Study by a rapid mixing method of the interaction between a fluorescent cholinergic agonist and membrane fragments rich in cholinergic receptors from Torpedo marmorata]

The analysis by stopped-flow of the interaction of fluorescent agonist (C5DACho1) with the acetylcholine receptor in its membrane-bound form reveals several kinetic steps: a fast one, in the millisecond range, associated with the binding of C5DACho1 to a high affinity state and a "medium" and "slow" one, the last one representing possibly an isomerisation of the receptor molecule towards the high affinity state.     

The genome of Theobroma cacao

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.     

Targets and dynamics of promoter DNA methylation during early mouse development

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.     

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell–cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.     

Interdependence of behavioural variability and response to small stimuli in bacteria

The chemotaxis signalling network in Escherichia coli that controls the locomotion of bacteria is a classic model system for signal transduction. This pathway modulates the behaviour of flagellar motors to propel bacteria towards sources of chemical attractants. Although this system relaxes to a steady state in response to environmental changes, the signalling events within the chemotaxis network are noisy and cause large temporal variations of the motor behaviour even in the absence of stimulus. That the same signalling network governs both behavioural variability and cellular response raises the question of whether these two traits are independent. Here, we experimentally establish a fluctuation-response relationship in the chemotaxis system of living bacteria. Using this relationship, we demonstrate the possibility of inferring the cellular response from the behavioural variability measured before stimulus. In monitoring the pre- and post-stimulus switching behaviour of individual bacterial motors, we found that variability scales linearly with the response time for different functioning states of the cell. This study highlights that the fundamental relationship between fluctuation and response is not constrained to physical systems at thermodynamic equilibrium but is extensible to living cells. Such a relationship not only implies that behavioural variability and cellular response can be coupled traits, but it also provides a general framework within which we can examine how the selection of a network design shapes this interdependence.     

Metabolic and redox signaling in the retina

Visual perception by photoreceptors relies on the interaction of incident photons from light with a derivative of vitamin A that is covalently linked to an opsin molecule located in a special subcellular structure, the photoreceptor outer segment. The photochemical reaction produced by the photon is optimal when the opsin molecule, a seven-transmembrane protein, is embedded in a lipid bilayer of optimal fluidity. This is achieved in vertebrate photoreceptors by a high proportion of lipids made with polyunsaturated fatty acids, which have the detrimental property of being oxidized and damaged by light. Photoreceptors cannot divide, but regenerate their outer segments. This is an enormous energetic challenge that explains why photoreceptors metabolize glucose through aerobic glycolysis, as cancer cells do. Uptaken glucose produces metabolites to renew that outer segment as well as reducing power through the pentose phosphate pathway to protect photoreceptors against oxidative damage.     

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP^C), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP₉₁. Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.     

Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior

The breast cancer resistance protein, also known as ABCG2, is one of the most highly studied ATP-binding cassette (ABC) transporters because of its ability to confer multidrug resistance. The lack of information on the physiological role of ABCG2 in humans severely limits cancer chemotherapeutic approaches targeting this transporter. We report here that ABCG2 comprises the molecular basis of a new blood group system (Junior, Jr) and that individuals of the Jr(a-) blood type have inherited two null alleles of ABCG2. We identified five frameshift and three nonsense mutations in ABCG2. We also show that the prevalence of the Jr(a-) blood type in the Japanese and European Gypsy populations is related to the p.Gln126* and p.Arg236* protein alterations, respectively. The identification of ABCG2(-/-) (Jr(a-)) individuals who appear phenotypically normal is an essential step toward targeting ABCG2 in cancer and also in understanding the physiological and pharmacological roles of this promiscuous transporter in humans.     

Functional impact of PTP1B-mediated Src regulation on oxidative phosphorylation in rat brain mitochondria

Given the presence of Src and PTP1B within rat brain mitochondria, we have investigated whether PTP1B regulates Src activity in mitochondria as in the cytosol. Results showed that Src was stimulated by in vitro addition of ATP to mitochondria, and this stimulation was reversed by a membrane-permeable allosteric inhibitor of PTP1B and by a potent selective Src inhibitor. They also indicated a direct action of PTP1B on phosphorylated tyrosine 527 residue of Src, thus implicating a role for PTP1B in the modulation of Src activity in mitochondria. Putative Src and PTP1B substrates were identified by liquid chromatography tandem mass spectrometry and two-dimensional blue native/SDS-PAGE. Both inhibitors inhibited ADP-stimulated respirations concurrently with Src activation and complex IV activation by ATP, while having no effect or increasing the activity of the other complexes. Our analysis emphasizes the regulatory function of Src and its modulation by PTP1B on oxidative phosphorylation in mitochondria.