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Biomedical subjects

O Kellermann

Publications and source records attributed to O Kellermann.

52 records · Page 3Linked to original sources
PubMed1987

Immortalization of precursors of endodermal, neuroectodermal and mesodermal lineages, following the introduction of the simian virus (SV40) early region into F9 cells.

F9 embryonal carcinoma cells were transfected with a hybrid plasmid containing the early genes of the simian virus SV40 under the control of the adenovirus type 5 E1A promoter [21]. These cells were induced to differentiate in aggregates in the presence of retinoic acid (RA). Unlike the derivatives of F9 that are usually obtained in this manner, the plasmid-containing cells were both programmed and immortalized; in addition, expression of the SV40 T antigen was now triggered. These immortalized cells could be separated into three classes: (1) extraembryonic derivatives, (2) embryonic differentiated tissues, (3) immature cells surrounding the differentiated cells. When injected into mice, the mixture of these cells gave rise to multipotential tumors. From the immature cells, committed precursors of the neuroectodermal, endodermal, and mesodermal pathways could be isolated by cloning and selection according to: (a) their specific pattern of differentiation in the tumors and (b) the occurrence of specific markers in the differentiated progeny. The isolation of stable immortalized cell lines corresponding to precursors of the three primitive germ layers and capable of differentiating reproducibly along a particular restricted pathway should facilitate molecular studies on early embryonic development in mouse.

Animals↗
PubMed1986

Immortalization of early embryonic cell derivatives after the transfer of the early region of simian virus 40 into F9 teratocarcinoma cells.

F9 embryonal carcinoma (EC) cells were transformed using a recombinant plasmid carrying simian virus 40 (SV40) early genes linked to the promoter-enhancer region of the early transcription unit 1A (E1A) of adenovirus type 5. One clone of transformed cells, F9-K4B2, contained several integrated copies of the plasmid and expressed SV40 early genes (T antigens) only when it was induced to differentiate in vitro by the addition of retinoic acid and dibutyryl-cAMP. From the cell population positive for T antigen, it is possible to isolate permanent cell lines with high efficiency. Most of these cells exhibit stable traits that are characteristic of parietal endoderm. We also obtained another cell type which did not express the specific markers of either undifferentiated EC cells or endoderm cells; this type might represent a different stage of commitment in the differentiation of F9 cells. All clones are tumorigenic when injected into irradiated syngeneic mice, and they maintain their phenotype after successive passages in vivo as well as in vitro. The introduction of SV40 early genes into other EC cell lines and early mouse embryos appears to be a promising approach for the immortalization of early embryonic cells.

Animals↗
PubMed1982

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. I. Species specificity of the polypeptide composition.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver. The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived. The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components. Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities. Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed. The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. II. Structural characterization of the polypeptide components and immunological identification of the methionyl-tRNA synthetase subunit.

The extensively purified multienzyme complexes from sheep and rabbit livers containing seven aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by two-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the two complexes fell within the pH range of 6.2 to 7.1, with the notable exception of the common polypeptide of Mr = 43,000 which was distinctly basic. The apparent molecular weight of each component from both complexes was determined by SDS-polyacrylamide gel electrophoresis. Four polypeptides, corresponding to molecular weights of 139,000, 129,000, 43,000, and 38,000 were common to both complexes. The other components from the two complexes displayed similar yet clearly distinct molecular weights. The molar ratios of the polypeptides, estimated by densitometry scanning of stained SDS-polyacrylamide gels, indicated that several components from each complex may be present as more than one copy. Following SDS-polyacrylamide gel electrophoresis, the methionyl-tRNA synthetase component from each complex was identified by the protein blotting procedure, using specific antibodies and 125I-labeled protein A. The unique labeled bands from the complexes of sheep and rabbit precisely matched the major polypeptides of Mr = 103,000 and 108,000, respectively. Mild trypsin treatment of the two native complexes generated fully active forms of methionyl-tRNA synthetase, with molecular weights of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver. Identification of the methionyl-tRNA synthetase component by affinity labeling.

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.

Affinity Labels↗
PubMed1979

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Amino Acyl-tRNA Synthetases↗
PubMed1978

Methionyl-tRNA synthetase from sheep mammary gland. Purification of a fully active monomeric enzyme derived from high-molecular-weight complexes by controlled proteolysis.

Methionyl-tRNA synthetase from sheep lactating mammary gland is found predominantly in the form of high-molecular-weight complexes. Controlled proteolysis of these aggregates generates a low-molecular-weight species of the enzyme with full maintenance of activity as assessed by the rate of aminoacylation of tRNA. The product of proteolysis, which has been purified to homogeneity with a yield of 23%, is a monomeric enzyme of molecular weight 78 000. It has a specific activity of 405 units/mg at 25 degrees C. These findings clearly demonstrate that the aggregated state of methionyl-tRNA synthetase is not a prerequisite for full expression of catalytic activity. Furthermore, the results emphasize the need to provide effective protection against proteolytic damage in studies dealing with the characterization of high-molecular-weight complexes of aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland.

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line.

Cells from the malignant teratocarcinoma line PCC4.azal were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Fifty-five clones were isolated from the surviving cells. Twelve clones are unable to form tumors in the syngeneic 129/Sv mice. However, these "tum-" clones form tumors as readily as the original cells when they are injected into irradiated mice. Moreover, they stimulate the production of immune memory cells, which protect the injected animals and confer resistance by adoptive transfer. The tum- clones are therefore unable to generate tumors in syngeneic mice because they elicit an immune rejection response.

Animals↗
PubMedDate not supplied

Lineage-specific patterns of p21ras proteins in immortalized cell lines derived from mouse teratocarcinoma.

The expression of proteins coded by the ras oncogene family was examined in mouse embryonal carcinoma (EC) cells and in immortalized cell lines derived from EC. These cell lines, which correspond to early stages of differentiation, express the simian virus 40 (SV40) T antigen and still proliferate. By 2-D gel electrophoresis of the immune complexes formed with monoclonal anti-ras antibodies, it was possible to distinguish the products of the Ha-, N- and Ki-ras genes and to correlate the observed patterns with the differentiation state of the cells. We show in this report (1) that the 2-D gel pattern of ras protein is identical for the various EC tested and is not influenced by SV40 transformation, (2) that p21Ki-ras is not detected in EC cells, although some EC cell lines are known to express a Ki-ras transcript, and (3) that the complex patterns of N- and Ha-ras observed in EC cells becomes simpler as differentiation proceeds, with a different, characteristic pattern for neuroectodermal, mesodermal and endodermal derivatives. Such patterns could prove useful as differentiation markers.

Animals↗
PubMedDate not supplied

Evolving views in prion glycosylation: functional and pathological implications.

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.

Animals↗
PubMedDate not supplied

[Results of magnetic resonance tomography (MRT) in 100 temporomandibular joints].

The results obtained by MRT of 100 temporomandibular joints in 12 probands with healthy dentitions and 38 patients are reported. The findings were dominated by dislocations of the articular disk. Diagnosis of this condition presented no difficulties, as the interarticular disk can be well differentiated from the adjoining bone structures and the bilaminar zone. The high quality of the soft tissue images renders MRT a valuable procedure completing other examination methods such as panoramic or Schüller radiographs for diagnosis of TMJ diseases.

Adult↗