ABSTRACT
Regulation of co-expression of three neuropeptide genes, i.e. genes encoding enkephalin, cholecystokinin, and gastrin-releasing peptide, was studied in human neuroepithelioma cells. In nondifferentiated state, the continuous cell line SK-N-MC displayed an equally high level of expression of the enkephalin, cholecystokinin, and gastrin-releasing peptide genes. By culturing in medium containing endothelial cell growth supplement the SK-N-MC cells differentiated morphologically into a cell type with neurite-like processes. After 3 days the expression of the enkephalin gene in endothelial cell growth supplement-differentiated cells was significantly reduced by 75% as compared to the nondifferentiated cells, while there was no change in the expression of the cholecystokinin and gastrin-releasing peptide genes during differentiation. The results show that the enkephalin gene is selectively down-regulated during differentiation of neuroepithelioma cells. It is suggested that the down-regulation is related to the transient expression of the enkephalin gene in developing brain and other organs. Thus the neuroepithelioma cell line may provide a cellular model to study the underlying molecular mechanism.
Subject(s)
Cholecystokinin/genetics , Enkephalins/genetics , Gene Expression Regulation, Neoplastic , Neuroectodermal Tumors, Primitive, Peripheral/genetics , Peptides/genetics , Protein Precursors/genetics , Blotting, Northern , Cell Differentiation , Cell Line , Gastrin-Releasing Peptide , Humans , In Vitro Techniques , RNA, Messenger/genetics , RNA, Neoplasm/geneticsABSTRACT
We have investigated the effects of soybean agglutinin on the cytoskeletal element actin in differentiated Caco-2 cells. The actin cytoskeleton of the cells was visualized by fluorescence microscopy using 7-nitrobenz-2-oxa-1, 3-diazole phallacidin as a specific marker for F-actin. Compared with control Caco-2 cells no changes in the fluorescence pattern were observed after incubation with soybean agglutinin. However, using the deoxyribonuclease-I inhibition assay a dose-related response was noted in the increase of intracellular G-actin after a 2-hour incubation period with soybean agglutinin. Already after exposure for 15 min to soybean agglutinin a decrease in intracellular F-actin was demonstrable. This apparent depolymerization could be prevented by incubating the Caco-2 cells with soybean agglutinin and the appropriate monosaccharide simultaneously. The increase in the amount of G-actin appeared to be correlated with a shortening of microvilli on the Caco-2 cells.
Subject(s)
Actins/analysis , Colonic Neoplasms/ultrastructure , Cytoskeleton/ultrastructure , Lectins/pharmacology , Soybean Proteins , Tumor Cells, Cultured/ultrastructure , Cell Differentiation , Colonic Neoplasms/pathology , Cytoskeleton/drug effects , Humans , Microscopy, Electron , Microscopy, Fluorescence/methods , Microvilli/drug effects , Microvilli/ultrastructure , Plant Lectins , Glycine maxABSTRACT
A test to determine quantitatively the lectin binding sites in brush-border membranes has been developed. Highly purified bovine small intestinal brush-border membranes were prepared, and subsequently coated directly to the bottom of a microtiter plate. Soybean agglutinin conjugated with peroxidase was coupled to its binding sites in the brush-border membranes and the peroxidase activity was determined in a spectrophotometer. The number of soybean agglutinin binding sites in the brush-border membranes has been established by means of iterized computer fit analysis of the data, indicating values for maximal binding of 7.10(-7) M soybean agglutinin per mg of brush-border membrane protein and a dissociation constant of 1.5.10(-5) M.
Subject(s)
Immunoenzyme Techniques , Immunosorbent Techniques , Intestine, Small/metabolism , Lectins/metabolism , Microvilli/metabolism , Plant Lectins , Receptors, Mitogen/analysis , Soybean Proteins , Animals , CattleABSTRACT
The acute phase SAA response was studied in hamsters. An SAA-stimulating factor (SAASF) was detected in the early acute phase blood plasma of hamsters which were subcutaneously injected with casein-LPS. The latter is routinely used in our laboratory for amyloid induction in hamsters. Acute (4 h) inflammatory exudates (greater than 80 per cent polymorphonuclear leukocytes) were produced by intraperitoneal injection with either casein-LPS, latex or Freund's incomplete adjuvant. Chronic inflammatory exudate macrophages (greater than 98 per cent) were elicited by intraperitoneal injection with Bacillus Calmette Guérin (BCG). Cells were stimulated in vitro with latex. SAASF was detected in the supernates and lysates of the acute exudate cells but not in those of the chronic peritoneal exudate macrophages. Lymphocyte activating factor (LAF), however, was evidently present in the latter samples, indicating that SAASF and LAF (IL-1) are functionally different substances in hamsters.