Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential

The present study addresses the effect of intracellular Na(+) and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K(i)) of DA in inhibiting the binding of cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na(+) to reduce DA K(i). No substantial effect on DA K(i) was observed upon gramicidin treatment in Na(+)-free buffer, membrane depolarization with high [K(+)](o), or elevation of [Na(+)](i) with monensin under non-depolarizing conditions. Elevation of DA K(i) was greater at more positive potentials when [Na(+)](i) was raised to a similar level, or at higher [Na(+)](i) when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K(i) was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA K(i) in cell-free membranes was insensitive to Na(+), gramicidin, and D313N mutation. The data suggest that (i) intracellular Na(+) plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na(+) and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.     

No‐analog climates and shifting realized niches during the late quaternary: implications for 21st‐century predictions by species distribution models

Empirically derived species distributions models (SDMs) are increasingly relied upon to forecast species vulnerabilities to future climate change. However, many of the assumptions of SDMs may be violated when they are used to project species distributions across significant climate change events. In particular, SDM's in theory assume stable fundamental niches, but in practice, they assume stable realized niches. The assumption of a fixed realized niche relative to climate variables remains unlikely for various reasons, particularly if novel future climates open up currently unavailable portions of species’ fundamental niches. To demonstrate this effect, we compare the climate distributions for fossil‐pollen data from 21 to 15 ka bp (relying on paleoclimate simulations) when communities and climates with no modern analog were common across North America to observed modern pollen assemblages. We test how well SDMs are able to project 20th century pollen‐based taxon distributions with models calibrated using data from 21 to 15 ka. We find that taxa which were abundant in areas with no‐analog late glacial climates, such as Fraxinus, Ostrya/Carpinus and Ulmus, substantially shifted their realized niches from the late glacial period to present. SDMs for these taxa had low predictive accuracy when projected to modern climates despite demonstrating high predictive accuracy for late glacial pollen distributions. For other taxa, e.g. Quercus, Picea, Pinus strobus, had relatively stable realized niches and models for these taxa tended to have higher predictive accuracy when projected to present. Our findings reinforce the point that a realized niche at any one time often represents only a subset of the climate conditions in which a taxon can persist. Projections from SDMs into future climate conditions that are based solely on contemporary realized distributions are potentially misleading for assessing the vulnerability of species to future climate change.     

药用真菌金耳的rDNA ITS序列分析与鉴别

研究了两个居群的金耳Tremella aurantialba以及近似品的rDNAITS区碱基全序列的特征及其差异,首次报道了金耳的ITS和5.8SrDNA完整序列,序列总长度为467~468,长度变异较少。聚类分析表明两个居群金耳亲缘关系非常密切,金耳药材和近似品金黄银耳形成一个稳定的独立分支,近似品黄金银耳形成另一个分支。ITS序列的差异为金耳的鉴别提供了可靠的分子标记,为金耳菌类药材基原入药建立了遗传基础。     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

A novel transition state analog inhibitor of guanase based on azepinomycin ring structure: Synthesis and biochemical assessment of enzyme inhibition

Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K_i of 16.7 ± 0.5 μM.     

小粒野生稻STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因结构中保守结构域,设计引物,以小粒野生稻基因组DNA为模板,扩增获得10条STK类抗病基因同源序列.同源性分析表明其都具有STK保守结构域,与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小粒野生稻中的STK类抗病基因提供依据.