Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Characterization of pea chloroplastic carbonic anhydrase. Expression inEscherichia coli and site-directed mutagenesis

A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed inE. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.     

Non-linear loss of suitable wine regions over Europe in response to increasing global warming

Evaluating the potential climatic suitability for premium wine production is crucial for adaptation planning in Europe. While new wine regions may emerge out of the traditional boundaries, most of the present-day renowned winemaking regions may be threatened by climate change. Here, we analyse the future evolution of the geography of wine production over Europe, through the definition of a novel climatic suitability indicator, which is calculated over the projected grapevine phenological phases to account for their possible contractions under global warming. Our approach consists in coupling six different de-biased downscaled climate projections under two different scenarios of global warming with four phenological models for different grapevine varieties. The resulting suitability indicator is based on fuzzy logic and is calculated over three main components measuring (i) the timing of the fruit physiological maturity, (ii) the risk of water stress and (iii) the risk of pests and diseases. The results demonstrate that the level of global warming largely determines the distribution of future wine regions. For a global temperature increase limited to 2°C above the pre-industrial level, the suitable areas over the traditional regions are reduced by about 4%/°C rise, while for higher levels of global warming, the rate of this loss increases up to 17%/°C. This is compensated by a gradual emergence of new wine regions out of the traditional boundaries. Moreover, we show that reallocating better-suited grapevine varieties to warmer conditions may be a viable adaptation measure to cope with the projected suitability loss over the traditional regions. However, the effectiveness of this strategy appears to decrease as the level of global warming increases. Overall, these findings suggest the existence of a safe limit below 2°C of global warming for the European winemaking sector, while adaptation might become far more challenging beyond this threshold.     

The Glucose-Related Decrease in Polar Auxin Transport During Ripening and its Possible Role in Grapevine Berry Coloring

Journal of Plant Growth Regulation - Auxin is a hormone that delays ripening in part by reducing anthocyanin content and impairing color development. Auxin content declines during the ripening...     

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-convertingenzyme (ACE) to study the involvement of its glycanic chainsin its physicochemical and catalytic properties. The effectsof endoglycosidases F₂ and H, and of N-glycanase were assessedby ACE mobility in SDS-PAGE. N-Glycanase only was completelyeffective with or without previous denaturation, leading toa shift in ACE M_r from 172 to 135 kDa; endoglycosidase F₂ producedthe same shift but only without previous denaturation. DeglycosylatedACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine,and an identical Stokes radius as measured by size-exclusionhigh performance liquid chromatography. Neuraminidase had noeffect on ACE Stokes radius but slightly decreased its kcatwhich could be related to variations in ionization of the activesite. The isoelectric point of ACE, as, determined by isoelectricfocusing, increased from 4.5–4.8 to 5.0–5.3 aftereither endoglycosidase F₂ or neuraminidase digestion, but stillwith microheterogeneities which thus did not seem to be relatedto ACE glycans. Deglycosylated ACE did not bind onto agaroselectinsin contrast to native ACE which bound strongly to concanavalinA showing interactions involving oligomannosidic or biantennaryand sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin,an inhibitor of N-glycosylation, did not modify ACE secretionby endothelial cells. Thus, ACE glycans have no drastic effectson structural and biological properties of the protein, butthey may have a functional role on intracellular targeting ofboth secreted and membrane-bound ACE isoforms, also for theprotection of the soluble plasma form against hepatic lectinsand the maintenance of its hydrosolubility. converting enzyme (peptidyldipeptidase EC.3.4.15.1) endothelium glycosidases lectins     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.