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Biomedical subjects

F Ramaekers

Publications and source records attributed to F Ramaekers.

At least 55 records · Page 3Linked to original sources
PubMed1988

Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript.

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against alpha A-crystallin and identified the antigenic determinant for two of these antibodies. Applying limited-digestion methods, we show that the region spanning residues 158-168 of alpha A-crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated alpha Ains-crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the alpha A-crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.

Animals↗
PubMed1988

A comparative immunohistochemical study of cytokeratin and vimentin expression in middle ear mucosa and cholesteatoma, and in epidermis.

Cytokeratin expression was studied in human middle ear cholesteatoma lesions, using a variety of immunohistological techniques and a wide range of polyclonal antisera and monoclonal antibodies against cytokeratin (CK) subgroups or individual CK polypeptides. The expression of the other cytoskeletal proteins, vimentin and desmin, was also investigated. Middle ear mucosa and epidermal tissues were used as reference tissues. Our investigations also included epithelial structures present in the cholesteatoma perimatrix and in dermal tissues. The results indicate that, compared with epidermal tissues, the expression profile of CKs in cholesteatoma matrix is representative of a hyperproliferative disease. Evaluating the presence of a marker of terminal keratinization - the 56.5 kD acidic CK n degrees 10 - we found supportive evidence of a pronounced retardation of its expression, which did not parallel histological differentiation. In epidermal tissues, the first prickle cell layers are CK10 positive whereas in many cholesteatomas this finding was observed near the stratum granulosum only. Probing the early stages of keratinization - the 58 kD basic CK n degrees 5 and the 50 kD acidic CK n degrees 14 - we regularly observed an extended staining area in the cholesteatoma matrix. In epidermal reference tissues, only the basal and nearest suprabasal layers were convincingly labeled. As a rule, non-epidermal CKs did not belong to the cholesteatoma CK set. However, exceptions to that rule were noticed as a focal or more extended expression of one or more non-epidermal CKs in about half of the cases. Together with the extended CK5 topography, this is further evidence that CK expression is seriously affected by the diseased state. CK expression in the perimatrix is limited to mucous glands, either normal, atrophic or hyperplastic. CKs n degrees 4, 5, 7, 14, 18 and 19, also displayed by middle ear mucosa, were consistently observed. Where ductal arrangements were present, CK10 was also detected, in analogy with the CK10 registration in ductal portions of mucous glands in the external ear canal skin. The absence of CK8 in mucous glands of the perimatrix, however, strongly differentiates these structures from the mucous gland acini and ducti in the external ear canal, where CK8 is systematically expressed. Vimentin staining was restricted to dendritic cells of the matrix (Langerhans cells) and to perimatrix fibroblasts, blood cells and vascular endothelium. Coexpression of CK and vimentin was not observed.

Animals↗
PubMed1988

Primary cutaneous adenoid cystic carcinoma: case report, immunohistochemistry, and review of the literature.

We report a case of primary cutaneous adenoid cystic carcinoma in a 75-year-old man. Electron microscopy revealed similar features to those previously described in adenoid cystic carcinomas of salivary gland origin: wide intercellular spaces, pseudocysts containing replicated basement membrane-like material and true lumina lined by cytoplasmic membranes with numerous microvilli. Immunohistochemistry using antibodies to several cytoskeletal proteins (keratins and actin) indicated the presence of two epithelial tumour cell populations, one with the phenotype of myoepithelial cells, lining the pseudocysts and the periphery of the tumour fields, and another with the phenotype of acinar cells of the secretory coil of sweat glands. In addition, the tumour showed immunoreactivity for epithelial membrane antigen, but not carcinoembryonic antigen. A review of the literature on other cases of primary cutaneous adenoid cystic carcinoma showed that this tumour generally affects older patients with a female to male ratio of 4:1. The most common tumour site appears to be the scalp (40%), and the recurrence rate is 50%. Surgical treatment with extensive resection margins is recommended.

Adult↗
PubMed1987

Tissue distribution of keratin 7 as monitored by a monoclonal antibody.

Monoclonal antibody (RCK 105) directed against keratin 7 was obtained after immunization of BALB/c mice with cytoskeletal preparations from T24 cells and characterized by one- (1D) and two-dimensional (2D) immunoblotting. In cultured epithelial cells, known from gel electrophoretic studies to contain keratin 7, this antibody gives a typical keratin intermediate filament staining pattern, comparable to that obtained with polyclonal rabbit antisera to skin keratins or with other monoclonal antibodies, recognizing for example keratins 5 and 8 or keratin 18. Using RCK 105, the distribution of keratin 7 throughout human epithelial tissues was examined and correlated with expression patterns of other keratins. Keratin 7 was found to occur in the columnar and glandular epithelium of the lung, cervix, breast, in bile ducts, collecting ducts in the kidney and in mesothelium, but to be absent from gastrointestinal epithelium, hepatocytes, proximal and distal tubules of the kidney and myoepithelium. Nor could it be detected in the stratified epithelia of the skin, tongue, esophagus, or cervix but strongly stained all cell layers of the urinary bladder transitional epithelium. When applied to carcinomas derived from these different tissue types it became obvious that an antibody to keratin 7 may allow an immunohistochemical distinction between certain types of adenocarcinomas.

Animals↗
PubMed1987

Detection of epithelial- and neural type of intermediate filament proteins in human lung tumors.

Five different types of lung cancers, i.e. squamous cell carcinomas, adenocarcinomas, small cell lung carcinomas, carcinoids and adenoid cystic carcinomas were examined for their intermediate filament constituents, with special emphasis on the different cytokeratin polypeptides and neurofilament proteins. Polyclonal as well as monoclonal antibodies to these proteins were used in immunocytochemical techniques applied to both tumor frozen sections and paraffin sections. Squamous cell carcinomas and adenocarcinomas could be shown to contain cytokeratins, which could be detected in both frozen sections and paraffin sections. Also small cell lung carcinoma (SCLC) and carcinoid lung tumors showed a positive staining reaction with polyclonal and monoclonal (cyto)keratin antibodies, but were negative with neurofilament antibodies, with the exception of one case of lung carcinoid, which co-expressed neurofilaments and cytokeratins. We have used antibodies to cytokeratin polypeptides, to neurofilament proteins and to a neuroendocrine related membrane antigen (MOC-1) to further subclassify heterogeneously composed squamous cell carcinomas. Using a monoclonal antibody to cytokeratin 18, normally present in glandular tissues and adenocarcinomas, we observed that more than 90% of the squamous cell carcinomas examined can be stained with this antibody. The percentage of tumor cells, however, positive for cytokeratin 18 varies between 1 and 100%. In these same tumors a monoclonal antibody to skin keratins, which is known to react specifically with keratinizing cells, also stained variable numbers of tumor cells. This finding confirms the presence of (keratinizing) squamous cell carcinoma elements in these tumors. Our data show that most lung tumors, heretofore considered pure squamous cell carcinomas, should be considered biologically adenosquamous carcinomas. Also areas positive with MOC-1 were found in these tumors, suggesting the presence of squamous cell carcinomas with neuroendocrine differentiation. Furthermore, in some poorly differentiated squamous cell carcinomas areas with neurofilament positive cells were detected, suggesting a neural differentiation within these neoplasms. Adenoid cystic carcinomas are shown to co-express cytokeratins and vimentin in the tumor cells. This phenomenon can be used to identify such tumors and to distinguish them from other lung tumors.

Adenocarcinoma↗
PubMed1986

Changing intermediate-sized filament patterns in metastatic hepatocellular carcinoma cells of the guinea pig.

Hepatocellular carcinoma cells obtained from ascitic fluid after diethylnitrosamine treatment of Sewall Wright strain-2 guinea pigs produce solid (primary) tumors, lymph-node metastases and malignant ascites when reinjected into animals of the same strain. When brought into culture the cells settle, form multilayer cultures and can be maintained in passage. In addition to epithelium-specific cytokeratin intermediate filaments (IF), these latter cells, like most cultured cells, also contain vimentin. Hepatocellular carcinoma cells in solid tumors and in metastatic tumors retain their original keratin IF and in general do not have an additional vimentin-IF system. When the tumor cells are present in ascites they develop vimentin-IF in addition to cytokeratin filaments. Vimentin is gradually lost when these cells sediment onto the peritoneal surface and proliferate continuously to form papillary projections, or when they are detected as circumscribed metastases. It seems likely, therefore, that in this system the synthesis of an additional vimentin cytoskeleton is related to reduced cell-to-cell contact and to the ability of the cells to survive individually or as cell clusters in body fluids, without being part of a cohesive tissue.

Animals↗
PubMed1986

The membrane-associated cytoskeleton in cultured lens cells. Electron microscopical visualization in cleaved whole-mount preparations and platinum replicas.

The membrane-bound cytoskeleton of bovine lens cells was investigated using different electron-microscopic methods. Cells were grown on glass coverslips and fixed in 0.5% glutaraldehyde, post-fixed and prepared for critical-point drying following standardized methods. Cells were broken open before post-fixation or after critical-point drying in order to expose the cortical cytoplasm. Cleaved, critical-point-dried cells were subsequently rotary replicated. Cells were also cleaved on grids before post-fixation. These cells were examined directly in the microscope. All methods showed the presence of a cytoskeletal network. Bundles and starlike foci of microfilaments and microtubules were usually found in close association with the membrane. Occasionally also intermediate filaments were observed, in a few cases running close to the microtubules. The reliability of the methods to visualize the membrane-bound cytoskeleton of lens cells is discussed.

Actin Cytoskeleton↗
PubMed1986

Protein composition of nuclear matrix preparations from HeLa cells: an immunochemical approach.

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with special emphasis on the use of various nucleases and detergents as well as on the ionic strength of the final salt extraction. The protein composition of the resulting nuclear matrix preparations was analysed by one- and two-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) and heterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to be present in such nuclear matrix preparations. The nature of some other protein components was elucidated using two-dimensional immunoblotting and immunofluorescence. For this purpose mouse monoclonal antibodies to cytoskeletal components (vimentin, cytokeratins), small nuclear RNP (70 X 10(3) Mr protein of U1-RNP), hnRNP (C1/C2) and the pore-complex lamina (lamins A, B and C) were used next to human autoimmune sera obtained from patients with connective tissue diseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodies enabled us to identify a number of proteins present specifically in the nuclear matrix and to show that part of the cytoskeletal proteins are still present in the isolated structures.

Cell Nucleus↗
PubMed1986

Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix.

Earlier studies suggested that the 70K (70 X 10(3) Mr) polypeptide is a nuclear matrix (associated) protein since it is the only U1 RNP-associated antigen that is not released from the nucleus after treatment of the cell with, successively, detergents, DNase I and/or RNase A and high salt. The possibility that the 70K protein functions in the binding of U1 RNP to the nuclear matrix is now further substantiated by the finding that U1 RNP particles that did or did not contain the 70K protein could be isolated, depending on the method of isolation. When U1 RNP particles were obtained by means of sonic disruption of the nucleus they contained the 70K polypeptide, whereas particles that were isolated by extraction at room temperature and a slightly alkaline pH lacked the 70K protein but contained the intact U1 RNA and the other U1 RNA-associated proteins. During interphase the localization of the 70K protein is restricted to the nucleus, giving a dot-like distribution pattern with exclusion of the nucleoli. During prophase to late anaphase the protein is dispersed throughout the entire cytoplasm with the exception of the chromatin regions. Immunofluorescence studies, using a monoclonal anti-70K antibody in combination with human autoimmune sera that react with U1 RNA-associated proteins, demonstrate that the 70K protein is localized in those areas of the cell where other U RNP proteins occur, also during mitosis. Topoisomerase I and nuclear lamins, typical nuclear matrix proteins, show completely different distribution patterns in all phases of the cell cycle. Assembly of the nuclear envelope is attended by the re-formation of the clustered appearance of the 70K antigen. These results suggest that, although associated with the nuclear matrix fraction in interphase cells, the 70K protein remains associated with the U1 RNP particles during cell division.

Animals↗
PubMed1985

Antibodies to cytokeratin and vimentin in testicular tumour diagnosis.

Thirteen primary and metastatic testicular germ cell tumours, including classical and anaplastic seminomas, and non-seminomatous testicular tumours were examined for their intermediate filament protein (IFP) types. The seminomas were shown to react with a monoclonal and a polyclonal antibody to bovine lens vimentin, while non-seminomatous germ cell tumours were strongly positive for a polyclonal and a monoclonal antibody to cytokeratin. In one case of seminoma with elevated serum levels of beta HCG and alpha FP, cytokeratin positive tumour cells were found. In the case of teratocarcinoma, several components of the tumour could be distinguished using a combination of antisera in double-label immunofluorescence microscopy. The glandular component of this tumour was positive with the polyclonal antikeratin, but also with the monoclonal cytokeratin antibody specific for glandular epithelia (RGE 53). However, the squamous component was negative with this latter antibody. Strikingly, the spindle cell component showed focal positivity for vimentin, with coexpression of cytokeratin and vimentin in some cells. Our data show that antibodies to cytokeratin and vimentin can be helpful in the diagnosis of testicular germ cell tumours, especially in the differentiation between seminomas and non-seminomatous tumours.

Antibodies, Monoclonal↗
PubMed1985

Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer.

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

Amino Acid Sequence↗
PubMed1985

Paget cells express cytokeratins typical of glandular epithelia.

The expression of cytokeratins in Paget cells in mammary and extramammary Paget's disease was studied using different keratin antibodies and immunofluorescence microscopy. Antibodies to epidermal keratin did not react with the Paget cells but stained the surrounding epidermis. Two monoclonal cytokeratin antibodies (PKKI and RGE 53), which reacted typically with simple glandular epithelia in normal tissues, brightly stained the Paget cells and left the surrounding epidermal cells unstained. The results indicate that Paget cells are derived from mammary or sweat duct epithelium rather than from epidermal cells.

Antibodies, Monoclonal↗
PubMed1985

Cytokeratin expression during neoplastic progression of human transitional cell carcinomas as detected by a monoclonal and a polyclonal antibody.

Antibodies to intermediate filament proteins were used to study different cell layers in normal human transitional epithelium, 16 human transitional cell carcinomas, and two cell lines derived from human bladder carcinomas. Conventional rabbit antisera to human skin keratins stained all layers of the transitional epithelium from bladder, ureter, and kidney. A slightly higher staining intensity was found in the basal and superficial layers as compared with the intermediate cell layers. A monoclonal antibody to cytokeratin 18 (RGE 53), however, stained only the superficial cell layer of transitional epithelium, the so-called umbrella cells. In well-differentiated (grade I) transitional cell carcinomas, RGE 53 stained only the superficial cells of papillary structures. In higher grade papillary tumors, RGE 53 also stained cells within the basal and intermediate layers, whereas in high-grade, invasive tumors almost all tumor cells were RGE 53 positive. These results show that monoclonal antibodies to cytokeratins can provide both an indication of processes involved in neoplastic progression of bladder tumors and a means of studying the molecular relationship of the tumor cells to normal cells.

Animals↗
PubMed1985

Characterization of three new variant type cell lines derived from small cell carcinoma of the lung.

Three new, well growing cell lines (GLC-1, GLC-2, and GLC-3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all metaphases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory granula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone markers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC-2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M13); and a combination of simple and squamous epithelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedifferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the clonal derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line.

Carcinoma, Small Cell↗
PubMed1984

Cytokeratin filament expression during in vitro teratocarcinoma cell differentiation as detected by a monoclonal antibody.

Undifferentiated F9 teratocarcinoma cells were induced to differentiate in culture using retinoic acid and cAMP. As a result, the morphology of the cultures changes dramatically. Using a monoclonal antibody directed against cytokeratin polypeptide 18 (RGE 53) in the indirect immunofluorescence technique we could show that this cytokeratin subunit is synthesized and assembled into a filamentous network upon differentiation in about 50% of the cells. Immunoblotting studies confirm these results.

Animals↗
PubMed1984

Cytokeratin filament expression during in vitro teratocarcinoma cell differentiation as detected by a monoclonal antibody.

Undifferentiated F9 teratocarcinoma cells were induced to differentiate in culture using retinoic acid and cAMP. As a result the morphology of the cultures changes dramatically. Using a monoclonal antibody directed against cytokeratin polypeptide 18 (RGF 53) in the indirect immunofluorescence technique we could show that this cytokeratin subunit is synthesized and assembled into a filamentous network upon differentiation in about 50% of the cells. Immunoblotting studies confirm these results.

Animals↗