Effect of Punicalagin and Resveratrol on Methionine Sulfoxide Reductase: A Possible Protective Contribution against Alzheimer's Disease.

Methionine sulfoxide reductase A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. MsrA has recently turned attention in coupling with the neurodegenerative disorders and in particular with Alzheimer disease. In fact this neurodegenerative disorder depends to a deposit of beta amyloid a peptide with an oxidizable methionine in position 35 which is proved able to modulate the expression to MsrA in neuronal cells. Here, we firstly provided evidence that pretreatment with Resveratrol and Punicalagin (a potent antioxidant extracted from pomegranate), up-regulate the expression and enzymatic activity of MsrA in human neuroblastoma IMR-32 cells with beta amyloid peptides. This effect determines a lowering of oxidative potential of the cells as demonstrated by the ROS measurement and a protective effect on cellular availability. Therefore we hypothesize a possible prevent role for these molecules in Alzheimer and in other neurodegenerative diseases.

Protective effect of rhubarb derivatives on amyloid beta (1-42) peptide-induced apoptosis in IMR-32 cells: a case of nutrigenomic.

Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease (AD). In the last years considerable attention has been focused on identifying natural food products, such as phytochemicals that prevent or almost retard the appearance of amyloid beta (1-42)-related neurotoxic effects. In this study, human neuroblastoma cells (IMR-32) was used as system model to evaluate the protective role of rhaponticin (3,3',5-trihydroxy-4'-methoxystilbene 3-O-d-glucoside) a stilbene glucoside extracted from rhubarb roots (Rhei rhizoma) and rhapontigenin, its aglycone metabolite, against amyloid beta (1-42)-dependent toxicity. The obtained results show that rhapontigenin maintains significant cell viability in a dose-dependent manner and it exerts a protective effect on mitochondrial functionality, as evidenced by mitochondrial oxygen consumption experiments. A similar behaviour, but to a lesser extent, has been shown by rhaponticin. The protective mechanism mediated by the two stilbenes could be related to their effect on bcl-2 gene family expression. Bax, a pro-apoptotic gene, resulted down-regulated by the treatment with rhaponticin and rhapontigenin compared with the results obtained in the presence of amyloid beta (1-42) peptide. Conversely, bcl-2, an anti-apoptotic gene, highly down-regulated by amyloid beta (1-42) treatment, resulted expressed in the presence of stilbenes similarly to that shown by control cells. The obtained results support the hypothesis that amyloid beta (1-42)-induced neurotoxicity occurs via bax over-expression, bcl-2 down-regulation, firstly indicating that rhaponticin and its aglycone moiety may alter this cell death pathway. Based on these studies, we suggest that rhaponticin and its main metabolite could be developed as agents for the management of AD.

Alzheimer's amyloid beta-peptide (1-42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing role for methionine 35.

The beta amyloid (Abeta), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals toxic to brain tissue and the redox state of Met-35 residue seems to play a particular and critical role in peptide's neurotoxic actions. In this study, we investigated, in human neuroblastoma cells (IMR-32), the relationship between the oxidative state of methionine, and both neurotoxic and pro-apoptotic actions induced by Abeta-peptide, comparing the effects of native peptide, in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 (Abeta(1-42)(35Met-ox)), as well as an Abeta-derivative with Met-35 substituted with norleucine (Abeta(1-42)(35Nle)). The obtained results show that Abeta induces a time-dependent decrease in cell viability; Abeta(1-42)(35Met-ox) was significantly less potent, though inducing a remarkable decrease in cell viability compared to control. On the contrary, no toxic effects were observed after treatment with Abeta(1-42)(35Nle). Abeta-peptide as well as the amyloid modified peptide with oxidized Met-35 induced the pro-apoptotic gene bax over-expression after 24 h, whereas Abeta(1-42)(35Nle) had no effect. Conversely, bcl-2, an anti-apoptotic gene, became highly down-regulated by Abeta peptide treatment, in contrast to that evidenced by the Abeta(1-42)(35Met-ox) peptide. Finally, Abeta caused an increase in caspase-3 activity to be higher with respect to that shown by Abeta(1-42)(35Met-ox) while Abeta(1-42)(35Nle) had no effect. These results support the hypothesis that Abeta-induced neurotoxicity occurs via bax over-expression, bcl-2 down-regulation, and caspase-3 activation, first indicating that methionine 35 redox state may alter this cell death pathway.

Methionine 35 oxidation reduces toxic and pro-apoptotic effects of the amyloid beta-protein fragment (31-35) on isolated brain mitochondria.

Amyloid beta-peptide (AbetaP), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of AD, since it represents the residue in AbetaP most susceptible to oxidation in vivo. In the present study, we used the fragment 31-35 of AbetaP, which has a single methionine at residue 35, in order to investigate the influence of the oxidation state of methionine-35 on the toxic and pro-apoptotic effects induced by Abeta(31-35) on isolated brain mitochondria. The obtained results show that exposure of isolated mitochondria from rat brain to AbetaP(31-35) determines (i) a large release of cytochrome c (ii) a significant reduction in mitochondrial respiration and (iii) a slight drop in the mitochondrial membrane potential (deltapsi). In contrast, the amplitude of these events resulted attenuated or completely abrogated in isolated brain mitochondria exposed to the AbetaP(31-35)Met35OX, in which methionine 35 was oxidized to methionine sulfoxide. We have further characterized the action of AbetaP(31-35) and Abeta(31-35)Met35OX peptide on PC12 cells. Although these two peptides, compromised mitochondrial function at a different extent as assessed by MTT reduction, neither one of them decreased cell viability as measured by Trypan Blue exclusion assay. The results obtained in this study support the hypothesis that the oxidative state of Met-35 may play a critical role in the mechanisms responsible of neurotoxicity exerted by this peptide.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes.

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37 C) of intact human red blood cells to doxorubicinol (40 M) and to aglycone derivatives of doxorubicin (40 M) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells...

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5.

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.

Molecular adaptation to hibernation: the hemoglobin of Dryomys nitedula.

The oxygen binding properties of Dryomys nitedula hemoglobin (Hb) were investigated as a function of pH both in the absence and in the presence of its physiological cofactors (i.e. chloride ions and 2,3-biphosphoglyceric acid) and at different temperatures. Moreover, the alpha- and beta-chains of the Dryomys Hb were partially sequenced. The results obtained show that the effects of Bohr protons, chloride ions, organic phosphates and temperature are significantly lower for Dryomys Hb than for human Hb. Thus, the increase in Hb oxygen affinity, resulting from the reduction of red cell organic phosphates and body temperature that occurs during hibernation, is advantageous for loading oxygen at the lung level without compromising oxygen release at the tissues, as could occur if Dryomys Hb had similar functional properties to those of other non-hibernating mammals. Furthermore, it is possible that the reduced Bohr effect may moderate the potential effects of increased CO(2) associated with prolonged apnea on the loading and unloading of oxygen. Moreover, the overall heat of oxygenation (Delta H) for Dryomys Hb is much less exothermic than that of the human Hb and it is completely independent of the 2,3-biphosphoglyceric acid concentration.

Functional modulation of the Thr72-->Ile mutant from Scapharca inaequivalvis homodimeric hemoglobin.

The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72-->Ile mutant of Scapharca inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8 degrees C and 36 degrees C, in comparison with the wild-type recombinant protein. Results demonstrate pH-independent O2-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication. Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural alterations of water molecules at the subunit interface occurring in the Thr72-->Ile mutant as well as with amino acid residues involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of (2) the heme propionates bridging the two subunits.

Functional modulation by lactate of myoglobin. A monomeric allosteric hemoprotein.

The effect of lactate on O2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O2 affinity (i.e. an increase of P50) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O2 affinity appears to differ for sperm whale and horse heart Mb, deltalogP50 being approximately 1.0 and approximately 0.4, respectively. From the kinetic viewpoint, the variation of O2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.

Oxygen transport by fetal bovine hemoglobin.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20 degrees C. However, on going from 20 degrees C to 37 degrees C, the difference in O2 affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.

A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.

The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E. coli after mutation of HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.

Hemoglobin function under extreme life conditions.

Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection between the binding of heterotropic effectors and the reaction with oxygen. The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation-deoxygenation cycle in dependence of the physiological needs of the particular organisms. Moreover they show that a thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural and functional characterization going deeper into the physiology of the specific organism.

Synergistic modulation by chloride and organic phosphates of hemoglobin from bear (Ursus arctos).

The oxygen binding properties of hemoglobin (Hb) from brown bear (Ursus arctos) have been studied focussing on the effect of heterotropic ligands, and the behaviour has been compared with that of human HbA, taken as a prototype of mammalian Hbs. It has been observed that in bear Hb chloride ions and 2,3-diphosphoglyceric acid (Gri(2,3)P2) can modulate the oxygen affinity in a synergistic way such that their individual effect is enhanced whenever they are both present in saturating amounts. The thermodynamic analysis of such a feature indicates that in bear Hb there are two classes of chloride binding sites, one acting synergistically with Gri(2,3)P2 and another one, which likely overlaps with the organic phosphate interaction cleft, and is therefore fully operative only in the absence of Gri(2,3)P2. The behaviour of the last site is similar to that observed in human HbA, where the effect of Cl- and Gri(2,3)P2 is mutually exclusive. The interaction energy between chloride and Gri(2,3)P2 synergistic binding sites appears to be O2-linked so that the interplay may have a relevant physiological role in modulating the oxygen transport in brown bear. This behaviour is associated with a marked pH-dependence of the oxygenation enthalpy in bear Hb, such that under acidotic and hypercloruremic conditions, oxygen supply to peripheral tissues could be maintained essentially unaltered even under low temperature conditions.

Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex).

The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals.

Physiological relevance of the overall delta H of oxygen binding to fetal human hemoglobin.

Human fetal hemoglobin is known to display, at 20 degrees C, a lower affinity than human adult hemoglobin for oxygen when both proteins are in the absence of organic phosphates. The physiologically important reverse situation is achieved at 37 degrees C upon addition of 2,3-bisphosphoglycerate (DPG), whose lower effect on fetal hemoglobin is related to some amino acid substitutions present in gamma-chains. However, the difference in oxygen affinity observed at 37 degrees C is not solely due to the different modulation power of DPG with respect to adult and fetal hemoglobins. In fact, the results presented here reveal new aspects linked to the interplay of temperature and organic phosphates. In particular, the lower effect of DPG on fetal hemoglobin renders almost identical the oxygen affinity of the two hemoglobins at 20 degrees C, abolishing the difference observed in the absence of the effector. Successively on going from 20 degrees C to 37 degrees C, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal hemoglobin when in the presence of DPG, adult hemoglobin shows a lower oxygen affinity, as it should if oxygen has to be transferred from maternal to fetal blood.

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.

A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.