Distinct roles of doublecortin modulating the microtubule cytoskeleton

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors.     

Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery

Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKalpha2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased ( approximately 35%) during postexercise recovery (P 相似文献   

Suppression of Escherichia coli O157:H7 by Dung Beetles (Coleoptera: Scarabaeidae) Using the Lowbush Blueberry Agroecosystem as a Model System

Wildlife as a source of microbial contamination is a food safety concern. Deer feces (scat) have been determined as a point source for Escherichia coli O157:H7 contamination of fresh produce. The ecological role of the scooped scarab (Onthophagus hecate (Panzer)), a generalist dung beetle species common in Maine blueberry fields, was explored as a biological control agent and alternatively as a pathogen vector between deer scat and food.A large-scale field survey of wildlife scat indicated that pathogenic E. coli O157:H7 was present, albeit at a low prevalence (1.9% of samples, n = 318), in the Maine lowbush blueberry agroecosystem. A manipulative field experiment verified that, should contact occur between deer scat and blueberry plants and fruit during the summer, contamination with E. coli O157:H7 can occur and persist for more than 72 h. For both the positive control and an experimental scat inoculation treatment, the levels of the bacterial population decreased over time, but at different rates (treatment x time interaction: F _(1.9,18.8) = 358.486, P < 0.0001). The positive control inoculation, which resulted in a higher initial E. coli level on fruit, decayed at a faster rate than inoculation of fruit via scat in the experimental treatment.We conducted 2 laboratory studies to elucidate aspects of dung beetle feeding ecology as it relates to suppression of E. coli O157:H7 from deer scat to lowbush blueberry fruit. In both experiments, dung beetles buried the same amount of scat whether or not the scat was inoculated with the pathogen (F _(1,6) = 0.001; P = 0.999 and (F _(2,17) = 4.10, P = 0.147). Beetles feeding on E. coli inoculated deer scat were not found to vector the pathogen to fruit. In two studies, beetles lowered the amount of pathogenic E. coli persisting in soils compared to soils without beetles (F _(2,9) = 7.757; P = 0.05 and F _(2,17) = 8.0621, P = 0.004).Our study suggests that the dung beetle species, Onthophagus hecate, has the potential to contribute to the suppression of E. coli O157:H7 in agricultural landscapes.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

Fluorometric measurement of tin-protoporphyrin in biological samples

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, can suppress neonatal and other forms of hyperbilirubinemia in laboratory animals, and represents a potential new approach to the treatment of neonatal jaundice in humans. In order to study the disposition of Sn-protoporphyrin in vivo we have developed a sensitive fluorometric method for the quantitation of this metalloporphyrin in biological samples. The method is sensitive to concentrations as low as 0.01 nmol/ml, and is specific for Sn-protoporphyrin even in the presence of other porphyrins such as protoporphyrin.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

BEAST: Bayesian evolutionary analysis by sampling trees

Background  

The evolutionary analysis of molecular sequence variation is a statistical enterprise. This is reflected in the increased use of probabilistic models for phylogenetic inference, multiple sequence alignment, and molecular population genetics. Here we present BEAST: a fast, flexible software architecture for Bayesian analysis of molecular sequences related by an evolutionary tree. A large number of popular stochastic models of sequence evolution are provided and tree-based models suitable for both within- and between-species sequence data are implemented.