De novo protein structure determination using sparse NMR data

We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.     

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.     

Potential seasonal ecological challenge of heat strain among Australian Aboriginal people practicing traditional subsistence methods: a computer simulation

It has been largely accepted that Australian Aboriginal people practicing hunting and gathering traditionally underused their objective economic possibilities by working short hours relative to nonhunter-gatherer populations. However, the possibility that their subsistence quest might have been limited by potential heat strain has not been considered for Australian hunter-gatherers. In this article the influence of work and heat load on the potential for heat strain among adult male Australian Aboriginal people is modelled. The possibility that the short working day of Arnhem Land adults reported in the literature might reflect ecologically limited work scheduling by way of potential heat strain is examined. Three climatic regions of the North of Western Australia and the Northern Territory were identified, using data available from the Australian Bureau of Meteorology. Data from the months of January, April, July, and October were used with the United States Army Heat Strain Model, along with assumptions with respect to work load and time scheduling. Predictive modelling indicates that a late start to the working day could carry considerable risks of potential heat strain during the summer, when humidity and maximum daily temperature are highest for all three climatic regions, but especially in the tropical coastal region. While extended work times may have been needed to acquire adequate food under traditional conditions, work output could have been limited by potential heat strain under some conditions likely to have prevailed.     

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat,trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.     

The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier‐limited apparent two‐state transition, analogous to its bacterial homologues. The high positive surface‐charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native‐state movement. The electrostatic strain was alleviated at high solution‐ionic‐strength by Debye‐Hückel screening. Differences in ionic‐strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade‐off between protein function and stability during protein evolution.     

Specific features of enteropeptidase hydrolysis of chimeric proteins at the specific linker (Asp)4Lys depending on the refolding conditions

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)₄Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)₄Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site’s conformation.     

液体复苏对于严重脓毒症患者生存率的预后分析

目的:严重脓毒症中液体复苏的生理病理学理论支持及动物实验证据不足,有必要进行相关临床试验来确定液体复苏对于严重脓毒症患者预后的影响。方法:将26名严重脓毒症和脓毒症休克患者分为液体复苏组和非液体复苏组。比较两组的28天生存率,ICU住院天数的差别,对两组在院生存天数进行生存函数分析。结果:液体复苏组的28天生存率为41.7%,未液体复苏组的28天生存率为50.0%,之间没有统计学差异(P=0.713)。液体复苏组的平均住院天数为24.7±6.0天,未液体复苏组的平均住院天数为17.7±3.4天,两组之间没有统计学差异(P=0.308)。液体复苏组的中位在院生存天数为38.0[28.0,48.0],液体复苏组的中位在院生存天数为25.0[22.8,27.2]。两组之间有显著统计学差异,(Log Rank P=0.044,Breslow P=0.025)。结论:在严重脓毒症及脓毒症休克患者中,采取液体复苏能够延长患者的中位在院生存天数,可见液体复苏在严重脓毒症的治疗中有重要意义。尚未发现液体复苏能提高严重脓毒症患者28天生存率的相关证据。采取液体复苏究竟能否改善严重脓毒症患者预后,还需要大样本随机临床对照试验来证明。     

多媒体教学软件结合带领教学法在心肺复苏培训中的应用研究

目的：探讨采用多媒体教学软件结合带领教学法对医护人员进行心肺复苏培训的效果。方法：将参加心肺复苏培训的120名医护人员随机分为对照组和观察组,对照组采用传统的教学法,观察组采用多媒体教学软件结合带领教学法,培训结束后,对培训时间、技能考核情况和学生的反馈意见进行分析和评估。结果：对照组和观察组每次培训时间分别为（3．23±1．13）h和（2．75±0．75）h,差异有统计学意义（P〈0．05）;两组学员的考核通过率分别为66．7％和83.3％,差异有统计学意义（P〈0．05）,但前者在完成颈动脉检查这一环节上优于后者,差异有统计学意义（P〈0．05）。就学员的反馈意见而言,后者在教学效果主观评价要高于前者。结论：通过多媒体教学软件结合带领教学法进行心肺复苏培训,医护人员能有效掌握心肺复苏技能,有利于开展较大规模的培训活动,值得在院内培训中推广应用。     

未培养环境微生物培养方法的研究进展

自然界中微生物种类繁多、功能多样、分布广泛,对人类健康安全、生态稳定和物种进化等发挥不可替代的作用。尽管微生物培养技术至今已有一百多年,然而由于各种限制因素的制约,目前成功分离培养的微生物仅占0.1%-1.0%,自然界中仍有十分丰富的微生物资源有待挖掘和开发利用。如何理解难培养微生物的制约因素并探索作用机制,同时借此开发新型微生物培养方法则尤为必要。本文首先分析了典型环境微生物生长的限制因素,然后介绍了目前利用传统培养改进措施进行分离筛选的研究成果,进一步综述了原位培养、共培养、微流控培养技术、细胞分选技术等新型培养技术在难培养微生物分离培养中的应用,最后对目前培养法存在的瓶颈问题进行深入分析,提出今后可在多技术联合使用、难培养微生物复苏机制、微生物互作机制、代谢途径与调控机制等方面进行研究,以期为今后微生物资源开发利用提供借鉴。     

Plasticity matches phenotype to local conditions despite genetic homogeneity across 13 snake populations

In a widespread species, a matching of phenotypic traits to local environmental optima is generally attributed to site-specific adaptation. However, the same matching can occur via adaptive plasticity, without requiring genetic differences among populations. Adult sea kraits (Laticauda saintgironsi) are highly philopatric to small islands, but the entire population within the Neo-Caledonian Lagoon is genetically homogeneous because females migrate to the mainland to lay their eggs at communal sites; recruits disperse before settling, mixing up alleles. Consequently, any matching between local environments (e.g. prey sizes) and snake phenotypes (e.g. body sizes and relative jaw sizes (RJSs)) must be achieved via phenotypic plasticity rather than spatial heterogeneity in gene frequencies. We sampled 13 snake colonies spread along an approximately 200 km northwest–southeast gradient (n > 4500 individuals) to measure two morphological features that affect maximum ingestible prey size in gape-limited predators: body size and RJS. As proxies of habitat quality (HQ), we used protection status, fishing pressure and lagoon characteristics (lagoon width and distance of islands to the barrier reef). In both sexes, spatial variation in body sizes and RJSs was linked to HQ; albeit in different ways, consistent with sex-based divergences in foraging ecology. Strong spatial divergence in morphology among snake colonies, despite genetic homogeneity, supports the idea that phenotypic plasticity can facilitate speciation by creating multiple phenotypically distinct subpopulations shaped by their environment.