Biomedical subjects

E Melloni

Publications and source records attributed to E Melloni.

At least 73 records · Page 4Linked to original sources

PubMed1994

Acute intravenous toxicity of dimethyl sulfoxide, polyethylene glycol 400, dimethylformamide, absolute ethanol, and benzyl alcohol in inbred mouse strains.

Acute intravenous toxicity of some solvents, i.e. dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG 400), dimethylformamide (DMF), absolute ethanol (EtOH) and benzyl alcohol (BeOH), was determined in three inbred (CD2F1, B6D2F1 and C57BL/6N) mouse strains used in many preclinical tests, mainly in oncology and toxicology. Haemolytic and precipitation potential tests in vitro were performed to assess the blood compatibility of the investigated solvents and its relationship with the observed symptoms. The single tested solvents did not show any major differences in acute toxicity in the three tested strains with the exclusion of DMSO (less toxic in CD2F1) and BeOH and EtOH (less toxic in B6D2F1). The tested dose ranges in the three strains (in ml/kg) were 1.0-5.66 for DMSO, 2.0-8.0 for PEG 400, 1.0-4.0 for DMF, 0.75-4.24 for EtOH, 0.025-0.4 for BeOH. The lowest tested dose was a safe dose and the highest one was the dose causing mortality in no more than half the animals in each group. The in vitro results suggest avoiding the use of BeOH (which also is more toxic than the other solvents in the in vivo test) and DMSO and using PEG400, EtOH and DMF even though the latter induced a body weight decrease in the B6D2F1 mouse strain. As a general conclusion, dilution of these solvents in water is suggested to ameliorate their blood compatibility and the use of doses not higher than the lowest dose tested in this study is recommended.

Animals↗

PubMed1993

Differentiation of HL60 promyelocytic cells is promoted by a 'differentiation enhancing factor' produced by erythroleukemia cells.

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12-myristate 13-acetate (20-fold and 40-fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

Cell Differentiation↗

PubMed1993

Modulation of rat liver protein kinase C during "in vivo" CC14-induced oxidative stress.

Rat intoxication with a single dose of the hepatotoxin carbon tetrachloride induces a significant modification of liver protein kinase C total activity which depends on the degree of the intrahepatocyte oxidative unbalance provoked by various concentrations of the haloalkane. Low carbon tetrachloride amounts stimulate total protein kinase C activity, while one order of magnitude higher amounts exert strong enzyme inhibition. The latter effect is due to an early inactivation followed with progress of time by a proteolytic degradation of the enzyme. A pathological recruitment of the calcium-dependent protein kinase C regulatory enzymes calpain and calpastatin appears responsible for protein kinase C loss. The prolonged excess of cytosolic calcium which characterizes the single high dose carbon tetrachloride poisoning also leads to inactivation of calpain II and calpastatin in a time-dependent manner.

Animals↗

PubMed1993

Characterization of the biological role of murine erythroleukemia cells "differentiation enhancing factor" using antisense oligodeoxynucleotides.

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL) cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a non-decapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and functionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Amino Acid Sequence↗

PubMed1993

Role of delta-PKC on the differentiation process of murine erythroleukemia cells.

In murine erythroleukemia (MEL) cells the length of the latent period before the onset of hexamethylenebisacetamide induced terminal erythroid differentiation is inversely correlated to the intracellular level of delta-PKC. This is supported by the following experimental evidence. V3.17[44] MEL cell line, characterized by a very high rate of differentiation, contains an amount of delta-PKC protein one third lower than that present in the N23 MEL cell line, characterized by a very low rate of differentiation. A similar difference in the amount of delta-PKC mRNA is present in the two cell lines. In N23 cells, following addition of HMBA, the amount of delta-PKC protein and delta-PKC mRNA is down-regulated to one third its original value, which now corresponds to that constitutively present in V3.17[44] cells. Furthermore, in these cells the levels of delta-PKC protein and of its specific mRNA are unaffected by treatment with HMBA. Following introduction of homologous purified delta-PKC both MEL cell variants display a longer latent period before the onset of differentiation from 50 to 75 hours in N23 cell line and from 20 to 40 hours in V3.17[44] cells, respectively. Taken together, these results suggest that a delta-PKC related signal plays a negative role in the early stages of MEL cell differentiation and that the level of the kinase is controlled through a down-regulation process upon exposure to the chemical inducer.

Acetamides↗

PubMed1993

Site-directed activation of calpain is promoted by a membrane-associated natural activator protein.

Human erythrocytes contain a calpain activator protein with a molecular mass of approx. 40 kDa. The activator is present in association with the plasma membrane and promotes expression of calpain activity at a concentration of Ca2+ close to physiological values. The initial step of the activating mechanism involves association of the activator with calpain, followed by autoproteolytic activation of the proteinase in the presence of 1 microM Ca2+, at a rate identical to that induced by 1 mM Ca2+. In a reconstituted system, the activator binds to erythrocyte membranes, but not to phospholipid vesicles, suggesting the participation of an intrinsic membrane protein(s). In its membrane-associated form the activator selectively binds calpain, thus favouring interaction of the proteinase with the inner surface of plasma membranes. These results further confirm the importance of a natural activator protein in promoting intracellular activation of calpain under physiological conditions through a site-directed mechanism, which explains the high specificity of the proteinase for membrane of cytoskeletal proteins.

Binding Sites↗

PubMed1993

[Peptic activity of gastric juice in chronic gastritis. Morpho-functional aspects].

The aim of this study has been to evaluate peptic activity in gastric juice and gastric peptic cell mass in chronic gastritis. As regard peptic activity, there is a close correlation between it and the peptic gastric cell mass considered globally and expressed as Peptic Gastric Index (PGI), resulting from the individual average between fundic peptic index (chief cells + fundic muco-peptic cells) and antral peptic index (antral muco-peptic cells), both obtained by multiplying the number of peptic cells per mm2 by the thickness of respectively fundic and antral gland layer). In particular fundic and antral superficiale gastritis does not involve changes in peptic activity in gastric juice. On the contrary, in case of fundic pre-atrophic or atrophic there is a significant drop of peptic activity in gastric juice, regardless of the histological condition of the antrum. The lowest value of peptic activity may be noticed in case of atrophic pan-gastritis. Pre-atrophic and atrophic gastritis limited to the antrum--with superficial fundic gastritis--does not involve significant decreases of peptic activity in gastric juice. In this experiences Helicobacter pylori seem to influence peptic secretion: in fact, there is an increases of peptic activity in gastric juice in case of chronic pre-atrophic gastritis HP+.

Adult↗

PubMed1992

Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation.

Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus mu-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forms are present in fresh rat muscle. Phosphorylation has been reproduced "in vitro" using a homologous Ca2+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered an important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form.

Alkaline Phosphatase↗

PubMed1992

Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.

Animals↗

PubMed1992

Mechanisms of perturbation of erythrocyte calcium homeostasis in favism.

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Calcimycin↗

PubMed1992

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.

Animals↗

PubMed1992

Echocardiographic parameters and indices in the normal beagle dog.

M-mode and two-dimensional echocardiographic measurements were made from the right sternal border of 50 healthy Beagles (25 males and 25 females) approximately 7 months old. The dogs were conscious and standing during the investigation. The following parameters, in systole and diastole, were measured on the echocardiographic images: left ventricular posterior wall thickness (LVWT); intraventricular septum thickness (IST); left ventricular internal dimension (LVID); and circumference (LVC). Fractional shortening (FS) and ejection fraction (EF) were also calculated. Mean, standard deviation, range and coefficient of variation are reported for each echocardiographic parameter and for body weight. Males and females were considered separately and together. Each parameter was analysed statistically to check for differences between the sexes and for correlations with body weight. A statistically significant difference between the sexes was only observed for LVWT in systole and diastole. A linear regression with body weight was obtained only for LVID in systole and in diastole. The results show that morphofunctional cardiac homogeneity is independent of size in dogs of this breed and age.

Analysis of Variance↗

PubMed1991

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.

Animals↗

PubMed1991

Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C.

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase. Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose-dependent fashion which results in a linearly correlated decrease in O2- production. Taken together, these results provide a direct demonstration that in neutrophils the intracellular level of protein kinase C represents one of the determinants of the rate and extent of O2- production.

Blotting, Western↗

PubMed1991

Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor.

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction. These results strongly support an important role for this natural protein factor in cell differentiation.

Acetamides↗

PubMed1991

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains.

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].

Animals↗

PubMed1991

The calpastatin defect in hypertension is possibly due to a specific degradation by calpain.

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Animals↗

PubMed1990

A vincristine-resistant murine erythroleukemia cell line secretes a differentiation enhancing factor.

A clone of vincristine resistant murine erythroleukemia cells V3.17[44], characterized by high sensitivity to terminal erythroid differentiation induced by hexamethylene bisacetamide, secretes into the extracellular medium a protein factor which partially reduces the latent period before commitment and accelerates the expression of the terminal differentiated phenotype in a slow responding murine erythroleukemia N23 cell variant. This differentiation enhancing factor increases the rate of protein kinase C down-regulation which occurs at slower rate during cell differentiation. The activity of the factor is detected either by coculture of the two cell line variants or by addition of conditioned medium from V3.17[44] cells to a culture of N23 cells in the presence of the inducer. In addition to being secreted by V3.17[44] cells, this factor can also be detected in the cytoplasm of both V3.17[44] and N23 cells, associated with a particulate fraction from which it can be released by sonication.

Animals↗