Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.     

Influence of vegetation on the accumulation of peat in southern Quebec

Mirabel bog (southern Quebec) was studied with the aim of specifying the past influence of local vegetation on peat accumulation. This influence was essentially translated by the reduction of minerotrophic inputs and peat accumulation induced by the highering of the peatland surface. Extremely rich initial conditions allow to explain the very high accumulations of the beginning of the Holocene, observed in various plant communities, among which Larix forests with ferns and Carex without modern analogues. Moreover, the structure (forest density) and the composition (dominance of Cyperaceae or Ericaceae) of these communities have locally controlled the dynamic of peat accumulation. These results point out the interest of the confrontation between palynological and macrofossil data and the spatialisation of palaeoecological studies.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

Arsenite oxidase,an ancient bioenergetic enzyme

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.     

Combining quantitative trait Loci analysis and an ecophysiological model to analyze the genetic variability of the responses of maize leaf growth to temperature and water deficit

Ecophysiological models predict quantitative traits of one genotype in any environment, whereas quantitative trait locus (QTL) models predict the contribution of alleles to quantitative traits under a limited number of environments. We have combined both approaches by dissecting into effects of QTLs the parameters of a model of maize (Zea mays) leaf elongation rate (LER; H. Ben Haj Salah, F. Tardieu [1997] Plant Physiol 114: 893-900). Response curves of LER to meristem temperature, water vapor pressure difference, and soil water status were established in 100 recombinant inbred lines (RILs) of maize in six experiments carried out in the field or in the greenhouse. All responses were linear and common to different experiments, consistent with the model. A QTL analysis was carried out on the slopes of these responses by composite interval mapping confirmed by bootstrap analysis. Most QTLs were specific of one response only. QTLs of abscisic acid concentration in the xylem sap colocalized with QTLs of response to soil water deficit and conferred a low response. Each parameter of the ecophysiological model was computed as the sum of QTL effects, allowing calculation of parameters for 11 new RILs and two parental lines. LERs were simulated and compared with measurements in a growth chamber experiment. The combined model accounted for 74% of the variability of LER, suggesting that it has a general value for any RIL under any environment.     

Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting

Ghrelin, a circulating growth-hormone releasing peptide derived from stomach, stimulates food intake through neuropeptide Y (NPY) neurons of the arcuate nucleus in the hypothalamus (ARC). We examined the effect of ghrelin microinjected into the ARC and the influence of intracerebroventricular (i.c.v.) pretreatment with a GHRH or NPY receptor antagonist on ghrelin-induced food intake in free-feeding male rats. Ghrelin (0.1-1 microg) stimulated food intake in a dose-dependent manner, and this effect was reduced by 55-60% by the Y(5) NPY receptor antagonist (10 microg i.c.v.), but not by the GHRH receptor antagonist MZ-4-71 (10 microg i.c.v.). We also evaluated the effects of passive ghrelin immunoneutralization by the microinjection of anti-ghrelin immunoglobulins (IgGs) intracerebroventricularly or directly into the ARC on food intake in free-feeding and fasted male rats. i.c.v. administration of anti-ghrelin IgGs decreased cumulative food intake over 24 h, whereas microinfusion of anti-ghrelin IgGs into the ARC induced only a short-lived (2 and 6 h) effect. Collectively, these data would indicate that centrally derived ghrelin has a major role in the control of food intake in rats and, in this context, blood-born ghrelin would be effective only in relation to its ability to reach the ARC, which is devoid of blood-brain barrier.     

Chl4p and iml3p are two new members of the budding yeast outer kinetochore

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.