Sub-lethal levels of amyloid ß-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons.

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aß) peptide. Current hypotheses regard soluble amyloid beta oligomers (AßOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aß fibrils and AßOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AßOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AßOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AßOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AßOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AßOs did not potentiate iron-induced cell death while iron chelation did not decrease AßOs-induced cell death. The lack of synergism between iron and AßOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.

[Febrile seizures in the first year of life: Dravet spectrum epilepsy?]. / Crisis con fiebre en el primer año de vida: ¿epilepsia del espectro Dravet?

INTRODUCTION: Dravet syndrome is a drug resistant epilepsy which starts in the first year of life with febrile seizures, followed by cognitive impairment and epilepsy with multiple seizure types. Diagnosis has been typically made at the age of three to four years, but earlier diagnosis is now possible as clinical features are better recognised and molecular diagnosis is available. PATIENTS AND METHODS: We studied a series of 14 children with Dravet syndrome or Dravet spectrum epilepsy. A screening test, developed by other authors to distinguish the febrile seizures in Dravet syndrome from febrile seizures from other origin, was applied to the clinical features of the seizures occurring during the first year of life in our patients. RESULTS: Clinical suspicion of Dravet spectrum epilepsy was possible in 100% of children in our series. Moreover, taking into consideration only the first seizure, 79% of patients scored sufficiently to detect Dravet syndrome. CONCLUSIONS: Dravet syndrome can be recognised during the first year of life. It is important that physicians are made aware of these clinical criteria capable to distinguish febrile seizures in Dravet syndrome from febrile seizures of other origin, and set up a protocol to collect appropriate data regarding febrile seizures occurring in the first year of life.

Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos.

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.

Iron dyshomeostasis in Parkinson's disease.

Owing to its ability to undergo one-electron reactions, iron transforms the mild oxidant hydrogen peroxide into hydroxyl radical, one of the most reactive species in nature. Deleterious effects of iron accumulation are dramatically evidenced in several neurodegenerative diseases. The work of Youdim and collaborators has been fundamental in describing the accumulation of iron confined to the substantia nigra (SN) in Parkinson's disease (PD) and to clarify iron toxicity pathways and oxidative damage in dopaminergic neurons. Nevertheless, how the mechanisms involved in normal neuronal iron homeostasis are surpassed, remain largely undetermined. How nigral neurons survive or succumb to iron-induced oxidative stress are relevant questions both to know about the etiology of the disease and to design neuroprotective strategies. In this work, we review the components of neural iron homeostasis and we summarize evidence from recent studies aimed to unravel the molecular basis of iron accumulation and dyshomeostasis in PD.

Copper overload affects copper and iron metabolism in Hep-G2 cells.

Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 microM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 microM Cu. (64)Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in K(m) compared with cells grown in 10 microM Cu. The decreased K(m) may represent an adaptive response to Cu deficiency. Cells treated with >50 microM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells.

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.

Iron-induced oxidative damage in colon carcinoma (Caco-2) cells.

Intestinal epithelial cells have an active apical iron uptake system that is involved in the regulated absorption of iron. By the action of this system, intestinal cells acquire increasing amounts of iron with time. Since intracellular reactive iron is a source of free radicals and a possible cause of colon carcinoma, this study analyzed the oxidative damages generated by iron accumulation in Caco-2 cells. Cells cultured with increasing concentrations of iron increased both total intracellular iron and the reactive iron pool, despite an active IRE/IRP system, which regulates intracellular iron levels. Increasing concentrations of iron resulted in increased protein oxidative damage, as shown by the immunoreactivity for 4-hydroxy-2-nonenal-modified proteins, and markedly induced DNA oxidation determined by 8-hydroxy-2'-deoxyguanidine production. Iron also impaired cell viability, resulting in increased cell death after 6 days of culture. In summary, iron accumulation by intestinal Caco-2 cells correlated with oxidative damage to proteins and DNA. Oxidative damage finally resulted in loss of cell viability. The Fe-induced oxidative damage observed may be relevant in understanding the cascade of events associated with iron-mediated colon carcinogenesis.

Prevalencia de las parasitosis intestinal en escolares a nivel de la comunidad / Prevalence of intestinal parasitosis in scholaships on community level

Estudio observacional de corte transversal, encuesta a niños escolares con edades comprendidas entre 8 y 10 años, en dos escuelas dela ciudad de hernandarias y dos de ciudad del Este durante los meses de mayo y agosto del año 2001. Se analizan 256 muestras de heces con los métodos, directo y de Kato-Katz. Resultado: De las 256 muestra 73 pto fueron positivas para algún tipo de parásitos. La edad promedio de los niños encuestados fue de 8,8 años. La prevalencia pra los gohelmintos encontrada fue de 5,08 pto, ascaris lumbricoides 2,34 pto y uncinarias 3,13pto. La prevalenica de otros helmintos como strongiloides stercolaris fue de 0,78pto, uxiurus vermicularis 1,56pto, himenolepis nana de 12, 80pto giardia lamblia de 42,18pto Conclusion: Los datos reflejan que la prevalencia de geohelmintos es baja, en cambio las parasitosis cosmopolitas como las giardias, himenolepsis nana y otros tienen prevalencia alta, semejante a la de otros centros urbanos de los países vecinos, según la epidemilogía de la literatura consultada

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells.

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.

The cellular mechanisms of body iron homeostasis.

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.

The cellular mechanisms of body iron homeostasis

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.

Transferrin stimulates iron absorption, exocytosis, and secretion in cultured intestinal cells.

The cellular mechanism by which basolateral transferrin (Tf) produces an increase in apical-to-basolateral Fe flux in Caco-2 cells was analyzed. After a pulse of 59Fe from the apical medium, three types of basolateral 59Fe efflux were found: a 59Fe efflux that was independent of the presence of Tf in the basolateral medium, a 59Fe efflux in which 59Fe left the cell bound to Tf, and a Tf-dependent 59Fe efflux in which 59Fe came off the cell not bound to Tf. Furthermore, addition of Tf to the basolateral medium doubled the exocytosis rate of Tf and increased the secretion of apolipoprotein A, a basolateral secretion marker. Both apotransferrin and Fe-containing Tf produced similar increases in 59Fe efflux, Tf exocytosis, and apolipoprotein A secretion. The Ca2+ channel inhibitor SKF-96365 inhibited both the Tf-mediated increase in transepithelial Fe transport and the secretion of apolipoprotein A. Thus the activation of transepithelial Fe transport by Tf seems to be mediated by Ca2+ entry into the cells.

Effect of copper, cadmium, mercury, manganese and lead on Fe2+ and Fe3+ absorption in perfused mouse intestine.

Body iron homeostasis is maintained by the regulation of iron absorption in the first portion of the small intestine. Previous studies have shown that some trace metal cations may have an inhibitory effect on inorganic iron absorption. In view of the possible nutritional and toxicological implications of this inhibition, we decided to study the effect of Cu2+, Cd2+, Hg2+, Pb2+, and Mn2+ on Fe2+ and Fe3+ intestinal uptake. To that end, the duodenal portion of the mouse intestine was perfused with 1 microM 55Fe either in the 2+ or 3+ redox state, in the absence or presence of a 10-fold molar excess of Cd2+, Cu2+, Hg2+, Mn2+, or Pb2+. It was found that Cd2+ and Cu2+, but not Hg2+, Mn2+, or Pb2+, significantly inhibited the uptake of Fe2+ (p < 0.001). Surprisingly, the observed inhibition by Cu2+ and Cd2+ was dependent on the iron redox state, since neither cation inhibited Fe3+ uptake by mouse duodenum. Similarly, no inhibition of Fe3+ uptake was observed with a 10-fold molar excess of Hg2+, Mn2+, or Pb2+. These results indicate that inhibition of iron uptake by Cu2+ and Cd2+ takes place only in a reducing environment. Therefore, under the common oxidative conditions found in nutrients no inhibition of non-heme Fe uptake by Cu2+ and Cd2+, as well as Mn2+, Hg2+ and Pb2+ is to be expected. We propose either that Fe2+ and Fe3+ are transported through two different mechanisms, or that the enterocyte-mediated reduction of Fe3+, and the ensuing transport of Fe2+ are processes coupled in such a way that they are refractory to inhibition by Cu2+ and Cd2+.

Apotransferrin and holotransferrin undergo different endocytic cycles in intestinal epithelia (Caco-2) cells.

Previous studies have demonstrated that diferric transferrin and apotransferrin compete for the binding to basolateral transferrin receptors and that transferrin-mediated iron uptake by Caco-2 cells is inhibited by apotransferrin to a larger extent than that predicted solely by receptor competition. This inhibition can have important implications in determining the net exchange of iron between intestinal cells and the basolateral milieu. Accordingly, we further characterized the endocytic cycles of apotransferrin and diferric transferrin in Caco-2 cells. We found that after internalization both apotransferrin and diferric transferrin recycled to the cell exterior, but that apotransferrin had a protracted endocytic cycle. Confocal microscopy studies revealed a different cellular distribution of apotransferrin and diferric transferrin; both were found in a compartment close to the basal membrane, but apotransferrin reached as well regions closer to the apical membrane. Moreover, the intracellular distribution of transferrin receptors was influenced by the iron load of transferrin; cells incubated with apotransferrin presented a more apical distribution of transferrin receptors than cells incubated with diferric transferrin. These results indicate for the first time that the endocytic cycle of transferrin receptors in intestinal epithelial cells is determined by the iron content of transferrin. They explain also the marked inhibitory effect of apotransferrin on transferrin-mediated iron uptake by Caco-2 cells, since incubation of cells with apotransferrin resulted in the actual sequestration of the receptor in the cell interior.

Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells.

In vertebrates, body Fe homeostasis is maintained through the regulation of its intestinal absorption. In addition, because Fe is both essential and toxic, intracellular Fe levels are tightly regulated. Consequently, intestinal epithelial cells are in the unique position of being responsible simultaneously for the regulation of body Fe absorption and the regulation of their intracellular Fe levels to remain viable. We tested the hypothesis that the regulation of transepithelial Fe transport and the regulation of intracellular Fe levels are sensitive to a common effector. To this end, we used a recently developed protocol to obtain cultured intestinal epithelial cells with defined intracellular Fe concentrations. In these cells we tested Fe absorption and Fe regulatory protein (IRP) activities. We found that transepithelial Fe transport was inversely related to 20-200 microM intracellular Fe and that Caco-2 cells expressed Fe regulatory protein-1 and Fe regulatory protein-2 activities. Fe regulatory protein-1 activity, Fe regulatory protein-2 mass, transferrin receptor density, and ferritin levels were regulated by intracellular Fe in the same range (20-200 microM) that affected transepithelial Fe transport. These results suggest that a common Fe-responsive factor regulates both intracellular Fe levels and Fe absorption by Caco-2 cells.

Intestinal epithelia (Caco-2) cells acquire iron through the basolateral endocytosis of transferrin.

Although the absorption of iron through the intestinal epithelia is inversely related to body iron stores, the mechanisms by which the enterocytes sense body iron stores are unknown. Polarized enterocytes have transferrin receptors in their basolateral surface; hence, we tested the hypothesis that the endocytosis of circulating transferrin may be part of the body's iron sensing mechanism. Particularly, we evaluated the contribution of basolateral transferrin to iron content of intestinal cells, and we investigated what factors modulate this contribution. For this purpose, we used the intestinal cell line Caco-2 grown on porous filters. When the cells were simultaneously offered equimolar amounts of iron, from the apical medium as 55Fe-nitrilotriacetate and from the basolateral medium as 59Fe-transferrin, most of the internalized iron came from the basolateral endocytosis of 59Fe-transferrin. Experiments of transferrin binding and internalization showed that holotransferrin and apotransferrin had similar numbers of basolateral receptors. The receptors had associated constants for diferric transferrin, monoferric transferrin, and apotransferrin of 2.69, 1.71 and 0.26 x 10(7) L/mol, respectively. The binding of diferric transferrin or monoferric transferrin to receptors was competitively inhibited by apotransferrin. Caco-2 cells, but not K562 cells, showed inhibition of basolateral, transferrin-mediated iron uptake by apotransferrin. This inhibition should regulate the net basolateral uptake of iron mediated by the endocytosis of Fe-containing transferrins. We propose that the basolateral endocytosis of transferrin forms part of the system by which intestinal epithelia cells sense plasma iron concentrations.

Regulation of Fe absorption by cultured intestinal epithelia (Caco-2) cell monolayers with varied Fe status.

Body Fe homeostasis is maintained through the regulation of Fe absorption by the intestinal epithelia. Working under the hypothesis that the intracellular concentration of Fe is instrumental in the control of its transepithelial flux, we investigated in vitro which steps in Fe absorption are regulated by cellular Fe content. For that study, Caco-2 cells containing different concentrations of intracellular 55Fe were grown in porous filters, and the apical-to-cell-to- basolateral flux of 59Fe was then determined. We found that 1) at low (up to 0.1 mM) intracellular Fe content the apical-to-basal Fe transport was primarily regulated by a decrease in apical Fe uptake (first stage of regulation), 2) at higher levels of intracellular Fe (0.1-1 mM) the transepithelial Fe flux was regulated by intracellular factors that sequester most of the Fe taken up at the apical surface (second stage of regulation), and 3) a fraction of the apical-to-basolateral Fe flux was not regulated by the intracellular concentration of Fe. Ferritin synthesis preceded the onset of the second stage of regulation, suggesting a causal relationship between intracellular Fe levels, ferritin levels, and regulation of Fe absorption.

Role of redox systems on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells.

Caco-2 cells were used as a model of human intestinal epithelium to investigate the role of redox systems in transepithelial transport of 59Fe3+. The cells reduced Fe3+ present in the apical medium; the reduction was 50% inhibited by adriamycin and p-chloromercuribenzoate. Addition of [14C]ascorbate to the basolateral medium resulted in accumulation of 14C radioactivity in both cells and apical medium; apical radioactivity increased with time and was probably caused by paracellular flux. The cells provided Fe3+ reduction capacity to the apical incubation medium. Addition of ascorbate to the basolateral medium increased this reduction capacity 2-fold and the cellular uptake of 59Fe3+ 1.8-fold. Adriamycin significantly inhibited both cellular 59Fe uptake and Fe transport into the basolateral side. The results indicate that Caco-2 cells reduce apical Fe3+ by two parallel mechanisms: by a plasma membrane ferrireductase and by the secretion of reductants of either cellular or basolateral origin. The data support a model for Fe3+ intestinal absorption in which cell-mediated Fe3+ reduction occurs before cellular Fe uptake.

Arginyl residues are involved in the transport of Fe2+ through the plasma membrane of the mammalian reticulocyte.

Sealed reticulocyte ghosts were treated with reagents that modify a variety of amino acid residues. Only ninhydrin and phenylglyoxal, both modifiers of arginyl residues, produced inhibition of the initial rate of 59Fe2+ uptake. The inhibition (i) was dependent on the concentration of ninhydrin or phenylglyoxal, (ii) increased from pH 7 to 9, a feature of the modification of arginine by ninhydrin or phenylglyoxal, and (iii) was blocked when Fe2+ was present during the modification step. A23187, an effective membrane Fe2+ transporter, diminished the inhibitory effect of ninhydrin and phenylglyoxal, indicative that the transport of iron through the membrane, and not a secondary process, was selectively inhibited. We conclude that the iron transporter from the plasma membrane of erythroid cells has one or more arginyl residues in a segment accessible to ninhydrin or phenylglyoxal, and that this residue is involved in the transmembrane transport of iron.