15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.     

An association analysis for 14 candidate genes mapping to meat quality quantitative trait loci in a Duroc pig population reveals that the ATP1A2 genotype is highly associated with muscle electric conductivity

In previous GWAS carried out in a Duroc commercial line (Lipgen population), we detected on pig chromosomes 3, 4 and 14 several QTL for gluteus medius muscle redness (GM a*), electric conductivity in the longissimus dorsi muscle (LD CE) and vaccenic acid content in the LD muscle (LD C18:1 n − 7), respectively. We have genotyped, in the Lipgen population, 19 SNPs mapping to 14 genes located within these QTL. Subsequently, association analyses have been performed. After correction for multiple testing, two SNPs in the TGFBRAP1 (rs321173745) and SELENOI (rs330820437) genes were associated with GM a*, whereas ACADSB (rs81449951) and GPR26 (rs343087568) genotypes displayed significant associations with LD vaccenic content. Moreover, the polymorphisms located at the ATP1A2 (rs344748241), ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) genes showed significant associations with LD CE. We made a second round of association analyses including the SNPs mentioned above as well as other SNPs located in the chromosomes to which they map. After performing a correction for multiple testing, the only association that remained significant at the chromosome-wide level was that between the ATP1A2 genotype and LD CE. From a functional point of view, this association is meaningful because this locus encodes a subunit of the Na⁺/K⁺-ATPase responsible for maintaining an electrochemical gradient across the plasma membrane.     

Expression patterns of two genes for the delta-subunit of mitochondrial F1-ATP synthase from sweet potato in transgenic tobacco plants and cells.

Two nuclear genes, F1 delta-1 and F1 delta-2, coding for the delta-subunit of mitochondrial F1-ATP synthase, which corresponds to oligomycin-sensitivity conferring protein in animal and yeast mitochondria, were isolated from sweet potato. The gene for the delta-subunit was composed of 6 exons and these two genes shared high sequence similarities to each other not only in exons but also in introns and in the 5'-upstream regions. However, the 5'-upstream regions of F1 delta-1 and F1 delta-2 were distinguishable by the presence of novel sequences, designated Ins-1 and Ins-2, respectively. Ins-1 and Ins-2 contained a terminal direct repeat of 10 bp and 12 bp, respectively, and various forms of repeat sequences. The promoter fusion of both F1 delta-1 and F1 delta-2 with the GUS coding sequence gave expression of GUS activity in transformed tobacco BY-2 cells, although the levels of GUS activity and the patterns of expression during the growth of cells were different between the two. In transgenic tobacco plants, the two fusion genes showed similar levels of expression in leaves and stems, while F1 delta-2:GUS gave significantly higher levels of expression in roots than F1 delta-1:GUS. Deletion of Ins-1 from the 5'-upstream region of F1 delta-1:GUS did not affect the expression of the fusion gene in various organs of transgenic plants. However, it caused significant enhancement of expression in transformed tobacco BY-2 cells.     

湖北利川水杉原生种群及其生境1948～2003年间变化分析

为保护和恢复水杉原生种群提供科学依据,对中国湖北利川水杉原生母树及其生境1948～2003年间的变化进行对比分析,指出了保护工作存在的问题及保护对策。主要结果有：①1948年初步调查为1000多株,1983年普查有5746株,涉及4个镇(场)、45个行政村,分布面积为6万hm^2;2003年普查为5388株,分布范围没有大的变化,有2种分布类型：散生分布的有896株,群落分布的有4492株,最大的种群为105和123株。②中国政府对水杉的保护取得了巨大成绩,但过去只重视对个体的保护,忽视了对群落及其栖息环境的保护,导致由混交林变为纯林、结构趋于简单、生物多样性减少、天然更新困难,1983～2003年的20年间已有386株水杉原生母树死亡,亟需对水杉原生种群及其栖息地进行保护。③应运用现代恢复生态学手段,开展水杉植被恢复重建理论研究,首先加强对集中分布种群及其栖息地进行就地保护,其次小河河谷实施种群及其栖息地恢复与重建。④人为活动对水杉的危害加重,应减少及停止一切有害的人为活动,改善水杉原生母树生境,提高居民、社区自主参与水杉保护的意识;进行生态移民和实施退耕还林等是一个可以考虑的方法。     

The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS‐790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N‐terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X‐ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand‐soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.     

Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes

We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants. Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection. Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants. With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision. These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation. We showed that the wild-type TPase transcribed by the 2 promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter. In contrast to what had been observed in tobacco and petunia, the truncated TPase (103–807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2 promoter.     

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.