Electronic nose and GC-MS analysis of volatile compounds in Tuber magnatum Pico: evaluation of different storage conditions.

The characteristic aromatic composition of white truffles (Tuber magnatum Pico) determines its culinary and commercial value. However modifications of truffle organoleptic proprieties occur during preservation. A study of headspace of white truffles by using Electronic nose (E-nose), gas chromatography-mass spectrometry (GC-MS) and sensory analyses was performed. Truffles were stored at different conditions for 7 days: +4 and +8°C wrapped in blotting paper or covered by rice or none of the above. Headspace E-nose measurements and sensory analyses were performed each day. Statistical multivariate analysis of the data showed the capability of E-nose to predict sensorial analysis scores and to monitor aroma profile changes during storage. Truffle's volatile molecules were also extracted by headspace solid phase microextraction technique and separated and identified by GC-MS. Partial Components Analysis of data was performed. E-nose and GC-MS results were in agreement and showed that truffle storage in paper at +8°C seemed to be the best storage condition.

Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions.

In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

Rhodiola rosea as antioxidant in red blood cells: ultrastructural and hemolytic behaviour.

Rhodiola rosea L. (Crassulaceae) is a plant that lives at high altitude in Europe and Asia, widely used for its high capacity to increase the organism resistance to different stress conditions. Although a few international literature supports these effects, today R. rosea has become a common component of many dietary supplements also in the Western world. The aim of the present study was to investigate the effect of the R. rosea roots aqueous extract on in vitro human erythrocytes exposed to hypochlorous acid (HOCl)-oxidative stress. Several damages occur in human erythrocytes exposed in vitro to HOCl, among these membrane protein and lipid modifications, shifting from the discocyte shape to the echinocyte one, and determining lysis ultimately. Therefore, in the present work, the evaluation of the antioxidant capacity of the Rhodiola extract has been carried out by means of scanning electron microscopy and of hemolytic behaviour on human erythrocytes exposed to HOCl in the presence of increasing doses of the aqueous extract in different experimental environments (co-incubation and subsequent incubations). The results obtained are consistent with a significant protection of the extract in presence of the oxidative agent, but a cautionary note emerges from the analysis of the data related to the cell exposition to the plant extract in the absence of any induced oxidative stress. In fact, the addition to erythrocyte of high doses of R. rosea extract always determines severe alterations of the cell shape.

Quercetin prevents glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells.

Exposure of rabbit red blood cells to dehydroascorbic acid (DHA) caused a significant decline in glutathione content which was largely prevented by quercetin, whereas it was insensitive to various antioxidants, iron chelators or scavengers of reactive oxygen species. This response was not mediated by chemical reduction of either extracellular DHA or intracellular glutathione disulfide. In addition, the flavonoid did not affect the uptake of DHA or its reduction to ascorbic acid. Rather, quercetin appeared to specifically stimulate downstream events promoting GSH formation.

Intracellular ascorbic acid enhances the DNA single-strand breakage and toxicity induced by peroxynitrite in U937 cells.

A well-established protocol to increase the intracellular content of ascorbic acid was used to investigate the effects of the vitamin on DNA single-strand breakage and toxicity mediated by authentic peroxynitrite (ONOO(-)) in U937 cells. This protocol involved exposure for 60 min to 100 microM dehydroascorbic acid, which was taken up by the cells and converted into ascorbic acid via a GSH-independent mechanism. At the time of exposure to ONOO(-), which was performed in fresh saline immediately after loading with dehydroascorbic acid, the vitamin present in the cells was all in its reduced form. It was found that, in cells that are otherwise ascorbate-deficient, an increase in their ascorbic acid content does not prevent, but rather enhances, the DNA-damaging and lethal responses mediated by exogenous ONOO(-). These results therefore suggest that acute supplementation of ascorbic acid can be detrimental for individuals with pathologies associated with a decrease in ascorbic acid and in which ONOO(-) is known to promote deleterious effects.

The wall paintings in the Oratorio of San Giovanni Battista in Urbino: a study finalized to a correct conservation project.

The walls of the Oratorio of San Giovanni Battista in Urbino are decorated with outstanding mural paintings dating back to the 15th century. Due to degradation processes that have occurred in the past years, such paintings require a conservative restoration project. In order to evaluate reasons of the decay, some scientific studies have been performed. They consist of macroscopic observations and chemical (EDS), morphological (SEM) and mineralogical (XRD) analyses of samples both from the original preparatory layers under the painted layers and from the restored plasters at the surbase of the wall. In addition, environmental studies have been performed to verify microclimatic conditions of the church in which the mural paintings are located. Finally, a conservative restoration project was proposed.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.