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Density Fractions (density + fraction)
Selected AbstractsTARPs ,-2 and ,-7 are essential for AMPA receptor expression in the cerebellumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2010Maya Yamazaki Abstract The ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, ,-2 and ,-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, ,-2 and ,-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in ,-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in ,-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in ,-2/,-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in ,-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber,Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber,Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in ,-7-KO granular layer reflected its loss at mossy fiber,granule cell synapses, whereas that of GluA1 and GluA4 in ,-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, ,-2 and ,-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression. [source] CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix protein CASTGENES TO CELLS, Issue 1 2004Maki Deguchi-Tawarada The cytomatrix at the active zone (CAZ) is thought to define the site of Ca2+ -dependent exocytosis of neurotransmitters. We have recently identified a novel CAZ protein from rat brain which we have named CAST (CAZ-associated structural protein). CAST forms a large molecular complex with other CAZ proteins such as Bassoon, RIM1 and Munc13-1, at least through direct binding to RIM1. Here, we have identified a rat protein that is structurally related to CAST and named it CAST2. Subcellular fractionation analysis of rat brain shows that CAST2 is also tightly associated with the postsynaptic density fraction. Like CAST, CAST2 directly binds RIM1 and forms a hetero-oligomer with CAST. In primary cultured rat hippocampal neurones, CAST2 co-localizes with Bassoon at synapses. Furthermore, immunoelectron microscopy reveals that CAST2 localizes to the vicinity of the presynaptic membrane of synapses in mouse brain. Sequence analysis reveals that CAST2 is a rat orthologue of the human protein ELKS. ELKS has also recently been identified as Rab6IP2 and ERC1. Accordingly, the original CAST is tentatively re-named CAST1. These results indicate that CAST2 is a new component of the CAZ and, together with CAST1, may be involved in the formation of the CAZ structure. [source] Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fractionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Huei-Hsuan Cheng Abstract A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for ,3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal. © 2006 Wiley-Liss, Inc. [source] Distribution of polycyclic aromatic hydrocarbons in particle-size separates and density fractions of typical agricultural soils in the Yangtze River Delta, east ChinaEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 6 2008J. Z. Ni Summary Soil organic matter can be divided into different organic carbon (C) pools with different turnover rates. The organic pollutants in soils associated with these organic C pools may have different bioavailability and environmental risks during the decomposition of soil organic matter. We studied the distribution patterns of 15 USEPA priority polycyclic aromatic hydrocarbons (PAHs) in different particle-size separates (clay, fine silt, coarse silt, fine sand and coarse sand) and density fractions (light and heavy fractions) of nine agricultural topsoils (0,20 cm depth) from a contaminated area in the Yangtze River Delta region of east China. There was a decreasing trend in PAH concentration in particle-size separates with decreasing particle size. However, the different particle-size separates had similar PAH composition. The concentration of PAHs in the light fraction ranged from 13 037 to 107 299 ,g kg,1, far higher than in the heavy fraction, which ranged from 222 to 298 ,g kg,1. Although the light fraction accounted for only 0.4,2.3% of the soils, it was associated with 31.5,69.5% of soil PAHs. The organic matter in coarse silt had the strongest capacity for enrichment with PAHs. Combining the distributions of PAHs and the turnover rates of organic matter in different soil fractions, the environmental risks of PAH-polluted soils may be due mainly to the PAHs associated with sand and the light fraction. [source] Soil organic carbon in density fractions of tropical soils under forest , pasture , secondary forest land use changesEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 2 2008S. Paul Summary Our knowledge of effects of land use changes and soil types on the storage and stability of different soil organic carbon (SOC) fractions in the tropics is limited. We analysed the effect of land use (natural forest, pasture, secondary forest) on SOC storage (depth 0,0.1 m) in density fractions of soils developed on marine Tertiary sediments and on volcanic ashes in the humid tropics of northwest Ecuador. The origin of organic carbon stored in free light (< 1.6 g cm,3) fractions, and in two light fractions (LF) occluded within aggregates of different stability, was determined by means of ,13C natural abundance. Light occluded organic matter was isolated in a first step after aggregate disruption by shaking aggregates with glass pearls (occluded I LF) and in a subsequent step by manual destruction of the most stable microaggregates that survived the first step (occluded II LF). SOC storage in LFs was greater in volcanic ash soils (7.6 ± 0.6 Mg C ha,1) than in sedimentary soils (4.3 ± 0.3 Mg C ha,1). The contribution of the LFs to SOC storage was greater in natural forest (19.2 ± 1.2%) and secondary forest (16.6 ± 1.0%) than in pasture soils (12.8 ± 1.0%), independent of soil parent material. The amount of SOC stored in the occluded I LF material increased with increasing silt + clay content (sedimentary soils, r = 0.73; volcanic ash soils, r = 0.58) and aggregation (sedimentary soils, r = 0.52; volcanic ash soils, r = 0.45). SOC associated with occluded I LF, had the smallest proportion of new, pasture-derived carbon, indicating the stabilizing effect of aggregation. Fast turnover of the occluded II LF material, which was separated from highly stable microaggregates, strongly suggested that this fraction is important in the initial process of aggregate formation. No pasture-derived carbon could be detected in any density fractions of volcanic ash soils under secondary forest, indicating fast turnover of these fractions in tropical volcanic ash soils. [source] Trafficking and localization of platinum complexes in cisplatin-resistant cell lines monitored by fluorescence-labeled platinum,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Xing-Jie Liang Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. Resistance to cisplatin represents a major obstacle to effective cancer therapy because clinically significant levels of resistance quickly emerge after treatment. Based on previous studies indicating abnormal plasma membrane protein trafficking in cisplatin-resistant (CP-r) cells, Fluorescence (Alexa Fluor)-labeled cisplatin was used to determine whether this defect altered the trafficking and localization of cisplatin by comparing drug sensitive KB-3-1 and KB-CP-r cells. Alexa Fluor,cisplatin was readily internalized and localized throughout the KB-3-1 cells, but overall fluorescence decreased in KB-CP-r cells, as detected by flow cytometry (FACS) and confocal microscopy. Only punctate cytoplasmic staining was observed in KB-CP-r cells with less fluorescence observed in the nucleus. Colocalization experiments with a Golgi-selective stain indicate the involvement of Golgi-like vesicles in initial intracellular processing of Alexa Fluor conjugated cisplatin complexes. As detected using an antibody to Alexa Fluor,cisplatin, cisplatin complex-binding proteins (CCBPs) were reduced in membrane fractions of single-step cisplatin-resistant KB-CP.5 cells, and increased in the cytoplasm of KB-CP.5 cells compared to KB-3-1 cells. CCBPs localized to lower density fractions in KB-CP.5 cells than in KB-3-1 cells as determined by iodixanol gradient centrifugation. In summary, inappropriate trafficking of CCBPs might explain resistance to cisplatin in cultured cancer cells, presumably because membrane binding proteins for cisplatin are not properly located on the cell surface in these cells, but are instead trapped in low density vesicles within the cytoplasm. © 2004 Wiley-Liss, Inc. [source] | ||||||||||