Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and Aβ(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40°. Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Aβ(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Aβ(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth     

Structure of Self-Aggregated Alamethicin in ePC Membranes Detected by Pulsed Electron-Electron Double Resonance and Electron Spin Echo Envelope Modulation Spectroscopies

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)^7,18,19]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70-1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n ≈ 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and ∼5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 ≈ TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)^18,19 residues are suggested to stabilize the self-aggregate superstructure.     

The dynamics of surface acoustic wave‐driven scaffold cell seeding

Flow visualization using fluorescent microparticles and cell viability investigations are carried out to examine the mechanisms by which cells are seeded into scaffolds driven by surface acoustic waves. The former consists of observing both the external flow prior to the entry of the suspension into the scaffold and the internal flow within the scaffold pores. The latter involves micro‐CT (computed tomography) scans of the particle distributions within the seeded scaffolds and visual and quantitative methods to examine the morphology and proliferation ability of the irradiated cells. The results of these investigations elucidate the mechanisms by which particles are seeded, and hence provide valuable information that form the basis for optimizing this recently discovered method for rapid, efficient, and uniform scaffold cell seeding. Yeast cells are observed to maintain their size and morphology as well as their proliferation ability over 14 days after they are irradiated. The mammalian primary osteoblast cells tested also show little difference in their viability when exposed to the surface acoustic wave irradiation compared to a control set. Together, these provide initial feasibility results that demonstrate the surface acoustic wave technology as a viable seeding method without risk of denaturing the cells. Biotechnol. Bioeng. 2009;103: 387–401. © 2009 Wiley Periodicals, Inc.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

大鼠脑片中细胞内游离钙动态变化的研究

目的：探索大鼠急性脑片中电刺激诱发的细胞内钙的动态变化规律。方法：采用表面灌流的急性脑片模型,结合电生理和激光共聚焦技术,利用细胞内钙荧光探针进行细胞内游离钙标记,观察电刺激诱发的脑片中神经细胞内游离钙的变化情况。结果：急性脑片组织中,钙标记染料的神经细胞内钙探针荧光强度,电刺激后出现显著增强,且具有波样特征,而Suramin明显抑制此反应,表现为钙探针荧光强度下降和钙反应时间出现延迟,两组之间差异具有统计学意义（P〈0．05）.结论：刺激诱发的大鼠急性脑片中瞬时动态钙信号变化具有一定的时空发生特征,且这种钙信号的时空变化过程可能与嘌呤能信号的作用有关。     

SynaptotagminI和IV在LβT2细胞中的定位和shRNA基因干扰研究

目的：研究Sytnaptotagmin（Syt）I和Ⅳ在LβT2细胞中的定位和shRNA干扰技术对这两种蛋白在促性腺激素释放细胞（LβT2）细胞中蛋白表达量的影响。方法：运用全内反射荧光显微系统,通过在LβT2细胞中共表达各种细胞器的蛋白标记物和Syt I、Ⅳ与荧光蛋白的融合蛋白,对SytI、Ⅳ进行细胞内定位。并运用shRNA干扰技术对sytI、Ⅳ在L8T2细胞中蛋白表达量进行了干扰。结果：发现了sytI、sytⅣ在LβT2细胞中和致密核心大囊泡（LDCV）都存在了一定的共存比例,而且shRNA技术对SytI和SytⅣ在LβT2细胞中的表达沉默产生了一定的效用。结论：SytI、SytⅣ在LβT2细胞中与致密核心大囊泡共存比例很大,shRNA干扰技术对SytI、SytⅣ的沉默具有高效性,为未来进一步对SytI、SytⅣ在LβT2细胞中的功能研究打下了基础。     

Aerenchyma and an Inducible Barrier to Radial Oxygen Loss Facilitate Root Aeration in Upland, Paddy and Deep-water Rice (Oryza sativa L.)

The present study evaluated waterlogging tolerance, root porosityand radial O₂ loss (ROL) from the adventitious roots, of sevenupland, three paddy, and two deep-water genotypes of rice (Oryzasativa L.). Upland types, with the exception of one genotype,were as tolerant of 30 d soil waterlogging as the paddyand deep-water types. In all but one of the 12 genotypes, thenumber of adventitious roots per stem increased for plants grownin waterlogged, compared with drained, soil. When grown in stagnantdeoxygenated nutrient solution, genotypic variation was evidentfor root porosity and rates of ROL, but there was no overalldifference between plants from the three cultural types. Adventitiousroot porosity increased from 20–26 % for plants grownin aerated solution to 29–41 % for plants grown instagnant solution. Growth in stagnant solution also induceda ‘tight’ barrier to ROL in the basal regions ofadventitious roots of five of the seven upland types, all threepaddy types, and the two deep-water types. The enhanced porosityprovided a low resistance pathway for O₂ movement to the roottip, and the barrier to ROL in basal zones would have furtherenhanced longitudinal O₂ diffusion towards the apex, by diminishinglosses to the rhizosphere. The plasticity in root physiology,as described above, presumably contributes to the ability ofrice to grow in diverse environments that differ markedly insoil waterlogging, such as drained upland soils as well as waterloggedpaddy fields.     

天然色素的超声提取研究

本文主要研究利用超声波提取叶绿素。并且对超声提取法和单纯浸泡法作了比较,同时,分析了叶绿素提取效率对时间的依赖性。实验结果显示,用超声来提取绿色植物中的叶绿素,可以增大提取率,缩短提取时间。