Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells

Vassy, Jany; Rigaut, Jean Paul; Hill, Anne-Marie; Foucrier, Jean

doi:10.1111/j.1365-2818.1990.tb02950.x

Cited by 21 publications

(12 citation statements)

References 26 publications

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“…As to vimentin, the amounts detected by immunoblotting are in agreement with the expression levels in lung and liver. In adult liver vimentin expression is limited to a few cell types (29). In contrast, lung expresses high levels of vimentin (30).…”

Section: Resultsmentioning

confidence: 99%

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Otero

1997

Journal of Biological Chemistry

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Chromatography on immobilized antibodies specific to nucleoside diphosphate (NDP) kinase was utilized for affinity purification of this enzyme from detergent extracts of frog heart post-mitochondrial fractions. SDSpolyacrylamide gel electrophoresis analysis of eluates from these supports shows that five polypeptides copurify with nucleoside diphosphate (NDP) kinase. Tryptic digests of each band were analyzed by mass spectrometric microsequencing. Data base searches by peptide mass matching and sequence homology led to the identification of these proteins as glyceraldehyde-3-phosphate dehydrogenase (40 kDa), creatine kinase (45 kDa), vimentin (55 kDa), pyruvate kinase (60 kDa), and a putative member of the antioxidant protein family (28 kDa). Distinct protein compositions were found in eluates of lung and liver extracts processed in a like manner. The 28-kDa band and vimentin were associated with NDP kinase from all tissues, but co-purification of pyruvate kinase was seen only in liver, while creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were absent from eluates from lung and liver. The results suggest that while NDP kinase is associated with vimentin intermediate filaments and an antioxidant protein in most tissues, it interacts with energy metabolism enzymes in a tissue-specific manner.In recent years NDP kinase 1 has been found to have unexpected roles, in addition to its well known ability to catalyze transfer of phosphate groups from trinucleotides to dinucleotides (1). NDP kinases are involved in growth, differentiation, development, tumor progression, metastasis, and apoptosis (reviewed in Ref. 2). NDP kinase participates in muscarinic K ϩ channel opening (3), is a transcription factor for c-myc (4) and a protein kinase as well (5, 6). These varied functions seem to be accomplished by a limited number of gene products: to date, complete cDNAs encoding 4 human NDP kinases (nm23-H1, nm23-H2, DR-nm23, and nm23-H4) have been identified (7-9).Also, in Dictyostelium a separate nuclear gene encodes a mitochondrial NDP kinase (10). Although other NDP kinase genes may be identified in the future, it is unlikely that each of the multiple functions associated with this protein is performed by a distinct isoform. Differential distribution of NDP kinase isoforms (11) may account for adaptation to the requirements of specific cell types. Additionally, interaction of distinct isoforms with cell-specific factors, conceivably other proteins, could govern NDP kinase function in different tissues. This in turn might explain apparently contradictory findings regarding the connection between NDP kinase and cancer (2): while in some cell types, studies of NDP kinase expression suggest that it has a metastasis suppressor function, in other systems this relation between expression of NDP kinase and metastatic potential is either nonexistent or operates in the opposite direction.The objective of the present work was to identify proteins that interact with NDP kinase in cardiac muscle. Immobilized antibodies to frog ...

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Section: Resultsmentioning

confidence: 99%

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Otero

1997

Journal of Biological Chemistry

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“…Cryostat slices can be used for CSLM instead of fixed blocks (65), but with a depth limitation of about 50 pm. The results of DNA quantitation in the liver after CA3 staining (not shown) are not statistically different (Smirnov test) from those after the en bloc staining technique described in the present article.…”

Section: Discussionmentioning

confidence: 99%

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

et al. 1991

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A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 Fm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-BOO (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.Key terms: DNA cytometry, confocal laser microscopy, histology, 3-D There is a considerable interest in DNA cytometry, notably because of the importance of searching for correlations of nuclear DNA content with cancer prognosis in individual patients (e.g., 22, 40, 62).The two methods commonly used a t present for nuclear DNA content analysis, image cytometry (ICM) and flow cytometry (FCM), are not devoid of limitations. Observing the tissue architecture may be important in identifying regions for cytometry and, therefore, the main shortcoming of ICM on smears or imprints and of FCM is obviously that they do not allow the study of DNA contents in situ. Even ICM on histological sections does not allow this, as the tissue structure is observed in projection and the resulting nuclear profiles are usually either incomplete if the section is thin or overlapping if the section is thick enough to include a sufficient proportion of complete nuclei. Finally, preparation procedures for ICM and FCM may produce a selection bias of a given cell type (9) and/or nuclear debris (27).'This paper is an expanded version of a work presented at the

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“…Such effects can be corrected by image processing (24). The contamination, however, arises only when the FITC labeling is very strong.…”

Section: Methodsmentioning

confidence: 98%

Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers

et al. 1993

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Immunolocalization of desmin in fetal rat livers shows that on day 12 of gestation a high number of liver cells express desmin. This number decreases from day 14 onward. On day 20 about the same density of desmin-containing cells is found in fetal rat livers as is found in adult rat livers. Desmin-containing cells show two types of labeling patterns, especially on days 12 and 13 of gestation: (a) a basketlike network of intermediate filaments throughout the whole cell, similar to the labeling pattern of cytokeratin in hepatocytes; and (b) more strongly labeled intermediate filaments developing long and slender processes between adjacent cells, close to the labeling pattern of Ito cells in adult rats. From day 14 of gestation, the first type becomes rare, and from day 18 only the second type remains. Double-labeling experiments show that coexpression of desmin and cytokeratin is found in cells of the first type on days 12, 13 and 14 of gestation. Cells containing desmin with the labeling pattern of the second type never express cytokeratin. Coexpression of vimentin and cytokeratin is never found in fetal hepatocytes, even on day 12 of gestation. Numerous nonhepatocyte cells coexpress desmin and vimentin, but some cells contain vimentin or desmin alone. In desmin-containing cells the labeling pattern is of the first type (basketlike network). These results suggest that in early stages of fetal liver development, desmin is found in two different types of liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells

Cited by 21 publications

References 26 publications

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers

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