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Biomedical subjects

L Cocco

Publications and source records attributed to L Cocco.

At least 109 records · Page 6Linked to original sources
PubMed1989

Changes in nuclear inositol phospholipids induced in intact cells by insulin-like growth factor I.

Swiss 3T3 cells were labelled for 36 hours with high levels of 3H-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 minutes, but not for 4 hours, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a slight decrease in PtdIns and more marked decreases in PtdInsP and PtdInsP2. These effects were not seen if isolated nuclei were incubated with IGF-I and bombesin. We interpret these results to mean that rapid mass changes occur in nuclear inositol phospholipids in the early stages of the mitotic response.

Animals↗
PubMed1989

Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement.

When Swiss 3T3 fibroblasts are treated with a combination of IGF-I2 and bombesin at mitogenic concentrations, in vivo phosphorylation of some nuclear proteins occurs within 45-90 min. Among these proteins, histone H1 and a 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, recently demonstrated, in nuclear polyphosphoinositide 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.

Animals↗
PubMed1989

Nuclear polyphosphoinositides during cell growth and differentiation.

When highly purified nuclei of Swiss mouse 3T3 cells are incubated with gamma-32P-ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and (4,5)P2. If the cells are pre-treated with IGF-I, the incorporation into the polyphosphoinositides is decreased. This effect is maximal by 2 min, is transient in that it disappeared by 1 hr, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. Friend cells exhibit a related phenomenon in that the labelling of PIP2 in isolated nuclei is increased by conditions which cause erythroid differentiation (DMSO addition). We suggest that some aspect of nuclear polyphosphoinositide metabolism is modified when the nucleus is induced to divide or to differentiate, and that this change in inositide metabolism is a very early event in the sequence leading to cell division or differentiation.

Animals↗
PubMed1988

Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulin-like growth factor I.

When highly-purified nuclei of Swiss-mouse 3T3 cells are incubated with [32P]-gamma ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and phosphatidylinositol(4,5)P2. If the cells are pre-treated with insulin-like growth factor I, this incorporation into the phospholipids is decreased. The effect is maximal by 2 minutes, is transient in that it has disappeared by one hour, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. We suggest that some aspect of polyphosphoinositide metabolism is altered in the nucleus (leading to a decreased radiolabelling) when the cells are treated with mitogenic growth factors, and that this change in inositide metabolism is a very early event in the sequence leading to cell division.

Adenosine Triphosphate↗
PubMed1988

Lipid mediated signal transduction in the cell nucleus.

Cell growth and differentiation can be affected by the transduction of extracellular signals involving cyclic nucleotides, inositol phospholipids and phospholipid dependent protein kinase C systems. Since we previously reported existence of lipids inside the nucleus and nuclear fractions, it seems of interest to examine the possible presence of the cascade of inositol lipids in isolated nuclei as well as the presence of the protein kinase C, whose activity is tightly related to the phosphoinositide cycle, and requires the presence of phosphatidylserine, which has been previously demonstrated to deeply affect nuclear structure and function. Here we show that highly purified nuclei from both rat liver and Friend cells, free of nuclear membrane, can incorporate radiolabel from ATP-[32P] into phosphatidic acid, phosphatidyl-inositol phosphate and phosphatidylinositol (4', 5')bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells. Moreover, a doublet of immunoreactive bands has been identified in rat liver nuclei by means of a polyclonal antibody against protein kinase C. The two polypeptides appear to be tightly bound to the nuclear matrix. These two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Evidence for a reduction of the replicative activity of matrix-bound DNA polymerase alpha following treatment with phospholipase C.

Since phospholipids have been suggested to play some role in the molecular organisation of the nuclear matrix, the effect of their removal by means of phospholipase C has been investigated in regenerating rat liver nuclear matrix. The matrix-bound DNA polymerase alpha shows an almost complete loss of activity following the digestion with phospholipase C. Since the polymerase activity is restored by adding exogenous DNA, we suggest that the effect is due to the removal of matrix residual DNA, which is in some way linked to the nuclear matrix by means of hydrophobic interactions.

Animals↗
PubMed1988

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Animals↗
PubMed1988

From animal to molecule: aspects of the biology of insulin-like growth factors.

The synthesis of IGF-II mRNA in sheep foetal tissues is considerably higher than IGF-I. IGF-II probably has a paracrine role in the foetus; however it is likely that IGF-I originates mainly from the foetal liver and has an endocrine function. Although in the adult system IGF-I is tightly bound to serum carrier proteins it is potentially biologically active. Galactopoiesis in the goat mammary gland provides a useful model for demonstrating the importance of circulating IGF-I as a mediator of GH action. Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by IGF-I, II and insulin were examined. It was found that the potency of binding to type I receptors was IGF-I greater than IGF-II much greater than insulin by competitive binding assays and chemical cross-linking studies, and that some cell lines secrete an IGF binding protein which is specific for IGF-I and II and which acts as an inhibitor in cellular binding assays. Maximal stimulation of DNA synthesis induced by IGF-I, II and insulin in the presence of synergising mitogens were similar. While the actions of the IGFs were consistent with type I receptor binding insulin appeared to act through its own receptor. The reduction of EGF receptor affinity following the addition of IGF-I and insulin to 3T3 cells may involve a protein kinase that is not sensitive to phorbol esters. 3T3 cell nuclei contain endogenous inositol phospholipids and their corresponding kinases and monoesterases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.

Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.

Animals↗
PubMed1987

6-Hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors.

Dopamine-sensitive adenylate cyclase and 3H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and/or dendrites of dopaminergic neurons; striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers.

Adenylyl Cyclases↗
PubMed1987

Unfolding of nucleosome core induced by phosphatidylserine.

The main experimental findings on the actual presence of lipids among the minor chromatin components are revised and discussed especially in the light of the reported effects that exogenous lipids induce in DNA and RNA synthesis by using purified templates. Moreover, all the available evidence of the influence of phospholipid liposomes on the activities and structure of isolated nuclei are reported. In order to further clarify the possible mechanism by which phospholipids could affect gene expression, the modifications at the nucleosome core level have been investigated by means of IAF staining and electron microscopy. The results obtained indicate that the increased transcriptional activity induced by PS MLV in isolated nuclei requires both the removal of histone H1, which causes the unfolding of the solenoid into the nucleosome fiber configuration of the chromatin, and the subsequent splitting of the H3 dimer. This latter process, monitored by IAF accessibility to H3 in isolated nucleosomes incubated with PS, causes the transition from the nucleosome to the lexosome structure, which is the configuration favoring the activity of RNA polymerases.

Animals↗
PubMed1987

Phospholipids as components of the nuclear matrix: their possible biological significance.

The phospholipid involvement in the regulation of the functional and structural properties of the nuclear matrix is discussed analysing the results obtained with the enzymic removal of these molecules. Namely phospholipids seem to mediate hydrophobic interactions between nucleic acids and matrix fibrils either directly or indirectly through an association with the non-histone proteins of the matrix.

Animals↗
PubMed1986

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy.

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.

Animals↗
PubMed1986

Changes in nucleosome structure and histone H3 accessibility. Iodoacetamidofluorescein labelling after treatment with phosphatidylserine vesicles.

The accessibility of the sulfhydryl-specific dye 6-iodoacetamidofluorescein (IAF) to H3 histone has been studied in isolated rat liver nuclei and mononucleosomal core particles after treatment with phosphatidylserine (PS) multilamellar vesicles (MLV). In isolated nuclei, despite the enhancement of total RNA synthesis and the massive chromatin decondensation produced by liposomes, the amount of histone H3 which can be labelled with the dye remains essentially the same in PS-treated as in control nuclei. However, when mononucleosomal core particles, treated with PS vesicles, are reacted with IAF, H3 becomes derivatized by the dye, while controls do not. These data provide additional evidence that the metabolic and structural changes observed in isolated nuclei treated with PS MLV, are due mainly to the reported removal of histone H1. Moreover, the experiments reported confirm the usefulness of IAF in studying the changes of nucleosome organization, since PS is able to affect the nucleosomal core configuration in isolated nucleosome particles, derivatizing the buried cysteine groups of H3 histone.

Animals↗
PubMed1986

Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei.

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultra-structural morphology. Total transcription in rat liver and beta-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC. The significance of these modifications in relationship to the possible involvement of phospholipids in the control of gene expression is discussed.

Animals↗
PubMed1985

Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase.

Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methotrexate concentration indicates that the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 X 10(7) M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 X 10(5) M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Folic Acid Antagonists↗
PubMed1985

Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand.

Stopped-flow spectrophotometry and stopped-flow fluorometry have been used to study the binding of methotrexate (MTX) and 3-deazamethotrexate (3-deazaMTX) to dihydrofolate reductase (DHFR) isoenzymes from Streptococcus faecium and from Lactobacillus casei. The absorbance change and fluorescence quenching that occur when MTX binds to DHFR isoenzyme II from S. faecium (SFDHFR II) are both biphasic and give similar apparent rate constants for both phases. The faster phase has an apparent rate constant that is dependent on MTX concentration and therefore corresponds to the initial binding reaction. From the concentration dependence it has been calculated that the association rate constant is 3.0 X 10(5) M-1 s-1 at 20 degrees C and pH 7.3, and the association constant (equilibrium constant) under these conditions is 5.8 X 10(5) M-1. By examination of the amplitude of the fast-phase absorbance change at various wavelengths, it has been determined that the absorbance change occurring in the fast phase is due to MTX protonation. Within the limits of the method it was thus not possible to detect a difference in the rates of binding and of protonation of MTX. The MTX association rate constant is pH dependent, decreasing 330-fold as the pH is decreased from 5.0 to 9.0. The data fit well to a curve generated by assuming a single ionization with a pKa of 6.0 and a pH-independent association rate constant 1000-fold greater for binding of protonated MTX to SFDHFR II than for binding of unprotonated MTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-Ion Concentration↗