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Membrane Fraction (membrane + fraction)
Kinds of Membrane Fraction Selected AbstractsPresence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx moriFEBS JOURNAL, Issue 15 2004Mohamed Elmogy Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1,m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol·mg,1 protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events. [source] Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulansCYTOSKELETON, Issue 2 2003Katrin V. Koch Abstract Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans,Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20°C but not at 37°C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria. Cell Motil. Cytoskeleton 55:114,124, 2003. © 2003 Wiley-Liss, Inc. [source] A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DEELECTROPHORESIS, Issue 23 2009Gloria Alvarez-Llamas Abstract With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBindŌ reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and well-resolved 2-DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano-LC coupled to an LTQ-Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated. [source] Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)FEBS JOURNAL, Issue 3 2006Sanjida Ahmed Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1. Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] The role of Sov protein in the secretion of gingipain protease virulence factors of Porphyromonas gingivalisFEMS MICROBIOLOGY LETTERS, Issue 2 2010Keitarou Saiki Abstract Porphyromonas gingivalis transports Arg-gingipains and Lys-gingipain across the outer membrane via an unknown pathway. Recently, we found that the sov gene of P. gingivalis W83 was required for this step. In the present study, we characterized the Sov protein. We constructed a P. gingivalis strain that expresses histidine-tagged Sov instead of Sov. Subcellular fractionations and a histidine-tag pulldown experiment showed that histidine-tagged Sov was present in an outer membrane fraction. Furthermore, antiserum raised against the terminal regions of Sov obstructed the secretion of Arg-gingipains from wild-type W83 cells. A deletion study showed that the region from Phe2495 to the C-terminus Gln2499 of Sov is essential for gingipain secretion. Anti-histidine-tag immunoglobulins interfered with the secretion of Arg-gingipains by P. gingivalis cells that expressed histidine-tagged Sov. In conclusion, we found that Sov is an outer membrane protein participating in the secretion of gingipains and that the C-terminal region of Sov is exposed to the extracellular milieu and involved in the modulation of Sov function. [source] Kir4.1 and AQP4 associate with Dp71- and utrophin-DAPs complexes in specific and defined microdomains of Müller retinal glial cell membraneGLIA, Issue 6 2008Patrice E. Fort Abstract The dystrophin-associated proteins (DAPs) complex consisting of dystroglycan, syntrophin, dystrobrevin, and sarcoglycans in muscle cells is associated either with dystrophin or its homolog utrophin. In rat retina, a similar complex was found associated with dystrophin-Dp71 that serves as an anchor for the inwardly rectifying potassium channel Kir4.1 and the aqueous pore, aquaporin-4 (AQP4). Here, using immunofluorescence imaging of isolated retinal Müller glial cells and co-immunoprecipitation experiments performed on an enriched Müller glial cells end-feet fraction, we investigated the effect of Dp71 deletion on the composition, anchoring, and membrane localization of the DAPs,Kir4.1 and/or ,AQP4 complex. Two distinct complexes were identified in the end-feet fraction associated either with Dp71 or with utrophin. Upon Dp71 deletion, the corresponding DAPs complex was disrupted and a compensating utrophin upregulation was observed, accompanied by diffuse overall staining of Kir4.1 along the Müller glial cells and redistribution of the K+ conductance. Dp71 deficiency was also associated with a marked reduction of AQP4 and ,-dystroglycan expression. Furthermore, it was observed that the Dp71,DAPs dependent complex could be, at least partially, associated with a specific membrane fraction. These results demonstrate that Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in Müller cell end-feet membranes. They also show that despite its close relationship to the dystrophin proteins and its correlated upregulation, utrophin is only partially compensating for the absence of Dp71 in Müller glial cells. © 2008 Wiley-Liss, Inc. [source] Processing of extended shelf life milk using microfiltrationINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 4 2006WOLFGANG HOFFMANN Extended shelf life (ESL) milk was processed with integrated microfiltration (pore size 1.4 µm). The germ-enriched retentate was not used for the final whole milk. Microfiltration led only to a negligible change in the content of the main components of the ESL product compared with the source milk. The total protein was only slightly decreased (0.02,0.03%) and the ratio of the protein fractions was unchanged within the measurement accuracy. The furosine content of the isolated fat globuline membrane fraction could be used as a diagnostic to prove cream had been subjected to high-temperature treatment. The shelf life of the ESL milk was distinctly prolonged compared to HTST-pasteurized milk. [source] The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow troutJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2007F. Dumetz Abstract Aims:, The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. Methods and Results:, The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the ,OmpA/MotB' domain/signature and five putative ,Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. Conclusions:, Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. Significance and Impact of the Study:, This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine. [source] High glucose inhibits fructose uptake in renal proximal tubule cells: Involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Su Hyung Park The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l -glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14,22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs. © 2004 Wiley-Liss, Inc. [source] Role of phospholipases A2 in growth-dependent changes in prostaglandin release from 3T6 fibroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2001Teresa Sįnchez Previously, we reported a growth-dependent change in prostaglandin production as a consequence of a marked growth-dependent alteration in arachidonic acid (AA) mobilization from phospholipids. Our present results show that fetal calf serum (FCS) and 4,-phorbol-12-myristate acetate (PMA) caused an enhancement of phospholipase A2 (PLA2) activity in the membrane fraction of non-confluent cells allowing PLA2 access to its substrate and the release of AA. Western blot analysis has shown that FCS and PMA increased secreted PLA2 (sPLA2) expression in non-confluent 3T6 fibroblast cultures. Moreover, FCS and PMA induced dithiothreitol-sensitive and bromoenol lactone-sensitive PLA2 activities in cytosol and membrane fraction. However, these stimuli did not modify significantly the PLA2 activity in both fractions when 3T6 fibroblasts reached a high cell density. This could be associated with the impairment of AA mobilization in these cell culture conditions. On the other hand, we observed that FCS and PMA induced the same prostaglandin H synthase-2 induction in non-confluent and confluent culture conditions. Moreover, the prostaglandin E2 levels reached in cell culture supernatants were independent of the degree of confluence when AA was added exogenously. These results suggest that the changes of intracellular distribution of PLA2 activity of sPLA2 and iPLA2 stimulated by exogenous stimuli may be controlled by cell density conditions which constitute an important mechanism in the regulation of prostaglandin release.© 2001 Wiley-Liss, Inc. [source] Subcellular redistribution of protein kinase C isozymes is associated with rat liver cirrhotic changes induced by carbon tetrachloride or thioacetamideJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2001Da-Hee Jeong Abstract Background and Aims: Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA). Methods and Results: Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear-cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment. Discussion: When the subcellular redistribution of PKC isozymes (PKC,, -,1, -,, and -,) was examined, all the PKC isozymes in CCl4 -treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment. Conclusions: From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis. [source] Temperature-dependent localization of GPI-anchored intestinal alkaline phosphatase in model rafts,JOURNAL OF MOLECULAR RECOGNITION, Issue 6 2007Marie-Cécile Giocondi Abstract In plasma membranes, most of glycosylphosphatidylinositol (GPI)-anchored proteins would be associated with rafts, a category of ordered microdomains enriched in sphingolipids and cholesterol (Ch). They would be also concentrated in the detergent resistant membranes (DRMs), a plasma membrane fraction extracted at low temperature. Preferential localization of GPI-anchored proteins in these membrane domains is essentially governed by their high lipid order, as compared to their environment. Changes in the temperature are expected to modify the membrane lipid order, suggesting that they could affect the distribution of GPI-anchored proteins between membrane domains. Validity of this hypothesis was examined by investigating the temperature-dependent localization of the GPI-anchored bovine intestinal alkaline phophatase (BIAP) into model raft made of palmitoyloleoylphosphatidylcholine/sphingomyelin/cholesterol (POPC/SM/Chl) supported membranes. Atomic force microscopy (AFM) shows that the inserted BIAP is localized in the SM/Chl enriched ordered domains at low temperature. Above 30°C, BIAP redistributes and is present in both the ,fluid' POPC enriched and the ordered SM/Chl domains. These data strongly suggest that in cells the composition of plasma membrane domains at low temperature differs from that at physiological temperature. Copyright © 2007 John Wiley & Sons, Ltd. [source] Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-,-ligand, in insulin-deficient diabetic ratsJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Shin-Ichiro Yamagishi Abstract Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-, ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-, in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-,. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-,. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway. [source] Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signalingJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Haruhiro Higashida Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+ -dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and ,-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed. [source] Exposure to lead elevates induction of zif268 and Arc mRNA in rats after electroconvulsive shock: The involvement of protein kinase CJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2002Kyung-Ah Kim Abstract Exposure to lead is well known to impair cognitive function in young children. Because of the importance of gene regulation for neurodevelopment, we examined the effect of lead on the induction of the mRNA of the immediate early genes zif268 and Arc. The time course for the induction of zif268 mRNA and Arc mRNA by electroconvulsant shock (ECS) was altered in the area of the dentate gyrus of the hippocampus in rats exposed to lead from postnatal days (PND) 1 to 28. Other areas of the hippocampus were not affected by lead. The effects on the induction of zif268 mRNA were observed at blood lead levels as low as 12 ,g/dl. No change in the induction of zif268 mRNA was observed in the hippocampus of rats exposed to lead from PND 28 to PND 56. Because of the possible involvement of protein kinase C (PKC) in the effect of lead, activation of different isoforms of PKC was investigated. An increase in the amount of PKC, and PKC, was observed at 60 min after ECS in the membrane fraction from hippocampus, indicating activation of these isoforms. The amount of PKC, in membranes was higher in rats exposed to lead than in rats not exposed to lead after ECS. Taken together, the data suggest that lead may disturb regulation of specific immediate early genes by activating PKC,. © 2002 Wiley-Liss, Inc. [source] Association of neuronal nitric oxide synthase (nNOS) with ,1-syntrophin at the sarcolemmaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2001Yuko Miyagoe-Suzuki Abstract ,1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. ,1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the ,1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with ,1-syntrophin. Microsc. Res. Tech. 55:164,170, 2001. © 2001 Wiley-Liss, Inc. [source] Kin1 is a plasma membrane-associated kinase that regulates the cell surface in fission yeastMOLECULAR MICROBIOLOGY, Issue 5 2010Angela Cadou Summary Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1-GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1, null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue-sensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membrane-associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen-activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen-activated protein kinase components reduces kin1, hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall. [source] Imp/OstA is required for cell envelope biogenesis in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 5 2002Martin Braun Summary In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane. [source] Bicarbonate-Induced phosphorylation of p270 protein in mouse sperm by cAMP-Dependent protein kinaseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008Masako Kaneto Abstract Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility. Mol. Reprod. Dev. 75: 1045,1053, 2007. © 2007 Wiley-Liss, Inc. [source] 1H- and 31P-MR spectroscopy of primary and recurrent human brain tumors in vitro: malignancy-characteristic profiles of water soluble and lipophilic spectral componentsNMR IN BIOMEDICINE, Issue 5 2001Fritz-Georg Lehnhardt Abstract In vitro NMR spectrocopy was performed on specimen of human brain tumors. From all patients, tissue samples of primary tumors and their first recurrences were examined. 31P- and 1H-spectra were recorded from samples of meningioma, astrocytoma and glioblastoma. A double extraction procedure of the tissue samples permitted acquisition of information from the membrane fraction and from the cytosolic fraction. 31P-spectra were used to analyze the lipophilic fraction (phospholipids of the membrane) of the tissue extracts, while the 1H-spectra reflected information on the metabolic alterations of the hydrophilic, cytosolic fraction of the tissue. The tumor types showed distinctive spectral patterns in both the 31P- and the 1H-spectra. Based on the total detectable 31P signal, the level of phosphatidylcholine was about 34% lower in primary astrocytomas than in primary glioblastomas (p,=,0.0003), whereas the level of sphingomyelin was about 45% lower in primary gioblastomas than in primary astrocytomas (p,=,0.0061). A similar tendency of these phospholipids was observed when comparing primary and recurrent astrocytoma samples from the same individuals [+15% (p,=,0.0103) and ,23% (p,=,0.0314) change, respectively]. 1H-spectra of gliomas were characterized by an increase of the ratios of alanine, glycine and choline over creatine as a function of the degree of malignancy. In agreement with findings in the 31P-spectra, the 1H-spectra of recurrent astrocytomas showed metabolic profiles of increased malignancy in comparison to their primary occurrence. Since gliomas tend to increase in malignancy upon recurrence, this may reflect evolving tumor metabolism. 1H-spectra of meningiomas showed the highest ratio of alanine over creatine accompanied by a near absence of myo-inositol. Phospholipid profiles of meningiomas showed higher fractional contents of phosphatidylcholine along with lower phosphatidylserine compared to astrocytomas, while higher phosphatidylethanolamine and sphingomyelin fractional contents distinguished meningiomas from glioblastomas. The extraction method being used in this study combined with high-resolution 1H- and 31P-MRS provides a wide range of biochemical information, which enables differentiation not only between tumor types but also between primary and recurrent gliomas, reflecting an evolving tumor metabolism. Copyright © 2001 John Wiley & Sons, Ltd. [source] High salt-treatment-induced Na+ extrusion and low salt-treatment-induced Na+ accumulation in suspension-cultured cells of the mangrove plant, Bruguiera sexangulaPLANT CELL & ENVIRONMENT, Issue 10 2001M. Kura-Hotta Abstract A suspension-cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non-adapted cells were transferred to media containing 0,200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na+, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na+ and Cl, concentrations. In the latter treatment, the intracellular concentration of Na+ returned to the original low level within 2 weeks. It took a longer time for Cl, to return to its original level. As a result, the Na+ and Cl, concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na+/H+ antiporter, suppressed the recovery of Na+ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate-dependent H+ -ATPase and the Na+/H+ antiporter in the plasma membrane fraction. [source] Island clustering analysis for the comparison of the membrane and the soluble protein fractions of human brain proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2008Kyung-Hoon Kwon Abstract A protein identified in multiple separate bands of a 1-D gel reflects variation in the molecular weight caused by alternative splicing, endoproteolytic cleavage, or PTMs, such as glycosylation or ubiquitination. To characterize such a protein distribution over the bands, we defined an entity called an ,island' as the band region including the bands of the same protein identified sequentially. We quantified the island distribution using a new variable called an Iscore. Previously, as described in Park et al.. (Proteomics 2006, 6, 4978,4986.), we analyzed human brain tissue using a multidimensional MS/MS separation method. Here, the new method of island analysis was applied to the previous proteome data. The soluble and membrane protein fractions of human brain tissue were reanalyzed using the island distribution. The proteome of the soluble fraction exhibited more variation in island positions than that of the membrane fraction. Through the island analysis, we identified protein modifications and protein complexes over the 1-D gel bands. [source] Exploring the proteome of meningococcal outer membrane vesicle vaccinesPROTEOMICS - CLINICAL APPLICATIONS, Issue 9 2007Jun X. Wheeler Dr. Abstract Neisseria meningitidis, one of the principal causes of bacterial meningitis and septicemia, continues to present a challenge for vaccine developers. While significant progress has been made in the development and implementation of conjugate vaccines, which are based on the capsular polysaccharide of the organism, this approach has failed to produce a vaccine against organisms expressing a serogroup B capsule. The completion of the first meningococcal genome sequences in 2000 provided new ways of meeting this challenge. One approach has been to learn more about meningococcal biology and pathogenesis through exploring its proteome. This article reviews the results of ten recent studies of the meningococcal proteome and compares the different methodologies employed. Not surprisingly, given the renewed impetus to develop a comprehensive vaccine and the continuing clinical development of outer membrane vesicle vaccines, many of these studies focus on the proteome of the outer membrane fraction. As in other areas of proteome research, the direct comparison of data from different studies is hampered by the lack of standardization of separation technologies and data formats. Nevertheless, proteomic analysis, especially when combined with detailed knowledge of meningococcal population structures, represents a powerful tool in the development of vaccines against this important pathogen. [source] Bioorganic studies on plant movement, from natural products to its receptorTHE CHEMICAL RECORD, Issue 6 2006Mitsuru Shoji Abstract The chemical aspects of the circadian leaf movement known as "nyctinasty" are discussed in this paper. Each of the nyctinastic plants of five different genera so far examined contained a pair of factors, one of which induced leaf closure and another induced leaf opening. The relative contents of the closing and opening factors changed correlating with the nyctinastic leaf movement. The use of fluorescence-labeled and photoaffinity-labeled factors revealed that the factors bind to specific cells, the motor cells, present in the pulvini, and that the membrane fraction of the motor cells contained two proteins of 210 and 180 kDa, which can bind to the factors. © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 344,355; 2006: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20102 [source] YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Ming-Shyan Chang YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-,, -,, -,, -, and -, isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-,, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-,. The MEK inhibitor, PD 98059 (10,50 ,M), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-, activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression. British Journal of Pharmacology (2002) 136, 558,567; doi:10.1038/sj.bjp.0704777 [source] Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C, activationCELL PROLIFERATION, Issue 6 2001P. W. Sylvester Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0,250 µm) of ,-, ,-, or ,-tocopherol or ,-, ,-, or ,-tocotrienol. Treatment with growth inhibitory doses of ,-tocopherol (100 µm), ,-tocotrienol (50 µm), or ,- or ,-tocotrienol (10 µm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC,. However, these treatments were found to inhibit EGF-induced PKC, activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 µm,- or ,-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC, activation. [source] Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-, activationCELLULAR MICROBIOLOGY, Issue 9 2008Ambrose Jong Summary Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood,brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKC, in HBMEC. During C. neoformans infection, phosphorylation of PKC, was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKC, isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKC, colocalized with ,-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKC, function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKC, and downstream actin filament activity during the fungal invasion into HBMEC. [source] Competitive MS Binding Assays for Dopamine D2 Receptors Employing Spiperone as a Native MarkerCHEMBIOCHEM, Issue 10 2005Karin V. Niessen Abstract A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays. [source] Protein kinase C mRNA and protein expressions in hypobaric hypoxia-induced cardiac hypertrophy in ratsACTA PHYSIOLOGICA, Issue 4 2010M. Uenoyama Abstract Aim:, Protein kinase C (PKC), cloned as a serine/threonine kinase, plays key roles in diverse intracellular signalling processes and in cardiovascular remodelling during pressure overload or volume overload. We looked for correlations between changes in PKC isoforms (levels and/or subcellular distributions) and cardiac remodelling during experimental hypobaric hypoxic environment (HHE)-induced pulmonary hypertension. Methods:, To study the PKC system in the heart during HHE, 148 male Wistar rats were housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level (10% O2). Results:, At 14 or more days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV): (1) the expression of PKC-, protein in the cytosolic and membrane fractions was increased at 3,14 days and at 5,7 days of exposure respectively; (ii) the cytosolic expression of PKC-, protein was increased at 1,5, 14 and 21 days of exposure; (3) the membrane expressions of the proteins were decreased at 14,21 (PKC-,II), 14,21 (PKC-,), and 0.5,5 and 21 (PKC-,) days of exposure; (4) the expression of the active form of PKC-, protein on the plasma membrane was increased at 3 days of exposure (based on semiquantitative analysis of the immunohistochemistry). In the left ventricle, the expressions of the PKC mRNAs, and of their cytosolic and membrane proteins, were almost unchanged. The above changes in PKC-,, which were strongly evident in the RV, occurred alongside the increase in PAP. Conclusion:, PKC-, may help to modulate the right ventricular hypertrophy caused by pulmonary hypertension in HHE. [source] | ||||||||||||||||||||||||||||||||||