Biomedical subjects

M Dachà

Publications and source records attributed to M Dachà.

At least 19 recordsLinked to original sources

PubMed1998

Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide: free radical scavenging versus iron chelating mechanism.

Although the antioxidant properties of flavonoids are well documented, it is still unclear whether these effects are dependent on radical scavenging or iron chelating activities. By using an experimental approach based on the notion that iron chelators suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide, whereas radical scavenging antioxidants prevent only the latter response, we provide experimental evidence indicating that the most prominent activity of the flavonoid quercetin resides in its ability to chelate iron. This experimental approach can be utilized for the assessment of iron chelation in the biological activity of flavonoids or other antioxidants.

Antioxidants↗

PubMed1997

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells.

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2-0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed at the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate the a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.

Animals↗

PubMed1996

Purification and characterization of the carboxyl-domain of human hexokinase type III expressed as fusion protein.

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study, we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.

Amino Acid Sequence↗

PubMed1996

Effect of 50 Hz sinusoidal electric and/or magnetic fields on the rate of repair of DNA single strand breaks in cultured mammalian cells exposed to three different carcinogens: methylmethane sulphonate, chromate and 254 nm U.V. radiation.

Treatment of cultured mammalian cells with three different carcinogens, namely methylmethane sulphonate (MMS), chromate and 254 U.V. radiation, produces DNA single strand breaks (SSB) in cultured mammalian cells. The rate of removal of these lesions is not affected by exposure to 50 Hz electric (0.2 - 20 kV/m), magnetic (0.0002-0.2 mT), or combined electric and magnetic fields. These results indicate that, under the experimental conditions utilized in this study, 50 Hz electric, magnetic and electromagnetic fields (over a wide range of intensities) do not affect the machinery involved in the repair of DNA SSBs generated by different carcinogens in three different cultured mammalian cell lines, making it unlikely that field exposure enhances the ability of these carcinogens to induce transformation via inhibition of DNA repair.

Animals↗

PubMed1995

Mitochondria-bound hexokinase from rabbit reticulocytes is resistant to the inactivation induced by Fe(II)/ascorbate.

Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.

Animals↗

PubMed1995

The effect of 50 Hz sinusoidal electric and/or magnetic fields on the rate of repair of DNA single/double strand breaks in oxidatively injured cells.

Exposure of cultured mammalian cells to 50 Hz electric (0.2-20 kV/m), magnetic (0.002-2 G), or combined electric and magnetic fields did not affect the rate of repair of DNA single strand breaks (SSB) induced by hydrogen peroxide. The same lack of effect was observed on the repair of both DNA SSBs and DNA double strand breaks (DSBs) in cells treated with the cocktail hydrogen peroxide/L-Histidine. These results indicate that exposure to electric and/or magnetic fields does not affect the machinery involved in the repair of DNA lesions in oxidatively injured cells thus suggesting that it is unlikely that field exposure might induce changes in the response of the cells to the tumor promoting or carcinogenic effects elicited by reactive oxygen species.

Animals↗

PubMed1994

High resolution of multiple forms of rabbit reticulocyte hexokinase type I by hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) has been employed extensively in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the distribution of multiple forms of rabbit reticulocyte hexokinase type I. These distinct forms of the enzyme, designated hexokinase Ia, Ia* and Ib, show similar kinetic and physical properties, similar molecular masses (ca. 100,000) and a different intracellular distribution. The results obtained using Toyopearl Phenyl 650 S of 20-50-microns particle diameter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of charging a higher amount of starting material even with a high protein concentration. These characteristics render Toyopearl Phenyl 650 S suitable for analytical and preparative purposes. Further, in the separation of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient separation of proteins with high molecular mass and their recovery in active forms. The Toyopearl Phenyl 650 S column was also shown to be more efficient than the ion-exchange chromatographic media previously used, allowing a quicker analysis of the multiple forms of rabbit reticulocyte hexokinase under different biological conditions.

Ammonium Sulfate↗

PubMed1994

Inactivation of rabbit red blood cell hexokinase activity promoted in vitro by an oxygen-radical-generating system.

Rabbit red blood cell hexokinase (EC 2.7.1.1) has been shown to be inactivated in vitro by incubating intact erythrocytes in the presence of an oxygen-radical-generating system represented by ascorbate and iron. It was interesting to note that among the glycolytic enzymes, only hexokinase was found to be susceptible to the action of oxygen radicals, suggesting that the loss of activity of this enzyme may be one of the first signals of cellular damage in rabbit red blood cells. This statement is supported by the fact that, under the experimental conditions used, we did not observe any significant plasma membrane lipid peroxidation nor intracellular proteolysis. Furthermore, mature erythrocytes are unable to synthesize hexokinase as well as other proteins de novo; therefore, the inactivation of this enzyme, which is both the first and one of the rate-limiting enzymes of the glycolytic pathway, could play an important role in determining metabolic impairment of red blood cells, with possible physiological implications. We also investigated the effect of various radical scavengers and antioxidants (glucose, vitamin E, dithiothreitol, flavonoids) which are able to influence the inactivation of hexokinase activity to different extents. Finally, under the experimental conditions used (90 min of incubation at 37 degrees C), we did not observe any difference in the hemolysis of rabbit red blood cells incubated in the presence or absence of ascorbate and iron (hemolysis was about 1% after 90 min of incubation), suggesting that the system used was able to furnish information about the cellular damage produced by oxygen radicals without provoking cell lysis.

Adenosine Triphosphatases↗

PubMed1993

Studies on the possible biological effects of 50 Hz electric and/or magnetic fields: evaluation of some glycolytic enzymes, glycolytic flux, energy and oxido-reductive potentials in human erythrocytes exposed in vitro to power frequency fields.

An attempt has been made to understand whether 50 Hz electric and magnetic fields (EMFs) are involved in producing bioeffects by exposing human erythrocytes in vitro. The study evaluated some key glycolytic enzymes, glucose consumption, lactate production, energy charge, 2,3-diphosphoglycerate, and reduced glutathione levels, all of which are biochemical parameters significant to erythrocyte function. Cells exposed to individual or superimposed EMFs have not shown any significant difference compared with the controls.

2,3-Diphosphoglycerate↗

PubMed1992

Electric and/or magnetic field effects on DNA structure and function in cultured human cells.

Exposure of cultured K562 cells to 50 Hz electric (0.2-20 kV/m), magnetic (0.002-2 G), or combined electric and magnetic fields for up to 24 h did not result in the production of detectable DNA lesions, as assayed by the filter elution technique. The rate of cell growth was also unaffected as well as the intracellular ATP and NAD+ levels. These results indicate that, under the experimental conditions utilized in this study, 50 Hz electric, magnetic and electromagnetic fields are not geno- and cyto-toxic in cultured mammalian cells.

Adenosine Triphosphate↗

PubMed1992

High resolution of multiple forms of red blood cell enzymes using a Toyopearl DEAE 650 S.

We have investigated a new anion exchange chromatographic support (Toyopearl DEAE 650 S) which simultaneously allows easily to remove hemoglobin from hemolysates and to obtain a very high resolution of enzymes present in multiple forms. The results obtained are better than those obtainable using an anion-exchange HPLC column. The data obtained at analytical level suggest a wider use of this new matrix also for preparative purposes without significant changes in the resolution.

Animals↗

PubMed1992

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.

Animals↗

PubMed1987

Adenine and pyridine nucleotides in the red blood cells of subjects with solid tumors.

The concentration of adenine (ATP, ADP, AMP) and pyridine (NADP+, NADPH, NAD+, NADH) nucleotides in the erythrocytes of subjects affected by solid tumors was evaluated using a method which allows their simultaneous extraction and reverse-phase high-performance liquid chromatographic analysis. The results showed a lower level of ATP in the erythrocytes of subjects affected by solid tumors, whereas no significant modifications were observed in the other compounds. In fact, the mean value of ATP in these subjects was 27% lower than that of normal adults. This fact is discussed in relation to other enzymatic and metabolic modifications previously observed in red blood cells.

Adenine Nucleotides↗

PubMed1987

Red blood cell glucose metabolism in Down's syndrome.

The specific activity of red blood cell glycolytic enzymes was determined in 20 Down's syndrome patients and compared with 20 normal controls. According to previous evidence, a 50% increase of phosphofructokinase and a 30% increase of glucose-6-phosphate dehydrogenase and glutathione peroxidase activity was found. Metabolic studies of the patients' erythrocytes revealed a decrease in fructose-6-phosphate and 2, 3-diphosphoglycerate concentrations, while fructose-1, 6-diphosphate and ADP both increased. Glucose utilization by intact erythrocytes from Down's syndrome patients did not differ from that of normal controls. However, addition of methylene blue or inorganic phosphate produced a higher stimulation of erythrocyte glycolysis in patients with Down's syndrome compared to controls. These metabolic abnormalities could be, at least in part, ascribed to the increased phosphofructokinase activity which is due to a gene-dosage effect.

Blood Glucose↗

PubMed1986

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation.

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.

Adenosine Triphosphate↗

PubMed1986

Rat red blood cell hexokinase purification, properties and age-dependence.

Rat erythrocytes, in contrast to red blood cells from other mammals, have been shown to contain only one hexokinase isozymic form identified as type I by chromatographic and kinetic properties. Rat reticulocytes contain 3.6-times the hexokinase activity found in mature erythrocytes but exactly the same isozyme. By a combination of ion-exchange chromatography, dye-ligand chromatography and high-pressure liquid chromatography the rat erythrocyte hexokinase was purified more than 84 000-fold to a specific activity of 143 units/mg protein and shown to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native protein showed a molecular weight of 100 000 by gel-filtration and an apparent molecular weight of 98 000 under denaturating conditions in sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was shown to be 6.3 pH units. This data provides evidence of only one form of hexokinase in the erythrocytes of a mammal.

Animals↗

PubMed1986

Glucose metabolism in fibroblasts from patients with erythrocyte hexokinase deficiency.

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Ki for glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.

Adenosine Triphosphate↗

PubMed1986

Human erythrocyte hexokinases are immunologically related.

Human erythrocytes contain three major hexokinase isoenzymes eluted by DE-52 chromatography between hexokinase type I and type II. Cross-reactivities of these isoenzymes were studied by means of a monospecific rabbit antibody against purified human placenta hexokinase type I. It was shown that the three hexokinase isoenzymes were immunologically related, supporting the concept of a postsynthetic mechanism(s) as their origin.

Animals↗