Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus.

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

Murine cell complementation of a cold-sensitive defect for simian virus 40 replication in simian cells.

Mouse 3T3 fibroblasts were found to complement, in simian cell variants semipermissive to simian virus 40, a cold-sensitive defect of an early function, but not a nonconditional defect of viral uncoating. The variant simian cells could rescue simian virus 40 from 3T3 transformants, and this capacity was not temperature dependent.

Modulation of laser-evoked potentials by experimental cutaneous tonic pain.

The present study aimed to investigate whether tonic cutaneous pain exerts any effect on the cortical processing of nociceptive input and if this effect may involve only body parts in pain. Tonic cutaneous pain was obtained in nine healthy human subjects by infusion of a hypertonic saline (5%) in the s.c. tissue over the hypothenar muscles (10 ml/h for 20 min). Nociceptive cutaneous CO2 laser-evoked potentials were recorded after stimulation of the right hand dorsum, which was adjacent to the painful area, and the right perioral region, corresponding to the adjacent cortical sensory area. Laser-evoked potentials were obtained before saline injection, at the peak pain and 20 min after pain disappeared. During saline infusion, the laser-evoked pain to right hand stimulation was reduced and the vertex laser-evoked potentials (N2a-P2, mean latency 181 ms and 319 ms for the N2a and the P2 potentials, respectively), which are generated in the anterior cingulate cortex, were significantly decreased in amplitude compared with the baseline. Moreover, the topography of these potentials was modified by cutaneous pain, shifting from the central toward the parietal region. Dipolar modeling showed that the dipolar source in the anterior cingulate cortex moved backward during saline infusion. This result suggests that cutaneous pain may modify the relative activities of the anterior and posterior anterior cingulate cortex parts, which are thought to be devoted to encode different aspects of pain sensation. No laser-evoked potential change was observed after stimulation of the right perioral region, suggesting that functional changes in the nociceptive system are selective for the painful regions and not for areas with cortical proximity.

Pain and motor complications in Parkinson's disease.

AIMS: To study the association of pain with motor complications in 117 patients with Parkinson's disease. METHODS: Patients were asked to refer any pain they experienced at the time of study and lasting since at least 2 months. Basic parkinsonian signs and motor complications (including motor fluctuations and dyskinesia) were assessed and Unified Parkinson's Disease Rating Scale (UPDRS) motor score part III (during on) and part IV were calculated. Information on age, sex, duration of disease, use of dopamine agonists and levodopa, years of levodopa treatment and current levodopa dosage, medical conditions possibly associated with pain, and depression were collected. Single and multiple explanatory variable logistic regression models were used to check the association of pain with the investigated variables. RESULTS: Pain was described by 47 patients (40%) and could be classified into dystonic (n.19) and non dystonic pain (n.16); in 12 patients both types coexisted. Multiple explanatory variable logistic regression models indicated a significant association of pain with motor complications (adjusted OR, 5.7; 95% CI, 2 to 16.5; p = 0.001). No association was found between pain, dystonic or non dystonic, and the other investigated variables including medical conditions known to be associated to pain in the general population. There was a significant correlation (r = 0.31, p<0.05) between severity of pain (measured on a Visual Analogue Scale) and severity of motor complications (UPDRS part IV). CONCLUSIONS: Pain may be a representative feature of Parkinson's disease frequently associated with motor complications. The association is independent of a number of potentially relevant demographic and clinical variables.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Molecular cloning and restriction mapping of a simian virus 40 deletion mutant derived from simian transformants expressing a non-karyophilic T antigen.

A replication-defective Simian virus 40 genome, with a deletion of about 120 nucleotides in the region encoding the N-terminal fourth of the large T antigen, has been isolated from the DNA of Simian cells transformed by SV40. Both the original transformants, and the murine transformants obtained by transfection with this cloned mutant DNA, produced a large T antigen displaying in immunofluorescence an exclusively cytoplasmic localization. The protein apparent molecular mass (83 kDa) was about 6% smaller than that of normal karyophilic large T. Restriction analysis showed that the deletion eliminated two close HinfI sites, at nucleotides 4459 and 4376 (map unit 0.50).

Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53.

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T-p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N-terminal half or truncated by various lengths at the C-terminal end, we found that a region between amino acids 114 and 152 and a C-terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C-terminal end up to amino acid 669 is essential for T-p53 complex formation but not the N-terminus up to amino acid 152.

MyoD activity upregulates E2F1 and enhances transcription from the cyclin E promoter in differentiating myoblasts lacking a functional retinoblastoma protein.

We investigated the mechanism leading to cyclin E accumulation when cultured mouse myoblasts, lacking functional Rb because of sequestration or deletion, are exposed to differentiating conditions (mitogen subtraction and cell-cell contact), which activate MyoD and normally downregulate factors involved in cell division. After excluding that stabilization might account for the observed cyclin-E mRNA accumulation, we found an induction of the cyclin-E promoter that correlated with E2F activity upregulation and depended on both MyoD activation and Rb inactivation. Analyses of the E2F1-promoter activity, in normal and Rb-deficient fibroblasts converted by MyoD, identified a MyoD function stimulating E2F1 expression. The E2F1 induction was very manifest in the Rb-/- cells, but also detectable, at the early stage of differentiation, in normal cells. Its effects, although not indispensable for myogenesis, presumably contribute to raise the concentration of Rb-E2F1 transcription-repressing complexes, since MyoD strongly induces also Rb in differentiating myocytes. The activity of an E2F1 promoter lacking the E2F sites indicated that E2F1 itself underwent self-repression by such mechanism at late stages of differentiation. In the absence of Rb, however, the induced E2F1 is left with only its activating role, reversing the normal effect of this MyoD function.

The cytoplasmic RNA of HeLa cells: new discrete species associated with mitochondria.

Two new species of cytoplasmic RNA with approximate sedimentation values of 12S and 21S, and which appear associated with mitochondria, are described. The species are unmethylated, are relatively metabolically stable, and have a 43 per cent G + C content. Their synthesis is uniquely resistant to inhibition by UV irradiation. The 12S and 21S RNA do not appear to be components of polysome-like structures.

Late modifications of simian virus 40 chromatin during the lytic cycle occur in an immature form of virion.

Two main modifications of the simian virus 40 chromatin were found to occur during the lytic cycle. One was the progressive increase in the acetylation level in the four non-H1 histones as the 75S deoxynucleoprotein complexes (minichromosomes) became assembled into heavier structures. The other was the final elimination from viral chromatin of histone H1. An important stage in the course of these changes was represented by an intracellular simian virus 40 particle, in which the virus-coded proteins were already assembled, but properties distinct from those of mature virions were still present. This particle resembled the mature virions in morphology, sedimentation rate, and buoyant density. It was distinguished by the instability, the presence of histone H1, the uptake of radioactive acetate, and the lower infectivity. Its significance appears to be that of an immature virion on the basis of these characters and of the consistent kinetic behavior during the lytic cycle.

A nonkaryophilic T antigen of SV40 can either immortalize or transform rodent cells, and cooperates better with cytoplasmic than with nuclear oncoproteins.

We investigated the cell growth alterations brought about by a mutant nonkaryophilic large T antigen (NKLT) of SV40, alone and in combination with other oncogenes. NKLT by itself exhibited bivalent functional competence: it induced immortalization in early-passage rat embryo fibroblasts (REFs) and transformation in established NIH3T3 cells, although it was totally unable to transform nonestablished REFs. The absence of a normal small T reduced but did not suppress such effects. Coexpression of NKLT with the nuclear oncoprotein Polyoma large T significantly increased the efficiency of the same activities sustained by NKLT alone, but did not confer the ability to transform nonestablished REFs. Similar results were also observed in cells cotransfected with NKLT and another nuclear oncoprotein, E1A of adenovirus. In contrast, NKLT coexpression with either of the cytoplasmic oncoproteins, Polyoma middle T or activated Ha-ras, produced full transformation of early passage REFs. Thus the NKLT stimulus has the following properties: (i) it straddles the immortalizing/transforming distinction of oncogenic competence, (ii) it potentiates the effects of some nuclear oncoproteins, and (iii) it complements certain cytoplasmic oncoproteins to promote transformation of nonestablished cells.

Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.