Cytoplasmatic domain of Na,K-ATPase α-subunit is responsible for the aggregation of the enzyme in proteoliposomes

We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes.     

荒漠景观下柠条豆象的空间分布及其对景观斑块格局的反应

柠条豆象Kytorhinus immixtus Motschulsky是危害柠条种子的主要种实害虫之一。本文应用了多个聚集度指标和回归分析方法,研究探讨了柠条豆象卵、幼虫在宁夏盐池、灵武、银川等荒漠景观中11个生境斑块中的空间分布型及种群对景观斑块格局的反应情况。结果表明:在11个景观样地中,柠条豆象卵和幼虫在各种生境斑块中均呈聚集型的负二项分布;卵和幼虫空间分布的Iwao回归方程分别为m*=2.23695+1.64742m(r=0.8273)和m*=2.24418+1.42084m(r=0.8889)、Taylor幂模型分别为logS2=0.51292+1.41118logm(r=0.9344)和logS2=0.59645+1.14736logm(r=0.9786),种群分布的基本成分为个体群,个体间相互吸引,有密度依赖性,聚集主要由环境因素和种间竞争引起的,聚集强度随种群密度的升高而增加。在荒漠景观下,柠条豆象的种群发生与寄主植物的种类、在斑块内的分布格局有着密切的关系,与斑块面积相关性较低(R2=0.1995)。主成分分析显示柠条林生长的土壤类型、生长方式是影响柠条豆象寄生的主要因素。     

Simultaneous quantification of 8-iso-prostaglandin-F2α and 11-dehydro thromboxane B2 in human urine by liquid chromatography-tandem mass spectrometry

Both F₂-isoprostanes (8-iso-PGF_2α), a well-known marker of oxidative stress, and thromboxanes A₂ (TXA₂) are involved in atherosclerosis through LDL oxidation and platelet activation. Different aspects of the pathology can be described by 8-iso-PGF_2α and TXA₂ so it is important to determine both their concentrations to monitor the disease progression and/or therapy effects. We developed a simple and sensitive method based on liquid chromatography-tandem mass spectrometry, using electrospray ionization in negative-ion mode, for the simultaneous measurement of the concentration of 8-iso-PGF_2α and 11-dehydro thromboxane B₂ (11-DH-TXB₂), a TXA₂ metabolite. This method was applied to analyze urine samples collected overnight from 15 atherosclerotic patients, with documented carotid artery sclerosis (CAS), and from 20 controls. The detection limit was 0.097 pg/μL for 8-iso-PGF_2α and 0.375 pg/μL for 11-DH-TXB₂, with a linear range of 0.78-25 pg/μL; the inter- and intraday imprecision was <5% for both metabolites. These analytes were higher in CAS (P < 0.005 vs controls) and were positively correlated in patients but not in controls, even after adjustment for age and gender (r = 0.60; P = 0.032). This highly sensitive, precise, and rapid method allows for the simultaneous determination of 8-iso-PGF_2α and 11-DH-TXB₂ in human urine samples in order to evaluate oxidative stress and platelet aggregation.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.     

IbpA the small heat shock protein from Escherichia coli forms fibrils in the absence of its cochaperone IbpB

Small heat shock proteins (sHsps) associate with aggregated proteins, changing their physical properties in such a way that chaperone mediated disaggregation becomes much more efficient. In Escherichia coli two small Hsps, IbpA and IbpB, exist. They are 48% identical at the amino acid level, yet their roles in stabilisation of protein aggregates are quite distinct. Here we analysed the biochemical properties of IbpA. We found that IbpA assembles into protofilaments which in turn form mature fibrils. Such fibrils are atypical for sHsps. Interaction of IbpA with either its cochaperone IbpB or an aggregated substrate blocks IbpA fibril formation.

Structured summary

MINT-7876715: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888427: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888448: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888434: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888459: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by fluorescence microscopy (MI:0416)     

Self‐recruitment and sweepstakes reproduction amid extensive gene flow in a coral‐reef fish

Identifying patterns of larval dispersal within marine metapopulations is vital for effective fisheries management, appropriate marine reserve design, and conservation efforts. We employed genetic markers (microsatellites) to determine dispersal patterns in bicolour damselfish (Pomacentridae: Stegastes partitus). Tissue samples of 751 fish were collected in 2004 and 2005 from 11 sites encompassing the Exuma Sound, Bahamas. Bayesian parentage analysis identified two parent–offspring pairs, which is remarkable given the large population sizes and 28 day pelagic larval duration of bicolour damselfish. The two parent–offspring pairs directly documented self‐recruitment at the two northern‐most sites, one of which is a long‐established marine reserve. Principal coordinates analyses of pair‐wise relatedness values further indicated that self‐recruitment was common in all sampled populations. Nevertheless, measures of genetic differentiation (F_ST) and results from assignment methods suggested high levels of gene flow among populations. Comparisons of heterozygosity and relatedness among samples of adults and recruits indicated spatially and temporally independent sweepstakes events, whereby only a subset of adults successfully contribute to subsequent generations. These results indicate that self‐recruitment and sweepstakes reproduction are the predominant, ecologically‐relevant processes that shape patterns of larval dispersal in this system.     

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation‐prone polyQ protein derived from human huntingtin. Expression of Q97‐GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97‐GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97‐GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post‐translational import of mitochondrial precursor proteins into mitochondria competes with aggregation‐prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate‐limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.     

Isolation by distance in a continuous population under stochastic demographic fluctuations

The local density of individuals is seldom uniform in space and time within natural populations. Yet, formal approaches to the process of isolation by distance in continuous populations have encountered analytical difficulties in describing genetic structuring with demographic heterogeneities, usually disregarding local correlations in the movement and reproduction of genes. We formulate exact recursions for probabilities of identity in continuous populations, from which we deduce definitions of effective dispersal () and effective density (D_e) that generalize results relating spatial genetic structure, dispersal and density in lattice models. The latter claim is checked in simulations where estimates of effective parameters obtained from demographic information are compared with estimates derived from spatial genetic patterns in a plant population evolving in a heterogeneous and dynamic habitat. The simulations further suggest that increasing spatio‐temporal correlations in local density reduce and generally decrease the product , with dispersal kurtosis influencing their sensitivity to density fluctuations. As in the lattice model, the expected relationship between the product and the genetic structure statistic a_r holds under fluctuating density, irrespective of dispersal kurtosis. The product D σ² between observed census density and the observed dispersal rate over one generation will generally be an upwardly biased (up to 400% in simulations) estimator of in populations distributed in spatially aggregated habitats.     

Validation and efficacy of transgenerational mass marking of otoliths in viviparous fish larvae

Transgenerational mass marking of viviparous fish larvae in vivo was validated by intra‐muscular injection of elemental strontium chloride (SrCl₂) in gestating females and detection of the Sr in the otoliths of developing larvae. All otoliths of brown rockfish Sebastes auriculatus larvae produced from SrCl₂‐injected females showed enriched Sr:Ca ratios near the otolith edges, and the signatures did not appear to be affected by the anterior, centre and posterior positions of larvae within the ovary. Results from the present study indicate that transgenerational marking is a highly reliable technique for marking large numbers of extremely small viviparous fish larvae.     

Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.