RESUMEN
The pure Fano effect in angle-integrated valence-band photoemission of ferromagnets has been observed for the first time. A contribution of the intrinsic spin polarization to the spin polarization of the photoelectrons has been avoided by an appropriate choice of the experimental parameters. The theoretical description of the resulting spectra reveals a complete analogy to the Fano effect observed before for paramagnetic transition metals. While the theoretical photocurrent and spin-difference spectra are found in good quantitative agreement with experiment in the case of Fe and Co, only a qualitative agreement could be achieved in the case of Ni by calculations on the basis of plain local spin-density approximation. Agreement with experimental data could be improved in this case in a very substantial way by a treatment of correlation effects on the basis of dynamical mean field theory.
RESUMEN
Utilizing a sum rule in a spin-resolved photoelectron spectroscopic experiment with circularly polarized light, we show that the orbital moment in LaTiO3 is strongly reduced from its ionic value, both below and above the Ne el temperature. Using Ti L2,3 x-ray absorption spectroscopy as a local probe, we found that the crystal-field splitting in the t2g subshell is about 0.12-0.30 eV. This large splitting does not facilitate the formation of an orbital liquid.
RESUMEN
We have performed an angle resolved photoemission study on a single crystal of the optimally electron doped (n-type) cuprate superconductor Nd2-xCexCuO4 (x=0.15) at a photon energy of 400 eV. The Fermi surface is mapped out and is, in agreement with earlier measurements, of hole-type with the expected Luttinger volume. However, comparing with previous low energy measurements, we observe a different Fermi surface shape and a different distribution of spectral intensity around the Fermi surface contour. The observed Fermi surface shape indicates a stronger electron correlation in the bulk as compared to the surface.
RESUMEN
Orbital ordering (OO) in the layered perovskite La0.5Sr1.5MnO4 has been investigated using the enhanced sensitivity of soft x-ray resonant diffraction at the Mn L edges. The energy dependence of an OO diffraction peak over the L(2,3) edges is compared to ligand-field calculations allowing a distinction between the influences of Jahn-Teller distortions and spin correlations. The energy dependence of the diffraction peak at the Mn L1 edge is remarkably different from that observed at the Mn K edge.
RESUMEN
Treatment of U937 cells with nontoxic concentrations of TNFalpha increased the DNA strand scission induced by a short-chain lipid hydroperoxide analogue, tert-butylhydroperoxide. The following lines of evidence suggest that the enhancing effects of TNFalpha are mediated by inhibition of complex III and by the ensuing formation of superoxides and hydrogen peroxide: (a) the effects of TNFalpha were mimicked by the complex III inhibitor antimycin A; (b) the effects of TNFalpha, or antimycin A, were abolished by the complex I inhibitor rotenone, or by myxothiazol, an agent which inhibits the electron flow from the reduced coenzyme Q to cytochrome c(1) and therefore prevents ubisemiquinone formation; (c) the effects of TNFalpha, or antimycin A, were not observed in respiration-deficient cells; and (d) the effects of TNFalpha, or antimycin A, were sensitive to catalase. The TNFalpha-dependent inhibition of complex III appears to be mediated by ceramide. Three lines of evidence support this inference: (a) a synthetic cell-permeable ceramide analogue reproduced all the effects of TNFalpha, (b) TNFalpha promoted the formation of ceramide via a mechanism sensitive to inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFalpha-mediated enhancement of the tert-butylhydroperoxide-induced DNA-damaging response was prevented under conditions in which ceramide formation was inhibited.
Asunto(s)
Ceramidas/metabolismo , Daño del ADN , ADN de Cadena Simple/efectos de los fármacos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Peróxido de Hidrógeno/metabolismo , Esfingosina/análogos & derivados , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , terc-Butilhidroperóxido/farmacología , Antimicina A/farmacología , Calcio/metabolismo , Transporte de Electrón , Inhibidores Enzimáticos/farmacología , Humanos , Mitocondrias/metabolismo , Esfingosina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Células U937 , terc-Butilhidroperóxido/metabolismoRESUMEN
Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-alpha (TNF-alpha) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-alpha, however, ceramide potentiated, whereas NO inhibited, TNF-alpha-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-alpha-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.
Asunto(s)
Apoptosis/fisiología , Ceramidas/metabolismo , Óxido Nítrico/fisiología , Penicilamina/análogos & derivados , Factor de Necrosis Tumoral alfa/farmacología , Antígenos CD/fisiología , Apoptosis/efectos de los fármacos , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , GMP Cíclico/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Donantes de Óxido Nítrico/farmacología , Oxadiazoles/farmacología , Penicilamina/farmacología , Proteínas/metabolismo , Quinoxalinas/farmacología , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , S-Nitroso-N-Acetilpenicilamina , Sistemas de Mensajero Secundario , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacología , Factor 1 Asociado a Receptor de TNF , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacocinética , Células U937RESUMEN
Tumor necrosis factor-alpha (TNFalpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide. We have now studied the effects and mechanisms of action of NO on endocytosis, investigated with fluorescein isothiocyanate-labeled dextran using human monocyte-derived DC, both immature and after treatment with TNFalpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNFalpha. The intracellular accumulation of ceramide induced by TNFalpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNFalpha-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C(2)-ceramide. These effects of NO were mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylyl cyclase. At variance with rodents, the inducible isoform of the NO synthase was expressed neither in immature human DC nor after cell treatment with TNFalpha, interferon-gamma, and lipopolysaccharide, suggesting that regulation of these cells depends on exogenous NO. NO, working through cyclic GMP, might therefore prolong the ability of human DC to internalize antigens at the site of inflammation and thus modulate the initial steps leading to antigen-specific immune responses.
Asunto(s)
GMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Endocitosis/fisiología , Óxido Nítrico/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Células Cultivadas , Ceramidas/farmacocinética , Técnicas de Cocultivo , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacocinética , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Esfingosina/análogos & derivados , Esfingosina/farmacocinética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.
Asunto(s)
Fertilización/fisiología , Isoenzimas/fisiología , Mitosis/fisiología , Óvulo/enzimología , Fosfolipasas de Tipo C/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario , Femenino , Glutatión Transferasa/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Masculino , Datos de Secuencia Molecular , Fosfolipasa C gamma , Pruebas de Precipitina , ARN Mensajero , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/fisiología , Erizos de Mar , Interacciones Espermatozoide-Óvulo/fisiología , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/aislamiento & purificación , Dominios Homologos srcRESUMEN
Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cgamma (PLCgamma), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCgamma revealed the presence of this protein in cortical regions. It was also observed that PLCgamma displayed calcium-sensitive activity. The present results suggest that PLCgamma may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.
Asunto(s)
Óvulo/enzimología , Erizos de Mar/embriología , Fosfolipasas de Tipo C/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Fertilización/fisiología , Inmunohistoquímica , Microscopía Electrónica , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/análisis , Erizos de Mar/inmunología , Transducción de Señal , Tirosina/fisiologíaRESUMEN
We have reported earlier that the polyphosphoinositide messenger system may control mitosis in sea urchin eggs. Besides phospholipase C activation and its second messengers, phosphatidylinositol (PI) 3-kinase has been proposed to affect a wide variety of cellular processes in other cellular systems. Therefore, we have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. Our data presented here suggest that PI 3-kinase is present in sea urchin eggs. We found that wortmannin, an inhibitor of PI 3-kinase, led to arrest of the cell cycle. Chromosome condensation, nuclear envelope breakdown, microtubular aster polymerization, protein and DNA synthesis were not affected when fertilization was performed in the presence of the drug. However, maturation-promoting factor (MPF) activation was inhibited and centrosome duplication was perturbed preventing the formation of a bipolar mitotic spindle in wortmannin treated eggs. We discuss how PI 3-kinase might be involved in the cascade of events leading to the first mitotic divisions of the fertilized sea urchin egg.
Asunto(s)
Androstadienos/farmacología , Mitosis/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Cigoto/citología , Cigoto/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN/biosíntesis , Activación Enzimática , Factor Promotor de Maduración/análisis , Factor Promotor de Maduración/metabolismo , Micotoxinas , Membrana Nuclear/metabolismo , Fosfatidilinositol 3-Quinasas/análisis , Fosfolipasas A/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteínas/metabolismo , Erizos de Mar , Huso Acromático/efectos de los fármacos , Wortmanina , Cigoto/enzimologíaRESUMEN
MAP kinases have been implicated in the control of a broad spectrum of cellular events in many types of cells. In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state (in G0 or G2) only and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. We have reinvestigated this problem in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. We used anti-ERK1 antibodies or substrates specific for different MAP kinases, and performed in-gel phosphorylation experiments. Our results suggest that an ERK1-like protein was responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Furthermore, we observed that wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibited the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. We also discuss how the activities of MPF and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Mitosis/fisiología , Factor de Transcripción Activador 2 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Glucógeno Sintasa Quinasa 3 , Leucina Zippers , Factor Promotor de Maduración/fisiología , Meiosis/efectos de los fármacos , Proteína Básica de Mielina/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Erizos de Mar , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cigoto/citología , Cigoto/efectos de los fármacos , Cigoto/enzimologíaRESUMEN
In most deuterostome eggs, a transient increase in the intracellular free calcium concentration (Cai) occurs after fertilization and is essential for egg activation. How one spermatozoo triggers this calcium signal remains an enigma. In the light of our own results, we suggest a model, leading to the calcium signal and common to invertebrate and mammalian systems, where phosphorylation on tyrosine and phospholipase C gamma (PLC gamma) are interconnected pathways stimulated by a multimolecular complex involving integrins.
Asunto(s)
Fertilización/fisiología , Modelos Biológicos , Oocitos/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/fisiología , Animales , Canales de Calcio/fisiología , Femenino , Integrinas/fisiología , Invertebrados , Isoenzimas/fisiología , Masculino , Mamíferos , Fosfolipasa C gamma , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/fisiología , Tirosina/fisiologíaRESUMEN
It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By performing immunofluorescence labelling and Western blots with antibodies directed against some of the alpha and beta subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, alpha 2 and alpha 5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that alpha 2 and alpha 5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich in three cytoskeletal proteins known to be linked to the beta chain of integrins: talin, vinculin and alpha-actinin. Moreover, we found that talin and alpha-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.