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Insoluble Fraction (insoluble + fraction)
Selected AbstractsDynamics of metal subcellular distribution and its relationship with metal uptake in marine musselsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2005Tania Y-T. Abstract We examined the dynamics of subcellular distribution of metals (Cd, Ag, and Zn) in the marine green mussel Perna viridis by partitioning the metals into the insoluble fraction (IF), heat-sensitive proteins (HSP), and metallothionein-like proteins (MTLP) during metal uptake and elimination. Variations in metal uptake and elimination then were correlated with the subcellular distributions of these metals. The IF and HSP were the first ligands to bind with the metals during the dissolved exposure, and more metals were found in the HSP when the metal influx rate was higher. However, to minimize toxicity, metals were redistributed from HSP to MTLP afterwards. The subcellular distribution of metals was dependent of the exposure route in the mussels. During dietary metal exposure, the metals attained equilibrium before they were assimilated and the metal assimilation efficiency was independent of the metal partitioning in different subcellular fractions. During the efflux, metals in the soluble fraction mediated depuration, whereas metals in the insoluble fraction acted as a final storage pool. Redistribution also may occur between the metal-sensitive and inactive pools without significant depuration as a secondary protective mechanism. We further demonstrated that the higher efflux rate of Ag and Cd was related to a higher partitioning in the MTLP and a lower partitioning in the IF. Our study shows that subcellular pools other than MTLP were involved in immediate metal handling in the bivalves. The wide dynamics of subcellular metal distribution suggests that the relevance of individual subcellular fractions is dependent on the exposure pathway. [source] Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406FEBS JOURNAL, Issue 11 2008Structural importance of the crown domain Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP. [source] Ser-59 is the major phosphorylation site in ,B-crystallin accumulated in the brains of patients with Alexander's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Kanefusa Kato The phosphorylation state of ,B-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against ,B-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25,28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of ,B-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. ,B-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. ,B-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, ,B-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in ,B-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59. [source] Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (Oryza sativa)PLANT CELL & ENVIRONMENT, Issue 12 2008SANG-KYU LEE ABSTRACT During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of 14C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants. [source] Formation of a fibrillar morphology of crosslinked epoxy in a polystyrene continuous phase by reactive extrusionPOLYMER ENGINEERING & SCIENCE, Issue 4 2004Françoise Fenouillot An immiscible polymer blend where the dispersed phase is fibrillar was prepared by in situ crosslinking of the minor phase. A model polystyrene/epoxy-amine blend was selected on the basis of rheological (achievement of the fibrillar structure) and reactivity (fast crosslinking) criteria. The system was a polystyrene/diglycidyl ether of bisphenol A (DGEBA)-aminoethyl piperazine (AEP) blend. At the temperature of extrusion, 180°C, the DGEBA is immiscible in PS and heterogeneous material is obtained. The elongational flow imposed by drawing the extrudate at the die exit permitted controlled generation of a fibrillar morphology of the dispersed epoxy phase, with a fiber diameter of 1 ,m and an aspect ratio greater than 100. It was shown that when the amine comonomer was injected into the extruder, its reactivity with DGEBA at high temperature was high enough to ensure partial crosslinking of the epoxy. The fibrils were formed even though the gel point of the epoxy phase was exceeded. However, above a certain critical insoluble fraction that we estimated to be between 45% and 70%, a coarsening of the structure appeared, caused by the decreasing deformability of the domains and their coalescence. Finally, for our system, the crosslinking of the dispersed phase up to 90% of insoluble fraction did not totally stabilize the morphology after the second processing step (injection molding). Polym. Eng. Sci. 44:625,637, 2004. © 2004 Society of Plastics Engineers. [source] Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009M. Paulina Dani Abstract A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti-aggregation activity in vitro and shares the same N-terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full-length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N-terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1,mM) and growth of the bacteria at 37°C for 5,h, or at 24°C for 20,h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc. [source] Functional characterization of the C-terminal domain of mouse capping enzymeCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2006Li Liu Abstract Mouse capping enzyme (Mce1) consists of two functional domains: the amino-terminal triphosphatase domain and the carboxyl-terminal guanylyltransferase (GTase) domain. The bifunctional Mce1 gene encodes 597,a.a. with a molecular weight ,68,kDa. Mce1,cDNA is located on chromosome 4A4,4A5 and is composed of 17 exons. To functionally characterize the C-terminus of Mce1, we generated four truncated proteins with 12, 30, 37, or 60,a.a. deletions from the C-terminus of either the wild type (Mce1) or the isolated GTase domain (211,597), respectively. Plasmid shuffling experiment with Saccharomyces cerevisiae GTase subunit gene CEG1 null mutant demonstrated that deletion mutants 211,567 and 211,585 were able to support cell viability in the presence of 5-fluoroorotic acid, whereas 211,537 and 211,560 were not. Consistent with the yeast genetic study, both 211,567 and 211,585 had significant GTase activity in vitro, while 211,537 and 211,560 that were only detected in the insoluble fraction in the bacterial expression system, were completely inactive. Overall, both in vivo and in vitro studies indicate that the functional domain of Mce1 is between a.a. 211 and 567, and the heptapeptide sequence between 561 and 567 may play an important role in the enzyme activity. Copyright © 2005 John Wiley & Sons, Ltd. [source] Proteome analysis of human nuclear insoluble fractionsGENES TO CELLS, Issue 8 2009Hideaki Takata The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus. [source] Ser-59 is the major phosphorylation site in ,B-crystallin accumulated in the brains of patients with Alexander's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Kanefusa Kato The phosphorylation state of ,B-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against ,B-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25,28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of ,B-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. ,B-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. ,B-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, ,B-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in ,B-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59. [source] The polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation of the c-Met receptor in prostate cancer cellsMOLECULAR CARCINOGENESIS, Issue 8 2010Damian Duhon Abstract The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4,h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-,-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts. © 2010 Wiley-Liss, Inc. [source] Sequential Effects of Ultraviolet Radiation on the Histomorphology, Cell Density and Antioxidative Status of the Lens Epithelium,An In Vivo Study ,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2003S. R. Kaid Johar ABSTRACT In vivo progressive effects of UV irradiation on the lens epithelium were studied using various histomorphological and biochemical parameters. Fifteen day old rat pups were exposed to 600 mW/m2 of radiation, including UV-A and UV-B, 12 h daily for 90, 120, 150 and 180 days. Biochemical parameters such as protein-bound and non,protein-bound sulfhydryl groups in both soluble and insoluble fractions and enzymes, which play an important role in combating the oxidative stress, were studied. Decreased cell density of lens epithelial cells (LEC) was observed in all three zones along with the decrease in the levels of soluble sulfhydryls (S-SH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). On the other hand, an increase in insoluble sulfhydryls was observed. Because of the decrease in S-SH and GR activities, the LEC became vulnerable to oxidative stress. Decreased activities of SOD, GPx and CAT suggest elevated oxidative stress. This effect of UV radiation may lead to cell death that may be responsible for the observed decrease in the cell density in all three zones of the lens epithelium. [source] Processing behavior of polycarbonate/functionalized-ethylene copolymer blendsPOLYMER ENGINEERING & SCIENCE, Issue 12 2000Marcos L. Dias The melt blending of polycarbonate (PC) and ethylene-methacrylic acid copolymers (EFC) either in the acid form (EFC-H) or partially neutralized with sodium (EFC-Na) or zinc (EFC-Zn) was investigated. Torque monitoring of the blending showed that the polymers are capable of reacting generating new chemical species that increase the melt viscosity. As general behavior, the torque curves pass by a maximum that takes place before 30 min, the final torque being higher than that of the individual polymers. SEC analyses reveal that PC degradation also occurs and is stronger in the case of blends with EFC-Na that acts to catalyze PC degradation, promoting CO2 formation. FTIR studies on chloroform insoluble fractions of the PC/EFC-Zn blends showed that in addition to a very small number of carbonate groups, feature absorption bands of aromatic ester and hydroxyl groups appear in the new chemical species formed during the reactive processing. [source] The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leavesBIOFACTORS, Issue 1-4 2006N. Wilhelmová Abstract Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species. [source] | |||||||||||||