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Biomedical subjects

M Piacentini

Publications and source records attributed to M Piacentini.

At least 109 records · Page 6Linked to original sources
PubMed1991

Retinoic acid and alpha-difluoromethylornithine induce different expression of neural-specific cell adhesion molecules in differentiating neuroblastoma cells.

Human neuroblastoma cells SK-N-BE(2) can be induced to differentiate towards a neuronal phenotype by retinoic acid (RA) or a schwannian/glial phenotype by alpha-difluoromethylornithine (DFMO), producing differential binding of 14 antibodies (MAbs). RA induced the expression of the neural cell adhesion molecule, NCAM (also confirmed by northern blot); whereas DFMO enhanced the binding of MAbs UJ181.4, UJ127.11 which recognise an identical protein doublet of 220-240 kDa, thought to be the L1 protein(s). The data presented demonstrate that neuroblastoma cells differentiate toward separate phenotypes associated with a specific induction of two different adhesion molecules, NCAM on neuronal cells and L1 on schwannian/glial cells.

Antibodies, Monoclonal↗
PubMed1990

Ca2(+)-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes.

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca2+ (low Ca2+) incorporated [1-14C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1-14C]AA and reincubated in label-free medium containing 1.2 mM Ca2+ (normal Ca2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca2+ stimulation of phosphoinositide turnover. Cell shift to normal Ca2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1-14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca2+ are discussed.

Animals↗
PubMed1990

Post-translational modification of apolipoprotein B by transglutaminases.

The major form of cross-link found in apolipoprotein B was identified as N1N12-bis-(gamma-glutamyl)spermine, a product known to be formed through the catalytic action of transglutaminases (EC 2.3.2.13). N1-(gamma-Glutamyl)spermine was present in a trace amount but epsilon-(gamma-glutamyl)lysine cross-links, which are formed during fibrin formation in plasma, were not detected. In the presence of catalytic amounts of plasma Factor XIIIa (a thrombin-dependent extracellular transglutaminase) or cellular transglutaminase (a cytosolic enzyme), apolipoprotein B and other plasma apolipoproteins (A-I, A-II and C) underwent covalently bridged polymerization and served as amine acceptor substrates. These results suggests that transglutaminases may participate in the covalent modification of apolipoproteins, either in the physiological state or during pathogenesis.

Animals↗
PubMed1990

Polyamine-dependent post-translational modification of proteins in differentiating mouse epidermal cells.

In order to get a better understanding of the role played by polyamines in calcium-induced epidermal cell differentiation, the time course of their metabolism was investigated. Results demonstrate that differentiating epidermal cells are characterized by time-dependent changes in polyamine concentrations. An early polyamine catabolic phase, characterized by increased total putrescine concentration and drastic reduction of both spermidine and spermine levels, is followed by active spermidine biosynthesis. The differences in putrescine and, in particular, spermidine metabolism are reflected in a time-dependent modulation of protein-bound polyamine derivatives. In fact, upon addition of calcium to the culture medium, hypusine N epsilon-(4-amino-2-hydroxybutyllysine) is rapidly reduced to undetectable levels. The very low hypusine level is paralleled by an increase in gamma-glutamyl putrescine derivatives and followed by a large increase in gamma-glutamyl spermidine derivatives; in addition, there is a remarkable concomitant biosynthesis of transglutaminase-catalyzed mono and bis gamma-glutamyl spermidine derivatives and epsilon(gamma-glutamyl)lysine cross-links. The effect of TPA and RA on hypusine formation is also reported.

Animals↗
PubMed1990

Arachidonic acid incorporation and redistribution in human neuroblastoma (SK-N-BE) cell phospholipids.

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.

Arachidonic Acid↗
PubMed1989

Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action.

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase-contrast microscopy and show irregular, globular morphology by scanning-electron microscopy. Part of their DNA content is cleaved into nucleosomal and oligonucleosomal fragments. Their insolubility, like that of the cornified envelope, is evoked by epsilon-(gamma-glutamyl)lysine and N1,N8-bis(gamma-glutamyl)spermidine protein cross-linking bonds formed by transglutaminase.

Animals↗
PubMed1988

Retinoic acid-induced modulation of rat liver transglutaminase and total polyamines in vivo.

The effect of a single intraperitoneal injection of retinoic acid on liver transglutaminase (EC 2.3.2.13) activity and total putrescine, spermidine and spermine was studied. The results demonstrate that: (1) transglutaminase activity is increased over control values as early as 4-6 h after treatment, reaching a maximum (2-fold increase) at 12 h and returning to control values at 36 h; (2) the retinoic acid-induced form of enzyme is the soluble tissue transglutaminase; (3) actinomycin D treatment does not completely inhibit the early (6 h) increase of activity, while suppressing that at 12 h; (4) the immunoassay of the soluble transglutaminase shows that, 6 h after treatment, there is no increase in the protein, whereas at 12 and 24 h a significant increase is observed; (5) putrescine, but not spermidine and spermine, increases (5-7-fold) 6 and 18 h after the retinoic acid treatment. The possibility also that the expression of soluble transglutaminase is modulated in vivo by retinoic acid and the relationship to polyamine levels are discussed.

Animals↗
PubMed1988

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid.

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.

Animals↗
PubMed1988

Covalent incorporation of polyamines as gamma-glutamyl derivatives into CHO cell protein.

The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [3H]putrescine. A single radiolabeled protein band with an apparent relative molecular mass of 18,000 Da was observed by SDS-polyacrylamide gel electrophoresis. Almost all the radioactivity covalently linked to this protein was recovered as hypusine. The labeling of this protein was increased several-fold when cells were cultured with alpha-difluoromethylornithine (DFMO) or with this drug plus methylglyoxal bis(guanylhydrazone) (MGBG), as a result of increase in specific radioactivity of the hypusine immediate precursor, spermidine. Also labeled under the latter condition were other cellular proteins. These were aggregates on the top both of the stacking gel and of the running gel, and protein-like materials with relative molecular masses of 36 and 8 kDa. The radioactivity covalently associated with these proteins was recovered after acid hydrolysis as polyamines. The identification of gamma-glutamylputrescine and gamma-glutamylspermidines in proteolytic digests of the acid-insoluble fraction of treated cells indicates that polyamines are covalently linked to these cellular protein. Several possible cellular functions of gamma-glutamylpolyamine protein components are discussed.

Animals↗
PubMed1988

gamma-Glutamylamine derivatives in isolated rat hepatocyte proteins.

Freshly isolated rat hepatocytes were found to contain a 3-fold higher level of putrescine than perfused liver. The bulk of this diamine was recovered in the acid-insoluble fraction of the cell. In order to determine the nature of the amine binding, the levels of gamma-glutamylamine derivatives were measured. The method used involves exhaustive proteolytic digestion of the acid-insoluble fraction of hepatocytes, followed by ion-exchange chromatography. For N1-(gamma-glutamyl)putrescine, a combined ion-exchange chromatographic and reverse-phase h.p.l.c. procedure was adopted. This allowed for the direct detection of less than 50 pmol of this derivative in enzymic hydrolysates. Several of the gamma-glutamylamines reported previously [Beninati, Piacentini, Argento-Ceru', Russo-Caia & Autuori (1985) Biochim. Biophys. Acta 841, 120-126] in the whole organ were found in the isolated liver cells. The elevated level of N1-(gamma-glutamyl)putrescine and the absence of bis-(gamma-glutamyl)spermine was noteworthy. The results suggest that, in rat hepatocytes, both polyamine-dependent post-translational modification of some proteins and cross-linking between proteins involving the glutamine and lysine residues occurs.

Animals↗
PubMed1988

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine.

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.

Cell Differentiation↗