Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases

A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.     

The roles of intracellular protein-degradation pathways in neurodegeneration

Many late-onset neurodegenerative diseases, including Parkinson's disease and Huntington's disease, are associated with the formation of intracellular aggregates by toxic proteins. It is therefore crucial to understand the factors that regulate the steady-state levels of these 'toxins', at both the synthetic and degradation stages. The degradation pathways acting on such aggregate-prone cytosolic proteins include the ubiquitin-proteasome system and macroautophagy. Dysfunction of the ubiquitin-proteasome or macroautophagy pathways might contribute to the pathology of various neurodegenerative conditions. However, enhancing macroautophagy with drugs such as rapamycin could offer a tractable therapeutic strategy for a number of these diseases.     

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.     

RNA toxicity is a component of ataxin-3 degeneration in Drosophila

Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of a CAG repeat encoding glutamine within the coding region of the respective genes. The molecular and cellular pathways underlying polyQ-induced neurodegeneration are the focus of much research, and it is widely considered that toxic activities of the protein, resulting from the abnormally long polyQ tract, cause pathogenesis. Here we provide evidence for a pathogenic role of the CAG repeat RNA in polyQ toxicity using Drosophila. In a Drosophila screen for modifiers of polyQ degeneration induced by the spinocerebellar ataxia type 3 (SCA3) protein ataxin-3, we isolated an upregulation allele of muscleblind (mbl), a gene implicated in the RNA toxicity of CUG expansion diseases. Further analysis indicated that there may be a toxic role of the RNA in polyQ-induced degeneration. We tested the role of the RNA by altering the CAG repeat sequence to an interrupted CAACAG repeat within the polyQ-encoding region; this dramatically mitigated toxicity. In addition, expression of an untranslated CAG repeat of pathogenic length conferred neuronal degeneration. These studies reveal a role for the RNA in polyQ toxicity, highlighting common components in RNA-based and polyQ-protein-based trinucleotide repeat expansion diseases.     

Generation of prion transmission barriers by mutational control of amyloid conformations

Self-propagating beta-sheet-rich protein aggregates are implicated in a wide range of protein-misfolding phenomena, including amyloid diseases and prion-based inheritance. Two properties have emerged as common features of amyloids. Amyloid formation is ubiquitous: many unrelated proteins form such aggregates and even a single polypeptide can misfold into multiple forms--a process that is thought to underlie prion strain variation. Despite this promiscuity, amyloid propagation can be highly sequence specific: amyloid fibres often fail to catalyse the aggregation of other amyloidogenic proteins. In prions, this specificity leads to barriers that limit transmission between species. Using the yeast prion [PSI+], we show in vitro that point mutations in Sup35p, the protein determinant of [PSI+], alter the range of 'infectious' conformations, which in turn changes amyloid seeding specificity. We generate a new transmission barrier in vivo by using these mutations to specifically disfavour subsets of prion strains. The ability of mutations to alter the conformations of amyloid states without preventing amyloid formation altogether provides a general mechanism for the generation of prion transmission barriers and may help to explain how mutations alter toxicity in conformational diseases.     

Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure

Amyloid or amyloid-like fibrils are elongated, insoluble protein aggregates, formed in vivo in association with neurodegenerative diseases or in vitro from soluble native proteins, respectively. The underlying structure of the fibrillar or 'cross-beta' state has presented long-standing, fundamental puzzles of protein structure. These include whether fibril-forming proteins have two structurally distinct stable states, native and fibrillar, and whether all or only part of the native protein refolds as it converts to the fibrillar state. Here we show that a designed amyloid-like fibril of the well-characterized enzyme RNase A contains native-like molecules capable of enzymatic activity. In addition, these functional molecular units are formed from a core RNase A domain and a swapped complementary domain. These findings are consistent with the zipper-spine model in which a cross-beta spine is decorated with three-dimensional domain-swapped functional units, retaining native-like structure.     

随机扰动下DNA三核苷酸重复序列的弯曲和柔性

利用统计力学模型讨论了与人类遗传病有关的DNA三核苷酸重复序列的弯曲和柔性,提出随机成动模型来近似表示DNA序列和蛋白质的相互作用,发现弯曲很小的三核苷重复序列在有随机扰动的情况下,可以出现大角度的弯曲,且随扰动强度的加大出现大角度弯曲的概率增加,求出了十二种三核苷重复序列的柔性及其长度依赖,发现当序列长度大于150 bp时,柔性的变化开始显,CTG和CGG具有最大的柔性,研究了柔性在有随机扰动时下的变化,发现在序列上不同位置施加扰动,柔性的变化程度不同,对于600bp长的序列,在390bp处的柔性变化程度最大。     

Folding proteins in fatal ways

Human diseases characterized by insoluble extracellular deposits of proteins have been recognized for almost two centuries. Such amyloidoses were once thought to represent arcane secondary phenomena of questionable pathogenic significance. But it is has now become clear that many different proteins can misfold and form extracellular or intracellular aggregates that initiate profound cellular dysfunction. Particularly challenging examples of such disorders occur in the post-mitotic environment of the neuron and include Alzheimer's and Parkinson's diseases. Understanding some of the principles of protein folding has helped to explain how such diseases arise, with attendant therapeutic insights.     

集料衰变规律及机理

为了研究集料的衰变机理及规律,为热再生混合料的级配恢复打下基础,通过旋转压实试验来模拟不同级配的集料破碎情况,结合集料受力模型分析集料的衰变机理及规律.试验结果表明:旋转次数对集料破碎影响不大,破碎在试验初期已经完成;连续级配的破碎程度明显小于骨架级配,但会形成较多的细集料;4.750 mm颗粒是稳定的受力点和粗细集料...     

关于细集料砂当量指标的探讨

通过加土的方法得到不同泥土含量的集料，进行砂当量试验，并用不同砂当量值的集料制成成心性稀浆封层混合料进行湿轮磨耗试验，试验结果表明，砂当量与集料中的泥土含量存在良好的线性关系，是评价细集料洁净程度的有效方法，试验中集料级配和取样均匀程度会对试验结果产生一定影响，砂当量低的集料会使改性剂无法发 对心性稀浆封层混合料的改性效果，建议用于心性稀浆封层的的细集料砂当量不低于60％。     

Towards a transgenic model of Huntington's disease in a non-human primate

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.     

矿料强嵌挤骨架密实级配的PFC2D数值试验研究

为了充分优化矿料级配,应用PFC2D构建了矿料密实度和加州承载比(CBR)的数值试验方法,在可靠性验证的基础上,分析了合成粗集料、合成细集料、粗细混合集料的密实度和CBR变化规律,提出了矿料强嵌挤骨架密实级配,并对其力学性能进行了室内试验验证.结果表明:19～31.5mm集料、9.5 ～ 19mm集料和4.75～ 9.5 mm集料3种规格粗集料的最佳质量比分别为70∶20∶10、60∶30∶10、60∶20∶20和50∶30∶20,2.36 ～4.75mm集料、0.6 ～2.36mm集料和0.6mm以下集料3种规格细集料的最佳质量比为38.5∶16.5∶45、33∶22∶45、36∶24∶40和30∶30∶40,粗细混合集料的最佳比例为65∶35;由矿料强嵌挤骨架密实级配组成的级配碎石的CBR值和抗压强度分别为规范级配的1.16倍和1.12倍以上,证明该级配具有良好的力学性能.     

Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders

The expansion of a CAG repeat coding for polyglutamine in otherwise unrelated gene products is central to eight neurodegenerative disorders including Huntington's disease. It has been well documented that expanded polyglutamine fragments, cleaved from their respective full-length proteins, form microscopically visible aggregates in affected individuals and in transgenic mice. The contribution of polyglutamine oligomers to neurodegeneration, however, is controversial. The azo-dye Congo red binds preferentially to beta-sheets containing amyloid fibrils and can specifically inhibit oligomerization and disrupt preformed oligomers. Here we show that inhibition of polyglutamine oligomerization by Congo red prevents ATP depletion and caspase activation, preserves normal cellular protein synthesis and degradation functions, and promotes the clearance of expanded polyglutamine repeats in vivo and in vitro. Infusion of Congo red into a transgenic mouse model of Huntington's disease, well after the onset of symptoms, promotes the clearance of expanded repeats in vivo and exerts marked protective effects on survival, weight loss and motor function. We conclude that oligomerization is a crucial determinant in the biochemical properties of expanded polyglutamine that are central to their chronic cytotoxicity.     

Rationalization of the effects of mutations on peptide and protein aggregation rates

In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimer's and Parkinson's diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.     

微细粒赤铁矿凝聚体的内部结构

以ＸＳＺ－Ｄ型生物显微镜为基础，设计了一个套适观察－显微照相装置，该装置可以方便地得到凝聚体的照片，利用图象分析仪测定了微一赤铁矿凝聚体的有关参数，并从分形理论出发，用分维数定量描述了凝聚体的内部结构。     

Two-headed binding of a processive myosin to F-actin

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.     

一类富足半群的好同余格

引入了可消幺半群的半格的好同余组概念,利用好同余组刻画了这类半群上的好同余.特别是确定了交换的可消幺半群的半格的好同余格上的交与并运算.