Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules

Tau is one of the most abundant microtubule-associated proteins involved in kinetic stabilization and bundling of axonal microtubules. Although intense research has revealed much about tau function and its involvement in Alzheimer's disease during the past years, it still remains unclear how exactly tau binds on microtubules and if the kinetic stabilization of microtubules by tau is accompanied, at least in part, by a mechanical reinforcement of microtubules. In this paper, we have used atomic force microscopy to address both aspects by visualizing and mechanically analyzing microtubules in the presence of native tau isoforms. We could show that tau at saturating concentrations forms a 1 nm thick layer around the microtubule, but leaves the protofilament structure well visible. The latter observation argues for tau binding mainly along and not across the protofilaments. The radial elasticity of microtubules was almost unaffected by tau, consistent with tau binding along the tops of the protofilaments. Tau did increase the resistance of microtubules against rupture. Finite-element calculations confirmed our findings.     

Establishment and characterization of a primary canine duodenal epithelial cell culture

Many mechanisms involved in the pathogenesis of chronic enteropathies or host–pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37° C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33° C. With this method, the cultures were growing to confluent monolayers within 5–6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected. Presented in part at the American College of Veterinary Internal Medicine Forum in Louisville, Kentucky, May 31–June 3, 2006. Julia L. Golaz and Nathalie Vonlaufen contributed equally to this work and are joint first authors. Supported in part by the Vetsuisse research foundation, the Foundation Research 3R (project No. 85/03), and the Swiss National Science Foundation (3100A0-112532).     

The effects of increased flow and fine sediment on hyporheic invertebrates and nutrients in stream mesocosms

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Development of a biotic index using stream macroinvertebrates to assess stress from deposited fine sediment

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New high affinity H3 receptor agonists without a basic side chain

In this study, we replaced the basic amine function of the known histamine H(3) receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H(3) receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H(3) receptor over the human H(4) receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H(3) receptor agonist (pK(i) = 8.0 and pEC(50) = 8.1) with a 320-fold selectivity at the human H(3) receptor over the human H(4) receptor.     

A potato model intercomparison across varying climates and productivity levels

A potato crop multimodel assessment was conducted to quantify variation among models and evaluate responses to climate change. Nine modeling groups simulated agronomic and climatic responses at low‐input (Chinoli, Bolivia and Gisozi, Burundi)‐ and high‐input (Jyndevad, Denmark and Washington, United States) management sites. Two calibration stages were explored, partial (P1), where experimental dry matter data were not provided, and full (P2). The median model ensemble response outperformed any single model in terms of replicating observed yield across all locations. Uncertainty in simulated yield decreased from 38% to 20% between P1 and P2. Model uncertainty increased with interannual variability, and predictions for all agronomic variables were significantly different from one model to another (P < 0.001). Uncertainty averaged 15% higher for low‐ vs. high‐input sites, with larger differences observed for evapotranspiration (ET), nitrogen uptake, and water use efficiency as compared to dry matter. A minimum of five partial, or three full, calibrated models was required for an ensemble approach to keep variability below that of common field variation. Model variation was not influenced by change in carbon dioxide (C), but increased as much as 41% and 23% for yield and ET, respectively, as temperature (T) or rainfall (W) moved away from historical levels. Increases in T accounted for the highest amount of uncertainty, suggesting that methods and parameters for T sensitivity represent a considerable unknown among models. Using median model ensemble values, yield increased on average 6% per 100‐ppm C, declined 4.6% per °C, and declined 2% for every 10% decrease in rainfall (for nonirrigated sites). Differences in predictions due to model representation of light utilization were significant (P < 0.01). These are the first reported results quantifying uncertainty for tuber/root crops and suggest modeling assessments of climate change impact on potato may be improved using an ensemble approach.     

Area, altitude and aquatic plant diversity

Several explanations have been given for the decline in species richness with altitude. However, separating the influences of altitude, area, and isolation is difficult because of the conical shape of mountains. We used species lists of aquatic plants from >300 lakes in a small geographical area to investigate the influence of altitude on species richness. Altitude and/or surface area were better predictors of species richness than any measure of water chemistry. The surface area and depth of individual lakes were not related to altitude, neither was the degree of isolation from other waterbodies. Although species range size increased with altitude, range sizes of all but the rarer species (in the data set) encompassed the lowest altitudes, indicating species loss rather than turnover and no influence of the Rapoport rescue effect. Nevertheless we found a decline in species richness with altitude, additive to the effect of area. Species were ascribed to attribute groups according to an a priori classification based on morphological and life-history traits. The number of attribute groups and number of species within each group increased with area, suggesting both increased diversity and coexistence within niches. With altitude, the number of attribute groups declined, but the number of species per group increased, consistent with a loss of richness and reduced competition. The species remaining at high altitudes were characterised by stress tolerant traits, associated with sites of low productivity.
Our results suggest an absolute effect of altitude on species richness, irrespective of other influences and consistent with a decline in productivity.     

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.     

Studies on the continuous production of (R)-(−)-phenylacetylcarbinol in an enzyme-membrane reactor

The optimization of a continuous enzymatic reaction yielding (R)-(−)-phenylacetylcarbinol ((R)-PAC), a key intermediate of the (1R,2S)-(−)-ephedrine synthesis, is presented. We compare the suitability of different mutants of the pyruvate decarboxylase (PDC) from Zymomonas mobilis with respect to their application in biotransformation using pyruvate or acetaldehyde and benzaldehyde as substrates, respectively. Starting from 90 mM pyruvate and 30 mM benzaldehyde, (R)-PAC was obtained with a space time yield of 27.4 g/(L·day) using purified PDCW392I in an enzyme-membrane reactor. Due to the high stability of the mutant enzymes PDCW392I and PDCW392M towards acetaldehyde, a continuous procedure using acetaldehyde instead of pyruvate was developed. The kinetic results of the enzymatic synthesis starting from acetaldehyde and benzaldehyde demonstrate that the carboligation to (R)-PAC is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Starting from an inlet concentration of 50 mM of both aldehydes, (R)-PAC was obtained with a space-time yield of 81 g/(L·day) using the mutant enzyme PDCW392M. The new reaction strategy allows the enzymatic synthesis of (R)-PAC from cheap substrates free of unwanted by-products with potent mutants of PDC from Z. mobilis in an aqueous reaction system.