Philippe Marin — Page 2

PubMed2002

High performance two-dimensional gel electrophoresis using a wetting agent Tergitol NP7.

Two-dimensional electrophoresis is an efficient method for the analysis of a broad range of complex protein samples. Current two-dimensional gel techniques are not suited for analysis of the small amount of proteins from tissue samples in the presence of high concentration of salts. Here we describe an improved two-dimensional gel electrophoresis procedure based on the use of a nonionic wetting agent, Tergitol NP7, in rehydration solution combined with the application of a linear potential sweep during isoelectrofocusing. This experimental approach yields a dramatic increase in the resolution and focusing of proteins visualized on two-dimensional gels. This technique is less time-consuming and laborious than the current techniques and can be used for a variety of two-dimensional electrophoresis applications, including proteome analysis.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Akt mediates the anti-apoptotic effect of NMDA but not that induced by potassium depolarization in cultured cerebellar granule cells.

Apoptosis of cultured cerebellar granule neurons (CGNs) deprived of serum is prevented by K+ depolarization or moderate concentrations of N-methyl-d-aspartate (NMDA). Here, we have examined the role of the serine/threonine kinase Akt in these protective effects. The exposure of mouse CGNs to NMDA or K+ depolarization increased the phosphorylation of Akt, compared with that measured in cells incubated in a physiological K+ concentration. Only the NMDA-evoked response was reduced by inhibitors of phosphatidylinositol 3-kinase (wortmannin and LY294002) and mitogen-activated protein kinase (PD98059 and U0126). Similarly, the capacity of NMDA to inhibit apoptosis of CGNs deprived of serum was greatly reduced by these inhibitors as well as by the transfection of neurons with a catalytically inactive mutant of Akt, whereas the protective effect of K+ depolarization remained unaffected. These findings indicate that K+ depolarization and NMDA activate Akt through different signalling pathways in CGNs. Moreover, Akt mediates the anti-apoptotic effect of NMDA, but not that evoked by K+ depolarization.

Animals↗

PubMed1991

Synergistic Regulation of Cytosolic Ca2+ Concentration by Adenosine and alpha1-Adrenergic Agonists in Mouse Striatal Astrocytes.

Adenosine has a broad array of actions on neurons but astrocytes also possess adenosine receptors. We have previously shown that adenosine, by acting on astrocytes in the striatum, can modulate neuronal responses mediated by receptors coupled to phospholipase C through an astrocyto - neuronal interaction. In addition, adenosine was found to potentiate the alpha1-adrenergic production of inositol phosphates in astrocytes. The mechanism involved in this potentiation was further investigated by examining the effects of adenosine and alpha1-adrenergic receptor agonists on cytosolic Ca2+ in cultured striatal astrocytes from the embryonic mouse in primary culture. When used alone, methoxamine, a selective agonist of alpha-adrenergic receptors or 2-chloroadenosine, a stable analogue of adenosine, induced a transitory increase in cytosolic Ca2+, but their combined addition led to a sustained increase in cytosolic Ca2+, which seems to be due to a Ca2+ influx, because it was not observed in the absence of external Ca2+. Voltage independent Ca2+ channels contribute to this process and different blockers of voltage-operated calcium channels, such as dihydropyridines, phenylalkylamines, La3+ or Co2+ were ineffective in suppressing the sustained cytosolic Ca2+ elevation. Three observations suggest the implication of arachidonic acid in the observed potentiation: (i) arachidonic acid induced a sustained elevation of cytosolic Ca2+ similar to that evoked by the coapplication of methoxamine and 2-chloroadenosine; (ii) the addition of arachidonic acid during the calcic plateau produced by the combined application of the agonists did not increase further cytosolic Ca2+ levels; (iii) in the presence of methoxamine, 2-chloroadenosine induced a release of arachidonic acid. The stimulation of phospholipase C and the resulting activation of protein kinase C induced by methoxamine seem to be required for the potentiating effect of 2-chloroadenosine on cytosolic Ca2+. In fact, the direct activation of protein kinase C by an exogenous diacylglycerol analogue mimicked the effect of methoxamine because, in this condition, 2-chloroadenosine alone evoked a sustained elevation of cytosolic Ca2+. Therefore, methoxamine, through the successive activation of phospholipase C and protein kinase C, could allow a lipase, probably phospholipase A2, to be stimulated by 2-chloroadenosine. Arachidonic acid has already been shown to trigger the opening of K+ channels and the formation of inositol phosphates in other cell types. Therefore, in striatal astrocytes, 2-chloroadenosine, through an arachidonic acid-mediated hyperpolarization, could increase the Ca2+ driving force and thus improve Ca2+ influx through inositol phosphate-gated channels. This hypothesis is further supported by the suppressing effect of a 50 mM KCI-induced depolarization on the long lasting elevation of cytosolic Ca2+ seen in the combined presence of 2-chloroadenosine and methoxamine.

Journal Article↗