Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.     

Antioxidant responses of cortex neurons to iron loading

Brain cells have a highly active oxidative metabolism, yet they contain only low to moderate superoxide dismutase and catalase activities. Thus, their antioxidant defenses rely mainly on cellular reduced glutathione levels. In this work, in cortical neurons we characterized viability and changes in reduced and oxidized glutathione levels in response to a protocol of iron accumulation. We found that massive death occurred after 2 days in culture with 10 microM Fe. Surviving cells developed an adaptative response that included increased synthesis of GSH and the maintenance of a glutathione-based reduction potential. These results highlight the fundamental role of glutathione homeostasis in the antioxidant response and provide novel insights into the adaptative mechanisms of neurons subjected to progressive iron loads.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Quiescence induced by iron challenge protects neuroblastoma cells from oxidative stress

The brain uses massive amounts of oxygen, generating large quantities of reactive oxygen species (ROS). Because of its lipid composition, rich in unsaturated fatty acids, the brain is especially vulnerable to ROS. Furthermore, oxidative damage in the brain is often associated with iron, which has pro-oxidative properties. Iron-mediated oxidative damage in the brain is compounded by the fact that brain iron distribution is non-uniform, being particularly high in areas sensitive to neurodegeneration. This work was aimed to further our understanding of the cellular mechanisms by which SHSY5Y neuroblastoma cells adapt to, and survive increasing iron loads. Using an iron accumulation protocol that kills about 50% of the cell population, we found by cell sorting analysis that the SHSY5Y sub-population that survived the iron loading arrested in the G(0) phase of the cell cycle. These cells expressed neuronal markers, while their electrical properties remained largely unaltered. These results suggest that upon iron challenge, neuroblastoma cells respond by entering the G(0) phase, somehow rendering them resistant to oxidative stress. A similar physiological condition might be involved in neuronal survival in tissues known to accumulate iron with age, such as the hippocampus and the substantia nigra pars compacta.     

Selective hypoxia-cytotoxins based on vanadyl complexes with 3-aminoquinoxaline-2-carbonitrile-N1,N4-dioxide derivatives

A new vanadyl complex with the formula VO(L1)2, where L1=3-amino-6(7)-chloroquinoxaline-2-carbonitrile N(1), N(4)-dioxide, has been synthesized and characterized by elemental analyses, conductometry, fast atom bombardment mass spectroscopy (FAB-MS) and electronic, Fourier transform infrared (FTIR), Raman, nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies. Results were compared with those previously reported for analogous vanadium complexes with other 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives as ligands. As an effort to develop novel metal-based selective hypoxia-cytotoxins and to improve bioavailability and pharmacological and toxicological properties of aminoquinoxaline carbonitrile N-dioxides bioreductive prodrugs, the new complex and VO(L)2 complexes, with L=3-amino-6(7)-bromoquinoxaline-2-carbonitrile N1,N4-dioxide (L2) and 3-amino-6(7)-methylquinoxaline-2-carbonitrile N1,N4-dioxide (L3), were subjected to cytotoxic evaluation in V79 cells in hypoxic and aerobic conditions. The complexes resulted in vitro more potent cytotoxins than the free ligands (i.e. potencies P(VO(L1)2)=3.0, P(L1)=9.0 microM) and Tirapazamine (P=30.0 microM) and showed excellent selective cytotoxicity in hypoxia, being no cytotoxic in oxia. In addition, the solubility in hydrophilic solvents resulted significantly higher for the vanadyl complexes than for the free ligands. These results could be indicative that complexation of the quinoxaline-2-carbonitrile N1,N4-dioxide derivatives with vanadium could improve their bioavailability. In addition, a new aspect of the series has been investigated. A detailed comparison of the electrochemical behavior of the free ligands and the complexes has been performed searching for a correlation between reduction potentials of the complexes and their activities and hypoxia selectivities.     

Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276–1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, β-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and β-2 microglobulin (β2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE–β2m to the apical membrane. The amount of HFE–β2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or β2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE–β2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.     

Iron toxicity in neurodegeneration

Iron is an essential element for life on earth, participating in a plethora of cellular processes where one-electron transfer reactions are required. Its essentiality, coupled to its scarcity in aqueous oxidative environments, has compelled living organisms to develop mechanisms that ensure an adequate iron supply, at times with disregard to long-term deleterious effects derived from iron accumulation. However, iron is an intrinsic producer of reactive oxygen species, and increased levels of iron promote neurotoxicity because of hydroxyl radical formation, which results in glutathione consumption, protein aggregation, lipid peroxidation and nucleic acid modification. Neurons from brain areas sensitive to degeneration accumulate iron with age and thus are subjected to an ever increasing oxidative stress with the accompanying cellular damage. The ability of these neurons to survive depends on the adaptive mechanisms developed to cope with the increasing oxidative load. Here, we describe the chemical and thermodynamic peculiarities of iron chemistry in living matter, review the components of iron homeostasis in neurons and elaborate on the mechanisms by which iron homeostasis is lost in Parkinson's disease, Alzheimer's disease and other diseases in which iron accumulation has been demonstrated.     

Novel vanadyl complexes with quinoxaline N(1),N(4)-dioxide derivatives as potent in vitro insulin-mimetic compounds

As a contribution to the development of novel vanadyl complexes with potential insulin-mimetic activity, three new oxovanadium(IV) complexes with the formula VO(L)(2), where L are 3-amino-quinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives, have been synthesized. Complexes have been characterized by elemental and thermal analyses, fast atom bombardment mass spectroscopy (FAB-MS), conductivity measurements and electronic, Fourier transform infrared (FTIR) and electron paramagnetic resonance (EPR) spectroscopies. The in vitro insulin-mimetic activity of the vanadyl complexes has been estimated by lipolysis inhibition tests, in which the inhibition of the release of free fatty acid from isolated rat adipocytes treated with epinephrine was determined. All the complexes showed inhibitory effects on free fatty acid release. [V(IV)O(3-amino-6(7)-bromoquinoxaline-2-carbonitrile N(1),N(4)-dioxide)(2)] exhibited higher in vitro insulin-mimetic activity than the very active bis(6-methylpicolinato)oxovanadium(IV), VO(6mpa)(2). This new vanadyl complex is expected to exhibit a higher blood glucose lowering activity than VO(6mpa)(2) in diabetic animals.     

hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein. Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-α, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling.