Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils,including amyloid pores

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.     

Direct measures of mechanical energy for knife mill size reduction of switchgrass, wheat straw, and corn stover

Lengthy straw/stalk of biomass may not be directly fed into grinders such as hammer mills and disc refiners. Hence, biomass needs to be preprocessed using coarse grinders like a knife mill to allow for efficient feeding in refiner mills without bridging and choking. Size reduction mechanical energy was directly measured for switchgrass (Panicum virgatum L.), wheat straw (Triticum aestivum L.), and corn stover (Zea mays L.) in an instrumented knife mill. Direct power inputs were determined for different knife mill screen openings from 12.7 to 50.8 mm, rotor speeds between 250 and 500 rpm, and mass feed rates from 1 to 11 kg/min. Overall accuracy of power measurement was calculated to be ±0.003 kW. Total specific energy (kWh/Mg) was defined as size reduction energy to operate mill with biomass. Effective specific energy was defined as the energy that can be assumed to reach the biomass. The difference is parasitic or no-load energy of mill. Total specific energy for switchgrass, wheat straw, and corn stover chopping increased with knife mill speed, whereas, effective specific energy decreased marginally for switchgrass and increased for wheat straw and corn stover. Total and effective specific energy decreased with an increase in screen size for all the crops studied. Total specific energy decreased with increase in mass feed rate, but effective specific energy increased for switchgrass and wheat straw, and decreased for corn stover at increased feed rate. For knife mill screen size of 25.4 mm and optimum speed of 250 rpm, optimum feed rates were 7.6, 5.8, and 4.5 kg/min for switchgrass, wheat straw, and corn stover, respectively, and the corresponding total specific energies were 7.57, 10.53, and 8.87 kWh/Mg and effective specific energies were 1.27, 1.50, and 0.24 kWh/Mg for switchgrass, wheat straw, and corn stover, respectively. Energy utilization ratios were calculated as 16.8%, 14.3%, and 2.8% for switchgrass, wheat straw, and corn stover, respectively. These data will be useful for preparing the feed material for subsequent fine grinding operations and designing new mills.     

Cloning of the bluetongue virus L3 gene.

The growth of phage T1 on cells tandemly lysogenic for heteroimmune lambdoid prophages leads to a nonrandom packaging of lambda DNA by T1. A site, called esp-lambda, is located between the P and Q genes of lambda and results in increased packaging to the left by T1. When cloned into pBR322, the esp-lambda site causes a significant increase in transduction of the plasmid by T1. The nin5 deletion inactivates esp-lambda.     

Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves

As many processes are regulated by both light and plant hormones, evaluation of diurnal variations of their levels may contribute to the elucidation of the complex network of light and hormone signal transduction pathways. Diurnal variation of cytokinin, auxin, and abscisic acid levels was tested in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under a 16/8 h photoperiod. The main peak of physiologically active cytokinins (cytokinin bases and ribosides) was found after 9 h of light, i.e. 1 h after the middle of the light period. This peak coincided with the major auxin peak and was closely followed by a minor peak of abscisic acid. Free abscisic acid started to increase at the light/dark transition and reached its maximum 3 h after dark initiation. The content of total cytokinins (mainly N-glucosides, followed by cis-zeatin derivatives and nucleotides) exhibited the main peak after 9 h of light and the minor peak after the transition to darkness. The main, midday peak of active cytokinins was preceded by a period of minimal metabolic conversion of tritiated trans-zeatin (less than 30%). The major cytokinin-degrading enzyme, cytokinin oxidase/dehydrogenase (EC 1.5.99.12), exhibited maximal activity after the dark/light transition and during the diminishing of the midday cytokinin peak. The former peak might be connected with the elimination of the long-distance cytokinin signal. These cytokinin oxidase/dehydrogenase peaks were accompanied by increased activity of beta-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of cytokinin O-glucosides and/or in fine-tuning of active cytokinin levels at their midday peak. The achieved data indicate that cytokinin metabolism is tightly regulated by the circadian clock.     

Hydrazyl-nitrones, novel hybrid molecules in free radical research

This work describes the synthesis and characterisation of some novel hybrid molecules which contains in the same molecule a free radical moiety of hydrazyl type and a spin-trap moiety of nitrone type. The new compounds synthesized have multiple and easy to follow spectroscopic properties, making them useful as sensors or probes in radical chemistry. The new class of hydrazyl-nitrone molecules can act, in a single step process, as both generator and spin-trap of short-lived radicals. The hybrid molecules can be also involved in acid-base or redox processes, and the chemical processes can be easily monitored by visible or electron paramagnetic resonance spectroscopy. The excellent generator and trap properties recommend them as valuable sensors and probes in radical chemistry.     

Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco

Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.     

A decoy set for the thermostable subdomain from chicken villin headpiece,comparison of different free energy estimators

Background  

Estimators of free energies are routinely used to judge the quality of protein structural models. As these estimators still present inaccuracies, they are frequently evaluated by discriminating native or native-like conformations from large ensembles of so-called decoy structures.     

Seed dispersal effectiveness of the western lowland gorilla (Gorilla gorilla gorilla) in Gabon

The quantitative and qualitative aspects of seed dispersal by the western lowland gorilla (Gorilla gorilla gorilla) were investigated in Gabon. Fresh faeces were collected and washed to identify and count the seeds. Seed germinability after gut passage was estimated with trials in a nursery at the study site. To assess the impact of gut passage on germination success and delay, comparative trials were run with four treatments: (i) gut passed seeds cleaned of faeces, (ii) gut passed seeds within a faecal matrix, (iii) seeds from fresh fruits surrounded by pulp, and (iv) seeds from fresh fruits cleaned of pulp. The analysis of 180 faecal units resulted in the identification of 58 species of seed. Germination trials were realized for 55 species and the mean germination success reached 46%. The impact of gut passage was investigated for Santiria trimera and Chrysophyllum lacourtianum; both species displayed higher germination success after ingestion. This study shows that gorillas effectively disperse seeds of numerous plant species, many of which provide timber or nontimber forest products or are typical of Gabonese forests. Considering the high‐quality of gorilla deposition sites, gorillas is thought to play a unique role in the dynamics of Central African forest.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.