Elena Piatti

Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO(-)), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO(-). Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO(-) or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.

Senescence delay and change of antioxidant enzyme levels in Cucumis sativus L. etiolated seedlings by ELF magnetic fields

We exposed cucumber Cucumis sativus L. etiolated seedlings to a 50 Hz, 1-Gauss magnetic field (MF) 24 h per day for 2 weeks in the dark, starting from complete divarication of cotyledons and observed quickened growth and prolonged life of EMF-exposed seedlings compared to the controls. Since this phenomenon may be due to MF-induced delay in the senescence process which, in turn, is believed to be dependent on the potency of the seedling oxidative defense system, we assayed some related enzymes and found that the combined action of higher levels of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) in MF-exposed seedlings may promote their survival and delay the appearance of signs of decay.

Anti-inflammatory activity of a honey flavonoid extract on lipopolysaccharide-activated N13 microglial cells.

Neuroinflammation is an important contributor to pathogenesis of age-related neurodegenerative disorders such as Alzheimers or Parkinsons disease. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for neurodegenerative processes. Flavonoids, widely distributed in the vegetable kingdom and in foods such as honey, have been suggested as novel therapeutic agents for the reduction of the deleterious effects of neuroinflammation. The present study investigated the potential protective effect of a honey flavonoid extract (HFE) on the production of pro-inflammatory mediators by lipopolysaccharide-stimulated N13 microglia. The results show that HFE significantly inhibited the release of pro-inflammatory cytokines such as TNF-α and IL-1β. The expressions of iNOS and the production of reactive oxygen intermediates (ROS) were also significantly inhibited. Accordingly, the present study demonstrates that HFE is a potent inhibitor of microglial activation and thus a potential preventive-therapeutic agent for neurodegenerative diseases involving neuroinflammation.

Antibacterial effect of a magnetic field on Serratia marcescens and related virulence to Hordeum vulgare and Rubus fruticosus callus cells

The exposure to a static magnetic field of 80+/-20 Gauss (8+/-2 mT) resulted in the inhibition of Serratia marcescens growth. Callus cell suspensions from Hordeum vulgare and Rubus fruticosus were also examined and only the former was found to be affected by the magnetic field, which induced a decreased viability. S. marcescens was shown to be virulent only toward H. vulgare and this virulence was reduced by the presence of the magnetic field. The modification of glutathione peroxidase activity under the different experimental conditions allowed us to speculate on the possibility of an oxidative-stress response of H. vulgare both to S. marcescens infection and magnetic field exposure. Since the control of microbial growth by physical agents is of interest for agriculture, medicine and food sciences, the investigation presented herein could serve as a starting point for future studies on the efficacy of static magnetic field as low-cost/easy-handling preservative agent.

Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate.

Hydroxytyrosol (HyT) is a polyphenol primarily released in olive mill wastewater and in olive oil. In animal and cell model studies, HyT and its metabolites have strong antioxidant and antimicrobial activities, as well as beneficial effects on the cardiovascular system and in several human diseases. Differently, many researchers reported that HyT down-regulates tumor cell viability and cell cycle progression, and induces reactive oxygen species (ROS) production and apoptosis. In this study we have investigated the effects of HyT and the corresponding ester hydroxytyrosyl laurate in U937 cells, a human monocytoid cell line, and in C2C12 myoblasts, a murine proliferating muscle cell model, after apoptotic death induction. Inverted, light and transmission electron microscopy have been utilized to characterize cell death patterns. H2O2, at the concentrations known to induce apoptosis, was utilized as cell death trigger. The results obtained show that laur-HyT has a protective antioxidant effect against H2O2 treatment, greater than HyT, so having a role in the prevention of apoptotic death in normal and tumor cells. These data suggest these compounds as good candidate for novel therapeutic strategies.

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with nH = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mM). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (Ki = 120 microM). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 mumol of glucose/h/ml red blood cells (RBC) compared with values of 0.56-2.85 mumol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mM against a plasma level of 5 mM). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.

Automated analysis of morphometric parameters for accurate definition of erythrocyte cell shape

Modification of erythrocyte morphology is clinically important in hematology and medicine. Its detection is routinely performed by subjective microscopic evaluation, which is difficult and strongly dependent on the operators expertise. We developed an original automated methodology to analyze erythrocyte cell shape modification to support and improve the operators capability and expedite measurements.

Honey Flavonoids, Natural Antifungal Agents Against Candida Albicans

Candida albicans is the most prevalent fungal pathogen in humans. In this study, flavonoids from unprocessed multifloral honey were extracted and investigated their anticandidal activity in vitro. These results indicated that honey flavonoids inhibited Candida growth; however, they did not kill the yeasts and did not directly affect the cytoplasmic membrane. The viability tests of cells yeast with the fluorescent probe Sybr green I in combination with propidium iodide suggested that antifungal activity of honey flavonoids depended on their relative lipophilic properties and that they may reach a possible intracellular site of action without compromising membrane-associated functions.

Honey flavonoids inhibit Candida albicans morphogenesis by affecting DNA behavior and mitochondrial function

AIM Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle, morphology and subcellular organelles. MATERIALS & METHODS HFE anticandidal properties were investigated using flow cytometry and scanning electron microscopy. RESULTS Flow cytometry and scanning electron microscopy analyses indicated that HFE may inhibit the growth of the three phenotypes displayed by C. albicans and reduce infection by affecting membrane integrity. HFE affects hyphal transition by reducing the G0/G1 phase and increasing the G2/M phase. Conversely, yeast and pseudohyphae do not appear to be affected. Modifications of vacuolization and mitochondrial activity, during yeast-hypha transition establish the involvement of vacuole and mitochondria. CONCLUSION HFE improved mitochondrial functionality and reduced the vacuolization, modifying the branching process associated with virulence. It is hypothesized that HFE induces changes in cell cycle progress, membrane integrity, mitochondrial function and biogenesis.

Glucose-1,6-P2 synthesis, phosphoglucomutase and phosphoribomutase correlate with glucose-1,6-P2 concentration in mammals' red blood cells

Glucose 1,6-biphosphate (G1,6P2) was measured in human, pig, cow, rabbit, rat and sheep red blood cells. Mean values are variable among the species and range from 33 to 122 nmol/ml RBC for pig and rabbit erythrocytes, respectively. The activities of G1,6P2 synthase, phosphoglucomutase (PGM) and phosphoribomutase (PRM) have also been assayed in red cell haemolysates of the same species. The correlations between the biphosphate content and the occurrence of the three enzymatic activities have been studied in order to gain an insight into the regulation of the G1,6P2 turnover in mammalian erythrocytes.