Histochemical studies of epithelial cell glycoproteins in normal rat colon

Two general classes of glycoproteins have been identified in the colonic epithelial cells of the Sprague Dawley rat. Glycoproteins belonging to the first of these classes contain sialic acids both with and without side chain o-acyl substituents, abundant o-sulphate ester and 'neutral sugars' (hexose, 6-deoxyhexose or N-acetyl hexosamine residues) with oxidisable vicinal diols and are located in the goblet cells of the descending colon and in goblet cells populating the upper halves of the crypts of the ascending colon. In the descending colon, the sulphosialoglycoproteins in the goblet cells in the base of the crypts contain sialic acids without side chain o-acyl substituents. It appears that as these cells migrate up the crypts, there is o-acylation of the side chains of the sialic acids of the glycoproteins and an increase in the quantity of 'neutral sugars' without a corresponding increase in sialic acid. Glycoproteins with similar properties to those of the goblet cells of the upper halves of the crypts of the descending colon, but containing less sulphate, are found in the goblet cells of the upper half of the crypts of the ascending colon. The second general class of glycoproteins contain sialic acids all, or almost all of which, are substituted at position C8 and only relatively small quantities of sulphate. They are located in the mucous cells of the descending colon, the deep crypt secretory cells of the ascending colon and the columnar absorptive cell brush border.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.     

Functional activity of ABC transporters (markers of multidrug resistance) in human colon adenocarcinoma and normal colonic mucosa]

Functional activity of multidrug resistance (MDR) markers (total activity of ABC-transporters, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) activities) in human colon adenocarcinoma and normal mucosa was examined. Functional activity of ABC-transporters was revealed in all colon tumors and in 70% of normal mucosa samples investigated. Expression of Pgp and MRP functional activity was determined in about 50% and 70% of colon tumors respectively. Pgp+MRP+ phenotype was determined in 36% of normal mucosa and adenocarcinoma samples. Expression of Pgp+MRP- phenotype was practically the same in normal mucosa and tumors (in 10 and 18% of samples respectively). Pgp-MRP+ phenotype was revealed two times more often in tumors than in mucosa--in 36 and 18% respectively. On the contrary, Pgp-MRP- phenotype was detected more rarely in tumors than in mucosa (in 10 and 36% of samples respectively). Transporters different from Pgp and MRP were also determined in some tumors and normal mucosa. At the patients with expression of Pgp function in normal mucosa the activity of the transporter was revealed in 25% of tumor samples only. On the contrary, at the patients with expression of MRP function in normal mucosa the activity of the transporter was revealed in 70% of tumor samples. At the patients with no expression of Pgp or MRP activity in normal mucosa the function of the transporters in tumors was determined in 60% and 70% of samples respectively. It is concluded that functional activity of various ABC-transporters (Pgp, MRP and other different from Pgp and MRP) is expressed in human colon adenocarcinoma; expression of ABC-transporters functional activity in normal mucosa does not predict MDR phenotype of the tumor.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.     

Isolation and characterization of epithelial cells of colon mucosa: preliminary results

A procedure for the isolation of epithelial cells from rat and human large bowel is described; the minced tissue was carefully washed with saline and incubated for 45 min in a collagenase-jaluronidase solution; the dispersion of the epithelial cells was achieved by a subsequent treatment with the calcium chelator EDTA. The isolated cells were characterized by cytological and histochemical (PAS, alkaline phosphatase, N-acetyl-D-glucosaminidase) procedures; viability index was assessed by the trypan blue exclusion test; the ability to grow in culture on both liquid and semisolid media was also tested. This method can be successfully applied either to normal or neoplastic colonic mucosa, the resulting cell suspension being suitable for further characterization.     

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1β, lysozyme, serotonin 5-HT antibodies was detected.     

Expression of cyclooxygenase-1 and cyclooxygenase-2 in normal and pathological human oral mucosa

Cyclooxigenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid (AA) to prostaglandins (PGs). Two isoforms of COX have been identified: COX-1 is constitutively expressed in many cells and is involved in cell homeostasis, angiogenesis and cell-cell signalling; COX-2 is not expressed in normal condition however it is strongly expressed in inflammation. The oral cavity is constantly exposed to physical and chemical trauma that could lead to mucosal reactions such as hyperplasia, dysplasia and cancer. Early diagnosis is the most important issue to address for a positive outcome of oral cancer; therefore it would be useful to identify molecular markers whose expression is associated with the various stages of oral cancer progression. Since COX enzyme has been involved, with different mechanisms, in the development and progression of malignancies we decided to investigate the expression and localization of COX-1 and COX-2 in normal human oral mucosa and three different pathologies (hyperplasia, dysplasia and carcinoma) by immunohistochemistry and RT-PCR. COX-1 mRNA and protein have been detected already in normal oral mucosa and their expression progressively increases from normal samples towards hyperplasia, dysplasia and finally carcinoma. On the contrary, COX-2 is not expressed in the normal tissue, starts to be expressed in hyperplasia, reaches the maximum activation in dysplasia and then starts to be downregulated in carcinoma.     

The actin cytoskeleton in normal and pathological cell motility

Cell motility is crucial for tissue formation and for development of organisms. Later on cell migration remains essential throughout the lifetime of the organism for wound healing and immune responses. The actin cytoskeleton is the cellular engine that drives cell motility downstream of a complex signal transduction cascade. The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. The multitude of proteins and regulatory mechanisms partaking in this system makes it vulnerable to mutations and alterations in expression levels that ultimately may cause diseases. The most familiar one is cancer that in later stages is characterized by active aberrant cell migration. Indeed tumor invasion and metastasis are increasingly being associated with deregulation of the actin system.     

Long-term culture of epithelial cells from the normal rat colon

Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures. Supported by National Cancer Institute Contract N01-CP-75914.     

Proteomic analysis of the aging-related proteins in human normal colon epithelial tissue

In order to screen the aging related proteins in human normal colon epithelia, the comparative proteomics analysis was applied to get the two-dimensional electrophoresis (2-DE) profiles with high resolution and reproducibility from normal colon epithelial tissues of young and aged people. Differential proteins between the colon epithelia of two age groups were found with PDQuest software. The thirty five differential protein-spots were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and database searching. Among them there are sixteen proteins which are significantly up-regulated in the colonic mucosal epithelia of young people group, which include ATP synthase beta chain, electron transfer flavoprotein alpha-subunit, catalase, glutathione peroxidase 1, annexin A2 and heat shock cognate 71 kDa protein, etc.; There are nineteen proteins which are significantly up-regulated in the colonic mucosal epithelia of aged people group, which include far upstream element-binding protein 1, nucleoside diphosphate kinase B, protein disulfide-isomerase precursor and VDAC-2, etc.. The identified differential proteins appear to be involved in metabolism, energy generation, chaperone, antioxidation, signal transduction, protein folding and apoptosis. The data will help to understand the molecular mechanisms of human colon epithelial aging.     

Lectins as markers of rumen epithelial cell differentiation

Summary Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B₄ (Bandeiraea simplicifolia, isolectin B₄)) were explored for use as differentiation markers of rumen epithelial cellsin vivo andin vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA),N-acetylglucosamine (WGA) or forN-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B₄ and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.