Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

Interferon affects the formation of adhesion plaques in human monocyte cultures

When monocytes isolated from human blood adhere to glass substratum, actin- and vinculin-containing punctate plaques rapidly appear at the ventral surface of the cells. We show here that highly purified human leukocyte interferon (IFN) can inhibit formation of these adhesion plaques in a dose-dependent manner. Complete inhibition was obtained when 300 IU/ml IFN were added into the cell-seeding medium. Plaques already formed in the absence of IFN were only partially affected by subsequent addition of IFN into the culture medium. Prevention by IFN of the formation of the adhesion plaques was associated with loosened attachment of the cells to the substratum. Effect of IFN on cellular morphology was complex. At higher doses, IFN added to the cultures within 24 h of seeding almost completely inhibited the differentiation of monocytes to macrophages and most of the cells remained rounded. At lower doses, however, an enhancement of the bipolar spreading was seen and the end result was a culture with predominantly elongated fibroblastoid cells. The latter cells, unlike the fibroblastoid cells in untreated monocyte-macrophage cultures, were completely devoid of the actin plaques, while the reorganization of vimentin-type intermediate filaments took place in a normal manner. These results further support the view that the actin- and vinculin-containing plaques have a role in mediating firm adherence of human monocytes to growth substratum.     

Organization of early region 1B of human adenovirus type 2: identification of four differentially spliced mRNAs.

The mRNAs from early region 1B of adenovirus type 2 have been studied by Northern blot, S1 nuclease, and cDNA analysis. Two novel mRNAs, designated 14S and 14.5S, have been observed in addition to the previously identified 9S, 13S, and 22S mRNAs. They are 1.26 and 1.31 kilobases long and differ from the 13S and 22S mRNAs in being composed of three exons instead of two. Their two terminal exons are the same as those present in the 13S mRNA, whereas the middle exon is unique to each of the two novel mRNA species. The structures of the 14S and 14.5S mRNAs allow the prediction of their coding capacities: both mRNA species, like the 22S and 13S mRNAs, contain an uninterrupted translational reading frame encoding a 21,000-molecular-weight (21K) polypeptide. The 14S mRNA can, in addition, encode a 16.5K polypeptide which shares N-terminal and C-terminal sequences with the 55K polypeptide, known to be encoded by the 22S mRNA. The 14.5S mRNA species encodes a hypothetical 9.2K polypeptide which has the same N terminus as the 55K polypeptide but a unique C terminus. The two mRNAs differ in their kinetics of appearance; the 14.5S mRNA is preferentially expressed late after infection in contrast to the 14S mRNA, which is present in approximately equal amounts early and late after infection. Taken together with previously published information the results suggest that early region 1B of adenovirus type 2 encodes five proteins in addition to virion polypeptide IX. These have predicted molecular weights of 55,000, 21,000, 16,500, 9,200, and 8,100.     

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Esterase-type of activity possessed by human plasma apolipoprotein C-II and its synthetic fragments

Human plasma apolipoproteins apoA-I, A-II, C-I, C-II and C-III (with the exception of apoE), porcine pancreatic colipase and procolipase hydrolyze 4-methylumbelliferyloleate. In all cases, liberation of 4-methylumbelliferone could be inhibited by phenylmethylsulfonylfluoride, thus suggesting the involvement of serine residues. To the best of our knowledge this is the first report on the esterase activities of these peptides. Synthetic fragments of the lipoprotein lipase activator, apoC-II, prepared according to the known sequence, also possessed this esterase-type of activity. Furthermore, the esterase-type of activities of the synthetic apoC-II fragments with different chain lengths bore a relatively good correlation to the reported abilities to these peptides to produce activation of lipoprotein lipase.We propose a model for the mechanism of activation of lipoprotein lipase by apolipoprotein C-II. ApoC-II would enhance the apparent catalytic rate constant of lipoprotein lipase by functioning as a specific acyl-enzyme hydrolase. A similar catalytic mechanism is suggested for other protein co-factors of hydrolytic enzymes.     

Psophocarpus tetragonolobus agglutinin reveals N-acetyl galactosaminyl residues confined to endothelial cells and some epithelial cells in human tissues

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.     

Glial fibrillary acidic protein and desmin in salivary neoplasms. Expression of four different types of intermediate filament proteins within the same cell type

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.