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121.
Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed. 相似文献
122.
Crystal N. Falco Kaitlyn M. Dykstra Bradley P. Yates Peter B. Berget Professor 《Biotechnology journal》2009,4(9):1328-1336
Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors. 相似文献
123.
Vladimir V. Shuvaev Jingyan Han Samira Tliba Evguenia Arguiri Melpo Christofidou-Solomidou Servio H. Ramirez Holly Dykstra Yuri Persidsky Dmitriy N. Atochin Paul L. Huang Vladimir R. Muzykantov 《PloS one》2013,8(10)
Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation. 相似文献
124.
Interaction between RNA polymerase and a ribosomal RNA promoter of E. coli. 总被引:1,自引:2,他引:1 下载免费PDF全文
The interaction between RNA polymerase and the E. coli ribosomal (r) RNA promoter(s) of the rrnE operon has been studied by the filter-binding method. The extent of complex formation between RNA polymerase and rrnE promoter(s) is salt-dependent; ppGpp specifically inhibits interaction of RNA polymerase with the rrnE promoter(s). A tentative model is proposed for the molecular events in the early steps of rRNA initiation: a transition of the primarily formed, labile RNA polymerase-rRNA promoter complex to a more stable form is the determining step. This step is salt-sensitive; ppGpp acts on this "isomerization". 相似文献
125.
Background
Cancer treatments are complex and involve different actions, which include many times a surgical procedure. Medical imaging provides important information for surgical planning, and it usually demands a proper segmentation, i.e., the identification of meaningful objects, such as organs and lesions. This study proposes a methodology to segment the liver, its vessels and nodules from computer tomography images for surgical planning. 相似文献126.
127.
Stuart Wagenius Amy B. Dykstra Caroline E. Ridley Ruth G. Shaw 《Restoration Ecology》2012,20(3):352-359
Establishment of persistent plant populations may be restricted by limitations on the numbers of seeds, emergence of seedlings, or survival to reproductive maturity. The relative importance of these phases in establishment of new populations, particularly restorations, is poorly understood. In an experiment to quantify seedling emergence and juvenile survival of Echinacea angustifolia during its reintroduction to previously agricultural sites, we evaluated effects of two types of vegetation and prescribed burning at four times relative to sowing. We collected achenes from prairie remnants in western Minnesota, United States, and, each October 2000–2002, overseeded them into nearby study plots either in recently planted stands of native grasses or in oldfields abandoned 40 years earlier. For each cohort, we determined germinability of achenes in the laboratory and, in the field, monitored seedling emergence the following spring and subsequent survival in annual censuses through summer 2009. Germinability ranged from 20 to 37%, varying significantly among collection years. Seedlings emerged in every treatment combination, but emergence rarely exceeded 8% of achenes sown. Burns during the spring prior to sowing tended to enhance emergence, but to differing degrees depending on the year and vegetation. Burning in the spring after sowing reduced emergence. Burning enhanced juvenile survival in restored plots but not in oldfields. Strategies to reintroduce this species should include burning in the spring before sowing, sowing large quantities of seed, avoiding burning in the spring following sowing, and burning at least once within the first 6 years. 相似文献
128.
Ortega Roldan JL Romero Romero ML Ora A Ab E Lopez Mayorga O Azuaga AI van Nuland NA 《Journal of biomolecular NMR》2007,39(4):331-336
CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney
function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin.
These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP.
To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have
started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C
domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure
was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of 1HN, 15N, 1Hα, 13Cα, 13CO and 13Cβ chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure
of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site
for the recognition of polyproline sequences. 相似文献
129.
Cell coats were cytochemically demonstrated for the first time in myxamebae of Fulig- septica, Didymium iridis, Dictyostelium discoideum, Cavostelium apophysatum, and amebae of Naegleria grubei. The stain enhances the cell coats of Physarum polycephalum plasmodia, Ceratiomyxa fruticulosa myxamebae, and Acanthamoeba sp. Cell coats usually unstained by cationic dyes stain intensely with the aid of the new cytochemical protocol utilizing 0.5% Alcian blue in the primary fixative and 0.05% ruthenium red in the secondary fixative. 相似文献
130.
Alzheimer and prion diseases are neurodegenerative disorders characterised by the abnormal processing of amyloid-β (Aβ) peptide and prion protein (PrPC), respectively. Recent evidence indicates that PrPC may play a critical role in the pathogenesis of Alzheimer disease. PrPC interacts with and inhibits the β-secretase BACE1, the rate-limiting enzyme in the production of Aβ. More recently PrPC was identified as a receptor for Aβ oligomers and the expression of PrPC appears to be controlled by the amyloid intracellular domain (AICD). Here we review these observations and propose a feedback loop in the normal brain where PrPC exerts an inhibitory effect on BACE1 to decrease both Aβ and AICD production. In turn, the AICD upregulates PrPC expression, thus maintaining the inhibitory effect of PrPC on BACE1. In Alzheimer disease, this feedback loop is disrupted, and the increased level of Aβ oligomers bind to PrPC and prevent it from regulating BACE1 activity.Key words: alzheimer disease, amyloid-β, Aβ oligomers, amyloid intracellular domain, BACE1, presenilin, prion protein 相似文献