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P Ninfali

Publications and source records attributed to P Ninfali.

At least 19 recordsLinked to original sources
PubMed2006

Characterization and biological activity of the main flavonoids from Swiss Chard (Beta vulgaris subspecies cycla).

The molecular components of a phenolic fraction (P2), obtained from liquid chromatography of a Swiss Chard (Beta vulgaris subsp. cycla) extract, were identified using HPLC-ESI-MS/MS. The primary P2 components were: vitexin-2''O-rhamnoside, its demethylated form 2''-xylosylvitexin, isorhamnetin 3-gentiobioside, and rutin. P2 "in toto" and the single components were characterized for antioxidant capacity, antimitotic activity on MCF-7 human breast cancer cells and for toxicity to human lymphocytes and macrophages. P2 inhibited MCF-7 cell proliferation (IC(50) value = 9 microg/ml) without inducing apoptosis, showed no toxicity to human lymphocytes and slight toxicity to macrophages. Vitexin-2''O-rhamnoside strongly inhibited DNA synthesis in MCF-7 cells, whereas 2''-xylosylvitexin and isorhamnetin 3-gentiobioside were activators; combinations of activators and inhibitors maintained the over-all inhibitory effect.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Rabbit brain glucose-6-phosphate dehydrogenase: biochemical properties and inactivation by free radicals and 4-hydroxy-2-nonenal.

Glucose-6-phosphate dehydrogenase (G6PD) purified from rabbit brain is composed of two identical subunits of 56 kDa. The enzyme exhibits biphasic pH curve, linear Arrhenius plot and elevated susceptibility to inactivation by metal catalyzed oxidation and thiol binding reagents. 4-Hydroxy-2-nonenal (HNE) is able to inactivate the enzyme after only a few minutes of incubation. Since reactive oxygen species and G6PD-HNE adducts form easily in brain under conditions of oxidative stress, these findings have important implications in the loss of active G6PD molecules in vivo, a process which lowers the antioxidant protection and may be critical for neuron survival.

Aldehydes↗
PubMed2001

Glucose-6-phosphate dehydrogenase supports the functioning of the synapses in rat cerebellar cortex.

This study investigates heterogeneous glucose-6-phosphate dehydrogenase (G6PD) expression in the rat cerebellar cortex. G6PD activity and its electrophoretic pattern, evaluated on the cerebellar homogenate, were found to be similar to those of other brain areas. However, histochemical and immunohistochemical analyses revealed that the highest expression of G6PD activity and protein was in Purkinje's cells, followed by the molecular and granular layers. Electron microscopy analysis showed that, in Purkinje's cells, the G6PD reaction products were concentrated in the neurites while in the basket cells in the cell body. The granules showed a weaker activity everywhere. The quantitative distribution of G6PD is discussed in the light of the neurochemical function of these cells.

Animals↗
PubMed2001

Electron paramagnetic resonance investigations of free radicals in extra virgin olive oils.

Free radicals in olive oils were identified and quantified by EPR, by means of the spin-trapping technique making use of alpha-phenylbutylnitrone (PBN) as spin trap. The radical species were identified as PBN-trapped hydroxyl radicals (PBN-*OH) in the water microdroplets inside the fat medium. The largest radical concentration was 12.5 microM identical with 100%. The following were the relative concentrations of the radicals under different conditions: (1) Two oils, produced by continuous centrifugation, aged for 1 year, showed a 25-30% increase in the radicals compared to nonaged oils; 1-year-old oil, produced by pressure, did not differ from the nonaged oil. (2) Radical production was markedly reduced by N(2) bubbling; it was increased by heating, whereas it showed a biphasic pattern by air bubbling over time. (3) Radical concentration as a function of the UV irradiation time increased up to a maximum, after which it decreased and finally remained constant. The phenolic and oxygen contents were related to the radical content. This study demonstrates that the EPR technique is suitably applied to the detection of free radicals in olive oil and that storage, handling, and stress conditions of the oils significantly influence the radical concentration.

Electron Spin Resonance Spectroscopy↗
PubMed2001

Glucose-6-phosphate dehydrogenase in small intestine of rabbit: biochemical properties and subcellular localization.

Biochemical properties and cellular and subcellular distribution patterns of glucose-6-phosphate dehydrogenase (G6PD) were investigated in small intestine of rabbits. The specific activity of G6PD in fresh homogenates of small intestine was 19 +/- 9 IU/g protein. This value did not change significantly after dialysis. The kinetic and electrophoretic properties of the partially purified enzyme were similar to those found in other rabbit tissues. Enzyme histochemical analysis of G6PD activity using the tetrazolium salt method showed high activity in epithelial cells of villi and crypts of Lieberkuhn. The activity in acinar cells of Brunner's glands was lower than that in epithelium, whereas cells of the muscularis externa showed a very low activity. Immunohistochemical analysis showed that the amounts of G6PD protein were lower in the epithelium than in Brunner's glands and muscularis externa. The differences between distribution patterns of activity and protein of G6PD may reflect the presence of inactive enzyme molecules in Brunner's glands and muscularis externa or posttranslational activation of G6PD in epithelium. Electron microscopic immunocytochemical analysis performed with gold-labelled antibodies showed the presence of G6PD protein throughout the cytoplasm and at smooth endoplasmic reticulum in enterocytes. In Paneth cells and cells of Brunner's glands, G6PD was found in the cytoplasm, at rough endoplasmic reticulum and Golgi complex. Immunolabelling was not found in mitochondria or nuclei. Our findings show that G6PD is heterogeneously distributed in cells of the small intestine and that the enzyme is associated with rough and smooth endoplasmic reticulum to support synthetic functions in these compartments by NADPH production.

Animals↗
PubMed2000

Iron release and oxidant damage in human myoblasts by divicine.

Divicine is an aglycone derived from vicine, a glucosidic compound contained in fava beans (Vicia faba major or broad beans). In this study, we investigated the effect of divicine on cultured human myoblasts from normal subjects, in order to see if the drug may induce signs of oxidant stress in these cells. Myoblasts incubated 24 hours in the presence of 1 mM divicine, showed an increase of carbonyl groups and 4-hydroxynonenal (4-HNE) bound to cell proteins, as well as a significant release of iron and lactate dehydrogenase in the culture medium. Desferrioxamine (DFO), an iron chelator, significantly prevented protein oxidation and formation 4-HNE adducts. Our results can be interpreted as indicating that divicine autooxidizes both at extracellular level and into myoblasts thus inducing the release of free iron, which initiates oxidation of cellular proteins and lipids. DFO protects the cells by subtracting the free iron both at intracellular and extracellular level.

Aldehydes↗
PubMed2000

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas.

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO(4), dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas.

Animals↗
PubMed2000

Heterogeneous distribution of glucose-6-phosphate dehydrogenase in lingual epithelium.

Lingual epithelium undergoes oxidative stress and apoptosis with consequent renewal of superficial keratinized cells by proliferation and differentation of the stem cells of the basal germinative layer. In 3 distinct areas of lingual epithelium of rat and rabbit, the anterior third, central third and posterior third, we determined the activity of hexose monophosphate shunt enzymes and antioxidant enzymes, which are essential for support of cell proliferation and differentation. Enzymatic assays of the epithelium showed that glucose-6-phosphate dehydrogenase (G6PD) activity was highest in the anterior third, whereas activity of glutathione peroxidase, 6-phosphogluconate dehydrogenase, glutathione reductase, superoxide dismutase and catalase was similar over all areas. Histochemical localization of activity and immunohistochemical localization of protein of G6PD showed that all types of papillae had a similar G6PD content; moreover, the presence of different G6PD isoforms in the 3 areas was excluded by electrophoretic analysis. We conclude that the higher G6PD activity in the anterior part of the epithelium is due only to the anatomical organization of the epithelial surface of this area, in which many filiform and fungiform papillae are arranged in a compact manner, which corresponds with a higher number of proliferating and differentiating cells. These processes need products of G6PD activity. This study indicates that G6PD is a good marker for the number of differentiating cells in tongue epithelium.

Animals↗
PubMed2000

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium.

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.

Animals↗
PubMed1999

High glucose-6-phosphate dehydrogenase activity contributes to the structural plasticity of periglomerular cells in the olfactory bulb of adult rats.

Glucose-6-phosphate dehydrogenase (G6PD) activity, assayed spectrophotometrically, was found to be higher in the olfactory bulb (OB) than in other brain areas of adult rats [P. Ninfali, G. Aluigi, W. Balduini, A. Pompella, Glucose-6-phosphate dehydrogenase is higher in the olfactory bulb than into other brain areas, Brain Res. 744 (1997) 138-142]. Histochemical demonstration of G6PD activity in cryostat sections of OB, analyzed with optical microscopy, revealed a marked and well defined line of formazan deposition in the internal part of the glomerular layer (Glm), indicating that G6PD was much higher in cells distributed along the glomeruli. Electron microscope analysis showed that G6PD activity was mainly concentrated in cytoplasm and dendrites of periglomerular cells, the interneurons which span glomeruli and connect olfactory nerves with mitral/tufted cells. Since G6PD regulates the flux through the hexose monophosphate shunt (HMS) pathway, which provides NADPH for reductive biosynthesis and pentose phosphates for nucleic acid formation, it can be concluded that high G6PD activity in periglomerular neurons is functional to their differentiating capability. This result is consistent with the occurrence of structural plasticity events in the OB of adult rats.

Animals↗
PubMed1998

Postnatal expression of glucose-6-phosphate dehydrogenase in different brain areas.

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differed from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.

Aging↗
PubMed1998

Variability of oxygen radical absorbance capacity (ORAC) in different animal species.

The oxygen radical absorbance capacity (ORAC) was measured both in whole (ORAC-T) and deproteinized (ORAC-AS) plasma samples of human, pig, cow, rabbit, dog, cat, sheep, horse, dolphin, turkey, guinea-hen and chicken. In the 12 species, ORAC-T data, expressed as micromoles of peroxyl radicals trapped by 11 of sample, were found scattered between 8,600 and 23,000 micromol/l. The species with the highest ORAC-T values were cat among mammals and chicken among avies. ORAC-AS values ranged between 600 and 2000 micromol/l, with the highest values found in dolphin and sheep among mammals, while chicken was first among avies. In the 12 species, the relative contribution of ORAC-AS in relation to ORAC-T ranged from 5% to 20%. Protein SH-groups and uric acid were measured in plasma of all species, but no significant correlation was found between thiols and ORAC-T values or between uric acid and ORAC-AS values. Our results show that: (1) the ORAC method is reproducible and sensitive enough to be used in the comparison of the peroxyl-radical absorbance capacity of protein and non-protein plasma components in different animal species; (2) both in mammals and in avies, there is a deep intra-class heterogeneity of ORAC-T and ORAC-AS values; (3) by considering most species, plasma proteins and lipoproteins account for about 85-90% of the overall peroxyl-radical trapping capacity. In the dolphin only, the protein contribution decreases to 80%; (4) uric acid accounts for about one-half of the ORAC-AS value in human, guinea-hen and for about one-third in chicken, while it provides a very limited contribution in other species. We conclude that species with the highest ORAC-T, like cat and chicken, or with the highest ORAC-AS, like dolphin, are interesting models to study the reasons of such a marked antioxidant defense in the plasma.

Absorption↗
PubMed1997

Glucose-6-phosphate dehydrogenase activity is higher in the olfactory bulb than in other brain areas.

The activity of antioxidant enzymes was measured in the olfactory bulb (OB) of rat and compared with cortex, hippocampus, striatum and septum. Glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase were not significantly different in the five brain areas, while glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase activities were four times higher in the OB than in the other areas. This picture prompted us to explore the reasons of the marked increase of G6PD, since it is the enzyme that regulates the operation of the hexose monophosphate shunt. A first approach was to analyze the G6PD electrophoretic pattern. The analysis revealed that the high G6PD activity of the bulb was neither due to new isoenzymes nor to a modification of the equilibrium between the G6PD dimers. We secondly hypothesized an induction of G6PD activity in the OB by oxidant stress. The assay of markers of the oxidant stress, such as thiobarbituric acid reactive substances, oxidized and reduced glutathione, did not confirm this hypothesis. A third approach was the cytochemical analysis of cryostat sections of OB. By this method we identified a particular cell type which was very rich in G6PD and located at the border of the glomerular layer. Thus, we attributed the high G6PD activity of the OB to the consistent presence of periglomerular cells, that probably need a high G6PD activity for their regulatory function in the neurochemical transmission.

Animals↗
PubMed1997

Methods for studying the glucose-6-phosphate dehydrogenase activity in brain areas.

This paper reports on the protocols for the spectrophotometric determination of the enzymatic activity, the electrophoretic pattern and the cytochemical assay of glucose-6-phosphate dehydrogenase (G6PD) in different areas of rat brain. For the spectrophotometric assay we used the method of Glock and McLean. Non-denaturing polyacrylamide gel electrophoresis was performed using 1.5 mm thick slab-gel, with a 7.5% acrylamide separating gel and a 4% acrylamide stacking gel. NADP (0.01 mM) was included at the cathode buffer. The cytochemical assay was performed on unfixed cryostat sections, by incubating the slides for 5 min at 37 degrees C in the staining medium. A combination of these techniques offers a means for the study of the effect of age, drugs, oxidizing or reducing agents on the G6PD activity in different brain areas.

Animals↗
PubMed1996

Interaction of ATP with erythroblast glucose-6-phosphate dehydrogenase.

ATP, complexed with magnesium and as free anion, binds to rabbit erythroblasts glucose-6-phosphate dehydrogenase (G6PD) inducing significant structural transitions in the enzyme, revealed by a different heat stability. ATP4- facilitates the process of thermal inactivation by inducing the presence of thermolable forms. On the contrary, ATP-Mg protects the enzyme from thermal inactivation. The protection is significant but less marked than that shown by NADP. Kinetic studies indicate a different type of binding between ATP-Mg and ATP4-. Uncomplexed ATP is a competitive inhibitor vs NADP or G6P and it shows a positive cooperative effect. ATP-Mg is an uncompetitive inhibitor vs G6P and competitive vs NADP and it shows a negative cooperativity. A similar behaviour is also shown by complexed and free CTP and GTP.

Adenosine Triphosphate↗