Biomedical subjects

L Cocco

Publications and source records attributed to L Cocco.

At least 91 records · Page 5Linked to original sources

PubMed1992

Mouse and human hemopoietic cell lines of erythroid lineage express lamins A,B and C.

Using monoclonal antibodies, we have studied the expression of lamins A,B,C and vimentin in mouse and human erythroleukemia cells. We have found that in contrast with previous reports these cells have all three lamins. Mouse cells lack vimentin, whereas human cells express it. Lamins B and C are the most abundant lamins, whereas considerably less lamin A is detectable. Our results argue that some mouse and human hemopoietic cells can express all three lamins and that production of vimentin does not necessarily precede that of lamins A/C, as other reports have suggested in the past. The data also show that the absence of a salt resistant inner nuclear matrix is not always related with the lack of lamins A/C and vimentin, as recently proposed.

Animals↗

PubMed1992

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.

Aphidicolin↗

PubMed1992

Age-related events in active T subpopulation. Changes in polyphosphoinositide metabolism during mitogenic activation.

The active E rosette test allows the characterisation of a lymphocyte subset with high affinity receptors for sheep red blood cells. In this study, we compared active T lymphocytes of healthy subjects over 65 and under 30 years of age after mitogenic stimulation. Active E rosettes increased in both age groups after 48 h of phytohemoagglutinin (PHA) stimulation, mainly in young subjects. In addition, analysing the in vitro phosphorylation of inositol lipids, changes occurred in the incorporation of radioactivity in both phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis phosphate (PIP2) in the early steps of the mitotic response in the elderly subjects as compared to young ones, hinting that modifications of this signal transduction system are related to mitogenic stimulation in this peculiar T subpopulation.

Adult↗

PubMed1992

Changes in inositol lipid metabolism and protein kinase C translocation in nuclei of mitogen stimulated Swiss 3T3 cells.

The correlation between changes in nuclear polyphosphoinositide levels preceding PKC translocation to the nucleus and the onset of DNA synthesis has been discussed. Using two different clones of Swiss 3T3 fibroblasts belonging to the same original cell line, one of which is unresponsive to mitogenic stimulation with IGF-I on its own or in combination with bombesin, it has been observed that a rapid and transient breakdown of nuclear PIP and PIP2 occurs only in responsive cells and this precedes the translocation of PKC to the nucleus, as evidenced by immunochemical analysis as well as by enzymatic activity. Therefore, it seems that a direct link exists between nuclear polyphosphoinositide metabolism, PKC translocation to the nucleus and cell division. Since IGF-I acts at the plasma membrane through a tyrosine kinase receptor it seems that the mitogenic stimulation induced by this factor utilizes different signalling pathways at the plasma membrane and at the nucleus. Because of the evidence that type I IGF receptor is expressed in both responsive and unresponsive cells and that the receptor machinery at the plasma membrane is active the lack of the transient changes in nuclear inositol lipids and of PKC translocation in unresponsive cells further suggests that the cell nucleus is capable of an autonomous signalling system based on polyphosphoinositide metabolism.

3T3 Cells↗

PubMed1992

Changes in polyphosphoinositide levels in rat liver nuclei in response to prolactin, a known hepatic mitogen.

The effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [gamma 32P] adenosinetriphosphate was observed in both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a maximum response at 10(-12) M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monoesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, this feature is strongly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.

Animals↗

PubMed1992

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.

Ammonium Sulfate↗

PubMed1991

Mitogen-stimulated events in nuclei of Swiss 3T3 cells. Evidence for a direct link between changes of inositol lipids, protein kinase C requirement and the onset of DNA synthesis.

Two different clones of Swiss 3T3 cells belonging to the same original cell line have been obtained, one of which was unresponsive to mitogenic stimulation (e.g. insulin-like growth factor-I, bombesin, insulin-like growth factor-I + bombesin), while the other clone showed a very high rate of DNA synthesis under identical conditions as demonstrated by 5-bromodeoxyuridine incorporation. Both types of cells expressed the IGF-I receptor and showed high contact inhibition. When highly purified nuclei from responsive cells, treated for a short time with bombesin and insulin-like growth factor-I or insulin-like growth factor-I alone, were incubated with [gamma-32P]adenosine triphosphate, the labelling of phosphatidylinositol-mono- and diphosphate decreased when compared to controls, while this transient effect did not appear in the nuclei from unresponsive cells. Similarly nuclear protein kinase C is activated only in responsive cells. Therefore, it seems that a direct link exists between polyphosphoinositide metabolism, protein kinase C activation and the early events leading to cell division, since the rapid changes in the labelling of both phosphatidylinositol mono- and di-phosphate occur only in nuclei from Swiss 3T3 cells, which respond to the mitogenic stimulus determined by insulin-like growth factor-I on its own, or in combination with bombesin.

Adenosine Triphosphate↗

PubMed1991

Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin-like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphosphoinositides.

Using a polyclonal antibody raised against a synthetic peptide of the catalytic region of protein kinase C, we have carried out a combined immunocytochemical and immunochemical analysis to follow the subcellular localisation of this enzyme in response to mitogenic stimulation with insulin-like growth factor I and bombesin. These investigations show a time dependent translocation of protein kinase C from the cytoplasm to the nucleus since 5 min stimulation reaching a maximal effect after 45 min. These results show clearly that mitogen induced translocation of protein kinase C to the nucleus follows temporally the earlier changes in nuclear polyphosphoinositide metabolism previously demonstrated.

Amino Acid Sequence↗

PubMed1991

Heat-induced stabilization of the nuclear matrix: a morphological and biochemical analysis in murine erythroleukemia cells.

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.

Animals↗

PubMed1991

Inositol lipid phosphorylation in the cell nucleus.

Inositol lipid metabolism has been analyzed in isolated rat liver nuclei and nuclear fractions, in order to determine the subcellular distribution of the sites of lipid phosphorylation and breakdown. Lipid kinases and phosphoesterases appear to be tightly bound nuclear components, and can utilize exogenous substrates administered to membrane-depleted structures. The possible involvement of specific carrier protein in the nuclear metabolism of inositol lipids has also been analysed by studying the uptake and processing of phosphatidylinositol transferred to the isolated nuclei by phosphatidylinositol transfer protein (PI-TP). PI-TP greatly stimulates the incorporation of phosphatidylinositol from microsomal membranes and synthetic vesicles, and the lipid taken up is available for phosphorylation and breakdown by enzymes associated to the nucleus. The results obtained support previous data on the metabolic and structural role of nuclear lipids, and suggest that the cell nucleus is a site of lipid phosphorylation, not necessarily involving enzymes and substrates located on the nuclear membrane. They also indicate that an integrated signalling pathway can exist at the nuclear level utilizing inositol lipid-derived second messengers and PKC to control replication and transcription.

Animals↗

PubMed1991

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells.

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.

Aphidicolin↗

PubMed1991

Interferon transiently modulates intranuclear signalling system in erythroleukemia Friend cells.

The effect of human recombinant DNA interferon alpha type A on nuclear inositol lipids, diacylglycerol (DAG) and DNA metabolism has been investigated in Friend erythroleukemia cells. A transient enhancement of phosphatidylinositol (4,5) - bisphosphate (PIP2) phosphorylation together with an increase of diacylglycerol mass were observed in nuclei isolated from cells treated with interferon alpha for 90 min. At the same time, a marked reduction of DNA polymerase alpha activity was observed, suggesting a possible involvement of nuclear inositol fraction in the response of the cell nucleus to interferon treatment.

Animals↗

PubMed1990

Calcium free inositol (1,4,5)-trisphosphate stimulates protein kinase C dependent protein phosphorylation in nuclei isolated from mitogen-treated Swiss 3T3 cells.

As a step towards the elucidation of the role played by nuclear polyphosphoinositides, we have investigated the effect of exogenous calcium free inositol (1,4,5)-trisphosphate on the in vitro phosphorylation of proteins in nuclei prepared from Swiss 3T3 cells treated with bombesin and insulin-like growth factor I. When present in combination with phosphatidylserine, inositol (1,4,5)-trisphosphate enhanced the phosphorylation of two nuclear proteins, Mr 21,000 and 31,000, as well as of exogenous histone H1, to the same extent as a combination of phosphatidylserine and diacylglycerol. Inositol (1,4,5)-trisphosphate alone had no effect. This stimulation could be abolished by the protein kinase C inhibitor sphingosine and by EGTA, while could be restored by a combination of phosphatidylserine and exogenous Ca+(+) ions. These results raise the possibility that inositol (1,4,5)-trisphosphate is capable of liberating Ca+(+) ions from a nuclear store thus stimulating protein kinase C activity.

Animals↗

PubMed1990

Transient shift of diacylglycerol and inositol lipids induced by interferon in Daudi cells. Evidence for a different pattern between nuclei and intact cells.

The effect of human recombinant DNA interferon-alpha type A on inositol lipid and diacylglycerol metabolism was investigated in Daudi lymphoma whole cells and isolated nuclei. In isolated nuclei after 90 min of interferon treatment an enhanced rate of PIP2 phosphorylation and an increase of DAG mass were observed. In whole cells, after 1 min of interferon treatment, there was a rapid and transient shift of DAG mass apparently not related to inositol lipid modifications, thus indicating the presence in nuclear and cytoplasmic compartments of inositol lipid fractions with different metabolic features in response to interferon-alpha.

Adenosine Triphosphate↗

PubMed1990

Temperature-dependent association of DNA polymerase alpha activity with the nuclear matrix.

We have investigated the effect of preincubating isolated nuclei at the physiological temperature of 37 degrees C on the recovery of DNA polymerase alpha and beta activities bound to the nuclear matrix. In HeLa cells, when purified nuclei are incubated for at least 30 min at 37 degrees C prior to extraction with 2 M NaCl and digestion with DNase I, about 30% of nuclear DNA polymerase alpha activity is associated with the final matrix along with about 20% of nuclear protein. If the preincubation is carried out at 0 degrees C, less than 5% of the enzyme activity is resistant to high salt extraction and the protein recovery drops to about 12%. On the contrary, the recovery of nuclear DNA polymerase beta activity bound to the matrix fraction is independent of the temperature at which the preincubation is performed. The same levels of DNA polymerase alpha activity are found to be matrix associated even if reducing and chelating agents are present during the exposure of isolated nuclei to 37 degrees C, suggesting that this phenomenon does not depend on the in vitro formation of disulfide bonds or on some metal ion-protein interaction. Our data could explain why, in the past, different results have been obtained when the association of DNA polymerase alpha with the nuclear matrix has been analyzed.

Cell Nucleus↗

PubMed1990

Nuclear inositol lipids. Relationship between growth factor induced metabolic changes and protein kinase C activity.

We have sought to establish the effect of mitogen treatment on nuclear inositol lipids and the relationship between inositol cycle products and hyperphosphorylation of nuclear proteins via PKC during the lag phase leading to the onset of DNA synthesis. Swiss 3T3 cells were labelled for 36 hr with high levels of [3H]-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 min, but not for 4 hr, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a marked decrease in PtdInsP and PtdInsP2. Moreover, in vivo phosphorylation of some nuclear proteins occurs later on. Among these proteins, histone H1 and 0.75 M PCA soluble polypeptide, with an apparent Mr of 21,000 as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes in nuclear polyphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.

Adenosine Triphosphate↗

PubMed1990

Probing the sulfhydryl groups of nuclear matrix proteins with 6-iodoacetamidofluorescein.

Rat liver nuclear matrices were reacted with the fluorescent dye 6-iodoacetamidofluorescein and the matrix proteins were then separated by one and two-dimensional polyacrylamide gel electrophoresis. Upon transillumination with U.V. light it was possible to see that several proteins had reacted with the dy, thus indicating the presence of free -SH groups. This labelling technique allowed the detection of a large number of proteins, being several folds more sensitive than conventional Coomassie Blue staining, as demonstrated by two-dimensional electrophoretical separation. If nuclear matrices were treated with reducing agents before being reacted with 6-iodoacetamidofluorescein, the fluorescence increased with about the same intensity in all the protein bands. It is proposed that 6-iodoacetamidofluorescein can be used as a specific and very sensitive probe to study the -SH groups of nuclear matrix proteins.

Animals↗

PubMed1990

Nuclear lipids in Friend cells. Shifted profile of diacylglycerol during erythroid differentiation induced by DMSO.

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.

Animals↗