Characterization of the bifunctional cytochrome c reductase-processing peptidase complex from potato mitochondria

In potato, cytochrome c reductase, a protein complex of the respiratory chain, exhibits processing activity toward mitochondrial precursor proteins. One of the two cooperating components of the processing peptidase was shown to be identical with subunit III of the complex. Here we report that two additional proteins of the complex (subunit I and II) share 40-50% sequence identity with the processing enhancing protein, the other component of the processing enzyme from fungi and mammals. Thus the composition and structure of the complex integrated processing peptidase seems to be different from its fungal and mammalian counterparts. Cytochrome c reductase from potato is extraordinarily stable, and separation of subunit III from the complex leads to aggregation of the remaining subcomplex and irreversible loss of processing activity. Expression of the three high molecular weight subunits of the complex allowed purification of each individual protein. Neither the individual subunits nor their combinations are active in in vitro processing assays suggesting that they may need the structural support of the complex for activity. In contrast to mitochondrial processing peptidases from other organisms, the purified potato enzyme is active in the presence of high salt (above 1 M NaCl) and works efficiently without addition of metal ions. These data indicate that potato cytochrome c reductase is a bifunctional protein complex with unique features. Possibly, there is a more general evolutionary relationship between cytochrome c reductases and mitochondrial processing peptidases than hitherto assumed.     

New insights into the photocycle of Ectothiorhodospira halophila photoactive yellow protein: photorecovery of the long-lived photobleached intermediate in the Met100Ala mutant

There are previously two known intermediates (I1 and I2) in the room-temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The three-dimensional structures of ground-state PYP and of I2 have shown that light-induced conformational changes are localized to the active site. Previous site-specific mutagenesis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala). We now report the construction and characterization of a mutant at a third active site position (Met100Ala) in order to establish the role of this residue in the photocycle. Met100Ala PYP has an absorption spectrum which is very similar to wild-type (WT) PYP, but exhibits very different kinetic properties. At pH 7.0, the light-induced bleaching reaction (I2 formation) has a half-life <1 microseconds and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 microseconds and 140 ms for the same reactions in WT PYP. The slow rate of recovery from I2 for Met100Ala results in the accumulation of the bleached intermediate even under room light illumination. These results are qualitatively similar to what has been observed with the Arg52Ala mutant of PYP, and with WT PYP in the presence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein. The midpoint for guanidine denaturation confirms this. The slow recovery of I2 in the Met100Ala mutant has allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 nm laser flash, with kinetics (half-life = 160 microseconds; k = 6300 s-1) which are 6 orders of magnitude faster than dark recovery. This implies that chromophore reisomerization limits the rate of conversion of I2 to the ground state in PYP. Met100 is in van der Waals contact with the chromophore in the I2 state, and we suggest that the sulfur atom catalyzes cis-trans isomerization in WT PYP.     

Effect of selegiline on mortality in patients with Parkinson''s disease: a meta-analysis

What is the role of selective attention in visual perception? Before answering this question, it is necessary to differentiate between attentional mechanisms that influence the identification of a stimulus from those that operate after perception is complete. Cognitive neuroscience techniques are particularly well suited to making this distinction because they allow different attentional mechanisms to be isolated in terms of timing and/or neuroanatomy. The present article describes the use of these techniques in differentiating between perceptual and postperceptual attentional mechanisms and then proposes a specific role of attention in visual perception. Specifically, attention is proposed to resolve ambiguities in neural coding that arise when multiple objects are processed simultaneously. Evidence for this hypothesis is provided by two experiments showing that attention-as measured electrophysiologically-is allocated to visual search targets only under conditions that would be expected to lead to ambiguous neural coding.     

Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.     

Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420,000; it consisted of subunits with molecular weights of 72,400 (alpha), 20,400 (beta), 13,900 (gamma) in an estimated (alpha beta gamma)4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420,000 to 100,000. In the native enzyme, 4.09 (+/- 0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.     

聚丙烯在国际纺织品市场上的拓展

10年前把尼龙挤到市场份额的第四位后,聚丙烯持续进行着有条不紊的拓展。在回顾聚丙烯成功的潜在原因后,用图表对市场份额最大的国家和产品进行说明。     

景观：实现一个有弹性的世界

我们生活的世界是有限的。我很惭愧的承认,如果全世界的人都像大多数英国人这样生活,需要3个地球提供资源才能维持我们的生活。斯蒂芬·霍金(Stephen Hawking)教授曾经预言:"为了我们的后代能够生存下去,我们必须在有生之年制定长远计划—逃离地球"。我们知道世界气候正在发生变化;幸运的是,英国的人们并没有承受过多气候变化带来的后果。但是,世界上其他地方的人们并非同样幸运。我们需要科学和艺术知识背景的景观行业的从业者;广博的知识使我们可以理解自然系统的敏感性、预想我们的专业行为对自然系统的长期影响。我预测,随着人口增长、土地资源减少以及不可再生资源的使用叠加在一起带来的压力,社会对风景园林师会有更大的需求。因此,迫切需要吸引年轻人加入到风景园林行业。我们的风景园林教育系统必须传授风景园林行业的新从业者应对不断变化的行业挑战的技能。我非常赞赏中国国家领导人习近平主席的重要观点:提高中国人生活质量具有重要意义。本篇文章传递的观点是:提高每个人的生活质量是风景园林专业的责任;同时,我希望这是中国与英国景观专业长期合作的一个开始,我们将以共赢的方式进行合作、提供服务。     

探索基于自然的解决方案中景观设计的角色变化——对20年专业实践的反思

基于自然的解决方案（NbS）是与自然栖息地相互促进的解决方案,利用健康的自然及人工生态系统的功能,封存碳并支持生物多样性恢复。通过对致力于与自然合作的职业生涯的反思,探讨了景观规划和设计方法的演变历程,从单一目标的解决方案到系统性思维及整体设计,从经验性/定性的决策到量化的解决方案。分3个阶段展开实践探讨：1）绿化灰色基础设施;2）将自然景观融入公共空间;3）自然向好的未来。探索如何将NbS理念传输给政策制定者并充分融入广大公众的社会意识与日常认知领域,继而促成更广泛、更长期的国际环境上的成功。