Michel Hugues

Quantitative autoradiographic mapping in rat brain of the receptor of apamin, a polypeptide toxin specific for one class of Ca2+-dependent K+ channels

The localization of the receptor for apamin, a specific toxin for one class of sensitive Ca2+-dependent K+ channel, was studied in rat brain using an in vitro autoradiographic technique. Radiolabeled monoiodoapamin binds specifically to rat brain sections with a high affinity (Kd = 25 pM) to a single class of sites. Autoradiograms demonstrated a very heterogeneous distribution of the apamin receptor throughout the brain. Very high grain densities were localized on the habenula, lateral septum, supraoptic and suprachiasmatic nuclei. Areas containing high levels of apamin binding sites included anterior olfactory nucleus, stratum oriens of hippocampus, pontine nuclei and granular layer of the cerebellar cortex and inferior olive. The thalamus, some nuclei of hypothalamus, hippocampus, tegmental area, red and oculomotor nuclei, vestibular nuclei and superior olive, among others, presented intermediate grain densities. In the other main areas, in particular basal ganglia, raphe, low to very low levels of apamin binding sites have been observed.

Identification of a protein component of the Ca2+-dependent K+ channel by affinity labelling with apamin

Apamin, a selective inhibitor of the Ca 2+ -dependent K + channel, was cross-linked to its receptor on the channel structure using disuccinimidyl suberate (DSS). This covalent labelling indicates that the Ca 2+ -dependent K + channel of synaptic membranes has a protein component having a MW of about 28,000.

Establishment and Characterization of a Betacyanin Producing Cell Line of Amaranthus tricolor: Inductive effects of Light and Cytokinin

Summary The cell line isolated from callus obtained from Amaranthus tricolor seedlings proved to be capable of producing betacyanins.HPLC analysis showed that in vitro, as in the seedling, amaranthin (associated with its isomer, isomaranthin) is synthesized.Studies of the influence of light and cytokinins on biosynthesis of these pigments in cell suspensions demonstrated the inductive role of these two factors.The synergistic effect of light and cytokinins seems to favour an interaction between the two inducing factors.However, the results obtained with DRB, which specifically inhibits kinetin-induced synthesis without modifying the photochemical response, suggest that light and cytokinins act independently on some stages of biosynthesis of these pigments.

Polypeptide constitution of receptors for apamin, a neurotoxin which blocks a class of Ca2+-activated K+ channels

Affinity labelling experiments with different azido‐125I‐apamin derivatives were carried out to identify polypeptide components of the apamin‐sensitive Ca2+‐activated K+ channel in brain and pheochromocytoma cell membranes. Different polypeptides were labelled with different apamin derivatives. The major component has a molecular mass of about 30 kDa but other components at 45, 58 and 86 kDa were also identified. Results obtained with brain membranes on one hand and pheochromocytoma cells on the other were not exactly identical and suggest that there are sub‐types of apamin receptors.

Mapacalcine Protects Mouse Neurons against Hypoxia by Blocking Cell Calcium Overload

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

Distribution of mapacalcine receptors in the central nervous system of rat using the [125I]-labeled mapacalcine derivative

Mapacalcine is a dimeric protein of Mr 19041 extracted from the marine sponge Cliona vastifica. Electrophysiological and pharmacological approaches have demonstrated that mapacalcine was blocking a calcium channel different from N-, L-, P-, T- or Q-type calcium channels on mouse intestinal smooth muscle. Recently a [125I]-labeled derivative of mapacalcine has been synthesized and characterized as a tool usable as a probe to investigate mapacalcine receptors. On rat brain membranes, it binds to its receptor with a K(d)=0.35 nM and a maximal binding capacity of 706 fmol/mg protein. We use here [125I]-mapacalcine to study the mapping of its receptors in the rat brain. Data obtained show a practically homogeneous labeling of the brain. Our experiments suggest that mapacalcine receptors are present on neuronal and glial cells. Interestingly, choroid plexus demonstrates a high density of mapacalcine receptors. These data would suggest that mapacalcine sensitive calcium channels could be involved in the control of calcium homeostasis of the cerebrospinal fluid.

Characterization of mapacalcine-sensitive Ca2+ channels in rat kidney

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.

Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.