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排序方式: 共有163条查询结果,搜索用时 15 毫秒
51.
Altschuler Y Kinlough CL Poland PA Bruns JB Apodaca G Weisz OA Hughey RP 《Molecular biology of the cell》2000,11(3):819-831
MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits. 相似文献
52.
Nascent actin requires interactions with the highly conserved and essential eukaryotic chaperonin-containing TCP-1 (CCT) for its correct folding to the native state in vivo. Biochemical and structural analysis of the interaction between actin and CCT has been studied extensively but the underlying energetics and kinetics of the CCT-dependent actin folding process are not understood. We investigated the unfolding and folding pathways of actin, using stopped flow fluorescence and biochemical techniques. By using very low concentrations of actin, taking account of temperature and ATP concentration dependences we were able to determine accurately the activation energy of unfolding to a stable intermediate, I(3). Use of the fluorescent calcium chelator Quin-2 and consideration of the ATP concentration dependence on the unfolding rate has allowed the intrinsic kinetics to be linked to the accepted reaction scheme for actin denaturation. A free energy of -28.7(+/-0.2) kJ mol(-1) was determined for the loss of ATP from Ca-free G-actin, in good agreement with previous studies. Understanding the K(eq) value for this step then allowed the temperature dependence of the unfolding reaction of co-factor-free actin to be evaluated, yielding an activation energy for the unfolding of G-actin of 81.3(+/-3.3) kJ mol(-1). By chemical coupling of the extrinsic probe, Alexa Fluor 488 to cysteine 374 of native alpha-actin, we were able to follow the binding and folding of I(3) by CCT, observing for the first time, in vitro re-folding of EDTA-denatured G-actin. The high value of the activation energy between native actin and a non-native folding intermediate (I(3)) is characteristic of a partially folded, molten globule state expected to contain partial secondary structure. 相似文献
53.
Holt AG Asako M Lomax CA MacDonald JW Tong L Lomax MI Altschuler RA 《Journal of neurochemistry》2005,93(5):1069-1086
The inferior colliculus (IC) is a major center of integration in the ascending as well as descending auditory pathways, where both excitatory and inhibitory amino acid neurotransmitters play a key role. When normal input to the auditory system is decreased, the balance between excitation and inhibition in the IC is disturbed. We examined global changes in gene expression in the rat IC 3 and 21 days following bilateral deafening, using Affymetrix GeneChip arrays and focused our analysis on changes in expression of neurotransmission-related genes. Over 1400 probe sets in the Affymetrix Rat Genome U34A Array were identified as genes that were differentially expressed. These genes encoded proteins previously reported to change as a consequence of deafness, such as calbindin, as well as proteins not previously reported to be modulated by deafness, such as clathrin. A subset of 19 differentially expressed genes was further examined using quantitative RT-PCR at 3, 21 and 90 days following deafness. These included several GABA, glycine, glutamate receptor and neuropeptide-related genes. Expression of genes for GABA-A receptor subunits beta2, beta3, and gamma2, plus ionotropic glutamate receptor subunits AMPA 2, AMPA 3, and kainate 2, increased at all three times. Expression of glycine receptor alpha1 initially declined and then later increased, while alpha2 increased sharply at 21 days. Glycine receptor alpha3 increased between 3 and 21 days, but decreased at 90 days. Of the neuropeptide-related genes tested with qRT-PCR, tyrosine hydroxylase decreased approximately 50% at all times tested. Serotonin receptor 2C increased at 3, 21, and 90 days. The 5B serotonin receptor decreased at 3 and 21 days and returned to normal by 90 days. Of the genes tested with qRT-PCR, only glycine receptor alpha2 and serotonin receptor 5B returned to normal levels of expression at 90 days. Changes in GABA receptor beta3, GABA receptor gamma2, glutamate receptor 2/3, enkephalin, and tyrosine hydroxylase were further confirmed using immunocytochemistry. 相似文献
54.
Benjamin Pavie Satwik Rajaram Austin Ouyang Jason M. Altschuler Robert J. Steininger III Lani F. Wu Steven J. Altschuler 《Journal of visualized experiments : JoVE》2014,(85)
Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens. 相似文献
55.
56.
EMMA L. WRIGHT COLIN R. BLACK ALEXANDER W. CHEESMAN TREVOR DRAGE DAVID LARGE BENJAMIN L. TURNER SOFIE SJÖGERSTEN 《Global Change Biology》2011,17(9):2867-2881
Tropical peatlands play an important role in the global carbon cycling but little is known about factors regulating carbon dioxide (CO2) and methane (CH4) fluxes from these ecosystems. Here, we test the hypotheses that (i) CO2 and CH4 are produced mainly from surface peat and (ii) that the contribution of subsurface peat to net C emissions is governed by substrate availability. To achieve this, in situ and ex situ CO2 and CH4 fluxes were determined throughout the peat profiles under three vegetation types along a nutrient gradient in a tropical ombrotrophic peatland in Panama. The peat was also characterized with respect to its organic composition using 13C solid state cross‐polarization magic‐angle spinning nuclear magnetic resonance spectroscopy. Deep peat contributed substantially to CO2 effluxes both with respect to actual in situ and potential ex situ fluxes. CH4 was produced throughout the peat profile with distinct subsurface peaks, but net emission was limited by oxidation in the surface layers. CO2 and CH4 production were strongly substrate‐limited and a large proportion of the variance in their production (30% and 63%, respectively) was related to the quantity of carbohydrates in the peat. Furthermore, CO2 and CH4 production differed between vegetation types, suggesting that the quality of plant‐derived carbon inputs is an important driver of trace gas production throughout the peat profile. We conclude that the production of both CO2 and CH4 from subsurface peat is a substantial component of the net efflux of these gases, but that gas production through the peat profile is regulated in part by the degree of decomposition of the peat. 相似文献
57.
58.
Yanqin Wang Chin-Jen Ku Elizabeth R. Zhang Alexander B. Artyukhin Orion D. Weiner Lani F. Wu Steven J. Altschuler 《Cell reports》2013,3(5):1607-1616
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59.
1. Benthic macroinvertebrates (MI) are commonly used to assess freshwater ecosystems with the reference condition approach. Such assessments necessitate control for natural community variation, either by categorical typologies or by predictive models that have been widely and successfully developed for running water biota but not previously for lake profundal invertebrates. 2. We evaluated four modelling techniques [multivariate regression tree (MRT), limiting environmental differences, nonparametric multiplicative regression (NPMR) and River Invertebrate Prediction And Classification System (RIVPACS) and the operative Finnish lake typology for assessing taxonomic completeness (observed‐to‐expected number of taxa, O/E) of profundal MI assemblages. We used data from 74 and 33 minimally disturbed reference lake basins for calibration and validation of the approaches, respectively, and 72 test basins subject to various anthropogenic pressures to evaluate sensitivity to detect impact. Either all predicted taxa (threshold probability of capture Pt = 0+) or only those predicted to be captured with ≥0.25 probability were used to calculate O/E. 3. With Pt = 0.25, all four modelling approaches were accurate (mean O/E = 0.966–1.053) but imprecise (SD of O/E = 0.279–0.304) in predicting the fauna actually observed in validation sites. All models were subtly more precise than a null model (mean 1.038, SD 0.343) or the typology (1.046, 0.327). The taxon‐specific NPMR model was slightly more precise than the other three models based on site groupings. 4. The O/E values correlated relatively weakly (r = 0.55–0.86) among the approaches, which thus produced contrasting lake‐specific assessments, despite their seemingly comparable performances. Indeed, typology, suggesting that MI assemblages were impaired in 56% of test sites, was more sensitive than the other approaches (26–46%) as an indicator of human‐induced deterioration. However, this greater ostensible sensitivity seemed to be biased, as lake morphometry, a main driver of natural community variation, remained uncontrolled by the typology. 5. Generally, our exercise illustrates the inconclusiveness of the common validation criteria for the assessment methods. The apparent poor predictability of the profundal fauna, irrespective of the method, may partly stem from large observation error, which could be alleviated by more intensive sampling. However, instead of an O/E‐taxa index, some other metric encompassing quantitative aspects might be preferable for assessing these species‐poor communities. 相似文献
60.
Hayley M Bennett Hoi Ping Mok Effrossyni Gkrania-Klotsas Isheng J Tsai Eleanor J Stanley Nagui M Antoun Avril Coghlan Bhavana Harsha Alessandra Traini Diogo M Ribeiro Sascha Steinbiss Sebastian B Lucas Kieren SJ Allinson Stephen J Price Thomas S Santarius Andrew J Carmichael Peter L Chiodini Nancy Holroyd Andrew F Dean Matthew Berriman 《Genome biology》2014,15(11)