Effects of pH, reducing and alkylating reagents on the binding and Ca2+ release activities of inositol 1,4,5-triphosphate in the bovine adrenal cortex

In a wide variety of cells, inositol 1,4,5-triphosphate (IP3) is a second messenger which interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. When bovine adrenal cortex microsomes were incubated in the presence of dithiothreitol [(DTT) IC50 = 50 mM] or n-ethylmaleimide [(NEM) IC50 = 0.5 mM], they lost their IP3 binding capacity. Scatchard analysis of the binding data revealed that DTT decreased the affinity while NEM decreased the number of binding sites for IP3. The effect of DTT was reversible whereas the effect of NEM was permanent. pH variations between 6.5 and 9 increased the IP3 binding capacity of the microsomes. The effects of DTT, NEM, and pH on IP3-induced Ca2+ release from the microsomes were consistent with their effects on IP3 binding. Our data show that the binding sites for IP3 in the bovine adrenal cortex are proteins containing disulfide bridges and free sulfhydryl group(s) which are essential features for the recognition of IP3. These results also suggest that the binding sites for IP3 are the physiological receptors through which IP3 triggers the mobilization of Ca2+ in adrenal cortex in response to angiotensin II and other Ca2+ mobilizing ligands.     

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.     

Agonist-dependent AT(4) receptor internalization in bovine aortic endothelial cells

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.     

Identification of stop codon readthrough genes in Saccharomyces cerevisiae

We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI⁺] and [psi^–] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.     

Hormonal changes during 17 days of head-down bed-rest

We investigated in six men the impact of 17 days of head-down bed rest (HDBR) on the daily rhythms of the hormones involved in hydroelectrolytic regulation. This HDBR study was designed to mimic a real space flight. Urine samples were collected at each voiding before, during and after HDBR. Urinary excretion of Growth Hormone (GH), Cortisol, 6 Sulfatoxymelatonin, Normetadrenaline (NMN) and Metadrenaline (NM) was determined. A decrease in urinary cortisol excretion during the night of HDBR was noted. For GH, a rhythm was found before and during HDBR. The rhythm of melatonin, evaluated with the urine excretion of 6 Sulfatoxymelatonin (aMT6S), the main hepatic metabolite, persisted throughout the experiment without any modification to the level of phase. A decrease during the night was noted for normetadrenaline urinary derivates, but only during the HDBR.     

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form

We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. Conclusions: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.     

Genetic diversity within human erythroviruses: identification of three genotypes

B19 virus is a human virus belonging to the genus Erythrovirus: The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.     

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.     

Towards a computational model for -1 eukaryotic frameshifting sites

MOTIVATION: Unconventional decoding events are now well acknowledged, but not yet well formalized. In this study, we present a bioinformatics analysis of eukaryotic -1 frameshifting, in order to model this event. RESULTS: A consensus model has already been established for -1 frameshifting sites. Our purpose here is to provide new constraints which make the model more precise. We show how a machine learning approach can be used to refine the current model. We identify new properties that may be involved in frameshifting. Each of the properties found was experimentally validated. Initially, we identify features of the overall model that are to be simultaneously satisfied. We then focus on the following two components: the spacer and the slippery sequence. As a main result, we point out that the identity of the primary structure of the so-called spacer is of great importance. AVAILABILITY: Sequences of the oligonucleotides in the functional tests are available at http://www.igmors.u-psud.fr/rousset/bioinformatics/.     

Direct haplotyping of kilobase-size DNA using carbon nanotube probes

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.