Biomedical subjects
Publications and source records attributed to P Traub.
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Polyribosomes were isolated from Mg2-stabilized, Triton X-100-resistant residual cell structures and from a postmitochondrial supernatant of hypotonically swollen and homogenized Ehrlich ascites tumor cells and analysed for their vimentin contents by polyacrylamide gradient slab gel electrophoresis. Whereas polyribosomes from detergent-resistant cell residues were accompanied by a small amount of vimentin on sucrose gradients, polyribosomes isolated from a hypotonic, postmitochondrial supernatant were free of vimentin. Since the presence of vimentin in cell residue-derived polyribosomes might be interpreted as the result of unspecific adsorption or cosedimentation of vimentin filament fragments, both due to the presence of high Mg2 concentration during cell extraction, the results suggest that there is no direct and stable interaction of polyribosomes with vimentin-type intermediate filaments in suspension-grown Ehrlich ascites tumor cells. This is in accordance with the electron microscopic observation that cytoplasmic regions rich in intermediate filaments are virtually devoid of polyribosomes. When the extraction of the same cells with Triton X-100 was carried out at low ionic strength and in the presence of small amounts of Ca2, residual cell structures were completely destabilized and clean nuclei without any attached cytoplasmic material were obtained. This is due to the degradation of the vimentin-type filaments by the Ca2-activated thiol proteinase highly specific for vimentin.
A Ca2+-activated neutral protease is described which, when tested against various native proteins, appears to be specific for vimentin, the 58,000-Mr subunit protein of intermediate-sized (7--11 nm) filaments in Ehrlich-ascites-tumour cells. The protein subunits of other classes of intermediate-sized filaments have been tested; neurofilament protein and glial fibrillary acidic protein are not degraded, however skeletin, the subunit protein of intermediate-sized filaments in smooth muscle, is degraded. The protease is found associated with the detergent-resistant cytoskeleton of Ehrlich-ascites-tumour cells; proteins, other than vimentin, present in this structure are not degraded. The protease is activated by Ca2+ and Sr2+ but not by other divalent cations tested: the Ca2+ concentration required for activation is 10 microM. The pH optimum is between pH 7.5 and 8.0, and the KCl concentration required for optimal activity is 100 mM. The protease is inhibited by 1-chloro-3-tosylamido-7-amino-L-2-heptanone hydrochloride and L-1-tosylamido-2-phenylethyl chloromethyl ketone but not by soybean trypsin inhibitor; inhibition by phenylmethylsulphonyl fluoride is moderate. The high substrate specificity of the protease suggests it may play a role in vimentin intermediate-sized filament protein turnover in Ehrlich-ascites-tumour cells.
A simple method of analytical subcellular fractionation, combined with a sensitive computational method for data analysis and presentation, has been used to reinvestigate the distribution and relative amounts of several enzymes in the cytoplasmic and plasma membranes of two different cell types: one is a neoplastic, transformed cell type (Ehrlich ascites tumour cells), the other an untransformed, highly differentiated cell type (liver hepatocytes plus Kupffer and endothelial cells). In general the distribution of the enzymes in particular membranes is similar in the two cell types, however the relative amounts differ. Ehrlich ascites tumour cells have a higher specific activity of galactosyltransferase and ouabain-sensitive (Na,K)ATPase, while liver cells have higher glucose-6-phosphatase, 5'-nucleotidase and succinate dehydrogenase activity. These differences appear to be correlated with morphological and, in some cases, functional differences between the two cell types.
Previous investigations of the filamentous network in eukaryotic cells have been based on observations by electron and fluorescence microscopy. In order to examined, in more detail, the interconnection of the various components of th filamentous network, we have treated Ehrlich ascites tumour cells with Triton X-100 in the presence of Mg++, disassembled the detergent-resistant, residual cell structure with Tris-EDTA and subjected the postnuclear supernatant to sucrose density gradient equilibrium centrifugation. Using this technique we are able to demonstrate 1) the association of the major part of intermediate-sized filament protein (vimentin) with unfolded ribosomal subunits, 2) the nearly identical sedimentation behavior of the boundary lamina and actin, and a minor part of the intermediate-sized filament protein respectively, and 3) the association of a Ca++-dependent protease specific for vimentin intermediate-sized filament protein with the Triton X-100 resistant, residual cell structure. Furthermore, we are able to confirm, by labelling intact Ehrlich ascites tumour cells with [3H] concanavalin A and recovering radioactivity in the lighter sucrose gradient fractions, that the detergent-resistant boundary lamina is derived from the plasma membrane. The presence of coated vesicles in Triton X-100-treated cells as well as of coated pits in the derived membrane point at the same origin of the boundary lamina. The results of the fractionation study are correlated with structures observed by electron microscopy of ultrathin sections of the intact filamentous network.
Several mammalian cell lines propagated in suspension and monolayer culture and some normal and cancerous tissues from rat, hamster and cat were screened for the presence of the Ca 2+ activated protease specific for the intermediate-sized filament protein vimentin. Gel permeation chromatography on Sephacryl S-300 of postnuclear supernatants, and sucrose density gradient centrifugation of extracts from Triton X-100-resistant residual cell structures revealed the presence of the enzyme in all cells and tissues tested. Its apparent molecular weight amounted to 100 000. Except in the cases of a spontaneous rat lung tumour and a rat hepatocellular carcinoma induced by diethylnitrosamine, most of the enzyme was released into the postnuclear supernatant during cell or tissue extraction, indicating that it is of cytoplasmic origin. There was no correlation between the enzyme level and the vimentin content of cells and tissues. Rat and hamster liver as well as cat kidney, in which vimentin has not been detected by polyacrylamide gel electrophoresis, were relatively rich in the Ca 2+ activated protease. The experimental results point at the widespread, if not general, occurrence of the enzyme in mammalian cells.
Mengovirus particles adsorbed to Ehrlich ascites tumour cells are redistributed into patches and caps without the action of antibodies. Thus capping may be an early signal in picornavirus infection.
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The effect of mengovirus infection on the extent of phosphorylation of histone H1 was studied in Ehrlich ascites tumor cells. After prelabeling of the nuclear protein with [32P] orthophosphate, the excorporation of radioactivity was followed as a function of time postinfection. Employing high-resolution polyacrylamide gradient slab gel electrophoresis and autoradiography, it was found that, compared to a relatively slow turnover of phosphate groups in histone H1 in mock-infected cells, in mengovirus-infected cells the excorporation of radiolabel from histone H1 was significantly enhanced. In the latter case, the decrease of histone-bound radioactivity was paralleled by a reduction of the band multiplicity in the histone H1 region of the electrophoresis profile. It was also shown that the microheterogeneity in the histone H1 complements isolated at various times postinfection was reduced to the same basal 3-band level by incubation of the nuclear protein fractions in the presence of alkaline phosphatase. After this treatment, the band multiplicity equaled that found in histone H1 from stationary cells.
The distribution of poly(A)+ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)+ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [3H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)+ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)+ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [3H]poly(U)hybridization experiment.
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Mengovirus, extracted from infected L-cell cultures with Freon 113 and concentrated from the aqueous phase with polyethyleneglycol, was chromatographed on protein-coated controlled pore glass (CPG). The covalent binding of protein to CPG is described. Further purification and concentration of mengovirus was achieved by isopycnic density gradient centrifugation in solutions of either CsCl or salts of iodinated benzoic acid derivatives. The described procedure is superior to conventional methods for the isolation and purification of large quantities of mengovirus. It yields highly purified virus preparations within a short time and a recovery of more than 50% of the starting infectivity.
Isolated Acetabularia crenulata nuclei were injected with Mengo virus RNA solution and then implanted into anucleate posterior Acetabularia mediterranea cell fragments or fused with Acetabularia ryukyuensis cytoplasts. The injected animal virus RNA was actively translated in the plant cell cytoplasm. Mengo virus proteins were detected and localized in Acetabularia cytoplasts by use of an immunofluorescence method on the first to fifth day after injection.
We have prepared homologous, fractionated, cell-free translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells in order to determine what alterations occur following virus infection in the translational machinery of the host cell. Two major differences distinguish the system developed from infected cells. First, it has a 40% lower rate of protein synthesis, primarily a consequence of the rate of chain elongation, which is depressed to 60 amino acids/min from 90 amino acids/min in the system from uninfected cells. Second, at supraoptimal concentrations of Mg2+ and K+ the system from virus-infected cells supports the translation of mengovirus RNA but not host mRNA. These differences between the two systems may reflect specific changes which are responsible for the selective translation of mengovirus RNA in the infected cell. In both systems the optimal concentrations of polyamines, monovalent and divalent cations, mRNA, and ribosomal subunits are the same for the translation of either host or viral RNA. This uniformity is useful in experiments, designed to investigate the selective translation of viral RNA, where various components of the two systems are interchanged.
The selective translation of viral RNA in mengovirus-infected Ehrlich ascites tumor cells was investigated using fractionated translational systems whose macromolecular components were derived entirely from uninfected or virus-infected cells. Both systems translate host mRNA from uninfected cells, host mRNA from virus-infected cells, and mengovirus RNA. In competition experiments, where viral RNA and host mRNA were translated together in systems from uninfected cells, the relative amounts of virus-specific and host-specific proteins synthesized were proportional to the relative concentrations of the RNA templates. In systems whose components were obtained from virus-infected cells, mengovirus RNA was preferentially translated. 70% of the selectivity found in the translational systems derived from infected cells was due to the initiation factor fraction, the remaining 30% to components of the pH 5 enzyme fraction. In addition, host mRNA isolated after virus infection is translated in vitro to a lower extent in the presence of mengovirus RNA than is host mRNA from uninfected cells.
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Employing high-resolution polyacryl-amide gradient slab-gel electrophoresis in 6M urea and 6% acetic acid, a distinct change in the microheterogeneity of histone H1 was detected after mengovirus infection of Ehrlich ascites tumor cells. Whereas in uninfected cells the band multiplicity was found to be 6, it was reduced to 4 in the course of the infectious cycle (8 to 9 h). It could be deomonstrated that, while the electrophoretically slowly moving subspecies H1e and H1f disappeared from the band profile of histone H1, the faster migrating bands H1a and H1b increased in relative intensity. The relative intensity of band H1d was also drastically reduced, that of band H1c stayed practically constant throughout infection. When Ehrlich ascites tumor cells were labeled with a mixture of [14C]amino acids prior to mengovirus infection, the radioactivity incorporated into histone H1 subspecies of low electrophoretic mobility was chased into histone H1 components of high mobility in the course of infection, suggesting a virus-induced, unidirectional interconversion of the multiple histone H1 subunits. With the exception of a histone H2B subspecies, which also decreased in amount, the relative quantities of the other histones stayed constant throughout infection.
After mengovirus infection of Ehrlich ascites tumor cells, an increased incorporation of radioactive phosphate into ribosomal protein S6 was detected. As judged from the shape of the radioautography spot and the location of the modified protein on the two-dimensional polyacrylamide gel, it must be concluded that more than one phosphate group was incorporated into ribosomal protein S6. Concomitantly, multiple cytoplasmic proteins, particularly those with high affinity for ribosomes, were phosphorylated. Compared with the controls (mock infection), mengovirus infection induced only a slight additional phosphorylation of the proteins of the ribosome-free supernatant. Employing the radio isotope dilution test with 3', 5'-cyclic AMP-binding protein, no enhancement of the intracellular concentration of 3', 5'-cyclic AMP in response to mengovirus infection could be deomonstrated.