly identified 200 kDa form of Tn in the small intestine is predominantly expressed in the villus under a developmentally regulated pattern.

Adult↗

PubMed1993

Basement membrane formation and re-distribution of the beta 1 integrins in a human intestinal co-culture system.

We have developed a co-culture system suited for the study of epithelial-mesenchymal interactions in the human fetal small intestine. As the epithelial component of this model, we used the human intestinal cell line Caco-2 that is unique in its property to differentiate in vitro into a mature fetal enterocyte-like cell type. A sheet of human intestinal mesenchymal cells, which we derived from an 18-week-old fetus, was used as mesenchymal element. Expression and distribution of cell-specific markers (cytokeratin 18 and dipeptidyl peptidase IV), major basement membrane components, and beta 1 integrins were analyzed. In 14-day co-cultures, Caco-2 cells formed a cytokeratin 18-positive epithelial-like sheet covering the vimentin-positive HIM cell layers. As assessed by brush border dipeptidyl peptidase IV expression, co-cultured Caco-2 cells achieved cytodifferentiation as when cultured on plastic. A complete deposition of all known major human fetal intestinal basement membrane components occurred at the Caco-2/HIM interface. Type IV collagen and tenascin were produced from the mesenchymal compartment, whereas laminin and fibronectin were contributed by both cell types. Interestingly, synthesis and deposition of basement membrane heparan sulfate proteoglycan were exclusively observed in co-cultures, suggesting modulation of epithelial expression of this molecule by HIM cells. Finally, we observed that epithelial integrin-beta 1 chains redistributed at the basal domain of co-cultured Caco-2 cells. Taken together, these observations indicate that the Caco-2/HIM co-culture model is a valuable system to study in vitro human basement membrane formation in the context of intestinal epithelial-mesenchymal interactions.

Basement Membrane↗

PubMed1993

Intramitochondrial localization of the main 70-kDa heat-shock cognate protein in Drosophila cells.

The main heat shock cognate protein (hsc) of 70 kDa in Drosophila, hsc 4, was localized in cultured cells using a specific affinity-purified antibody and colloidal gold immunoelectron microscopy. This constitutively expressed member of the heat shock protein (hsp) 70 family is found in the cytosol, in mitochondria, and in the nucleus of unstressed cells. The identity of hsc 4 in these three cellular compartments was confirmed by two-dimensional gel immunoblots and partial proteolytic digestion patterns. In mitochondria, the colloidal gold particles are observed in close proximity to or on the inner membranes. The intramitochondrial localization of this hsc was confirmed by density gradient purification and by resistance of hsc 4 to externally added trypsin. In the nucleus, the labeling is found on nucleo-plasmic perichromatin RNP fibrils and is not detected in the nucleolus. Heat shock induces an intracellular redistribution of hsc 4 with an enrichment in the nucleus. The localization of this hsc in different cellular compartments is consistent with the previously suggested functions of some members of this family of proteins in basic cellular processes such as protein folding. Moreover, the present data suggests that the main constitutively expressed member of the hsp 70 family, hsc 4, functions both within the mitochondrial compartment and in the nucleus.

Animals↗

PubMed1993

Relationship between tenascin and alpha-smooth muscle actin expression in the developing human small intestinal mucosa.

The expression of tenascin (Tn) and alpha-smooth muscle actin (alpha-SMA) was analyzed in the developing and adult human small intestine by means of double immunofluorescent staining with specific antibodies. By 7 weeks of gestation, the gut anlage has a simple tubular shape and is formed of a stratified undifferentiated epithelium surrounded by a poorly organized mesenchyme. Both Tn and alpha-SMA were found exclusively at the periphery of the tissue, corresponding to the presumptive muscularis propria. By 9 weeks, villus rudiments had formed but Tn and alpha-SMA remained restricted to the muscularis propria. Tn was first detected in the mesenchyme at 11 weeks. By 13 weeks, a preferential distribution of Tn in the subepithelial region of the mesenchyme was readily observed while alpha-SMA was still absent. From this stage to 20 weeks, Tn gradually concentrated in this region that, as determined by alpha-SMA detection, corresponded to the future muscularis mucosa area. As shown by double staining of Tn and alpha-SMA, deposition of Tn also preceded the appearance of the other alpha-SMA-expressing cells in the mucosa. These observations suggest that Tn could have a role in the differentiation of intestinal contractile cells.

Actins↗

PubMed1993

An intestinal secretory protein is found in most glands associated with the gastrointestinal tract: von Ebner's and salivary glands, gallbladder, and pancreas.

We have previously shown that monoclonal antibodies (MAb) prepared against the duodenal mucosa of 4-day-old mice disclosed the presence of two antigens associated with the formation of intestinal crypts. One of these, MIM-1/39, was found in the apical cytoplasm of undifferentiated epithelial crypt cells of the duodenum and colon. We report here the immunolocalization of MIM-1/39 in different glands associated with the gastrointestinal tract, using a polyclonal antibody produced against antigen MIM-1/39. By indirect immunofluorescence on 1-micron thick Lowicryl K4M sections, MIM-1/39 was detected in secretory granules of serous cells in lingual (von Ebner's gland), sublingual, submandibular and parotid glands, and in pancreas; it was also found in epithelial cells of the gallbladder and in the secretory granules of chief cells of gastric glands. Liver, kidney, and Brunner's glands were not immunoreactive. Immunocytochemistry revealed the presence of antigen MIM-1/39 in small secretory granules of the gallbladder, duodenum, colon, and in the large secretory granules in serous cells of lingual and parotid glands, in pancreas, and in gastric chief cells. In Western blotting, the MIM-1/39 MAb revealed two bands (330 and 350 KD) in adult mouse duodenal mucosa, gallbladder and stomach, whereas only one (330 KD) was disclosed in pancreatic juice. However, two bands (330 and 350 KD) were detected in pancreatic juice with the polyclonal antibody. The distribution of MIM-1/39 was different from that reported for IgA, bound and free secretory components, cryptdin, and Tamm-Horsfall protein. Therefore, MIM-1/39 appears to be a unique protein. Its exact role remains to be elucidated.

Animals↗

PubMed1992

Developmental expression of two antigens associated with mouse intestinal crypts.

Two monoclonal antibodies were prepared against the duodenal mucosa of four-day-old mice (MIM-1/39 and MIM 1/130). The expression of the antigens was associated with the crypts of the small and large intestine in the fetus and adult. MIM-1/39 was present in epithelial cells of the intervillous areas in the small intestine at 17 and 18 days of gestation; afterwards its expression was detected only in crypt cells from birth to adulthood. Transition from the mouth of the crypts to intestinal villi was abrupt. Expression of MIM-1/39 was first detected at time of birth in the colon: In the adult, only crypt cells expressed the antigen and goblet cells were negative. Antigen MIM-1/130 was detected from 16 to 18 days of gestation in the small intestine, in the mesenchymal matrix lying under the intervillous epithelium. After birth, it was present in the pericryptal mesenchymal matrix. This antigen was also expressed at birth in the colon and remained in the pericryptal matrix in the adult. In vivo, multiple injections of an organic extract of rat amniotic fluid to mothers, starting at 14 days of gestation, induced a profound modulation in the pattern of expression of both antigens at 17 days of gestation: The pattern of expression was comparable to that observed at least 5 days after birth in untreated animals. The expression of both antigens before crypt appearance may reflect some molecular differentiation in preparation for the formation of crypts, while their association with differentiated crypts may indicate that they have a role in the maintenance of crypt functional and/or morphological integrity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.

To gain further insight on the mosaic expression of specific functional intestinal markers (such as sucrase-isomaltase) in postconfluent Caco-2 cells, a human colon cancer cell line unique in its property to differentiate in vitro into a mature enterocyte-like cell type, a comparative study was undertaken to examine the morphological and functional differentiation of Caco-2 cells at various culture stages. The observations clearly indicate that Caco-2 cells can exist only in three different states in culture: homogeneously undifferentiated (at subconfluence), heterogeneously polarized and differentiated (between 0 and 20 days after confluence), and homogeneously polarized and differentiated (after 30 days). Indeed, in the intermediate state, a strong discrepancy is found among adjacent differentiating cells throughout the monolayer relative to sucrase-isomaltase expression as well as to cell morphology and brush border organization. Back-scattered electron imaging analysis showed a lack of correlation between these parameters at the cellular level. These observations indicate that morphological and functional differentiations of Caco-2 cells progress concomitantly according to a transient mosaic pattern, thus providing evidence that these two processes are not coupled.

Cell Differentiation↗

PubMed1992

Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine.

Regulation of epithelial cell proliferation, migration and differentiation under physiological conditions remains poorly understood. Interaction of the cells with their underlying basement membrane through integrins, a specific subset of cell surface binding proteins, is one potential mechanism. In the present work, I examined this hypothesis by investigating the distribution of a variety of epithelial basement membrane proteins and the expression of the members of the VLA family of integrins in the adult intestinal epithelium. Indeed, this rapidly renewing simple epithelium contains within its functional unit, the crypt-villus axis, essentially two distinct cell populations: the proliferative and undifferentiated crypt cells and the mature enterocytes on the villus. Although immunolocalization of basement membrane molecules revealed that laminin, type IV collagen and heparan sulfate proteoglycan are distributed homogeneously all along the crypt-villus axis, other non-exclusive basement membrane components were found differentially expressed. Tenascin was concentrated at the base of both villus and lower crypt cells while cellular fibronectin was mostly detected in association with the crypt cells. Moreover, VLA beta 1 as well as 5 of the 6 VLA alpha subunits tested were expressed by intestinal epithelial cells under specific patterns of staining. The beta 1 and alpha 6 subunits were strongly detected at the base of all enterocytes while alpha 5, also detected all along the crypt-villus axis, was weaker and consistently appeared with a punctated/interrupted pattern. On the other hand, the VLA alpha 1, alpha 2 and alpha 3 were expressed at the basolateral domains of enterocytes under distinctive crypt-villus gradients. The alpha 1 subunit was detected at the base of all epithelial cells but lateral staining was only observed in differentiating cells (middle and upper crypt). Finally, in most specimens, alpha 2 and alpha 3 displayed strictly complementary staining patterns for the lower crypt region (alpha 2+, alpha 3-) and the upper crypt-to-villus region (alpha 2-, alpha 3+). Taken together, these data emphasize that proliferation, migration and differentiation in the normal state are susceptible to various influences including compositional changes in the basement membrane and differential expression of receptors for these components.

Basement Membrane↗

PubMed1991

Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

Adenocarcinoma↗

PubMed1991

Immunolocalization of extracellular matrix components during organogenesis in the human small intestine.

The expression and distribution of several major extracellular matrix macromolecules were investigated at the epithelial-mesenchymal interface of the human fetal small intestine from 8 to 20 weeks of gestation. Localization of heparan sulfate proteoglycan, type-IV collagen and laminin, three basement membrane components, as well as fibronectin and tenascin, were assessed by indirect immunofluorescence staining on cryostat sections, and correlated to morphogenesis and epithelial cell differentiation. Basement membrane components and fibronectin were all detected as early as 8 weeks (a time when the epithelium is still stratified and does not express sucrase-isomaltase). Tenascin appeared only after short villi had developed (around 10 weeks) and was restricted to the connective tissue at the tip of villus rudiments. At 18 weeks, well-formed villi and crypts were apparent. The antibody against heparan sulfate proteoglycan stained exclusively the epithelial basement membrane. Anti-type-IV collagen and anti-laminin antibodies stained the epithelial basement membrane and also cellular and fibrillar structures in the lamina propria. Fibronectin was found uniformly distributed over the lamina propria except in the upper third position of the villus core. On the contrary tenascin was mainly localized in the stroma at the tip of the villi. Staining for tenascin was also detected at the epithelial-mesenchymal interface of the villus and in the mesenchyme immediately surrounding budding crypts. These results provide basic data concerning the development of the human gut, and suggest that extracellular matrix components could be involved in the remodelling process of the intestinal mucosa.

Cell Adhesion Molecules, Neuronal↗

PubMed1990

Detection and characterization of sucrase-isomaltase in adult human colon and in colonic polyps.

A panel of monoclonal antibodies specific for sucrase-isomaltase, but differing in their ability to stain the proliferative crypt cells in human jejunum, was used to investigate expression of this enzyme in adult human colon and colonic tumors. Immunofluorescence staining on cryostat sections demonstrated the presence of sucrase-isomaltase in the apical region of normal colonic crypt cells but not on surface epithelium. Colonic sucrase-isomaltase was purified by immunoprecipitation with selected monoclonal antibodies and identified predominantly as high-mannose and complex glycosylated single-chain precursors endowed with relatively low levels of enzyme activities. Most polyps examined (10/16) were also found to express significant amounts of sucrase-isomaltase. In contrast, only 3 of 45 adenocarcinomas were positive by immunofluorescence staining; no correlation was found between enzyme expression and tumor classification either by "Dukes" stage or degree of histological differentiation. These results demonstrate that colonic crypt cells and some benign tumor cells synthesize and express at their cell surface a form of sucrase-isomaltase immunologically distinct from that present in the brush borders of small intestinal villose cells.

Adenocarcinoma↗

PubMed1989

Posttranslational regulation of sucrase-isomaltase expression in intestinal crypt and villus cells.

Expression and synthesis of sucrase-isomaltase (SI) were studied in human jejunum and in the colon tumor cell lines Caco-2 and HT-29. Twelve monoclonal antibodies produced against the adult human intestinal enzyme were shown to recognize specifically SI by immunoprecipitation of 14C-labeled membrane proteins, analysis of enzyme activities in the immunoprecipitates, and immunoblotting. These antibodies produced markedly different patterns of immunofluorescent staining of the intestinal mucosa. Three of them were specific for the absorptive villus cells, while the other nine also stained the luminal membrane of the proliferative crypt cells, with different intensities which paralleled their ability to recognize SI in immunoblots. Sequential immunoprecipitation of SI solubilized from purified brush borders or entire jejunum with four selected antibodies demonstrated the presence of different forms of the enzyme, expressed by either villus or crypt cells. Two immunologically distinct forms of high mannose precursor (hmP1 and hmP2) were also identified in both jejunal mucosa and colon tumor cells. They were present as monomers and their immunological differences were preserved under various ionic and pH conditions. Pulse-chase studies indicated that, in Caco-2 cells, hmP1 is converted into hmP2 within 30 min of chase, and hmP2 is then processed into the complex-glycosylated precursor destined for the brush border membrane. hmP1 was immunologically related to the mature SI present in crypt cells and lacked the epitopes specific for mature SI expressed by villus cells. These results demonstrated that sucrase-isomaltase is synthesized by both crypt and villus cells, but processing of the cotranslationally glycosylated high mannose precursor is dependent on the state of differentiation of the enterocytes. This may represent a general mechanism for the regulation of expression of differentiated cell products at the post-translational level.

Adult↗

PubMed1989

Interaction of Drosophila 27,000 Mr heat-shock protein with the nucleus of heat-shocked and ecdysone-stimulated culture cells.

The intracellular localization and expression of hsp27 (heat-shock protein 27) were investigated by cellular fractionation and immunofluorescence microscopy in Drosophila S3 cells. In unstressed cells, hsp27 is expressed in only 2% of the cells, whereas following heat shock, during recovery or after induction by ecdysone, the protein is detected in all cells. Under all these conditions, hsp27 appears to be concentrated in the nuclear region as revealed by immunofluorescence. During heat shock, this hsp is localized primarily in the nucleus with an enrichment in the perinucleolar region. However, the cellular fractionation data indicate that the nature of hsp27 interaction with nuclear components greatly differs depending on whether or not cells were subjected to elevated temperatures. After heat shock, hsp27 is resistant to non-ionic detergent extraction. In cells allowed to recover at normal temperature and in those where its synthesis was induced by the molting hormone, ecdysone, this hsp is readily solubilized by detergent. These data suggest that, following heat shock, hsp27 may become physically associated with some nuclear component(s) that are resistant to detergent extraction.

Animals↗

PubMed1988

Members of the Drosophila HSP 70 family share ATP-binding properties.

In Drosophila, the hsp 70 family consists of a group of proteins of similar molecular masses (hsps 68, 70 and 72) that exist as multiple isoforms. In this report, it is shown that hsps 68, 70 and 72 from Drosophila cells can be purified by affinity chromatography on ATP-agarose. Furthermore it is demonstrated that the multiple members of the hsp 70 family, which accumulate in large amounts in the nucleus during a heat shock, can be specifically solubilized from the isolated nuclei fraction by ATP. One of the major cognate proteins (hsc 70) also shows similar behavior. These data suggest that most, if not all, of the related Drosophila hsps 70 possess, like their mammalian counterparts, an ATP-binding site which could be related to their function in the stress response.

Adenosine Triphosphate↗

PubMed1987

Hormonal regulation of epithelial cell proliferation in the fetal mouse duodenum in vitro.

In adult rodents, radioautographic studies have shown that cell proliferation in the small intestinal epithelium is confined to the crypts. However, before birth DNA synthesis occurs also on villi. The role of extrinsic factors in the establishment of the adult pattern of enterocyte proliferation was studied in 17-day fetal mouse duodenal explants cultured with a completely synthetic medium. The effect of dexamethasone (30 and 300 ng/ml), thyroxine (10 nM), and insulin (125 mU/ml) on labelling indices was determined after 48 hours of culture. At the onset of the culture period close to 60% of the cells located in the first five cell positions in the intervillous areas became labelled after 4 hours of culture in presence of 3H-thymidine; 8% of the other epithelial cells found higher in the intervillous areas and on villi were also labelled. After 48 hours of culture, well differentiated crypts have developed in all the explants cultured with or without the hormones. The addition of thyroxine or insulin did not modify the labelling indices. However, after 48 hours of culture, the presence of dexamethasone (both concentrations) significantly reduced the labelling index of the cells found over cell position 5 but had no effect on the labelling index of cells located at the bottom of the crypts. When labelling index was determined for each cell position in the crypts at the end of the culture period, it appeared that dexamethasone (300 ng/ml) had no effect thereon for the first five cell positions but had a significant influence on the transitional zone from cell position 6 through 15.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Role of thyroxine and insulin on the development of the fetal mouse duodenum in organ culture.

The role of thyroxine and insulin in the regulation of proliferation and differentiation of the immature duodenal epithelium of the fetal mouse was investigated using an organ culture method with a serum-free medium. Thyroxine (10 nM) stimulates specifically the activity of maltase. Insulin (125 mU/mL) remains without effect on the maturation of all hydrolytic functions studied. Each hormone significantly increases the percentages of brush border enzyme activities liberated in the medium and reduces the amount of glucose released in the medium. In the presence of dexamethasone (76 nM) the effect of thyroxine on maltase activity is still observed. Finally, thyroxine and insulin do not modify the labelling index in the duodenal crypts of the explants in the presence or absence of dexamethasone. These findings indicate that thyroxine and insulin can act directly on the development of the fetal mouse duodenum at the end of gestation. Nevertheless, their implication in prenatal development of the gut functions appears to be of minor importance.

Animals↗