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O Barnabei

Publications and source records attributed to O Barnabei.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1991

Postnatal maturation of cholinergic markers in forebrain regions of C57BL/6 mice.

The maturation of some neurochemical markers linked to cholinergic function (choline acetyltransferase, acetylcholinesterase, muscarinic binding sites) has been studied from 10 to 150 days of age in mice belonging to C57BL/6 strain. Previous studies had suggested that part of the cholinergic neurons of the basal forebrain undergo degeneration during juvenile stages of life in these rodents. Our data showed a delayed maturation of the cholinergic levels in the cortex and hippocampus, the main targets of the forebrain cholinergic neurons, but not in the striatum and superior colliculus. In none of these regions was any clear trend towards a decrement of cholinergic levels observed during the lifespan considered. In the medial septum-diagonal band area, an actual decrease of cholinergic levels was observed between 60 and 150 days of age. A side experiment based on daily administration of GM1 ganglioside during juvenile life, showed no effect of this treatment on the maturation of cholinergic markers.

Acetylcholinesterase↗
PubMed1991

Temporal, regional and cellular selectivity of neonatal alteration of the thyroid state on neurochemical maturation in the rat.

The effects of alteration of thyroid state on neurochemical maturation have been studied in rats made hypothyroid by daily injections of methimazole or hyperthyroid by daily supplementation with thyroid hormone (T3) from postnatal days 1 to 27. Biochemical assays on seven brain regions plus the spinal cord were carried out on 14 and 28 day-old rats as well as in adult rats after at least 40 days of recovery. 2',3'cyclic nucleotide phosphohydrolase (CNPase), a specific marker for oligodendrocytes and myelination was significantly decreased in all regions except the spinal cord of hypothyroid rats. The astrocytic marker glutamine synthetase (GS) was slightly increased in the hippocampus of hypothyroid rats. Choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, was decreased in the prefrontal and visual cortices, the striatum and the superior colliculus and increased in the cerebellum of hypothyroid rats; in addition, the enzyme activity was increased in the prefrontal cortex and striatum and decreased in the cerebellum of hyperthyroid rats. Acetylcholinesterase (AChE) activity was decreased in the prefrontal cortex and in the striatum of hypothyroid rats while 3H-quinuclidinyl benzilate (QNB) muscarinic binding was decreased in all cortical areas and in the hippocampus of hypothyroid rats. Glutamate decarboxylase (GAD), a specific marker for GABAergic neurons, was decreased in the cortical areas of hypothyroid rats. Aromatic amino acid decarboxylase (AAD), a general marker for monoaminergic neurons, was unaffected. Alteration of neurochemical parameters was never observed in the spinal cord. Under our experimental conditions, the effects of alteration of thyroid state appeared graded and selective with respect to temporal, regional and cellular parameters.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
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

Adenosine and glutamate modulate each other's release from rat hippocampal synaptosomes.

In rat hippocampal synaptosomes, adenosine decreased the K+ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A1 receptor antagonist 8-cyclopentyltheophylline. In the same synaptosomal preparation, K+ (30 mM) strongly stimulated the release of the preloaded [3H]adenosine in a partially Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both L-glutamate and L-aspartate enhanced the release of [3H]adenosine derivatives ([3H]ADD). The glutamate-evoked release was dose dependent and appeared to be Ca2+ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers D-glutamate and D-aspartate were able to stimulate [3H]ADD release. In contrast, the specific glutamate agonists N-methyl-D-aspartate, kainate, and quisqualate failed to stimulate [3H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg2+ and L-glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6-7-dinitroquinoxaline-2,3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as gamma-aminobutyric acid stimulated [3H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still unknown, could help in the understanding of excitatory amino acid neurotoxicity.

Adenosine↗
PubMed1990

Effects of short- and long-term ganglioside treatment on the recovery of neurochemical markers in the ibotenic acid-lesioned rat striatum.

The striatum of adult rats was bilaterally lesioned with stereotaxic injections of ibotenic acid in a dose (16 nmoles) that resulted in subtotal lesions. Some rats received systemic ganglioside treatment starting the day before operation and lasting for 6 or 24 days after operation; they were compared with lesioned rat receiving systemic saline injections as well as with corresponding groups of sham-operated animals. Specific neurochemical markers for cholinergic neurons (choline acetyltransferase, ChAT), GABAergic neurons (glutamate decarboxylase; GAD), and astrocytes (glutamine synthetase; GS) were assayed to asses the neurochemical recovery promoted by ganglioside treatment. Twenty-four, but not six, days after operation a significant increase of ChAT and GAD was measured in the striatum of lesioned rats treated with gangliosides in comparison with the saline group. Furthermore, a significant increase of both enzymes occurred in the striatum of lesioned rats receiving ganglioside treatment for 24 days in comparison with rats receiving ganglioside treatment for 6 days only. A small but significant increase of ChAT was measured in the striatum of sham-operated rats after 24 days of ganglioside treatment in comparison with the corresponding saline group. Finally, the increase of GS caused by the glial reaction to the ibotenic acid lesion was not affected by ganglioside treatment. The results indicate that a relatively long-lasting ganglioside treatment stimulates the recovery of specific neuronal transmitter markers and that some effect is, in addition, exerted on unlesioned cholinergic striatal neurons.

Animals↗
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

Regional maturation of neurotransmitter-related and glial markers during postnatal development in the rat.

Neurotransmitter-related (choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, L-glutamate and GABA high affinity uptake) and glial neurochemical markers (glutamine synthetase, beta-alanine uptake and 2',3' cyclic nucleotide phosphohydrolase) have been quantitatively assayed in various regions of the rat CNS during normal postnatal development: spinal cord, cerebellum, superior colliculus, hippocampus, striatum, visual cortex, frontal sensory-motor cortex and prefrontal cortex. In general, neurochemical markers show an obvious trend toward increasing levels in parallel with brain maturation. However, some relevant exceptions have been observed and discussed. Detailed knowledge of regional neurochemical brain maturation is important since it gives us information concerning some key events of brain development. In addition, this knowledge is the essential pre-requisite for studies aimed at the alteration of specific regional and temporal parameters through experimental manipulation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Developmental profiles of cholinergic activity in the habenulae and interpeduncular nucleus of the rat.

Choline acetyltransferase (ChAT) was measured in the habenula and in the interpeduncular nucleus of rats from 1 to 12 weeks of age. A remarkable degree of parallelism was shown by the developmental curves in the two nuclei. In both cases the highest level of enzyme activity was reached at 3 weeks of age and was followed by some decrease towards adult values. A statistically highly significant correlation was demonstrated between ChAT levels in the two nuclei at the various developmental stages. The rise of the cholinergic marker was slightly advanced in the habenula in comparison with the interpeduncular nucleus. The present data may be useful for studies focused on neonatal synaptogenesis, plasticity and synaptic neurochemistry of this relatively simple model of brain connections.

Aging↗
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

Depletion of cholinergic habenulo-interpeduncular neurons by selectively timed methylazoxymethanol acetate (MAM) treatment during pregnancy.

Methylazoxymethanol acetate (MAM) was injected to female rats at the beginning of the 17th day of gestation. Resulting offspring showed a remarkable decrease in the size of the medial habenula while the interpeduncular nucleus, whose neurons are generated before the time of MAM treatment, appeared anatomically unaffected. Choline acetyltransferase was significantly reduced in the habenulae and in the interpeduncular nucleus suggesting that MAM treatment had depleted a portion of the cholinergic neurons of the medial habenula which project to the interpeduncular nucleus. Aromatic amino acid decarboxylase significantly increased in the interpeduncular nucleus, a likely effect of monoaminergic hyperinnervation in response to partial cholinergic deprivation. MAM strategy can be usefully adopted for the study of general aspects of brain development when connected nuclei showing no overlapping in neuronal generation times are involved.

Acetylcholinesterase↗
PubMed1988

Neurotoxic effect of 1-methyl-4-phenylpyridinium ion on dopaminergic neurons of the retina of goldfish.

Dopaminergic neurons of the goldfish retina were selectively destroyed after a single intravitreal injection of 1-methyl-4-phenylpyridinium ion (MPP+). The ultrastructural analysis of the retina 3 days after toxin administration shows darkening of some retinal neurons present in the inner nuclear layer including their cytoplasmic processes. Both uptake and release of dopamine were reduced in the toxin-injected retina, whereas choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, as well as the uptake of D-[3H]aspartate were not affected.

1-Methyl-4-phenylpyridinium↗
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↗
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↗
PubMed1985

[3H] kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain.

Binding of [3H]kainic acid to the synaptosomal-mitochondrial fraction (P2) of the goldfish brain was studied. Specific binding to this fraction represents about half of the total binding capability of the homogenate particulate material and is enriched in synaptic membranes; it is greater by about two orders of magnitude than those given for rat brain and pigeon optic tectum membranes. Association of the ligand-site complex has a time constant lower than 1 min and the same is true for the main component of the dissociation process. The binding equilibrium is apparently not affected by substances contained in the fraction material. The analysis of the dose-response data showed a main receptor population (B max = 139 pmol/mg protein) which displayed positive cooperativity (nH = 1.29). The same behaviour was shown by washed membranes from the same fraction but, in this case, the affinity for the ligand was lower (apparent affinity constants: K'D = 0.28 nM for the intact fraction and K'D = 0.38 nM for membranes). A smaller population of sites with higher affinity was also detected both in the intact fraction and in membranes. Among the substances tested as displacers of kainic acid from the synaptosomal sites, the most effective were quisqualate and L-glutamate. Folic acid and its dihydro and tetrahydro derivatives were half as potent as glutamate whereas methyltetrahydrofolic acid and folinic acid had a very weak action. The difference between these sites and those detected on rat brain membrane preparations is discussed.

Animals↗
PubMed1985

Kainic acid differentially affects the synaptosomal release of endogenous and exogenous amino acidic neurotransmitters.

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.

Animals↗