Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression.

PrPC contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrPC expression and zinc metabolism remains elusive. Here we studied the relationship between PrPC and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrPC gene. No significant difference in 65Zn2+ uptake was observed in cells expressing PrPC when compared with control cells. However, PrPC-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrPC. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrPC expression. Finally, we demonstrated that PrPC expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrPC modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrPC to PrPSc may deregulate zinc homeostasis mediated by metallothionein.

Neuroprotective and cardioprotective actions of an association of pyruvate, vitamin E and fatty acids.

Oxidative stress may involve overproduction of hydrogen peroxide which can generate highly cytotoxic hydroxyl radicals in the presence of ferrous ions. This work demonstrates that TCAT (Tricomponent Antioxidant Therapy), an association of pyruvate, vitamin E and fatty acids, provides neuronal and cardiac protection in oxidative stress, ex vivo. Mouse P19 neuron cultures were exposed for 30 min to low millimolar H2O2 concentrations in the absence or presence of Fe2+. Concentrations 1X (10 mmol/L pyruvate, 0.1 U/mL vitamin E and 0.1% fatty acids) and 3X of TCAT, respectively, prevented neuronal death caused by these treatments. Analysis of the contribution of TCAT components to neuroprotection showed that vitamin E and fatty acids enhanced pyruvate action whereas they displayed no neuroprotection by themselves. In contrast, vitamin E and fatty acids were as potent antioxidants as pyruvate in an in vitro cell-free assay, indicating that TCAT protection is modulated by the existence of the cellular membrane barriers. Isolated rat hearts were perfused under electrolysis or subjected to regional ischemia-reperfusion. TCAT 1X prevented the electrolysis-induced decrease in left ventricular pressure, heart rate and coronary flow. At 0.25X concentration, TCAT abolished the incidence of irreversible ventricular fibrillation in ischemia-reperfusion. The results indicate that TCAT could have a broad therapeutic utility in neurological and cardiac injuries associated with oxidative stress. The protective action of TCAT can surpass that of its components, revealing a benefit of the association.

Resveratrol enhances UVA-induced DNA damage in HaCaT human keratinocytes.

Resveratrol, a polyphenolic phytoalexin, is a very effective antioxidant that also exhibits strong antiproliferative and anti-inflammatory properties. Recent studies have provided support for the use of resveratrol in human cancer chemoprevention, in combination with either chemotherapeutic drugs or cytotoxic factors for a most efficient treatment of drug refractory tumor cells. Resveratrol is also widely used in topical preparations, as a chemoprotective compound against development of several cutaneous disorders, including skin cancer. Nevertheless, the combined effect of resveratrol and UVA irradiation on cellular toxicity and DNA damage has never been assessed. The aim of this work was to investigate the effect of resveratrol on cell fate in immortalized human keratinocytes HaCaT cells. The results indicated that resveratrol potentiates the production of significant amounts of 8-oxo-7,8-dihydro-2'-deoxyguanosine in UVA-irradiated genomic DNA. Moreover, the combination of resveratrol with UVA significantly enhances the induction of DNA strand breaks and cell death in HaCaT keratinocytes. The conclusion is a potential hazardous effect of topical application of resveratrol, particularly on regions exposed to sunlight.

Oxidative aggregation of ceruloplasmin induced by hydrogen peroxide is prevented by pyruvate.

Ceruloplasmin (CP) is a blue copper glycoprotein with multiple physiological functions including ferroxidase and oxidase activities. CP is also an important serum oxygen free radical (OFR) scavenger and antioxidant, exerting cardioprotective and antifibrillatory actions. Although it has been reported that CP activities can be inhibited by OFR, the intimate mechanism of this inactivation is still not clear. Exposure of bovine CP to H2O2 induced inactivation of the protein as well as structural alterations as indicated by loss of protein bands by SDS-PAGE. Both phenomena were H2O2 concentration and time dependent. HPLC gel filtration and capillary electrophoresis analysis of CP treated with H2O2 revealed an aggregation of the protein. Quantification of dityrosine formation by fluorescence indicated the involvement of dityrosine bridging, which could be responsible for aggregation of CP under oxidative attack. Oxidative damage to CP under H2O2 treatment was completely prevented by pyruvate, suggesting that the association of CP with antioxidants could extend the range of the protective action of this protein.

The blue copper ceruloplasmin induces aggregation of newly differentiated neurons: a potential modulator of nervous system organization.

Ceruloplasmin (CP) is a copper-dependent ferroxidase. It regulates iron metabolism and is involved in inflammation, angiogenesis, and protection against oxidative stress. CP also modulates K(+) channel activity in neuroblastoma cells and affects cardiodynamics of isolated hearts. Considering the presence of CP in the nervous system and the importance of iron ions and K(+) channels in neuronal activity, we postulated a role of CP in neuronal development. This hypothesis was tested using the P19 mouse embryonal carcinoma cell line, a model of neuronal differentiation. Addition of CP to the culture medium of newly differentiated P19 neurons induced cell aggregation within 24 h. This effect was concentration-dependent half-maximal at 50 nM, and not associated with necrosis, apoptosis or changes in secretory function. Deglycosylated CP was aggregative but not denatured CP, copper salts, His(2)Cu complex, or other copper enzymes or serum proteins. CP-induced aggregation was less pronounced with aging neurons and seemed not to involve K(+) channels. Immunocytofluorescence analysis demonstrated that digoxigenin-labeled CP bound to P19 neurons and the proportion of responding neurons decreased with aging. The interaction of digoxigenin-labeled CP with neurons was half-maximal at 120 nM by enzyme-linked immunosorbent assay and displaced by unlabeled CP. Our data indicate a specific aggregative action of CP on young neurons in vitro, possibly involving CP receptors. A potential developmental role of CP in nervous system organization is thus demonstrated.

Zinc homeostasis-regulating proteins: new drug targets for triggering cell fate.

Zinc is an essential trace element for life. Zinc is not only an important nutrient, cofactor of numerous enzymes and transcription factors, but also it acts as an intracellular mediator, similarly to calcium. The recent discovery of its intracellular molecular pathways opens the door to new fields of drug design. Zinc homeostasis results from a coordinated regulation by different proteins involved in uptake excretion and intracellular storage/trafficking of zinc. These proteins are membranous transporters, belonging to the ZIP and ZnT families, and metallothioneins. Their principal function is to provide zinc to new synthesized proteins, important for several functions such as gene expression, immunity, reproduction or protection against free radicals damage. Zinc intracellular concentration is correlated to cell fate, ie proliferation, differentiation or apoptosis, and modifications of zinc homeostasis are observed in several pathologies affecting humans at any stage of life. Two zinc-related diseases, acrodermatitis enteropathica and the lethal milk syndrome, have been recently related to mutations in zinc transporters, SLC39A4 and ZnT-4, respectively. Zinc acts as an inhibitor of apoptosis, while its depletion induces programmed cell death in many cell lines. However, excess zinc can also be cytotoxic, and zinc transporters as well as metallothioneins serve as zinc detoxificating systems. Several zinc channels, controlling the intracellular zinc movements and the free form of the metal, maintain the intracellular zinc homeostasis, and thus the balance between life and cell death. Apart from these general activities, zinc has particular biological roles in some specialized cells. It acts as a paracrine regulator in pancreatic cell, neuron or neutrophil activity by a mechanism of vesicles-mediated metal excretion and uptake. A well knowledge on zinc transporters will be useful to develop new molecular targets to act on these zinc-regulated biological functions.

Molecular characterization of human and bovine ceruloplasmin using MALDI-TOF mass spectrometry.

Using SDS-PAGE and MALDI-TOF mass spectrometry, we investigated the difference in the molecular structure between human and bovine ceruloplasmin. In both cases, we found that the protein is present in two majors forms of different molecular mass. The difference between human and bovine ceruloplasmin was more obvious when characterized by MALDI-TOF than with the SDS-PAGE analysis. Furthermore, we established that the N-glycoside content of both enzymes is dissimilar and that the N-glycosyl moieties are distributed in a distinctive fashion in two glycoproteins. Finally, it appeared that both proteins exhibited different cleavage patterns after treatment with trypsin. This study indicates that human and bovine ceruloplasmin differ not only in sugar composition but also in primary structure.

Deglycosylated ceruloplasmin maintains its enzymatic, antioxidant, cardioprotective, and neuronoprotective properties.

Ceruloplasmin (CP), an important serum antioxidant, is a blue copper glycoprotein with ferroxidase and oxidase activities. Among other physiological actions, plasma CP was shown to protect isolated rat hearts and cultured P19 neurons exposed to oxidative stress conditions, raising the possibility of using this protein in the treatment of cardiac and neuronal diseases related to oxidative damage. However, since therapeutic applications of CP must be compatible with restrictions in the administration of blood derivatives to humans, there is a need to produce the protein by genetic engineering. To help in the choice of adequate expression systems, we undertook this study to determine if the carbohydrate moiety on the protein is essential for its functions. CP was completely deglycosylated using N-glycosidase F under nondenaturing conditions. Deglycosylated CP was found to retain most of the conformational, antioxidant, and enzymatic properties of the native protein in vitro. Moreover, both forms of the protein had similar cardioprotective and neuronoprotective effects against oxidative stress as evaluated with isolated rat hearts undergoing ischemia-reperfusion and with cultured P19 neurons exposed to xanthine-xanthine oxidase. The data thus indicate that the carbohydrate moiety of CP is not essential for its enzymatic and protective actions. Accordingly, even the use of expression systems that do not glycosylate mammalian proteins could provide a recombinant CP that retains its therapeutic potential.

Novel cardiac protective effects of urea: from shark to rat.

1. This study was carried out to investigate novel cardioprotective effects of urea and the underlying mechanisms. The cardiac functions under oxidative stress were evaluated using Langendorff perfused isolated heart. 2. Isolated dogfish shark hearts tolerated the oxidative stress generated by electrolysis (10 mA, 1 min) of the perfusion solution (n=4), and also showed normal cardiac functions during post-ischaemia reperfusion (n=4). The high concentration of urea (350 mM) in the heart perfusate was indispensable for maintaining the normal cardiac functions of the shark heart. 3. Urea at 3 - 300 mM (n=4 for each group) protected the isolated rat heart against both electrolysis-induced heart damage and post-ischaemia reperfusion-induced cardiac injury. 4. A concentration-dependent scavenging effect of urea (3 - 300 mM, n=4 for each group) against electrolysis-induced reactive oxygen species was also demonstrated in vitro. 5. Urea derivatives as hydroxyurea, dimethylurea, and thiourea had antioxidant cardioprotective effect against the electrolysis-induced cardiac dysfunction of rat heart, but were not as effective as urea in suppressing the post-ischaemia reperfusion injury. 6. Our results suggest that urea and its derivatives are potential antioxidant cardioprotective agents against oxidative stress-induced myocardium damage including the post-ischaemia reperfusion-induced injury.