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F Marcheselli

Publications and source records attributed to F Marcheselli.

At least 19 recordsLinked to original sources
PubMed2004

Crystalline silica induces apoptosis in human endothelial cells in vitro.

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.

Annexin A5↗
PubMed1998

Melatonin increases the intensity of respiratory burst and prevents L-selectin shedding in human neutrophils in vitro.

Effects of melatonin priming of neutrophils and subsequent increase of phorbol 12-miristate 13-acetate stimulated respiratory burst were investigated on the modulation of L-selectin shedding and MAC-1 upregulation. Respiratory burst related H2O2 production and adhesion molecule expression were quantified by flow cytometry. Phorbol 12-miristate 13-acetate dose dependence of intracellular oxidation and adhesion molecule expression showed no relationship between respiratory burst intensity and MAC-1 expression or L-selectin shedding. Treatment of cells with 12.5 nM phorbol 12-miristate 13-acetate resulted in less than 20% of the respiratory burst response, however it induced 91.7% of total MAC-1 expression and 62.8% of L-selectin shedding. Melatonin priming experiments showed also no connection between the extent of respiratory burst and MAC-1 expression, however melatonin priming almost completely prevented L-selectin down-regulation elicited by phorbol 12-miristate 13-acetate, without affecting MAC-1 expression. It is suggested that melatonin may inhibit metalloproteases responsible for L-selectin cleavage.

Dose-Response Relationship, Drug↗
PubMed1998

Melatonin regulates the respiratory burst of human neutrophils and their depolarization.

The effect of different doses of melatonin on the respiratory burst as well as on the membrane potential changes of human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) was investigated. The intracellular production of reactive oxygen species (ROS) in stimulated neutrophils was quantified in individual cells by flow cytometry, measuring the oxidation of nonfluorescent dihydrorhodamine 123 to the green fluorescent rhodamine 123. The transmembrane potential change was measured using the fluorescent probe oxonol. Preincubating the cells with micromolar concentrations of the indole resulted in an increase of the response to PMA. In two of six subjects investigated, the respiratory burst was also increased by a 10 nM concentration of the indole, but when the melatonin concentration was increased to 2 mM the respiratory burst was inhibited. The change in the transmembrane potential of neutrophils paralleled the respiratory burst. Indeed, the treatment of the cells with doses of melatonin up to 0.5 mM increased the depolarization occurring subsequent to PMA stimulation, whereas 2 mM melatonin concentration decreased the extent of depolarization. To investigate whether melatonin could directly affect the transmembrane potential changes of neutrophils, the extent of depolarization, induced by increasing the extracellular potassium concentration, was measured in cells preincubated with 2 mM melatonin. This treatment resulted in a decrease of the extent of depolarization, which suggests that melatonin can directly alter membrane ion conductance in human neutrophils.

Antioxidants↗
PubMed1996

Effect of acetylcholine on the electrophysiology and proliferative response of human lymphocytes.

Using the patch-clamp technique, we determined that 1-15 mM extracellular acetylcholine reduced whole-cell n-type K+ currents in human peripheral blood lymphocytes and accelerated their inactivation. The percentage increase in K+ channel inactivation rate and the degree of drug induced block were independent of membrane potential. In flow cytometric membrane potential measurements with the oxonol dye similar doses of acetylcholine depolarized the lymphocyte population. Both acetylcholine induced K+ channel block and depolarization fully developed within 2 minutes. The depolarizing and K+ channel blocking effects of acetylcholine are in concert. [3H]thymidine incorporation experiments proved that the proliferative response of PHA stimulated peripheral blood lymphocytes was decreased by increasing concentrations of acetylcholine in the 1-50 mM range.

Acetylcholine↗
PubMed1995

The impairment of mitochondrial membrane potential and mass in proliferating lymphocytes from vitamin E deficient animals is recovered by glutathione.

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on splenocytes from control and vit. E deficient rats, stimulated to proliferate with Concanavalin A, in the presence and absence of reduced glutathione (GSH, 5 mM). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific probes to monitor the membrane potential and mass of mitochondria, respectively, by means of flow cytometry. Rh-123 uptake was high in an increasing number of cells from normally fed animals during the three-day culture period. On the contrary, splenocytes from vitamin E deficient rats showed a biphasic pattern. The number of cells showing a high uptake of Rh-123 increased after 24 hrs. from mitogenic stimulation, then it decreased at the other two time points considered. In parallel, a continuous increase of the number of cells with depolarized organelles (up to 60% by 72 hrs.) has been observed in vit. E deficiency. This impairment was fully prevented by GSH supplementation to the culture medium. In the presence of the thiol, about 80-85% of cells showed activated mitochondria, whereas the number of splenocytes with depolarized organelles did not exceed 17%, irrespective of the diet applied to the animals. The same pattern was observed considering the changes of mitochondrial mass, measured using NAO as a probe. Present results support that GSH may substitute vitamin E in protecting mitochondria from peroxidative damage.

Acridine Orange↗
PubMed1994

Melatonin: a peroxyl radical scavenger more effective than vitamin E.

We have compared the peroxyl radical scavenger ability to melatonin with that of vitamin E, vitamin C and reduced glutathione (GSH). In the assay system, beta-phycoerythrin (beta-PE) was used as fluorescent indicator protein, 2-2'-azo-bis(2-amidinopropane)dihydrochloride as a peroxyl radical generator and the water soluble vitamin E analogue. Trolox, as reference standard. Results are expressed as oxygen radical absorbing capacity (ORAC(perox)) units, where 1 ORAC unit equals the net protection produced by 1 microM Trolox. A linear correlation of ORAC values with concentration (0.5-4 microM) of all the substances tested has been observed. However, on molar basis, the relative ORAC(perox) of Trolox, vitamin C, GSH and melatonin was 1:1.12:0:68:2.04, respectively. Thus, melatonin, which is a lipid-soluble compound, was twice more active than vitamin E, believed to be the most effective lipophilic antioxidant.

Amidines↗
PubMed1992

The response of human lymphocytes to phytohemagglutinin is impaired at different levels during aging.

Several parameters generally believed to be necessary for the activation and progression of proliferation of human lymphocytes have been investigated and compared with special reference to aging. The responding capacity of plasma membrane potential to depolarizing and also repolarizing conditions induced by exposure to mitogens like PHA was lower in lymphocytes from old donors as compared to those of young ones. This indicates a significant age-dependent difference in the readiness to respond to channel-activating perturbations. As an early signal of activation, after one hour PHA stimulation the merocyanine 540 uptake by the lipid regions was chosen, based on the property of this fluorescent probe to bind to loosely packed lipids of the plasma membrane. The proteins encoded by the c-myc and c-myb genes were chosen as markers of the G0/G1 and G1/S phased transition, respectively. The mean number of cells that increased the uptake of MC 540 following mitogenic stimulation did not differ in young vs. old individuals. However, 4 samples out of 10 from the old population showed lower MC 540 fluorescence than the lowest signal from the young population. The number of responding cells was decreased during aging when the presence of the c-myc protein was taken as its measure; and this decrease was further accentuated, determining the expression of the c-myb protein. This frequently encountered age-dependent pattern, however, was not followed by the lymphocytes of all old donors. One example is reported in which the MC 540 uptake, the c-myc and c-myb expression in the cells from one old subject fell in the range of the young subjects. However, even in this case, the response of the lymphocytes as measured by 3H-thymidine incorporation was only 64% of that of young subjects. For this sample, we found an impairment of the response at the mitochondrial level. In addition to these parameters, the amount of 3H-thymidine incorporated by the cells expressing the c-myb protein was calculated. The values in old individuals were lower than those in the young, suggesting that not all the cells expressing the c-myb protein were able to synthesize DNA in lymphocyte populations from the elderly. Our data support the view that the age-dependent decline of lymphocyte responsiveness to mitogens can be accounted for by impairments at different levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1992

Cholesterol-rich rabbit serum modulates beta-adrenergic receptor density of human lymphocytes. A possible role of LDL-cholesterol.

The effect of in vitro treatment of human lymphocytes with rabbit cholesterol-rich serum (RCS) on the membrane microviscosity as well as on the beta-adrenergic receptor density has been investigated. RCS treatment of cells resulted in a 30% decrease of receptor density without any effect on membrane microviscosity. A complete recovery was observed incubating the RCS cells either with the "Active Lipids" (AL) or with heparin. The AL are a mixture of neutral lipids, phosphatidylcholine and phosphatidylethanolamine from hen egg yolk known to fluidify the cell membrane. The AL modified membrane microviscosity of control lymphocytes without altering their beta-receptor number. These observations support the proposition that beta-receptor density of human lymphocytes is not regulated by membrane microviscosity and suggest that probably low density lipoprotein-cholesterol complex is involved in such a regulation.

Animals↗
PubMed1992

Phytohemagglutinin induced changes of membrane lipid packing, c-myc and c-myb encoded protein expression in human lymphocytes during aging.

Three parameters which signal different stages of cell activation were analyzed in lymphocytes from young and old subjects. Merocyanine 540 (MC-540) incorporation into the membrane lipid phase was used as a very early marker of activation and was measured after 1 h of phytohemagglutinin (PHA) stimulation. The proteins coded by c-myc and c-myb protooncogenes were determined by appropriate antibodies and were taken as markers of the G0/G1 and G1/S phase transition, respectively. The number of cells which increased the uptake of MC-540 following PHA stimulation did not differ when comparing young and old individuals. Both the number of the responding cells and the size of the response were decreased during aging when the presence of the c-myc protein was taken into account. A consistent decrease of the percentage of lymphocytes able to express the c-myb protein was observed in the cells from old donors as compared to those from the young ones, but the amount of detectable protein per cell remained unchanged. Our data suggest that the deficiency of responsiveness which accompanies aging is due to impairments at different points of the cell cycle. The very low number of cells expressing the c-myb protein is likely the result of step by step elimination of those cells not able to fulfill the requirements to progress along the cell cycle.

Adult↗
PubMed1992

A sodium channel opener inhibits stimulation of human peripheral blood mononuclear cells.

The role of membrane potential changes in T cell activation was studied on human peripheral blood lymphocytes stimulated with phytohemagglutinin. Addition of bretylium tosylate, a sodium channels opener, to PHA treated lymphocytes modified the membrane potential and consequently blocked cell activation in a dose-dependent fashion. BT was non-toxic even in long-term (72 hr) incubations. It was reversibly removable, and the removal restored the stimulatory effect of PHA. 3H-thymidine incorporation was blocked if BT was present during the first 20-24 hr of the mitogenic activation. The later BT was added after PHA, the less inhibition of proliferation was observed. BT hyperpolarized the lymphocytes also in the presence of PHA. BT hindered the depolarizing effect of high extracellular potassium concns. The sustained polarized state of the lymphocytes did not influence the intracellular calcium increase upon PHA treatment. IL-2 and transferrin receptor expression was not hindered by BT during PHA stimulation of lymphocytes. Addition of rIL-2 did not abolish the inhibitory effect of BT. According to cell-cycle analysis BT arrested the majority of the cells in G1 phase. It is suggested that cell activation demands the flexible maintenance of a relatively narrow membrane potential "window". Any sustained and significant hyper-, or depolarization, may dramatically decrease the effectivity of transmembrane signalling.

Bretylium Tosylate↗
PubMed1992

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1991

Influence of membrane fluidity of 5'-nucleotidase activity in isolated hepatocyte plasma membrane.

The influence of membrane microviscosity on 5'-nucleotidase activity has been investigated on liver plasma membrane preparations from rats during aging and following diet restriction. In addition the microviscosity of membranes from old rats was changed in vitro by the Active Lipids. During aging the membrane microviscosity increased progressively and in parallel the activity of 5'-nucleotidase decreased. Diet restriction was able to slow down the modification of both parameters. The experiment performed with the Active Lipids further supports that membrane microviscosity modulated the enzyme activity.

5'-Nucleotidase↗
PubMed1990

Antioxidant enzymes in erythrocytes from old and diet restricted old rats.

Diet restriction, prolonging the lifespan of rodents, represents an interesting model for gerontological studies. We analyzed the activity of antioxidant enzymes, Superoxide Dismutase, Catalase and Glutathione Peroxidase in erythrocytes from young, old and old food restricted Wistar rats. Diet restriction was applied feeding the animals on every-other-day schedule starting from the age of 3.5 months. The age-dependent decrease of Catalase and Glutathione Peroxidase activities was prevented by food restriction, whereas Superoxide Dismutase activity was not influenced either by aging and dietary intervention. Present results support the hypothesis that diet restriction increases the protection of cell structure against the peroxidative damage, preserving the activity of antioxidant enzymes.

Aging↗
PubMed1990

Diet restriction decreases the membrane microviscosity of cerebellar membranes of old female Wistar rats.

The microviscosity of cerebellar membranes from female Wistar rats during aging and subsequent to diet restriction has been investigated. Diet restriction was applied feeding the animals on every-other-day schedule starting from the age of 3.5 months. Undernutrition increased the lifespan of the animals and retarded the appearance of the age-dependent increase of microviscosity of cerebellar membranes.

Aging↗
PubMed1989

Chronic dietary choline influences the permeability of nerve cell membranes as revealed by in vivo Rb+ uptake and release.

The effect of chronic choline supplementation on the in vivo Rb+ uptake and release of rat brain cortical cells was investigated. Adult (11 months old) and old (22 months old) controls as well as old (22 months old) female rats treated with choline (approximately 100 mg/day in the drinking water) by the age of 11 months, were used. All the animals received a daily dose of 30 mg RbCl/100 g body weight for 14 days, given intraperitoneally, half in the morning, half in the evening. After discontinuation of the RbCl treatment, the animals were killed at intervals of 2, 4, 9 and 16 days, respectively. The intracellular Rb+ and K+ contents were analyzed by energy dispersive X-ray microanalysis, whereas concentrations of these two elements were determined by atomic absorption spectrophotometry in the cerebrospinal fluid. Old animals accumulate more Rb+ than the adult ones at any time taken into account. Choline treatment was able to reduce the amount of accumulated Rb+ in the old rats. Rb+-discrimination ratios calculated on the basis of Rb+ and K+ contents of both cortical cells and cerebrospinal fluid, is higher in old rats as compared to both adult and old choline treated rats. Present findings support that chronic choline treatment is effective improving the passive membrane permeability of nerve cells for Rb+ (and K+) in the old animals.

Animals↗