Effect of consumption of dark chocolate on oxidative stress in lipoproteins and platelets in women and in men.

The goal of this research was to investigate the effects of 3 weeks consumption of 50 g flavonoid-rich dark chocolate on lipoprotein oxidative stress in vitro and in vivo in 25 women compared to 25 men. Levels of thiobarbituric acid-reactive substances, conjugated dienes and hydroperoxide levels in HDL and LDL before and after consumption of dark chocolate were determined. Moreover in platelets of the same subjects NO and peroxynitrite levels were studied. TBARs concentration in women's HDL decreased by 26.7% while in men's HDL 23.4%; lipid hydroperoxides decreased in women's HDL by 62.8% while in men's HDL they decreased by 21.1%. Conjugate diene formation decreased in women's HDL by 55.9%, while in men's HDL it decreased by 49.2%. Moreover TBARs concentration decreased in women's LDL by 26.7% after supplementation and in men's LDL by 21.6%; lipid hydroperoxides decreased in women's LDL by 83.6% while in men's LDL they decreased by 64.7%. Moreover conjugate diene formation decreased in women's LDL by 48.2%, while in men's LDL it decreased by 21.6%. After supplementation peroxynitrite values decreased in women by 24% and in men by 18.6% while NO increased after supplementation by 15.7% compared to basal determination in women, and by 32.2% in men. This study showed that a short-term intake of dark chocolate might improve the lipoprotein profile in healthy humans, more so in women than in men, and this might exert a protective effect on the cardiovascular system.

Vitamin E succinate enhances steatotic liver energy status and prevents oxidative damage following ischemia/reperfusion.

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.

Expression of basic fibroblast growth factor, its receptors and syndecans in bladder cancer.

Basic fibroblast growth factor (bFGF) is a heparin-binding cationic protein involved in a variety of pathological conditions including angiogenesis and solid tumour growth. The basic fibroblast growth factor receptor (FGFR) family comprises at least 4 high affinity tyrosine kinase receptors that require syndecans for their function. Mounting evidence indicates that syndecans, that bind both bFGF and their FGFRs, will act as stimulators, whereas syndecans that only bind bFGF will act as inhibitors of signaling by sequestering the growth factor. Recent findings have highlighted the importance of syndecans in urological cancers. The aim of this study is to investigate the expression of bFGF, its receptors (R1 and R2) and syndecans (1-4) in invasive urothelial carcinoma and normal-looking urothelium by Western blotting, RT-PCR, and immunohistochemistry analyses. Interestingly, bFGF, FGFR1 and FGFR2 protein levels statistically increased in bladder cancer tissues. mRNA of FGFR1 and syndecans (1-4), showed a statistically significant increase while an mRNA increase in the other molecules analysed was not significant. bFGF, its receptors and syndecan immunostaining were mainly present in the urothelium both in normal-looking tissues and urothelial neoplastic cells. In conclusion, our data report that the bFGF, FGFR and syndecan expressions are altered in bladder tumours.

On the importance of anandamide structural features for its interactions with DPPC bilayers: effects on PLA2 activity.

The acylethanolamide anandamide (AEA) occurs in a variety of mammalian tissues and, as a result of its action on cannabinoid receptors, exhibits several cannabimimetic activities. Moreover, some of its effects are mediated through interaction with an ion channel-type vanilloid receptor. However, the chemical features of AEA suggest that some of its biological effects could be related to physical interactions with the lipidic part of the membrane. The present work studies the effect of AEA-induced structural modifications of the dipalmitoylphosphatidylcholine (DPPC) bilayer on phospholipase A2 (PLA2) activity, which is strictly dependent on lipid bilayer features. This study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene fluorescence, demonstrates that the effect of AEA on PLA2 activity is concentration-dependent. In fact, at low AEA/DPPC molar ratios (from R = 0.001 to R = 0.04), there is an increase of the enzymatic activity, which is completely inhibited for R = 0.1. X-ray diffraction data indicate that the AEA affects DPPC membrane structural properties in a concentration-dependent manner. Because the biphasic effect of increasing AEA concentrations on PLA2 activity is related to the induced modifications of membrane bilayer structural properties, we suggest that AEA-phospholipid interactions may be important to produce, at least in part, some of the similarly biphasic responses of some physiological activities to increasing concentrations of AEA.

A new melanoma diagnosis active support system.

The aim of this paper is to present the operational performance of a new MDASS (Melanoma Diagnosis Active Support System) prototype able to distil optimal knowledge from acquired data to automatically capture and reliably discriminate and quantify the stage of disease evolution. Automated classification dermatoscopical parameters can be divided into two main classes: Size Descriptor (point size, local, and global) and Intrinsic Descriptor (morphological, geometrical, chromatic, others). Usually elementary geometric shape robust and effective characterization, invariant to environment and optical geometry transformations, on a rigorous mathematical level is a key and computational intensive problem. MDASS uses GEOGINE (GEOmetrical enGINE), a state-of-the-art OMG (Ontological Model Generator) based on n-D Tensor Moment Invariants for shape/texture effective description. MDASS main results show robust disease classification procedure with distillation of minimal reference grids for pathological cases and they ultimately achieve effective early diagnosis of melanocytic lesion. System results are validated by carefully designed experiments with certified clinical reference database. Overall system operational performance is presented. Finally, MDASS error analysis and computational complexity are addressed and discussed.

Oxidative response and membrane modification of diabetic platelets challenged with PAF.

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.

Plasma membrane polarity of polymorphonuclear leucocytes from children with primary ciliary dyskinesia.

BACKGROUND: Polymorphonuclear leucocytes (PMN) from subjects with primary ciliary dyskinesia (PCD) can have abnormal locomotory systems. The locomotory activity of PMN is the result of biochemical events mediated by the plasma membrane. In this study we investigated plasma membrane polarity of PMN from children with PCD. DESIGN: Membrane polarity was studied in 11 children with PCD and in healthy controls by measuring the steady-state fluorescence excitation and emission spectra of 2-dimethylamino[6-lauroyl]naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission red shift in polar solvents, with respect to nonpolar solvents. Moreover, the effect of the microtubule disassembling agent colchicine on PMN membrane polarity was evaluated. RESULT: Our results show a red shift of the fluorescence excitation and emission spectra of Laurdan in PMN from the PCD group with respect to the control group. These data indicate an increase in membrane polarity of PMN from the PCD group. Treatment of PMN with colchicine induced a red shift in the Laurdan excitation and emission spectra with the same trend observed in PMN from the PCD group. CONCLUSION: PMN from children with PCD are characterized by an increased plasma membrane polarity. These changes could be the basis of the modifications in the locomotory activities of PMN. The observed alterations may be attributed to abnormalities in the cytoskeleton.

Plasma membrane fluidity and polarity of polymorphonuclear leukocytes from children with type I diabetes mellitus.

Polymorphonuclear leukocytes (PMN) from diabetic subjects have been found to be abnormal in various functional activities. These activities are mediated by the plasma membrane. This study was designed to evaluate plasma membrane fluidity and polarity in children with type I diabetes mellitus using fluorescence spectroscopy. PMN membrane fluidity and polarity were assessed in a group of 32 diabetic children. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy and fluorescence decay of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH), whereas membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). TMA-DPH and Laurdan are known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer. Our data show a significant increase in steady-state fluorescence anisotropy in diabetic PMN that reflects a decrease in membrane fluidity, and a decrease in TMA-DPH lifetime distribution indicating a decrease in membrane heterogeneity. Laurdan shows a blue shift of the fluorescence emission and a red shift of the excitation spectra in diabetic PMN with respect to the control group, indicating a decrease in membrane polarity. The results demonstrate a decrease in the phospholipid order at the membrane surface and a decrease in membrane polarity in diabetic PMN. These alterations in the physico-chemical properties of the plasma membrane could be the basis of the modifications in functional activities of PMN. The changes in the plasma membrane of PMN could be the result of metabolic and chemical modification associated with type I diabetes.

Role of guanine nucleotides as endogenous ligands of a kainic acid binding site population in the mammalian cerebellum.

An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.

Interaction of trout hemoglobin with H2O2: a chemiluminescence study.

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H2O2 using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H2O2 produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H2O2.

Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx.

The effects of the insecticide lindane (the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) on membrane potential, cytosolic free Ca2+ concentration ([Ca2+]i) and surface biophysical properties were studied in human spermatozoa. The insecticide induces rapid, transient and reproducible membrane depolarization and opening of voltage-dependent Ca2+ channels leading to an increase in [Ca2+]i. In contrast with the effect in somatic cells, lindane did not affect gamma-aminobutyric acid receptor-linked Cl- currents. Ca2+ and K+ currents were found to drive lindane-induced membrane depolarization and repolarization respectively, whereas Na+ and Cl- fluxes appear not to have a role in the phenomenon. The insecticide was still able to produce membrane depolarization both in the combined absence of extracellular Ca2+ and Na+ and in high-K+ buffer, suggesting that lindane alters the membrane dipole potential. In agreement with this, Laurodan and Prodan fluorescence spectroscopy revealed that lindane partition into the sperm plasma membrane lowers water molecular dynamics in the uppermost region of the membrane external leaflet, probably as the result of reordering of water dipoles. We propose that the first effect of lindane partitioning into the sperm plasma membrane is a change in the membrane dipole potential, which results in the activation of membrane-located Ca2+-influx pathways.

Platelet plasma membrane in subjects with primary nocturnal enuresis: effect of desmopressin.

Plasma membrane fluidity of platelets (PLT) obtained from subjects with primary nocturnal enuresis (PNE) and healthy controls was investigated before and after addition of desmopressin (DDAVP). Membrane fluidity was studied by measuring steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene incorporated into PLT plasma membrane. Our results show an increase in membrane fluidity at the surface level of PLT from subjects with PNE. Moreover, the addition of DDAVP induces a stable and significant decrease of membrane fluidity in both groups. These results suggest alterations of the lipid order in the exterior part of the PLT plasma membrane from patients with PNE.

A sensitive detection of neutrophil activation by fluorescence quenching of membrane inserted singlet oxygen probe.

Fluorescence emission intensity of 1,3-diphenylisobenzofuran incorporated in polymorphonuclear granulocytes plasma membranes was investigated in basal conditions and during stimulation with different activators. Phorbol myristate acetate, known as the most effective "oxygen burst" inducer, produced a larger decrease in 1,3-diphenylisobenzofuran fluorescence intensity than 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and N-formyl-methionyl-leucyl-phenylalanine, known as weak stimulants of oxygen uptake. Diphenyl iodonium an inhibitor of leukocyte NADPH oxidase, and the singlet oxygen selective trap alpha-terpinene inhibited the quenching effect of phorbol myristate acetate on 1,3-diphenylisobenzofuran fluorescence. These data suggest formation of singlet oxygen in activated leukocytes and demonstrate that measurement of 1,3-diphenylisobenzofuran fluorescence intensity provides a new sensitive method of detection of neutrophils activation.

Polymorphonuclear leukocyte-generated oxygen metabolites decrease beat frequency of human respiratory cilia.

We investigated the effect of polymorphonuclear leukocyte (PMN)-generated oxygen metabolites on the ciliary beat frequency. PMNs were incubated with human respiratory cilia obtained by nasal brushing. The oxidative metabolism was stimulated by opsonized zymosan, and ciliary beat frequency was evaluated before and after activation of PMNs. Ciliary beat frequency was studied using video microscopy. Our results demonstrate a significant decrease in ciliary beat frequency after activation of PMNs. This effect was reduced by catalase. These data suggest that the PMN-generated oxygen metabolites, particularly H2O2, decrease beat frequency of human respiratory cilia.

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane.

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.

Probing the Interaction of PAF with Human Platelet Membrane Using the Fluorescent Probe Laurdan.

Changes in membrane polarity of human platelets during the interaction with PAF were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl)naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission red-shift in polar solvents, with respect to non-polar solvents. Our results demonstrate that platelet activation factor (PAF) (10(-7) M) induces a red-shift of the fluorescence emission spectra of Laurdan. These changes were not observed in the presence of the PAF antagonist, L-659,989. These data suggest that the interaction of PAF with its specific receptor and the signalling pathways involved in platelet activation are accompanied by an increase in polarity at the hydrophobic-hydrophilic interface of human platelet membranes.

Membrane fluidity of polymorphonuclear leukocytes from children with primary ciliary dyskinesia.

Plasma membrane fluidity and heterogeneity of polymorphonuclear leukocytes (PMN) were investigated in seven children with primary ciliary dyskinesia (PCD) and 17 healthy controls. Membrane fluidity and heterogeneity were studied by measuring the steady state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into PMN plasma membrane. Our results show an increase in membrane fluidity at the surface level of PMN from patients with PCD. Distribution analysis of TMA-DPH lifetime values indicate an increase in membrane heterogeneity in subjects with PCD. The observed changes in the physicochemical properties of the membrane could lead to alterations in the function of PMN from children with PCD.

[Oxidative metabolism in polymorphonuclear granulocytes of children with trisomy 21]. / Metabolismo ossidativo in granulociti polimorfonucleati di bambini con trisomia 21.

In response to certain stimuli, polymorphonuclear leukocytes (PMNs) undergo an oxidative burst during which a series of reactive oxygen metabolites are generated. The importance of the release of these oxygen metabolites by polymorphonuclear leukocytes, is recognized to be a key event in the function of these cells during infection and inflammation. We have evaluated the release of reactive oxygen species during the activation of respiratory burst (RB) of PMNs obtained from children with trisomy 21 using chemiluminescence techniques. As chemiluminogenic probes we have employed lucigenin and luminon that are know to be sensitive to the superoxide anion and the H2O2-myeloperoxidase-halide system of PMNs, respectively. Activated PMNs from children with trisomy 21 exhibited a low level of superoxide and a reduced activity of H2O2-myeloperoxidase-halide system compared to the control group. No significant difference in extracellular H2O2 release was observed. It seems likely that alterations in the enzymatic activities of the Cu/Zn-Superoxide dismutase and myeloperoxidase induce imbalance in the release of reactive oxygen species in activated PMNs from children with trisomy 21. This imbalance could be on the basis of the increased oxidative injury reported in trisomy 21.

Use of Laurdan fluorescence in studying plasma membrane organization of polymorphonuclear leukocytes during the respiratory burst.

The changes in plasma membrane polarity of polymorphonuclear leukocytes (PMN) during the activation of the respiratory burst were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission blue shift in nonpolar solvents, with respect to polar solvents. Our results show a blue shift of the fluorescence emission spectra of Laurdan during activation of PMN with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. These results suggest that the activation of the respiratory burst of PMN is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.

Effect of PAF on polyrnorphonuclear leucocyte plasma membrane polarity: a fluorescence study.

The effect of PAF on the plasma membrane polarity of polymorphonuclear leukocytes (PMNs) was investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-1auroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surrounding. Laurdan shows a marked steady-state emission blue-shift in non-polar solvents, with respect to polar solvents. Our results demonstrate that PAF (10(-7) M) induces a blue shift of the fluorescence emission spectra of Laurdan. These changes are blocked in the presence of the PAF antagonist, L-659,989. Our data indicate that the interaction between PAF and PMNs is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.