An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.     

The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone,and not mimicked by rh-TNF-alpha,IL-1beta and IFN-gamma

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS-induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti-inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti-inflammatory drug that interferes with many of the features characterizing pro-inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS-injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro-inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS-induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF-alpha/IL-1 beta/IFN-gamma.     

Slight influence of the estrous cycle stage on the mucosal and systemic specific antibody response induced after vaginal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis in mice

Since the immune response appears to be variable according to the hormonal stage of the mammalian female, the aim of this study was to determine whether estrous cycle stage modifies the mucosal and systemic immune responses induced by intraperitoneal and vaginal immunization of mice with protoxin Cry1Ac. We tested the influence of three immunizations on the specific antibody response elicited at estrus and diestrus, that were the same estrous cycle stages at which the antigen was applied. Both intraperitoneal and vaginal immunization of mice with Cry1Ac either at estrus or diestrus induces specific antibody responses at serum, vagina and large intestine. The stage of the estrous cycle have little or non influence in the magnitude of the response induced, since only at serum the IgM was slightly higher at estrus than at diestrus by both routes. At the large intestine only the IgA response elicited via the intraperitoneal route changed, being higher at diestrus, whereas at the vagina IgA response induced did not change significantly due to the cycle stage. Present results suggest that Cry1Ac may be used as an antigen carrier as it can elicit antibody responses at systemic level and at several mucosal sites including the vagina that are not modified significantly throughout the reproductive cycle.     

Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.