Biomedical subjects

E Melloni

Publications and source records attributed to E Melloni.

At least 91 records · Page 5Linked to original sources

PubMed1990

Identification of a protein kinase C activating factor from murine erythroleukemia cells: characterization of the activation kinetics.

A protein kinase C (PKC) activating factor (AF) has been identified in the extracellular medium of V3.17 vincristine resistant murine erythroleukemia (MEL) cells clone. The factor is a protein that stimulates the activity of PKC alpha and beta isozymes isolated from MEL cells, rat and mouse brain approximately 2 to 2.5 fold over the Vmax, respectively. AF promotes an identical activation in the presence of all the effectors but also when the amount of Ca2+ is reduced to microM concentration and in the absence of diacylglycerol (DAG). The factor shows a greater activating efficiency with PKC beta isozymes. AF binds to PKC presumably at the DAG binding site as suggested by the competition between phorbol dibutyrate and AF for binding to the kinase. Moreover, AF promotes the selective binding of PKC beta to natural or artificial membranes in the presence of microM concentrations of Ca2+. Altogether these results suggest the presence in MEL cells of a protein factor that can promote association of PKC to the membranes together with activation of the kinase, without the requirement for DAG formation. This could be visualized as a new mechanism for prolonged and selective activation of PKC.

Animals↗

PubMed1990

Inactivation of hepatocyte protein kinase C by carbon tetrachloride: involvement of drug's metabolic activation and prooxidant effect.

The involvement of CCl4 biotransformation mechanism in decreasing the Protein Kinase C activity has been analyzed in hepatocytes isolated from phenobarbital-pretreated rats. A significant inhibition (55%) and an almost total disappearance (87%) of the enzyme activity were observed at 15 min and at 30 min incubation with CCl4, respectively. Cell preincubation with Trolox or desferrioxamine allowed a marked whilst not complete protection of both cytosolic and particulate Protein Kinase C activity. These results show that the CCl4 reactive metabolites play a primary role in hepatocyte Protein Kinase C impairment and suggest that besides lipid peroxidation other mechanisms -possibly a derangement of Ca2+ homeostasis- may be involved in this process.

Animals↗

PubMed1990

Identification of an endogenous activator of calpain in rat skeletal muscle.

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.

Animals↗

PubMed1990

Photodynamic therapy by topical meso-tetraphenylporphinesulfonate tetrasodium salt administration in superficial basal cell carcinomas.

The efficacy of an originally developed photodynamic approach, using topical administration of tetraphenylporphinesulfonate as the photosensitizer, was evaluated in a series of 292 basal cell carcinoma lesions (less than 2-mm thick) in 50 treated patients. The lack of indication for conventional therapies was the main selection criterion. The photosensitizing agent (2% solution) was topically applied at 0.1 ml/cm2, followed by light irradiation with a dye laser emitting at 645 nm (120 or 150 J/cm2). After initial treatment, all lesions responded, with 273 (93.5%) complete responses. Recurrences were observed in 29 (10.6%). A second application of photoradiation was performed in 15 persistent lesions and 11 relapsed lesions, producing 19/26 complete responses. Our results suggest that this technique can be considered a promising alternative treatment modality in selected cases of superficial basal cell carcinomas.

Aged↗

PubMed1990

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms.

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.

Animals↗

PubMed1990

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases.

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.

Animals↗

PubMed1990

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity. These differences are related to the differences in biochemical responses, reported earlier, in neutrophils exposed to these two activators.

Antibodies, Monoclonal↗

PubMed1990

Introduction of the beta isozyme of protein kinase C accelerates induced differentiation of murine erythroleukemia cells.

Induction of differentiation in murine erythroleukemia cells (MELCs) involves a protein kinase C (PKC)-mediated step. Vincristine-resistant cells respond more rapidly to hybrid polar/apolar inducers than the parental cells. These vincristine-resistant MELCs contain elevated levels of the beta isozyme of PKC (PKC-beta). Exogenous homologous murine PKC-beta, incorporated into permeabilized MELCs, accelerates induced differentiation. Neither rat PKC-beta, nor mouse PKC-alpha, nor rat PKC-alpha, incorporated into permeabilized MELCs, is effective in altering the kinetics of induced differentiation. This provides direct evidence for a rate-limiting role for this PKC isozyme during N,N'-hexamethylenebisacetamide-mediated induced differentiation of a transformed cell.

Acetamides↗

PubMed1990

Epidemiological study of urinary tract stones in a northern Italian city.

An epidemiological study of stone disease in a Northern Italian city was carried out by means of a postal questionnaire mailed to 6000 individuals (2.5% of the entire population). It was found that the incidence of stone disease was comparable to that of industrialised Western Europe. There was a relationship between stone disease and gout and stone disease and a positive family history. The frequency of uric acid stones was high (26.5%). Stone-formers showed no alimentary differences from non-stone formers apart from the use of spices and herbs. Stone-formers used less water from public aqueducts and more uncarbonated mineral water, but only 19% of these drank at least 2 litres a day.

Adult↗

PubMed1989

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation.

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.

Acetamides↗

PubMed1989

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe.

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles. When both monoclonal antibody and calpain were present in these vesicles, a pronounced inhibition of the membrane bound proteinase activity was observed. These results provide an explanation for the previously observed inhibitory effect of the monoclonal antibody on intracellular calpain activity and on the concomitant inhibition of granule exocytosis. The activated calpain associated with the plasma membrane compartment is therefore identified as the form specifically involved in mediating the physiological responses.

Antibodies, Monoclonal↗

PubMed1989

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients.

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors. These results are consistent with the hypothesis that differences in the functional organization of the oxidase at the membrane level in neutrophils of hypertensive are responsible for the enhanced O2- production following agonist stimulation.

Cell-Free System↗

PubMed1989

Effect of low-energy laser irradiation on colony formation capability in different human tumor cells in vitro.

Fibroblasts and lymphocytes are the most widely used cells for studying the so-called biostimulative effect of low-power laser in vitro. In contrast, stimulation of cancer cells by laser light has not been investigated extensively. The present study attempted to evaluate whether or not human tumor cells could exhibit an increase in colony-forming capability following low-watt laser irradiation. LoVo and HT29 (colon carcinoma), MCF7 (breast carcinoma), M14 and JR1 (malignant melanoma) cell lines were irradiated at different doses of light delivered from an argon or an argon-dye laser. Radiant exposures between 4.2 and 150 kJ/m2 at irradiances ranging from 35 to 500 W/m2 were delivered. Results were mixed. Of the 41 experiments performed, five showed a significant statistical increase in the number of colonies (P less than 0.05), whereas three showed a decrease (P less than 0.05). Nevertheless, the trend of most data was toward an increase in colony formation, and Wilcoxon's signed-ranks test suggested that light increases tumor cell culture growth (P less than 0.03).

Breast Neoplasms↗

PubMed1989

The calpains.

In recent years interest has increased concerning the characterization of the structural-functional properties and the identification of the physiological role of non-lysosomal intracellular proteinases. Among these, calpain, a calcium-dependent cysteine proteinase ubiquitously present in a variety of tissues and cells, has been most extensively investigated in terms of activation, regulatory mechanisms, specificity and biological function. This review discusses each of these points on the basis of the most recent results concerning the general characteristics of calpain activity, and its preferential site of action within the cell as related to the specific functions of the proteinase in different cell types. As with other proteinases, calpain has to be under a continuous spatial and temporal control, and the structural and functional properties of the natural calpain inhibitor, calpastatin, must also be considered. The calpain-calpastatin system is the functional proteolytic unit that governs the activity of this intracellular proteolytic system, which is tightly correlated to the control of calcium homeostasis and thereby to the biological process of transmembrane signalling.

Calpain↗

PubMed1989

The role of intracellular proteinases in human neutrophil activation.

In addition to other proteinases human neutrophils contain two non granular neutral endopeptidases: a serine proteinase and a cysteine Ca2+ dependent proteinase named calpain. Serine proteinase localized in association with the cytoskeleton-membrane proteins, apparently exerts a dual role: it is partially released into the medium during neutrophil stimulation by phorbol myristate acetate (PMA), presumably acting as one of the cytotoxic factors; and in its intracellular localization is presumably involved in the process of down regulation of native protein kinase C (PKC). Calpain, predominantly present in resting conditions in an inactive form, becomes activated in the course of neutrophil stimulation and appears to be involved both in the down regulation of native PKC as well as in the formation of a proteinase-activated kinase form, presumably derived from PKC and defined as PKC-M. Calpain once activated appears to be also involved in cytoskeleton rearrangement, through proteolytic degradation specifically oriented by substrate phosphorylation. Activation, down regulation of PKC, formation of the proteinase-activated kinase, as well as proteolytic processing of cytoskeleton have been demonstrated to be correlated to those biochemical responses which characterize neutrophil activation.

Calpain↗

PubMed1988

Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension.

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.

Antibodies, Monoclonal↗

PubMed1988

Anti HLA class I monoclonal antibody effect on PKC kinetics in PHA activated human peripheral blood mononuclear and E+ cells.

Cytoplasmic protein kinase C (PKC) has been studied in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) and macrophage depleted E+ cell culture. Within 10' after contemporanous addition of PHA and anti HLA class I monoclonal antibody 01.65 (MoAb) PKC is depleted in both cell types. Enzyme activity recovers in the following hours however at 72 hours is at control values in E+ cultures while in PBMC cultures it is still depleted at 68% of the control. Anti HLA class I MoAb induced tritiated lymidine (3H-TdR) incorporation inhibition appears to be related to low levels of PKC activity.

Antibodies, Monoclonal↗

PubMed1988

Carbon tetrachloride-induced inhibition of protein kinase C in isolated rat hepatocytes.

Isolated rat hepatocytes exposed to CCl4 showed a dramatic decrease in [32P] incorporation into proteins which was evident as early as 5 min after the haloalkane addition. DEAE cellulose separation of protein kinases present in both particulated and cytosolic fractions of hepatocytes revealed that only the calcium and phospholipids dependent protein kinase C was affected by the treatment with CCl4, while kinases not requiring these factors for their activity were unmodified. Several 4-hydroxyunsaturated aldehydes known to be produced during CCl4-stimulated lipid peroxidation were found to inhibit protein kinase C at micromolar concentrations, suggesting the possibility that peroxidative events might be responsible for the impairment of protein kinase C during CCl4 intoxication.

Aldehydes↗