MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'BJ Cell' source. Sun, 21 Mar 2010 16:57:40 +0100 http://www.medworm.com/index.php?rid=3377653&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091604 Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase that is suggested to play a role in cell cycle progression and cellular differentiation. However, it is not known if the function of ERK3 is regulated during the cell cycle. Here, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3 deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using mass spectrometry analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Se... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3377653 Thu, 18 Mar 2010 00:00:00 +0100 3377653 http://www.medworm.com/index.php?rid=3330725&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100096 In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gµ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gµ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly i... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3330725 Thu, 04 Mar 2010 00:00:00 +0100 3330725 http://www.medworm.com/index.php?rid=3326313&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091469 The prion diseases occur following the conversion of the cellular prion protein (PrPC) into an alternatively folded, disease-related isoform (PrPSc). However, the spread of PrPSc from cell to cell is poorly understood. Here we report that soluble PrPSc bound to and replicated within both GT1 neuronal cells and primary cortical neurones. The capacity of PrPSc to bind and replicate within cells was significantly reduced by enzymatic modification of its glycosylphosphatidylinositol (GPI) anchor. Thus, PrPSc that had been digested with phosphatidylinositol-phospholipase C bound poorly to GT1 cells or cortical neurones and did not result in PrPSc formation in recipient cells. PrPSc that had been digested with phospholipase A2 (PrPSc-G-lyso-PI) bound readily to GT1 cells and cortical neurones bu... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3326313 Wed, 03 Mar 2010 00:00:00 +0100 3326313 http://www.medworm.com/index.php?rid=3326312&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091005 Signal peptide peptidase (SPP) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of endoplasmic reticulum (ER) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP specifically and tightly interacts with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both, SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Pr... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3326312 Wed, 03 Mar 2010 00:00:00 +0100 3326312 http://www.medworm.com/index.php?rid=3321595&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090867 Urotensin II (UII) and its paralog urotensin II-related peptide (URP) are two vasoactive neuropeptides whose respective central actions are currently unknown. Here, we have compared the mechanism of action of URP and UII on cultured astrocytes. Competition experiments performed with [125I]UII showed the presence of very high- and high-affinity binding sites for UII, and a single high-affinity site for URP. Both UII and URP provoked a membrane depolarization accompanied by a decrease of input resistance, stimulated the release of endozepines, neuropeptides specifically produced by astroglial cells, and generated an increase in cytosolic calcium concentration ([Ca2+]c). The UII/URP-induced [Ca2+]c elevation was pertussis toxin (PTX)-insensitive, and was blocked by the PLC inhib... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3321595 Tue, 02 Mar 2010 00:00:00 +0100 3321595 http://www.medworm.com/index.php?rid=3321594&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090977 Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 a-subunit and the KCNE1 b-subunit assemble with their membrane-spanning segments interacting resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. Here we provide evidence characterizing a physical interaction between the C-termini of KCNQ1 and KCNE1. When expressed in cultured cells the C-terminus of KCNE1 (KCNE1-CT) co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1 C-terminus (KCNQ1-CT) and KCN... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3321594 Tue, 02 Mar 2010 00:00:00 +0100 3321594 http://www.medworm.com/index.php?rid=3317940&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091690 The voltage-dependent anion channel (VDAC) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteines in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane permeable N-ethylmaleimide (NEM), bulky, charged 5-fluorescein-maleimide (5FM), and the cross-linking reagent, BMEO. Bilayer-reconstituted VDAC conductance was decreased by 5-FM b... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3317940 Mon, 01 Mar 2010 00:00:00 +0100 3317940 http://www.medworm.com/index.php?rid=3301489&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091383 MK-STYX is a pseudophosphatase member of the dual specificity phosphatase sub-family of the protein tyrosine phosphatases. MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic Cys residue and the adjacent His, which are conserved in all active dual specificity phosphatases, are replaced by Ser and Phe, respectively, in MK-STYX. Mutations to introduce His and Cys residues into the active site of MK-STYX generated an active phosphatase. Using mass spectrometry, we identified Ras-GTPase activating protein SH3 domain binding protein-1 (G3BP1), a regulator of RAS signaling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant, catal... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3301489 Wed, 24 Feb 2010 00:00:00 +0100 3301489 http://www.medworm.com/index.php?rid=3297771&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091681 We report here the identification of the ARF exchange factor GBF1 as a Golgi phosphoprotein. GBF1 is phosphorylated by CDK1-cyclin B in mitosis, which results in its dissociation from Golgi membranes. Consistent with a reduced level of GBF1 activity at the Golgi membrane there is a reduction in levels of membrane-associated GTP-bound ARF in mitotic cells. Despite the reduced levels of membrane bound GBF1 and ARF, COPI binding to the Golgi membrane appears unaffected in mitotic cells. Surprisingly, this pool of COPI is dependent upon GBF1 for its recruitment to the membrane, suggesting a low level of GBF1 activity persists in mitosis. We propose that the phosphorylation and membrane dissociation of GBF1 and the consequent reduction in ARF-GTP levels in mitosis are important for changes in G... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3297771 Tue, 23 Feb 2010 00:00:00 +0100 3297771 http://www.medworm.com/index.php?rid=3275321&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091594 Endostatin, the C-terminal domain of collagen XVIII, binds to transglutaminase-2 (TG-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (KD = 6.8 nM). Enzymatic assays indicated that, in contrast to other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, R27 and R139, are crucial for its binding to TG-2. They are also involved in the binding to heparin (Sasaki et al., EMBO J 18:6240-6248), and to α5β1 and αvβ3 integrins (Faye et al., J Biol Chem 284:22029-22040), suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3275321 Mon, 15 Feb 2010 00:00:00 +0100 3275321 http://www.medworm.com/index.php?rid=3262161&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100071 Attachment to the substrate is essential for both survival and differentiation of various kinds of cells, such as neurons and epithelial cells. We recently found a small synthetic molecule, adhesamine, that boosts adhesion and growth of mammalian cells. In the present study, we applied adhesamine to primary-cultured hippocampal neuronal cells and compared its effects with those of poly-L-lysine (PLL), which is widely used as a substrate for cell cultures. Neurons grown on adhesamine-coated coverslips survived for up to 1 month without a feeder layer of glial cells, and had greater viability than cells grown on PLL-coated coverslips. Morphological analysis revealed that neurons cultured with adhesamine exhibited earlier differentiation, i.e., earlier axonal outgrowth and dendritic maturatio... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3262161 Thu, 11 Feb 2010 00:00:00 +0100 3262161 http://www.medworm.com/index.php?rid=3262160&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091344 In this study, we use both electron microscopy and biochemistry to investigate AMPA receptor localization in synaptic membrane subfractions prepared in two different ways, by Triton X-100 detergent treatment or without detergent by sonication at high pH. Immunogold electron microscopy shows that a detergent-resistant synaptosomal membrane subfraction consists of empty vesicles 0.1 – 1.0µm in diameter. A sub-population of these vesicles labeled for glycosphingolipid GM1 ganglioside, a marker of lipid rafts, and 46 percent of the labeled vesicles also labeled for the AMPA receptor subunit GluR2. This co-segregation into specific vesicles does not depend on effects of detergent because a similar distribution of label was found in vesicles isolated without the use of detergent. O... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3262160 Thu, 11 Feb 2010 00:00:00 +0100 3262160 http://www.medworm.com/index.php?rid=3250584&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091451 Retroviral proteases have been shown previously to be only active as homodimers. They are essential to form the separate and active proteins from the viral precursors. Spumaretroviruses produce separate precursors for Gag and Pol, rather than a Gag and a Gag-Pol precursor. Nevertheless, processing of Pol into a protease-reverse transcriptase (PR-RT) and integrase is essential in order to obtain infectious viral particles. We showed recently that the PR-RT from a simian foamy virus as well as the separate protease domain (PRshort) exhibit proteolytic activities, although only monomeric forms could be detected. Here, we demonstrate that PRshort and PR-RT can be inhibited by the putative dimerization inhibitor cholic acid. Various other inhibitors, including darunavir and tipranavir, known... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3250584 Mon, 08 Feb 2010 00:00:00 +0100 3250584 http://www.medworm.com/index.php?rid=3250583&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091834 >200 phosphorylated 14-3-3-binding sites in the literature were analyzed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate though the +2 proline occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the disrupted-in-schizophrenia 1 (DISC1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different sub... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3250583 Mon, 08 Feb 2010 00:00:00 +0100 3250583 http://www.medworm.com/index.php?rid=3243109&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091813 In order to redefine the mannitol pathway in the necrotrophic plant pathogen Botrytis cinerea, we used a targeted deletion strategy of genes encoding two proteins of mannitol metabolism, a mannitol dehydrogenase (BcMTDH), and a mannitol-1-phosphate dehydrogenase (BcMPD). Mobilization of mannitol and quantification of Bcmpd and Bcmtdh gene transcripts during development and osmotic stress confirmed a role for mannitol as temporary and disposable carbon storage compound. In order to study metabolic fluxes, we followed conversion of labelled hexoses by wild type and Bcmpd and Bcmtdh mutant strains by in vivo NMR spectroscopy. Our data revealed that glucose and fructose were metabolized via the BcMPD and BcMTDH pathways, respectively. Existence of a novel mannitol phosphorylation pathway was s... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3243109 Fri, 05 Feb 2010 00:00:00 +0100 3243109 http://www.medworm.com/index.php?rid=3235199&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091845 In this study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the association rate of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings it is concluded that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=3235199 Wed, 03 Feb 2010 00:00:00 +0100 3235199 http://www.medworm.com/index.php?rid=3222727&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091521 Synopsis; Accumulating evidence suggests that hepcidin, a 25 residue peptide hormone, is the master regulator of iron metabolism. Further evidence suggests that the five N-terminal amino acids are crucial for mediating its biological function. With a histidine residue at position 3, this region also has the potential to bind divalent metal ions. To characterise this hepcidin-metal interaction in detail this study utilises electrospray mass spectrometry to measure the binding of a range of metal ions to wild type and mutant human and murine hepcidins. In addition the biological effects of these point mutations were tested on Caco-2 and HEK 293T human cell lines and in mice. Our results show that hepcidin-25 can form complexes with copper, nickel and zinc, though we failed to detect any hepc... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3222727 Fri, 29 Jan 2010 00:00:00 +0100 3222727 http://www.medworm.com/index.php?rid=3211616&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091512 Using human mesenchymal stem cells lacking vascular endothelial growth factor (VEGF) receptors, we show that the pro-angiogenic receptor neuropilin-1 associates with phosphorylated platelet-derived growth factor (PDGF) receptors, thereby regulating cell signaling, migration, proliferation and network assembly. Neuropilin-1 co-immunoprecipitated and co-localized with phosphorylated PDGF receptors in the presence of growth factors. Neuropilin-1 knockdown blocked PDGF-AA-induced PDGF receptor-alpha phosphorylation and migration, reduced PDGF-BB induced PDGF receptor-beta activation and migration, blocked VEGF-A activation of both PDGF receptors, and attenuated proliferation. Neuropilin-1 prominently co-localized with both PDGF receptors within mesenchymal stem cell networks assembled in matri... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3211616 Tue, 26 Jan 2010 00:00:00 +0100 3211616 http://www.medworm.com/index.php?rid=3204358&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091380 Vesicular V-type H+-ATPase (V-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed solid supported membrane (SSM)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-s... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3204358 Mon, 25 Jan 2010 00:00:00 +0100 3204358 http://www.medworm.com/index.php?rid=3197136&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091225 Junctophilins (JPs) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the transverse (t)-tubule and sarcoplasmic reticulum (SR) membranes. In humans with hypertrophic cardiomyopathy (HCM), mutations in JP2 are linked to altered Ca2+ signaling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. Herein, we tried to identify the dominant negative role of a JP2 mutation (S165F which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, over-expression of S165F mutant in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2&#x... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3197136 Fri, 22 Jan 2010 00:00:00 +0100 3197136 http://www.medworm.com/index.php?rid=3188532&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091641 Mutations in the gene encoding alpha skeletal muscle actin (ACTA1) account for around 20% of patients with the muscular disorder nemaline myopathy. Nemaline myopathy is a muscular wasting disease similar to muscular dystrophy, but distinguished by deposits of actin and actin-associated proteins near the z-line of the sarcomere. Around 1/3 of the over 140 myopathy actin mutations have been characterized either biochemically or in cultured cells to determine their effects on the actin cytoskeleton. However, the actin defects causing myopathy are likely to be heterogeneous, with only a few common trends observed among the actin mutants, such as reduced polymerization capacity or inability to fold properly. Notably, the transcriptional programme regulated by serum-response factor, which is ins... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3188532 Wed, 20 Jan 2010 00:00:00 +0100 3188532 http://www.medworm.com/index.php?rid=3188534&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091349 Activation of initiator caspases is dependent on interacting proteins and Ipaf (NLRC4), an inflammasome component, is involved in caspase-1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that it’s C-terminal LRR domain, through intramolecular interaction, negatively regulates its apoptosis inducing function. In A549 lung carcinoma cells, expression of LRR deleted Ipaf (Ac-Ipaf) induced cell death dependent on caspase-8, and not on caspase-1. Yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1, a component of 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91-253 of Ipaf which is also the region that LRR binds to. It potentiates cell death induced by... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3188534 Tue, 19 Jan 2010 00:00:00 +0100 3188534 http://www.medworm.com/index.php?rid=3188533&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091529 Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of the glucose transporter isoform 4 (GLUT4) to the plasma membrane (PM) from specialized GLUT4 storage vesicles (GSVs). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. Here we study the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs, and fluorescence re... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3188533 Tue, 19 Jan 2010 00:00:00 +0100 3188533 http://www.medworm.com/index.php?rid=3171204&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091329 Chromatin modifications and chromatin modifying enzymes are believed to play a major role in the process of DNA repair. The Tip60 histone acetyl transferase is physically recruited to DNA double strand breaks (DSB) where it mediates histone acetylation. Here, we show using a reporter system in mammalian cells that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through Homologous Recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiations, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11, Rad50, Nbs1) complex to DNA DSB. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a compl... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3171204 Wed, 13 Jan 2010 00:00:00 +0100 3171204 http://www.medworm.com/index.php?rid=3166488&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090990 Transforming growth factor-β (TGF-β) induces a cytostatic response of most normal cell types. In cancer cells, however, it often promotes metastasis, and its high expression is correlated with poor prognosis. We here show that S100A4, a metastasis-associated protein, also called metastatin-1, can physically and functionally interact with Smad3, an important mediator of TGF-β signaling. In agreement with its known property, S100A4 binds to Smad3 in a Ca2+-dependent manner. The S100A4-binding site is located in N-terminal region of Smad3. S100A4 can potentiate transcriptional activity of Smad3 and related Smad2. When exogenously expressed in MCF10CA1a.cl1, an MCF10-derived breast cancer cell line, S100A4 increases TGF-β-induced metalloproteinase-9 (MMP-9) e... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3166488 Wed, 13 Jan 2010 00:00:00 +0100 3166488 http://www.medworm.com/index.php?rid=3114428&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091525 Distal renal tubular acidosis (dRTA)2 and hereditary spherocytosis (HS) are two diseases that can be caused by mutations in the gene encoding the anion exchanger 1 (AE1, Band 3). dRTA is characterized by defective urinary acidification, leading to metabolic acidosis, renal stones and failure to thrive. HS results in anemia, which may require regular blood transfusions and splenectomy. Mutations in the gene encoding AE1 rarely cause both HS and dRTA. Here, we describe a novel AE1 mutation, Band 3 Edmonton I, which causes dominant HS and recessive dRTA. The patient is a compound heterozygote with the new mutation, C479W and the previously described mutation, G701D. Red blood cells from the patient presented a reduced amount of AE1. Expression in a kidney cell line showed that kAE1 C479W is r... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3114428 Wed, 23 Dec 2009 00:00:00 +0100 3114428 http://www.medworm.com/index.php?rid=3108060&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091491 Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. Here, we show that the expression level and/or subcellular localization in S. cerevisiae of an heterologous type of multispanning membrane protein, the H+-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimeric proteins were constructed by adding the putative N-terminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptid... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3108060 Mon, 21 Dec 2009 00:00:00 +0100 3108060 http://www.medworm.com/index.php?rid=3099872&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091277 N-terminal signal peptides direct secretory proteins into the endoplasmic reticulum (ER) of eukaryotes or the periplasmic space of prokaryotes. A hydrophobic core (h-region) is important for signal sequence function; however, the mechanism of h-region action is not resolved. To gain new insight about signal sequences, bioinformatic analysis of h-regions from humans, Saccharomyces cerevisiae, Trypanosoma brucei, and Escherichia coli was performed. Each species contains a unique set of peptide motifs (h-motifs) characterized by identity components (i.e. sequence of conserved amino acids) joined by spacers. Human h-motifs have four identity components, while those from the other species utilize three identity components. Example of h-motifs are human Hs3, (L-x(2)-[AGILPV]-L-x(0,2)-L); S. cere... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3099872 Fri, 18 Dec 2009 00:00:00 +0100 3099872 http://www.medworm.com/index.php?rid=3096008&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091448 Hepatocyte growth factor (HGF) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by the urokinase-type plasminogen (uPA), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPA receptor (uPAR) or heparin. By contrast the catalytic domains of the type-II transmembrane serine proteases (TTSPs) matriptase and hepsin were highly efficient activators (50% activation a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3096008 Thu, 17 Dec 2009 00:00:00 +0100 3096008 http://www.medworm.com/index.php?rid=3084331&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091459 Mitogen-activated protein kinase kinase 1 (MEK-1) is an important signal transducing enzyme that is implicated in many aspects of cellular functions. Here, we report that cellular polyamines regulate MEK-1 expression at the posttranscriptional level through the RNA-binding protein HuR in intestinal epithelial cells (IECs). Decreasing the levels of cellular polyamines by inhibiting ornithine decarboxylase (ODC) stabilized MEK-1 mRNA and promoted its translation through enhancement of HuR interaction with the 3’-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing [HuR/MEK-1 mRNA] complex; neither intervention changed MEK-1 gene transcription via its promoter. Hu... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3084331 Mon, 14 Dec 2009 00:00:00 +0100 3084331 http://www.medworm.com/index.php?rid=3062810&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091439 Mouse and human embryonic stem (ES) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the “stemness” of ES cells; however, little is known about how the cell cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of embryonic stem cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3062810 Mon, 07 Dec 2009 00:00:00 +0100 3062810 http://www.medworm.com/index.php?rid=3055935&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091372 Activation of AMPK by phosphorylation at Thr-172 is catalyzed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and calmodulin-dependent protein kinase kinase-β (CaMKKβ). The sequence around Thr-172 is highly conserved between the two catalytic subunit isoforms of AMPK and the twelve AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. We now report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKβ, although we did observe a slow phosphorylation and activation of BRSK1 by CaMKKα. Despite recent reports, we could not find any evidence that the α and/or β subunits of AMPK formed a stable complex with CaMKKβ. We also ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3055935 Fri, 04 Dec 2009 00:00:00 +0100 3055935 http://www.medworm.com/index.php?rid=3051594&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091321 Uncoupling protein 3 (UCP3) and its homologs UCP2 and UCP1 are regulators of mitochondrial function. UCP2 is known to have a short half-life of about one hour, owing to its rapid degradation by the cytosolic 26S proteasome, whereas UCP1 is turned over much more slowly, by mitochondrial autophagy. Here we investigate whether UCP3 also has a short half-life, and whether the proteasome is involved in UCP3 degradation. UCP3 half-life was examined in the mouse C2C12 myoblast cell line by inhibiting protein synthesis with cycloheximide and monitoring UCP3 protein levels by immunoblot. We show that UCP3 has a short half-life of 0.5-4 h. Rapid degradation was prevented by a cocktail of proteasome inhibitors, supporting a proteasomal mechanism for turnover. In addition, this phenotype is recapitula... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3051594 Thu, 03 Dec 2009 00:00:00 +0100 3051594 http://www.medworm.com/index.php?rid=3022406&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091121 The biosynthesis of asparagine-linked glycans occurs in an evolutionary conserved manner with the assembly of the unique lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol at the endoplasmic reticulum (ER). Here we characterize Alg11 from yeast as a mannosyltransferase catalyzing the sequential transfer of two α1,2-linked mannose residues from GDP-mannose to Man3GlcNAc2-PP-Dol and subsequently to Man4GlcNAc2-PP-Dol forming the Man5GlcNAc2-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic transmembrane spanning helices. Using Alg11 topology reporter fusion constructs, we show that only the N-terminal domain fulfils this criterion. Surprisingly, this domain can be deleted without disturbi... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3022406 Tue, 24 Nov 2009 00:00:00 +0100 3022406 http://www.medworm.com/index.php?rid=3007414&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090762 Synapsins are abundant synaptic vesicle (SV)-associated phosphoproteins that regulate synapse formation and function. The highly conserved COOH-terminal domain E was shown to contribute to several synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. We used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyze the role of domain E in regulating the interactions between synapsin I and liposomes mimicking the phospholipid composition of SVs (SV-liposomes) and other presynaptic protein partners. In affinity chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous synapsin I a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3007414 Thu, 19 Nov 2009 00:00:00 +0100 3007414 http://www.medworm.com/index.php?rid=3007413&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091414 Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating reactive oxygen species (ROS). 3OC12 is a quorum-sensing signaling molecule which regulates bacterial gene expression and modulates host immune responses. Paraoxonase-2 (PON2) is an esterase that inactivates 3OC12 and potentially attenuates P. aeruginosa virulence. Because increased intracellular calcium initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic calcium, we hypothesized that 3OC12 would also downregulate PON2. 3OC12 and the calcium ionophore A23187 caused a rapid cytosolic calcium influx and downregulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3007413 Thu, 19 Nov 2009 00:00:00 +0100 3007413 http://www.medworm.com/index.php?rid=3007412&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090976 We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2, by inducing oxidative stress that causes ER stress leading to autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP attenuated MCP-1 induced cell death. It caused induction of iNOS, NADPH oxidase subunit phox47, its translocation from the cytoplasm to the membrane and production of reactive oxygen species (ROS), and induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. It also caused autophagy as indicated by beclin-1 induction, cleavage of LC3 and autophagolysosome formation and apoptosis. Inhibitors of oxidative stress, inhibited ROS formation, ER stress, autophagy and cell death. Specific inhibitors of ER stress and knockdown of... BJ Cell journals http://www.medworm.com/rss/comments.php?id=3007412 Thu, 19 Nov 2009 00:00:00 +0100 3007412 http://www.medworm.com/index.php?rid=2962567&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091050 Dpl is a paralog of the PrPC, whose misfolded conformer (PrPSc) is responsible for the onset of transmissible spongiform encephalopathies (TSEs), or prion diseases. It has been shown that the ectopic expression of Dpl in the brain of some lines of PrP knock out mice provokes cerebellar ataxia that can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where and under which conditions, this event may occur. In the present study we addressed this issue by analyzing the intracellular localization and the interaction between Dpl and PrPC in FRT cells stably expressing the two proteins separately, or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2962567 Thu, 05 Nov 2009 00:00:00 +0100 2962567 http://www.medworm.com/index.php?rid=2962568&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091388 It has been supposed that the hemagglutinin (HA) of influenza virus must be recruited to membrane-rafts to perform its function in membrane fusion and virus budding. Here, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan-fluorescent protein Cerulean to the cytoplasmic tail of HA. Upon expression in CHO cells HA-Cer was glycosylated and transported to the plasma membrane similarly as authentic HA. We measured Förster’s resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) and showed strong association of HA-Cer with Myr-Pal-YFP, an established marker for rafts of the inner-leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2962568 Wed, 04 Nov 2009 00:00:00 +0100 2962568 http://www.medworm.com/index.php?rid=2953908&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091127 The interaction of human topoisomerase I and erybraedin C, a pterocarpan purified from the plant Bituminaria bituminosa that has been shown to have an antitumor activity, has been investigated through enzymatic activity assays and molecular docking procedures. Erybraedin C is able to inhibit both the cleavage and the religation steps of the enzyme reaction. In both cases, preincubation of the drug with the enzyme is required to have a complete inhibition. Molecular docking simulations indicate that, when interacting with the enzyme alone, the preferential drug binding site is localized in proximity of the active Tyr723 residue with one of the two prenilic groups close to the active site residues Arg488 and His632, essential for the catalytic reaction. When interacting with the cleavable co... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2953908 Tue, 03 Nov 2009 00:00:00 +0100 2953908 http://www.medworm.com/index.php?rid=2926772&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091237 PBP (Peroxisome proliferator-activated receptor Binding Protein) (Med1/TRAP220) is essential for mammary gland development. We established a mammary epithelial cell line with genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signaling through nuclear receptors is unlikely responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely turn down the expression of SOX10 (Sry-related HMG box gene 10). The re-expression of Sox10 was capable of reversing the growth inhibition due to PBP... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2926772 Mon, 26 Oct 2009 00:00:00 +0100 2926772 http://www.medworm.com/index.php?rid=2919484&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091179 The X-linked disease retinoschisis is caused by mutations in the RS1 gene encoding retinoschisin, most commonly missense mutations leading to a lack of secretion of functional protein. One potential approach to treat this disease would be the introduction of the wild-type protein by gene therapy in affected individuals. Retinoschisin normally forms homooctamers so co-expression of the wild-type protein with the mutant could result in their co-assembly. Here we show that retinoschisin assembles into an octamer prior to transport from the endoplasmic reticulum and that co-assembly of wild-type and mutant protein can occur when they are co-expressed in the same cell. This co-assembly results in the retention of some but not all expressed wild-type retinoschisin. Moreover, when the wild-type p... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2919484 Wed, 21 Oct 2009 23:00:00 +0100 2919484 http://www.medworm.com/index.php?rid=2911761&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091051 Targeting of inositol 1,4,5-trisphosphate receptors (IP3R) to membranes of the endoplasmic reticulum (ER) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of type 1 IP3R (IP3R1) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2. Replacement of these post-TMD1 residues with unrelated sequences of similar length (24-36 residues) partially mimicked the native residues. We conclude that for IP3R abou... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2911761 Tue, 20 Oct 2009 23:00:00 +0100 2911761 http://www.medworm.com/index.php?rid=2911760&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091122 Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. We assess here the impact of actin-modifying drugs and report that in the presence of cytochalasin D, mast cells stop membrane ruffling but instead bleb. Bleb formation is reversible following washout of the cytochalasin D and occurs in an actin polymerization-dependent manner. Bleb formation is inhibited by expression of dominant-negative ezrin-T567D. Blebbing is also inhibited by blebbistatin, a myosin II inhibitor, implying myosin II activation in the process. We used a selection of inhibitors and observed that myosin II activation is dependent mainly on calcium-calmodulin with only a small contribution from Rho kinase. The signalling pathways stimulated by antigen include phospholipase C and D. Bleb for... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2911760 Tue, 20 Oct 2009 23:00:00 +0100 2911760 http://www.medworm.com/index.php?rid=2911759&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090907 We present evidence that HspB8 binds to Bag3 through the hydrophobic groove formed by its strands β4 and β8, a region previously known to be responsible for the formation and stability of higher order oligomers of many sHsp. Moreover, we demonstrate that two conserved IPV motifs in Bag3 mediate its binding to HspB8 and that deletion of these motifs suppresses HspB8 chaperone activity towards mutant Htt43Q. In addition, we show that Bag3 can bind to the molecular chaperone HspB6. The interaction between HspB6 and Bag3 requires the same regions involved in HspB8-Bag3 association and promotes clearance of aggregated Htt43Q. Our findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their subst... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2911759 Tue, 20 Oct 2009 23:00:00 +0100 2911759 http://www.medworm.com/index.php?rid=2911763&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090884 Ca2+ entry through store-operated Ca2+ channels involves the interaction at endoplasmic reticulum-plasma membrane (ER-PM) junctions of STIM and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and following depletion of ER Ca2+, they oligomerise and translocate to ER-PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with plasma membrane phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. We investigated the role of phosphoinositides in the formation of STIM1 puncta and store-operated Ca2+ entry (SOCE) in response to store depletion. Trea... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2911763 Mon, 19 Oct 2009 23:00:00 +0100 2911763 http://www.medworm.com/index.php?rid=2911762&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090704 Plastoglobules, lipid-protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. Here we show that in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial plastoquinol-9 (PQ-9), the redox component of photosystem II, and of the cyclized product of PQ-9, plastochromanol-8 (PC-8), a tocochromanol with a structure similar to γ-tocopherol and γ-tocotrienol, but with a C40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1 overexpression plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules w... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2911762 Mon, 19 Oct 2009 23:00:00 +0100 2911762 http://www.medworm.com/index.php?rid=2898122&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090332 Cyclosporin A is a hydrophobic undecapeptide that inhibits cyclophilins, a family of peptidylprolyl cis-trans-isomerases. In some experimental models, cyclosporin A offers partial protection against lethal cell injury brought about by transient ischaemia; this is believed to reflect inhibition of cyclophilin-D, a mitochondrial isoform that facilitates formation of the permeability transition pore in the mitochondrial inner membrane. To evaluate this further, we have targeted cyclosporin A to mitochondria so that it becomes selective for cyclophilin-D in cells. This was achieved by conjugating the inhibitor to the lipophilic triphenylphosphonium cation, enabling its accumulation in mitochondria due to the inner membrane potential. In a cell-free system and in B50 neuroblastoma cells the nov... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2898122 Wed, 14 Oct 2009 23:00:00 +0100 2898122 http://www.medworm.com/index.php?rid=2889849&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090455 The alkyl-lysophospholipid (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was previously found impaired in ALP uptake via lipid raft-mediated endocytosis. Here, we report that these cells display cross-resistance to Fas/CD95 ligation (FasL), and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD, cFLIP or the caspase 8 inhibitor IETD. ALP-resistant cells showed decreased Fas expression, both at the mRNA and protein level, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2889849 Tue, 13 Oct 2009 23:00:00 +0100 2889849 http://www.medworm.com/index.php?rid=2915446&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090815 Increased polyamine concentrations play an important role in the development of cancer at all stages from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their polyamine transport system (PTS). The PTS is promiscuous and will transport a range of polyamine based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilising the PTS. The aim of this study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2915446 Wed, 07 Oct 2009 23:00:00 +0100 2915446 http://www.medworm.com/index.php?rid=2875138&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091064 The thiol-disulfide oxidoreductases of the protein disulfide isomerase (PDI) family assist disulfide-bond formation in the endoplasmic reticulum (ER). Here, we show that the previously uncharacterized PDI-family member TMX4 is an N-glycosylated type I membrane protein that localizes to the ER. We also demonstrate that TMX4 contains a single ER-lumenal thioredoxin-like domain, which – in contrast to similar domains in other PDIs – is mainly oxidized in living cells. The TMX4 transcript displays a wide tissue distribution, and is strongly expressed in melanoma cells. Unlike many type I membrane proteins, TMX4 lacks a typical C-terminal di-lysine retrieval signal. Instead, the cytoplasmic tail harbors a conserved di-arginine motif of the RXR type. We show that mutation of the RQ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2875138 Wed, 07 Oct 2009 23:00:00 +0100 2875138 http://www.medworm.com/index.php?rid=2871162&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090815 Increased polyamine concentrations play an important role in the development of cancer at all stages from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their polyamine transport system (PTS). The PTS is promiscuous and will transport a range of polyamine based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilising the PTS. The aim of this study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2871162 Wed, 07 Oct 2009 23:00:00 +0100 2871162 http://www.medworm.com/index.php?rid=2871161&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091112 The transcription factor SREBP1c is highly expressed in adipose tissue and plays a central role in several aspects of adipocyte development including the induction of PPARγ, the generation of an endogenous PPARγ ligand and the expression of several genes critical for lipid biosynthesis. Despite its significance, the regulation of SREBP1c expression during adipogenesis is not well characterised. We noted that in several models of adipogenesis SREBP1c expression closely mimics that of known C/EBPβ targets. Inhibition of C/EBP activity during adipogenesis by expressing either the dominant negative C/EBPβ LIP isoform, the co-repressor ETO, or using siRNAs targetting either C/EBPβ or C/EBPδ significantly impaired early SREBP1c induction. Furthermore, ch... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2871161 Wed, 07 Oct 2009 23:00:00 +0100 2871161 http://www.medworm.com/index.php?rid=2868466&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091283 Cell-derived giant plasma membrane vesicles (GPMVs) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and were used to investigate the cholesterol dependence of domain formation and stability. Cholesterol level is directly related to the abundance of the liquid ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. T... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2868466 Tue, 06 Oct 2009 23:00:00 +0100 2868466 http://www.medworm.com/index.php?rid=2865098&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090856 The activity of 5-lipoxygenase (5-LO), which catalyzes two initial steps in biosynthesis of proinflammatory leukotrienes (LT), is strictly regulated. One recently discovered factor, Coactosin-like protein (CLP), binds 5-LO and promotes LT formation. Here we report that CLP also stabilizes 5-LO and prevents non-turnover inactivation of the enzyme in vitro. Mutagenesis of Trp residues in the 5-LO ß-sandwich showed that 5-LO-W102 is essential for binding to CLP, and for CLP to support 5-LO activity. In addition, also the stabilizing effect depended on binding between CLP and 5-LO. After mutations which prevent interaction (5-LO-W102A, or CLP-K131A), the protective effect of CLP was absent. A calculated 5-LO-CLP docking model indicates that CLP may bind to additional residues in both do... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2865098 Mon, 05 Oct 2009 23:00:00 +0100 2865098 http://www.medworm.com/index.php?rid=2845795&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090825 The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly, and importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP. The 1.63Å structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild type caspase-3. Structural modeling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to fo... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2845795 Tue, 29 Sep 2009 23:00:00 +0100 2845795 http://www.medworm.com/index.php?rid=2845794&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091269 In eukaryotic cells, most membrane and secretory proteins are modified posttranslationally in the endoplasmic reticulum (ER) for correct folding and assembly. Disulfide bond formation is one of the important modifications affecting folding and is catalyzed by protein disulfide isomerase (PDI) family proteins. ERdj5 (also known as JPDI) is a member of the PDI family proteins and has been reported to act as a reductase in ER-associated degradation (ERAD). However, the role of ERdj5 at the whole-body level remains unclear. Therefore, we generated ERdj5 knockout mice and analyzed them. Although ERdj5 knockout mice were viable and healthy, the ER stress response was activated in the salivary gland of the knockout mice more than that of control mice. Furthermore, in ERdj5 knockout cells, the exp... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2845794 Tue, 29 Sep 2009 23:00:00 +0100 2845794 http://www.medworm.com/index.php?rid=2831482&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091286 The redox status of the extracellular compartment has only just been elucidated as a mechanism controlling intracellular signaling transduction and correlates with aging, diabetes, heart disease and lung fibrosis. Here, we describe a mechanism by which oxidizing extracellular environments, as maintained by the cysteine/cystine (Cys/CySS) redox couple, induce mitochondria-derived reactive oxygen species (ROS) generation and cause the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), inducing an antioxidant response. 3T3 cells were cultured in media with extracellular Cys/CySS redox potentials (Eh), ranging from 0 to -150 mV. Cellular and mitochondrial ROS production significantly increased in cells incubated at more oxidizing extracellular conditions (0 and -46 mV). Thioredo... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2831482 Thu, 24 Sep 2009 23:00:00 +0100 2831482 http://www.medworm.com/index.php?rid=2922964&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091140 Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 gene are the predominant cause of inherited nonsyndromic deafness and cause disfiguring skin disorders. Mass spectrometry was used to identify posttranslational modifications (PTMs) of Cx26 and determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem carboxyl-terminal hemagglutinin epitope and hexa(histidine-asparagine) sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase-GluC. Peptides were fractionated using reversed-phase matrix by stepwise elution with increasing concentrations of orga... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2922964 Tue, 22 Sep 2009 23:00:00 +0100 2922964 http://www.medworm.com/index.php?rid=2823412&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091140 Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 gene are the predominant cause of inherited nonsyndromic deafness and cause disfiguring skin disorders. Mass spectrometry was used to identify posttranslational modifications (PTMs) of Cx26 and determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem carboxyl-terminal hemagglutinin epitope and hexa(histidine-asparagine) sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase-GluC. Peptides were fractionated using reversed-phase matrix by stepwise elution with increasing concentrations of orga... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2823412 Tue, 22 Sep 2009 23:00:00 +0100 2823412 http://www.medworm.com/index.php?rid=2818905&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090576 We report for the first time that Ran interacts with a linker histone -histone H1- in vitro and that the two proteins co-localise at the parasite nuclear rim. Interaction of Ran with core histones H3 and H4, creating in metazoans a chromosomal Ran-GTP gradient important for mitotic spindle assembly, is speculative in Leishmania spp., not only because this parasite undergoes a closed mitosis but also because the main localisation of LdRan is different from that of core histone H3. Interaction of Ran with the leishmanial linker histone H1 (LeishH1), suggests that this association maybe involved in modulation of other pathways than those documented for the metazoan Ran-core histone association. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=2818905 Mon, 21 Sep 2009 23:00:00 +0100 2818905 http://www.medworm.com/index.php?rid=2803387&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091152 Cellulosomes, synthesized by anaerobic microorganisms such as Clostridium thermocellum, are remarkably complex nanomachines that efficiently degrade plant cell wall polysaccharides. Cellulosome assembly results from the interaction of type I dockerin domains, present on the catalytic subunits, and the cohesin domains of a large non-catalytic integrating protein that acts as a molecular scaffold. In general, type I dockerins contain two distinct cohesin binding interfaces that appear to display identical ligand specificities. Inspection of the Clostridium thermocellum genome reveals 72 dockerin-containing proteins. In four of these proteins, Cthe_0258, Cthe_0435, Cthe_0624 and Cthe_0918, there are significant differences in the residues that comprise the two cohesin-binding sites of the typ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2803387 Wed, 16 Sep 2009 23:00:00 +0100 2803387 http://www.medworm.com/index.php?rid=2792709&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090766 It has been reported that the C2-L1Tc protein located in the T. cruzi LINE L1Tc 3´ terminal end has nucleic acid chaperone (NAC) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in the transcription factor TFIIIA. The cysteine motifs are flanked by positively charged amino acid regions. The data presented in this paper shows that the C2-L1Tc recombinant protein has at least 16-fold higher affinity for single stranded than for double stranded nucleic acids and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-cooperative binding model. The zinc fingers present in C2-L1Tc have different binding affinity... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2792709 Sun, 13 Sep 2009 23:00:00 +0100 2792709 http://www.medworm.com/index.php?rid=2792708&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091071 Long-term changes of synaptic plasticity depend on protein synthesis and transcription. Neurogranin (Ng) is a small protein concentrated at dendrites and spines of forebrain neurons, involved in synaptic plasticity through the regulation of calmodulin (CaM) mediated signalling. Ng presents a central IQ motif that mediates its binding to CaM and phosphatidic acid (PA) and that can be phosphorylated by protein kinase C (PKC). Here, we report that Ng displays a strong nuclear localization when expressed in cell lines and hippocampal neurons, either alone or fused to green fluorescent protein (GFP-Ng). Further, using subcellular fractionation and immunocytochemical techniques, we were able to localize endogenous Ng in the nuclei of rat forebrain neurons. Nuclear localization of Ng depends on i... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2792708 Sun, 13 Sep 2009 23:00:00 +0100 2792708 http://www.medworm.com/index.php?rid=2785388&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090643 We previously reported a nongenomic action of T3, which stimulates the phosphoinositide-3-kinase (PI3K)-Akt pathway via p85α, the regulatory subunit of PI3K, in human skin fibroblasts. The goal of this study was to elucidate the mechanism by which T3 activates PI3K, and to investigate the physiological role of this T3 action in neuronal cells. We found that T3 activates PI3K-Akt through Src. First, T3 rapidly induced the activation of Src and Akt in N2a cells expressing thyroid hormone receptor (TR)-α1 (N2aTRα), and both were attenuated by either addition of a Src inhibitor or Src siRNA. In contrast, the PI3K inhibitor could only block the activation of Akt. Second, T3 enhanced TRα1/p85α/Src complex formation, which was also abrogated by Src inhibitor. Th... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2785388 Thu, 10 Sep 2009 23:00:00 +0100 2785388 http://www.medworm.com/index.php?rid=2785387&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082196 This study was to evaluate the possible function of mesothelin. Real-time PCR, RT-PCR, cytotoxicity assay, proliferative assay, apoptotic assay by Hoechst staining, detection of active caspases 3 and 7 by flowcytometric analysis, and immuno-precipitation and immuno-blotting were performed. Cancer tissues in paclitaxel-resistant ovarian cancer patients expressed higher levels of mesothelin by real-time PCR than paclitaxel-sensitive ovarian cancer patients (Mean crossing point value change of mesothelin 26.9±0.4 in resistant group and 34.3±0.7 for sensitive group, p<0.001). Mesothelin also protected cells from paclitaxel-induced apoptosis. The protein expression of the Bcl-2 family, such as Bcl-2 and Mcl-1, was significantly increased regardless cells treated with exogenous ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2785387 Thu, 10 Sep 2009 23:00:00 +0100 2785387 http://www.medworm.com/index.php?rid=2765354&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090697 Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary Heavy-chain antibodies, have a size that matches closely that of scorpion toxins. Therefore they might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple Nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These Nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants, kon, koff, or the equilibrium dissociation constant, KD. One particular Nanobody, referred to as N... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2765354 Thu, 03 Sep 2009 23:00:00 +0100 2765354 http://www.medworm.com/index.php?rid=2765353&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090730 Cry11Aa of Bacillus thuringiensis subsp. israelensis is most active toxin to Aedes aegypti in this strain. We previously reported that in addition to a 65 kDa GPI-anchored alkaline phosphatase (ALP) the toxin also binds a 250 kDa membrane protein. Since this protein is of the size of cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250-kDa protein in immunoblots of larval brush border membrane vesicles (BBMV). The antibody inhibits Cry11Aa toxin binding to BBMV and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the fu... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2765353 Thu, 03 Sep 2009 23:00:00 +0100 2765353 http://www.medworm.com/index.php?rid=2761257&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090699 Ceramide metabolism has come under recent scrutiny because of its role in cellular stress responses. Ceramide synthase 2 (CerS2) is one of the six mammalian isoforms of ceramide synthase, and it is responsible for the synthesis of very-long chain (VLC) ceramides, e.g. C24, C24:1. To study the role of CerS2 in ceramide metabolism and cellular homeostasis, we down-regulated CerS2 using siRNA and examined several aspects of sphingolipid metabolism and cell stress responses. CerS2 down-regulation had a broad effect on ceramide homeostasis, not just on VLC ceramides. Surprisingly, CerS2 down-regulation resulted in significantly increased long-chain (LC) ceramides, e.g. C14, C16, and our data suggested that the increase was due to ceramide synthase-independent mechanism. CerS2-down-regulation-in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2761257 Wed, 02 Sep 2009 23:00:00 +0100 2761257 http://www.medworm.com/index.php?rid=2756940&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090957 ESCRT-III mediates budding and scission of intralumenal vesicles into multivesicular endosomes. For the main ESCRT-III subunit Snf7, an additional role in activation of the transcription factor Rim101 ("Rim pathway") is now firmly established. Here, we investigate how the two Snf7 functions are related to each other. By generating SNF7 mutations that severely affect endocytic trafficking, but leave the Rim pathway function intact, we show that the two functions of SNF7 can be separated genetically. We analyzed in detail, how the SNF7 mutations affect the interaction of Snf7 with its various binding partners. While interactions with Rim-related functions (Rim13, Rim20) were not altered by the mutations, there was a strong effect on interactions with components of the ESCRT pathway. The inte... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2756940 Tue, 01 Sep 2009 23:00:00 +0100 2756940 http://www.medworm.com/index.php?rid=2756939&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090998 Trace amine-associated receptors (TAARs) are G protein-coupled receptors that respond to low abundance, endogenous amines such as tyramine and tryptamine, and represent potential targets for neuropsychiatric diseases. However, some members of this receptor subfamily either have no ligand identified or remain difficult to express and characterize using recombinant systems. Here we report the successful expression of human and mouse TAAR1, and the characterization of their responses to various natural and synthetic agonists. In HEK293/CRE-bla cells, mouse TAAR1 showed robust response to trace amines as measured by either cAMP assay or β-lactamase reporter assay, while human TAAR1 showed weaker but still measurable response. When certain fragments of human TAAR1 were replaced with the ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2756939 Tue, 01 Sep 2009 23:00:00 +0100 2756939 http://www.medworm.com/index.php?rid=2737154&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090794 The plasminogen/plasmin system is involved in a variety of normal physiological and pathological processes, including tissue remodelling, angiogenesis and tumour metastasis. Plasminogen activators and receptors for plasminogen/plasminogen activators are essential for the processing of plasminogen to the active serine protease, plasmin. Plasmin can in turn positively or negatively regulate further plasminogen activation via plasmin-mediated cleavage of receptors and activators. Histidine-rich glycoprotein (HRG), a relatively abundant (~100-150 μg/ml) plasma glycoprotein, has a multi-domain structure that can interact with many ligands including Zn2+, heparin, heparan sulfate (HS) and plasminogen. HRG has been shown to function as an adaptor molecule to tether plasminogen to gl... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2737154 Wed, 26 Aug 2009 23:00:00 +0100 2737154 http://www.medworm.com/index.php?rid=2737153&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091094 Exocytosis proceeds through prefusion stages such as hemifusion, but hemifusion is still an elusive intermediate of unknown duration. Using darkfield and fluorescence microscopy in alveolar type II (ATII) cells containing large secretory vesicles ("lamellar bodies", LBs), we show that exocytotic fusion events were accompanied by a mostly bi-phasic scattered (darkfield) light intensity decrease (SLID) originating from the vesicle border. Correlation with the diffusional behavior of fluorescence markers for either content or membrane mixing revealed that the onset of the fast, second, phase of SLID corresponded to fusion pore formation, which was followed by vesicle swelling. In contrast, a slow, first, phase of SLID preceded pore formation considerably but could still be accompanied by diff... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2737153 Wed, 26 Aug 2009 23:00:00 +0100 2737153 http://www.medworm.com/index.php?rid=2587620&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090542 The interactions of extra-cellular matrix (ECM) protein asporin with ECM components have previously not been investigated. Here we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2587620 Wed, 08 Jul 2009 23:00:00 +0100 2587620 http://www.medworm.com/index.php?rid=2569158&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090624 Insulin stimulates GLUT4 translocation in adipocytes and muscles. An emerging picture is that Rab10 could bridge the gap between insulin signaling cascade and GLUT4 translocation. In the present study, two potential effectors of Rab10, GDP dissociation inhibitor 1 and 2 (GDI-1 and GDI-2), are characterized in respect to their roles in insulin-stimulated GLUT4 translocation. It is shown that both GDI-1 and GDI-2 exhibit similar distribution to GLUT4 and Rab10 in the trans-Golgi network (TGN) and periphery structures. Meanwhile, GDI-1 is manifested to interact with Rab10 with higher affinity evidenced by both immunoprecipitation and in vivo Fluorescence Resonance Energy Transfer (FRET). In addition, the participation of GDIs in GLUT4 translocation is illustrated by that overexpression of eit... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2569158 Wed, 01 Jul 2009 23:00:00 +0100 2569158 http://www.medworm.com/index.php?rid=2503406&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090737 The natural polyamines are ubiquitous multifunctional organic cations which play important roles in regulating cellular proliferation and survival. Here we present a novel approach to investigate polyamine functions by using optical isomers of α-methylspermidine (MeSpd) and α,w-bismethylspermine (Me2Spm), metabolically stable functional mimetics of natural polyamines. We studied the ability of α-MeSpd and α,w-Me2Spm to alter the normal polyamine regulation pathways at the level of polyamine uptake and the major control mechanisms known to affect the key polyamine metabolic enzymes. These include: 1) ornithine decarboxylase (ODC), which catalyzes the rate limiting step of polyamine synthesis, 2) ODC antizyme, an inhibitor of ODC and polyamine uptake, 3) spermidin... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2503406 Thu, 11 Jun 2009 23:00:00 +0100 2503406 http://www.medworm.com/index.php?rid=2455847&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090513 C. elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, perception of which enables worms to enter the dauer state for long-term survival in the adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal β-oxidation of very long-chain fatty acid (VLCFA), we sought to investigate the physiological consequences of deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared to levels in wild type N2 worms. Furthermore, the deficiency in daumone b... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2455847 Thu, 04 Jun 2009 04:00:00 +0100 2455847 http://www.medworm.com/index.php?rid=2455846&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081189 We examined the localization and function of each ZnT2 isoform in cells generated to express ZnT2-HA fusion proteins. The 42 kDa isoform was localized primarily to the endosomal/secretory compartment, and over-expression resulted in increased zinc vesicularization and secretion. In contrast, the 35 kDa isoform was associated with the plasma membrane. Importantly, zinc transport was higher in cells over-expressing the each isoform indicating that both proteins are functional. Endogenous expression of the secretory vesicle-associated ZnT2 isoform predominates in mammary cells and expression is higher is secreting cells, directly reflecting the secretory function of the mammary gland. Together our data shed further light on the complex integration of cellular zinc transport mechanisms which m... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2455846 Thu, 04 Jun 2009 04:00:00 +0100 2455846 http://www.medworm.com/index.php?rid=2413016&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090484 The growth-associated protein-43 (GAP-43) is a dually palmitoylated protein in cysteine residues at positions 3 and 4 that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in Chinese hamster ovary (CHO)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and trans-Golgi network (TGN). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of cysteine 3 and 4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome associati... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2413016 Thu, 14 May 2009 04:00:00 +0100 2413016 http://www.medworm.com/index.php?rid=2413017&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090345 This study investigates the interaction of GPA with AE1 in K562 cells, a human erythroleukemic cell line that expresses GPA, and the role of GPA in the cell surface expression of AE1. In K562 cells, GPA was dimeric and N- and O-glycosylated similar to erythroid GPA. GPA was localized at the cell surface, but also to the Golgi. AE1 expressed in K562 cells contained both complex and high mannose oligosaccharides, and co-localized with GPA at the cell surface and in the endoplasmic reticulum (ER). The Wrb antigen was detected at the cell surface of AE1-transfected K562 cells, indicating the existence of an AE1-GPA complex. Immunofluorescence and co-immunoprecipitation studies using AE1 and an ER-localized Hereditary Spherocytosis mutant (R760Q AE1) showed that GPA and AE1 could interact in th... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2413017 Wed, 13 May 2009 04:00:00 +0100 2413017 http://www.medworm.com/index.php?rid=2295038&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082057 Statins are lipid lowering drugs that may help limit cancer occurrence in humans. They drive blockage of the mevalonate pathway, trigger cancer cell apoptosis in vitro and reduce tumour incidence in animals. We show here that statins induced apoptosis in HGT-1 human gastric cancer cells, and this was prevented by intermediates of the cholesterol synthetic pathway. In addition, similarly to what we reported previously for caspase-2 (Logette, E., Le Jossic-Corcos, C., Masson, D., Solier, S., Sequeira-Legrand, A., Dugail, I., Lemaire-Ewing, S., Desoche, L., Solary, E. and Corcos, L. (2005) Mol Cell Biol 25, 9621-9631), caspase-7 may also be induced by statins and is under the positive control of Sterol Regulatory Element Binding Proteins-1 & -2, major activators of cholesterol and fatty acids... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2295038 Wed, 25 Mar 2009 04:00:00 +0100 2295038 http://www.medworm.com/index.php?rid=2295040&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090402 Actin, the major component of cytoplasmic skeleton, has been shown to exist in the nucleus. Nuclear actin functions in several steps of transcription process, including chromatin remodeling, transcription initiation and elongation. However, as a part of PICs (pre-initiation complexes), the role of actin remains to be elucidated. Here, we identified RHA (RNA helicase A) as an actin-interacting protein in PICs. Using immunoprecipitation and immunofluorescence techniques, we showed that RHA associated with β-actin in the nucleus. GST pull-down assay using different deletion mutants revealed that RGG region of RHA was responsible for the interaction with β-actin, and this dominant-negative mutant reduced the recruitment of Pol II (RNA polymerase II) into PICs. Moreover, overexpre... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2295040 Mon, 23 Mar 2009 04:00:00 +0100 2295040 http://www.medworm.com/index.php?rid=2295039&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090051 In this study, we used a combination of epifluorescence and two-photon microscopy to visualize and quantify whether the detergent insolubility reflects a pre-existing organization of the plasma membrane (PM). We found that treatment of cells with cold Triton X-100 (TX) promotes a profound remodelling of the PM including a rapid rearrangement of GM1 and cholesterol into newly formed structures, only partial solubilization of fluid domains and the formation of condensed domains that cover 51% of the remaining membrane. Rather than inducing the coalescence of pre-existing domains, the remaining domains after TX treatment appear to be newly formed with a higher degree of condensation than those observed in native membranes. However, when cholesterol was physically complexed with a second deter... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2295039 Mon, 23 Mar 2009 04:00:00 +0100 2295039 http://www.medworm.com/index.php?rid=2238572&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090095 Chlorella virus PBCV-1 encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In the current manuscript, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisae mutants that lack endogenous K+ uptake systems. Compared to the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, b... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2238572 Fri, 06 Mar 2009 05:00:00 +0100 2238572 http://www.medworm.com/index.php?rid=2231098&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090005 Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antibacterial function of PAP has remained unclear. In the present study we demonstrate that N-terminal cleavage of PAP by trypsin at the Arg37-Ile38 peptide bond or by elastase at the Ser35-Ala36 peptide bond is a prerequisite for binding to the peptidoglycan of the Gram positive bacterium Bacillus subtili... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2231098 Tue, 03 Mar 2009 05:00:00 +0100 2231098 http://www.medworm.com/index.php?rid=2221051&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081905 The asparaginyl hydroxylase FIH was first identified as a protein that inhibits transcriptional activation by hypoxia inducible factor (HIF), through hydroxylation of an Asn residue in the C-terminal activation domain (HIF-CAD). More recently, several Ankyrin Repeat Domain (ARD) containing proteins were identified as FIH substrates using FIH interaction assays. Although the function(s) of these ARD hydroxylations is unclear, expression of the ARD protein Notch 1 was shown to compete efficiently with HIF-CAD for Asn hydroxylation, and thus to enhance HIF activity. The ARD is a common protein domain with over 300 examples in the human proteome. However, the extent of hydroxylation among ARD proteins, and the ability of other members to compete with HIF-CAD for FIH, is not known. Here we assa... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2221051 Fri, 27 Feb 2009 05:00:00 +0100 2221051 http://www.medworm.com/index.php?rid=2221050&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082135 The control of glycolysis in contracting muscle is not fully understood. The aim of the present study was to examine whether activation of glycolysis is mediated by factors related to energy state or by a direct effect of Ca2+ on the regulating enzymes. Extensor digitorum longus muscles from rat were isolated, treated with cyanide to inhibit aerobic ATP production and stimulated (0.2s trains every 4s) until force was reduced to 70% of initial force (Con). Muscles treated with BTS, an inhibitor of cross-bridge cycling without affecting Ca2+ transients, were stimulated for an equal time as Con. Energy utilization by the contractile apparatus (estimated from the observed relation between ATP utilization and force-time integral) was 60% of total. In BTS force-time integral and AT... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2221050 Fri, 27 Feb 2009 05:00:00 +0100 2221050 http://www.medworm.com/index.php?rid=2215197&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082140 The cellular Inhibitor of Apoptosis factor c-IAP1 has recently emerged as a negative regulator of the non-canonical NF-κB signalling cascade. While synthetic IAP inhibitors have been shown to trigger the autoubiquitinylation and degradation of c-IAP1, less is known about the physiological mechanisms by which c-IAP1 stability is regulated. Here we describe two distinct cellular processes that lead to the targeted loss of c-IAP1. Recruitment of a TRAF2:c-IAP1 complex to the cytoplasmic domain of the Hodgkin’s/anaplastic large cell lymphoma-associated receptor, CD30, leads to the targeting and degradation of the TRAF2:c-IAP1 heteromer through a mechanism requiring the RING domain of TRAF2, but not c-IAP1. In contrast, the induced autoubiquitinylation of c-IAP1 by IAP antagonists... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2215197 Wed, 25 Feb 2009 05:00:00 +0100 2215197 http://www.medworm.com/index.php?rid=2215196&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081659 The F-box domain is a degenerated motif consisting of ~40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. Here using TAP purification coupled to MudPIT analysis (Multidimensional Protein Identification Technology), we have identified a novel conserved protein, Sip1 in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT-repeats conta... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2215196 Wed, 25 Feb 2009 05:00:00 +0100 2215196 http://www.medworm.com/index.php?rid=2198877&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082385 In exercising muscle, acute reduction in ambient oxygen impairs muscle contraction due to effects of hypoxia on mitochondrial ATP supply. The less marked impairment reported after long-term exposure to hypoxia points to changes in regulation of the energetic system of contraction in hypoxic conditioned animals. This energetic system was conceptually defined here as two modules, ATP/PCr-producer and ATP/PCr-consumer connected by energetic intermediates. Modular control analysis (MoCA) that combines top-down control analysis with non-invasive 31P-NMR spectroscopy was used to describe in vivo the effects of hypoxia and adaptation to hypoxia on each module. Modulations of steady levels of ATP turnover (indirectly assessed as force output) and muscle PCr were obtained in hypoxic conditioned (HC... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2198877 Thu, 19 Feb 2009 05:00:00 +0100 2198877 http://www.medworm.com/index.php?rid=2186532&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090042 The exact mechanisms by which cell penetrating peptides such as oligoarginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. Here, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell penetrating peptide octaarginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 µM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the studied suspension cells but a ten-fold higher concentration was required to observe similar results in adh... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2186532 Fri, 13 Feb 2009 05:00:00 +0100 2186532 http://www.medworm.com/index.php?rid=2177028&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082127 ADAM15 (a disintegrin and metalloprotease 15) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM15. Here, we purified soluble recombinant ADAM15 to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM15. Overexpression of full-length membrane-anchored ADAM15 or the catalytically inactive ADAM15 E>A together with various membrane proteins uncovered increased release of the extracellular domain of the Fibroblast Growth Factor Re... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2177028 Wed, 11 Feb 2009 05:00:00 +0100 2177028 http://www.medworm.com/index.php?rid=2177027&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090037 In this report we identify the non-receptor tyrosine kinase Pyk2 as the adaptor protein that links Vav1 with Αvβ3. The association of Pyk2 and Vav1 with beta3 relies on the presence of β3 tyrosine residue 747, the primary site of β3 phosphorylation. However, association of Pyk2 with Vav1 is independent of β3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and the Pyk2 proline residue 717 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 in vitro. Cells transfected with GFP-Pyk2 P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhe... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2177027 Wed, 11 Feb 2009 05:00:00 +0100 2177027 http://www.medworm.com/index.php?rid=2159685&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082293 Interaction of Sec1/Munc18 (SM) proteins with their cognate syntaxins represents an important regulatory mechanism of SNARE-mediated membrane fusion. Understanding the conserved mechanism by which SM proteins function in this process has proved challenging, largely due to an apparent lack of conservation of binding mechanisms between different SM/syntaxin pairs. Here we have identified a hitherto uncharacterised mode of binding between syntaxin 4 and Munc18c that is independent of the binding mode previously shown to utilise the N-terminal peptide of syntaxin 4. Our data demonstrate that syntaxin 4/Munc18c interact via two distinct modes of binding, analogous to those employed by syntaxin 1a/Munc18a and syntaxin 16/Vps45. These data support the notion that all syntaxin/SM proteins bind usi... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2159685 Wed, 04 Feb 2009 05:00:00 +0100 2159685 http://www.medworm.com/index.php?rid=2107126&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081949 A vast color palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility, and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolor labeling and whole body imaging techniques. Here we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-infrared f... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2107126 Thu, 15 Jan 2009 05:00:00 +0100 2107126 http://www.medworm.com/index.php?rid=2103748&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082270 Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carry their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 and Hsp90 complex with carrier precursors and interact specifically with the Tom70 import receptor to promote import. We analysed how the presequences of the phosphate carrier (PiC) and citrate carrier (CIC) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2103748 Wed, 14 Jan 2009 05:00:00 +0100 2103748 http://www.medworm.com/index.php?rid=2100297&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081736 The K+ channel interacting proteins (KChIPs) are EF hand-containing proteins required for the traffic of channel-forming Kv4 K+ subunits to the plasma membrane. KChIP1 is targeted, through N-terminal myristoylation, to intracellular vesicles that appear to be intermediates in traffic from the ER to the Golgi but differ from those underlying conventional ER-Golgi traffic. To define KChIP1vesicles and the traffic pathway followed by Kv4/KChIP1 traffic, we examined their relationship to potential SNARE proteins mediating the traffic step. To distinguish Kv4/KChIP1 from conventional constitutive traffic we compared it to the traffic of the vesicular stomatitis G-protein (VSVG). Expression of KChIP with single or triple EF hand mutation quantitatively inhibited Kv4/KChIP1 traffic to the cell su... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2100297 Tue, 13 Jan 2009 05:00:00 +0100 2100297 http://www.medworm.com/index.php?rid=2100296&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081387 In this study, we selected a set of potential CIPP interactors that are directly or indirectly involved in mechanisms of cytoskeletal remodeling and membrane protrusions formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in mammalian cultured cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to IRSp53 carboxy-terminus. Analysis of the punct... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2100296 Tue, 13 Jan 2009 05:00:00 +0100 2100296 http://www.medworm.com/index.php?rid=2097454&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082090 Besides functioning as the plasma transporter of retinol and thyroxine, transthyretin (TTR) is a protease, cleaving apolipoproteinA-I (apoA-I) after a Phe residue. In this work we further investigated TTR substrate specificity. Both by using P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a Lys residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We followed by investigating whether TTR proteolytic activity is involved in these functions. Both wt TTR and proteolytically inactive TTR (TTRprot-) had a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2097454 Mon, 12 Jan 2009 05:00:00 +0100 2097454 http://www.medworm.com/index.php?rid=2089230&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081364 MsbA is an essential ATP-binding cassette (ABC) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA-MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA-MIANS was saturably quenched by... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2089230 Thu, 08 Jan 2009 05:00:00 +0100 2089230 http://www.medworm.com/index.php?rid=2083326&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081866 The inositol phosphatase, MTMR4, was identified as a novel interactor of the ubiquitin ligase Nedd4. hMTMR4 and Nedd4 co-immunoprecipitated and co-localized to late endosomes. The PY-motif of hMTMR4 binds to WW domains of hNedd4. MTMR4 expression was decreased in atrophying muscle while Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting this novel interaction may underlie the biological process of muscle breakdown. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=2083326 Tue, 06 Jan 2009 05:00:00 +0100 2083326 http://www.medworm.com/index.php?rid=2038759&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081973 SNARE proteins contribute to specific recognition between transport vesicles and target membranes and are required for fusion of membranes. The SNARE Vti1p is required for several transport steps between late Golgi, endosomes and the vacuole in the yeast Saccharomyces cerevisiae. Here we identified the late Golgi membrane protein TVP23 as a multicopy suppressor of the growth defect in vti1-2 cells. By contrast, the growth defect in vti1-11 cells was not suppressed by TVP23 overexpression. Deletion of TVP23 aggravated the growth defect in vti1-2 cells. Genetic interactions between TVP23 and vti1-2 were not found in transport from the late Golgi via the late endosome to the vacuole or in transport from the Golgi directly to the vacuole. These data suggest that Tvp23p is not involved in forwa... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2038759 Mon, 15 Dec 2008 05:00:00 +0100 2038759 http://www.medworm.com/index.php?rid=2031998&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081179 Proteoglycans (PGs) are proteins acquiring long, linear, and sulphated glycosaminoglycan (GAG) chains during Golgi passage. In Madin-Darby canine kidney (MDCK) cells, most of the chondroitin sulphate (CS) PGs are secreted apically, while most of the heparan sulphate (HS) PGs are secreted basolaterally. The apical and basolateral secretory routes differ in their GAG synthesis, since a protein core that traverses both routes acquires shorter chains, but more sulphate, in the basolateral pathway than in the apical counterpart [Tveit, Dick, Skibeli and Prydz (2005) J. Biol. Chem. 280, 29596-29603]. Golgi cisternae and the trans-Golgi network have slightly acidic lumens. We therefore investigated how neutralization of endomembrane compartments with the vacuolar H+-ATPase inhibitor Bafilomycin A... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2031998 Thu, 11 Dec 2008 05:00:00 +0100 2031998 http://www.medworm.com/index.php?rid=2025522&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081556 Modification with small ubiquitin-like modifiers (SUMOs) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The estrogen receptor-related receptor γ (ERRγ, ERR3, NR3B3) is a constitutively active orphan nuclear receptor. A phosphorylation-dependent sumoylation motif (PDSM) is located in close vicinity of the amino-terminally located ERRγ2-specific activation function 1 (AF-1). Its function can be substituted by a negatively charged amino acid-dependent sumoylation motif (NDSM). A mutational analysis reveals that ERRγ2-activity is modulated through sumoylation of a lysine residue at position 40 which in turn is regulated by phosphorylation. Phosphorylation in the +5-position relative to the s... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2025522 Wed, 10 Dec 2008 05:00:00 +0100 2025522 http://www.medworm.com/index.php?rid=2025521&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081402 The orphan nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. We studied the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy, the shortest member of the protein inhibitor of activated STAT family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 sumoylation status. In addition, histone deacetylase activit... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2025521 Wed, 10 Dec 2008 05:00:00 +0100 2025521 http://www.medworm.com/index.php?rid=2025520&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081434 KLF6 is a transcription factor and tumor suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of transforming growth factor-β1. KLF6 can be alternatively spliced, generating lower molecular weight isoforms that antagonize the full-length, wild type protein and promote growth. A key target gene of full length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-β auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-β on KLF6 expression and splicing, and to define the contribution of TGF-β on ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=2025520 Wed, 10 Dec 2008 05:00:00 +0100 2025520 http://www.medworm.com/index.php?rid=1995301&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081630 Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterised in recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterise for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability; furthermore, a myc-tagged form of this novel hexosaminid... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1995301 Fri, 28 Nov 2008 05:00:00 +0100 1995301 http://www.medworm.com/index.php?rid=1989081&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081662 Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin loop topology: it is anchored to the membrane with an N-terminal domain and N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 from C. elegans, stomatin-like protein (SLP) 3, and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in detergent resistant membrane domains (DRMs) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved proline residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulat... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1989081 Tue, 25 Nov 2008 05:00:00 +0100 1989081 http://www.medworm.com/index.php?rid=1989080&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081956 SNAP receptors (SNAREs) are widely accepted to drive all intracellular membrane fusion events. Sec1/Munc18 (SM) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least 3 modes: to a closed conformation of syntaxin; to the syntaxin N-terminus; and to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1989080 Tue, 25 Nov 2008 05:00:00 +0100 1989080 http://www.medworm.com/index.php?rid=1973803&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081388 In this study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK293 cell lysates, these two docking sites are not available in cytosolic Alix but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix’s involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=1973803 Wed, 19 Nov 2008 05:00:00 +0100 1973803 http://www.medworm.com/index.php?rid=1967554&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081597 In this study, we purified epitope-tagged forms of human PSS 1 and PSS 2. The purified PSS 2 was shown to catalyze the conversion of PE, but not PC, to PS, this being consistent with the substrate specificity observed in intact cells. On the other hand, the purified PSS 1 was shown to catalyze the conversion of both PC and PE to PS, although PSS 1 in intact cells had been shown not to contribute to the conversion of PE to PS to a significant extent. Furthermore, we found that the purified PSS 2, but not the purified PSS 1, was inhibited on the addition of PS to the enzyme assay mixture, raising the possibility that there was some difference between the mechanisms of the inhibitory actions of PS toward PSS 1 and PSS 2. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=1967554 Tue, 18 Nov 2008 05:00:00 +0100 1967554 http://www.medworm.com/index.php?rid=1967553&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081781 Transglutaminases (TGases) are a class of calcium-dependent enzymes that catalyze the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalyzed the incorporation of putrescine in the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high molecular mass aggregates of actin. Use of fluorescein-cadaverine showed that the labeled polyamine was incorporated in actin by pollen TGase, comparably to guinea pig liver TG... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1967553 Tue, 18 Nov 2008 05:00:00 +0100 1967553 http://www.medworm.com/index.php?rid=1950929&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081798 In this study, to reveal the roles of the C-terminus, we analyzed b0,+AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII cells and non-polarized HEK 293 cells and HeLa cells. Although the deletion of C-terminus of b0,+AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function due to the failure of the plasma membrane targeting of rBAT-b0,+AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence V480P481P482 was identified in the C-terminus of b0,+AT to be responsible for the C-terminus action in promoting the trafficking of rBAT-b0,+AT heterodimeric complex from ER to Golgi apparatus. This is the first report to demonstrate the activ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1950929 Tue, 11 Nov 2008 05:00:00 +0100 1950929 http://www.medworm.com/index.php?rid=1950928&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081288 The recruitment of clathrin to the membrane and its assembly into coated pits result from its interaction with endocytic adapators and other regulatory proteins in the context of a specific lipid microenvironment. Dab2 is a mitotic phosphoprotein and a monomeric adaptor for clathrin mediated endocytosis. In the present study we employed GFP-fusion constructs of different isoforms and mutants of rat Dab2 and characterized their effect on the size, distribution and dynamics of clathrin assemblies. Enhanced levels of expression of the p82 isoform of Dab2 in Cos7 cells induce enlarged clathrin assemblies at the plasma membrane. p82-clathrin assemblies, which concentrate additional endocytic proteins such as AP2 and Epsin, are dynamic structures in which both p82 and clathrin actively exchange... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1950928 Tue, 11 Nov 2008 05:00:00 +0100 1950928 http://www.medworm.com/index.php?rid=1937640&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080170 Paxillin, a major focal adhesion complex component belongs to the subfamily of LIM domain proteins and participates in cell adhesion–mediated signal transduction. It is implicated in cell motility responses upon activation of cell surface receptors and can recruit among others the GIT1/PIX/PAK1 complex. Several adhesion proteins including zyxin, Hic5 and Trip6 are also nuclear and can exert transcriptional effects. Here we show endogenous paxillin shuttles between the cytoplasm and nucleus, and have used a variety of tagged paxiilin constructs to mapped the nuclear export signal. This region overlaps an important LD4 motif that binds GIT1 and FAK1. We provide evidence that phosphorylation of Ser272 within LD4 blocks nuclear export, and show that this modification also reduces GIT1 b... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1937640 Thu, 06 Nov 2008 05:00:00 +0100 1937640 http://www.medworm.com/index.php?rid=1937639&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081575 In rat liver RL-34 cells, endogenous Nrf1 (NF-E2-related factor 1) is localized in the endoplasmic reticulum (ER) where it exists as a glycosylated protein. Electron microscopy has demonstrated that ectopic Nrf1 in COS-1 cells is located in the ER and the nuclear envelope (NE). Subcellular fractionation, together with a membrane proteinase protection assay, revealed that Nrf1 is an integral membrane protein with both luminal and cytoplasmic domains. The N-terminal 65 residues of Nrf1 direct its integration into the ER and NE membranes and tether it to a Triton X-100-resistant membrane microdomain that is associated with lipid rafts. The activity of Nrf1 was increased by the electrophile tert-butyl hydroquinone (tBHQ) probably through an N-terminal domain-dependent process. We found that th... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1937639 Thu, 06 Nov 2008 05:00:00 +0100 1937639 http://www.medworm.com/index.php?rid=1933010&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081268 Neutrophils release reactive oxygen species (ROS) as part of the innate inflammatory immune response. Phosphoinositide 3-kinase γ (PI3Kγ), which is induced by the bacterial peptide N-formyl-Met-Leu-Phe (fMLP), has been identified as an essential intracellular mediator of ROS production. However, the complex signalling reactions that link PI3Kγ with ROS synthesis by NADH oxidase have not yet been described in detail. We found that activation of neutrophils by fMLP triggers the association of PI3Kγ with protein kinase C α (PKCα). Specific inhibition of PI3Kγ suppresses fMLP-mediated activation of PKCα activity and ROS production, suggesting that the protein kinase activity of PI3Kγ is involved. Our data suggest that the direct in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1933010 Wed, 05 Nov 2008 05:00:00 +0100 1933010 http://www.medworm.com/index.php?rid=1920304&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071382 In endocrine cells, prohormones and granins are segregated in the trans-Golgi network from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner upon specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures which resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of chromogranin A is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP or a short epitope tag was expressed in COS-1 fibroblast cells and analyze... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1920304 Wed, 29 Oct 2008 04:00:00 +0100 1920304 http://www.medworm.com/index.php?rid=1893937&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081885 A family of anti-apoptotic regulators known as inhibitor of apoptosis (IAP) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c IAP1 and c IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated signaling complexes where they mediate receptor-induced NF κB activation. Additionally, through their ubiquitin E3 ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NF κB-inducing kinase, NIK, and regulate the non-canonical NF-κB pathway. Herein, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated domain) of IAPs is located between the baculovirus IAP repeat (BIR) domains and the caspase activation and recruitment domain (CARD) or the RING domain of c-I... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1893937 Wed, 22 Oct 2008 04:00:00 +0100 1893937 http://www.medworm.com/index.php?rid=1891343&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081258 Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose deprivation-induced cytotoxicity. To determine if oxidative stress mediated by O2•- and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, oxidation of dihydroethidine (DHE; for O2•-) and 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2; for hydroperoxides) were measured in human colon and breast cancer cells (HT29, HCT116, SW480, MB231) and compared to normal human cells (FHC, 33Co, HMEC). Cancer cells showed significant increases in DHE (2-20 fold) and CDCFH2 (1.8-10 fold) oxidation, relative to normal cells that were more pronounced in the presence of the mitochondrial electron transport chain blocker,... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1891343 Tue, 21 Oct 2008 04:00:00 +0100 1891343 http://www.medworm.com/index.php?rid=1882923&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081218 The rhdA gene of Azotobacter vinelandii codes for a rhodanese-domain protein (RhdA) with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, we undertook a study of in vivo functions of RhdA by using an A.vinelandii mutant strain (MV474) in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection of Fe-S enzymes which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the current study we analyzed the defects of the RhdA null strain by exploiting growth conditions able to enhance the cataly... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1882923 Fri, 17 Oct 2008 04:00:00 +0100 1882923 http://www.medworm.com/index.php?rid=1875413&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071563 Myeloperoxidase (MPO) catalyzes the oxidation of chloride, bromide and thiocyanate to their respective hypohalous acids. We have investigated the generation of HOBr by human neutrophils in the presence of physiological concentrations of chloride and bromide. HOBr was trapped with taurine and detected by monitoring the bromination of 4-hydroxyphenylacetic acid (4-HPAA). With 100 uM bromide and 140 mM chloride, neutrophils generated HOBr and it accounted for approximately 13% of the hypohalous acids they produced. Addition of superoxide dismutase (SOD) doubled the amount of HOBr detected. Therefore, we investigated the reaction of superoxide radicals with a range of bromamines and bromamides and found that superoxide radicals stimulated the decomposition of these species, with this occurring... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1875413 Tue, 14 Oct 2008 04:00:00 +0100 1875413 http://www.medworm.com/index.php?rid=1840246&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081640 Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc, on pathogen surfaces. From previous structural and functional studies of the M-ficolin fibrinogen-like domain (FD1)2, we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pKa of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S., and Kohno, T. (2007) J. Biol. Chem. 282, 3889-3895]. To investigate the origins of the pH dependency, mutational analyses wer... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1840246 Wed, 01 Oct 2008 04:00:00 +0100 1840246 http://www.medworm.com/index.php?rid=1836482&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080781 Transforming growth factor-β (TGF-β) pathway represents an important signaling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-β in inflammatory responses, its role in regulating NF-κB-dependent inflammatory response still remains unknown. Here we show that TGF-β1 synergizes with proinflammatory cytokine TNF-α to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. TGF-β1 synergistically enhances TNF-α-induced NF-κB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-α-induced RelA acetylation and DNA binding activity by TGF-&#x... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1836482 Tue, 30 Sep 2008 04:00:00 +0100 1836482 http://www.medworm.com/index.php?rid=1823594&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081013 During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. Herein we examine the relative roles of VEGFR (VEGF receptors) and associated signaling pathways mediating the effects of VEGF165 on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAEC (uterine artery endothelial cells) from NP (nonpregnant) vs P (pregnant) sheep, VEGF165 activates eNOS at a greater level in P- vs NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF-1 (placental growth factor) elicits only a modest activation of eNOS in P-UAEC compared to VEGF165, while the VEGFR-2 kinase inhibitor blocks VEGF165-stimulated eNOS activation, suggesting VEGF165 predominantly activates e... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1823594 Thu, 25 Sep 2008 04:00:00 +0100 1823594 http://www.medworm.com/index.php?rid=1814706&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080650 Cardiolipin (CL) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S phase in human cells and if CL or CL synthesis was required to support nucleotide synthesis in S phase. Hela cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-3H]thymidine incorporation into cells compared to serum starved cells by 8 h confirming entry into the S phase. CL mass was unaltered at 8 h but increased 2-fold by 16 h post serum addition compared to serum starved cells. The reason for the increase in CL mass upon entry into S phase was an increase in activity and expression of CL de novo biosynthetic and remodeling enzymes and this paralleled the in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1814706 Tue, 23 Sep 2008 04:00:00 +0100 1814706 http://www.medworm.com/index.php?rid=1797270&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081213 In this study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular amino (N)- and carboxyl (C)-termini of Kv4.2 glutathione S-transferase (GST) fusion protein revealed that the Kv4.2 C-terminal was phosphorylated by PKC, while the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Serine (Ser) 447 and Ser537. A phospho-site specific antibody showed that phosphorylation at the Ser537 site increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that alanine mutation to block phosphorylation at both of the PKC sites increased surface expression compared to wildtype Kv4.2. Electrophysiological... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1797270 Tue, 16 Sep 2008 04:00:00 +0100 1797270 http://www.medworm.com/index.php?rid=1738296&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080972 Permeabilization of the mitochondrial membrane has been extensively associated to necrotic and apoptotic cell death. Similarly that had been previously observed for B16F10-Nex2 melanoma cells, palladacycle compounds (PdC) obtained from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with the 1 or 2-ethanebis(diphenylphosphine) (dppe) were able to induce apoptosis in HTC cells, presenting anticancer activity in vitro. To elucidate cell site specific actions of dmpa:dppe that could respond for the induction of apoptosis in cancer cells, in this study, we investigated the effects of PdC on isolated rat liver mitochondria (RLM). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. How... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1738296 Thu, 28 Aug 2008 04:00:00 +0100 1738296 http://www.medworm.com/index.php?rid=1704205&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080617 D-Tyr-tRNATyr deacylase (DTD) is known able to deacylate D-aminoacyl-tRNAs into free D-amino acids and tRNAs and therefore contributes to cellular resistance against D-amino acids in E.coli and Yeast. We have found that human D-Tyr-tRNATyr deacylase (h-DTD) is enriched in the nuclear envelope region of mammalian cells. Treatment of HeLa cells with D-Tyr resulted nuclear accumulation of tRNATyr. D-Tyr treatment and h-DTD silencing caused tRNATyr downregulation. Furthermore, inhibition of protein synthesis by D-Tyr treatment and h-DTD silencing were also observed. D-Tyr, D-Asp and D-Ser treatment inhibited mammalian cell viability in a dose-dependent manner; overexpression of h-DTD decreased the inhibition rate while h-DTD-silenced cells became more sensitive to the D-amino acids treatment. ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1704205 Thu, 14 Aug 2008 04:00:00 +0100 1704205 http://www.medworm.com/index.php?rid=1695477&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080746 Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and β-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines R87 and R303 in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without si... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1695477 Mon, 11 Aug 2008 04:00:00 +0100 1695477 http://www.medworm.com/index.php?rid=1695476&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080775 Paraoxonase-2 (PON2) is a ubiquitously expressed anti-oxidative protein, which is largely found in the endoplasmic reticulum (ER). Addressing the cytoprotective functions of PON2, we observed that PON2 overexpression provided significant resistance to ER stress-induced caspase-3 activation when the ER stress was induced by interference with protein modification (by tunicamycin or dithiotreitol), but not when ER stress was induced by disturbance of Ca2+ homeostasis (by thapsigargin or A23187). When analyzing the underlying molecular events, we found an activation of the PON2 promoter in response to all tested ER stress-inducing stimuli. However, only tunicamycin and dithiotreitol resulted in increased PON2 mRNA and protein levels. In contrast, when ER stress was caused by thapsigargin or A2... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1695476 Mon, 11 Aug 2008 04:00:00 +0100 1695476 http://www.medworm.com/index.php?rid=1695475&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081372 Two major isoforms of protein 4.1R, a 135kDa isoform (4.1R135) and an 80kDa isoform (4.1R80), are expressed at distinct stages of terminal erythroid differentiation. 4.1R135 isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes while 4.1R80 isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R135 isoform includes an additional 209 amino acids designated as head-piece (HP) at the NH2 termini of 4.1R80. In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform specific differences. While both 4.1R135 and 4.1R80 bound to cytop... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1695475 Mon, 11 Aug 2008 04:00:00 +0100 1695475 http://www.medworm.com/index.php?rid=1695474&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080165 This study describes the identification of a novel mammalian major facilitator superfamily domain-containing protein, referred to as Mfsd2a, and an additional closely related protein, designated Mfsd2b. Most intron/exon junctions are conserved between the two genes suggesting that they are derived from a common ancestor. Mfsd2a/b share a 12 transmembrane α-helical domain structure that shows greatest similarity to that of the bacterial Na+/melibiose symporters. Confocal microscopy demonstrated that Mfsd2a localizes to the endoplasmic reticulum. Mfsd2a is expressed in many tissues and is highly induced in liver and brown adipose tissue (BAT) during fasting. Mfsd2a displays an oscillatory expression profile in BAT and liver consistent with a circadian rhythm. While the basal level of ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1695474 Mon, 11 Aug 2008 04:00:00 +0100 1695474 http://www.medworm.com/index.php?rid=1595516&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080724 In conclusion, PKC mediated phosphorylation increased capsid assembly, stability and structural stability. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595516 Tue, 08 Jul 2008 04:00:00 +0100 1595516 http://www.medworm.com/index.php?rid=1595519&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080471 Aminopeptidase A (EC 3.4.11.7, APA) is a membrane-bound zinc metallopeptidase, also activated by Ca2+, involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure in hypertensive animals. In the three-dimentional model of the ectodomain of mouse APA, we docked the specific APA inhibitor glutamate phosphonate, in the presence of Ca2+. The model showed the presence of one Ca2+ atom into an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of inhibitor is pointing. In this pocket, the Ca2+ atom was hexacoordinated with the acidic side chains of Asp-213 and Asp-218, the carbonyl group of Glu-215 and three water molecules, one of them being engaged in a hydrogen bond with the negatively charged... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595519 Fri, 04 Jul 2008 04:00:00 +0100 1595519 http://www.medworm.com/index.php?rid=1595518&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080209 Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including re-modelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 physically interacts with the plasma-membrane associated phosphatidylinositol 4-kinase Stt4. Here we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphatidylinos... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595518 Fri, 04 Jul 2008 04:00:00 +0100 1595518 http://www.medworm.com/index.php?rid=1595517&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071625 CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin α3β1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin α3β1 remains poorly understood. Here, we show that a panel of anti-human CD151 monoclonal antibodies (mAbs) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin α3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin α3 under both low- and high-stringency detergent conditions, Group-2 mAbs co-precipitate integrin α3 under the low-stringency condition and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 m... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595517 Fri, 04 Jul 2008 04:00:00 +0100 1595517 http://www.medworm.com/index.php?rid=1595520&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081237 Small G-proteins belonging to the Arf family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. Here we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane curvature dependent manner and stabilize or further facilitate changes in membrane curvature through the insertion of an amphipathic helix. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595520 Thu, 03 Jul 2008 04:00:00 +0100 1595520 http://www.medworm.com/index.php?rid=1595521&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080797 In this study we report that GKβ exerts its regulatory role by association with the yeast transcriptional repressor Mig1; the presence of Mig1 allows GKβ to bind to the SUC2 promoter, helping in this way in the maintenance of the repression of the SUC2 gene under high-glucose conditions. Since a similar mechanism has been described for the yeast Hxk2, our findings suggest that the function of the regulatory domain present in these two proteins has been conserved throughout evolution. In addition, we report that GKβ is enriched in the yeast nucleus of high-glucose growing cells, whereas it shows a mitochondrial localization upon removal of the sugar. However, GKβ does not exit the nucleus in the absence of Mig1, suggesting that Mig1 regulates the nuclear exit of ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595521 Mon, 30 Jun 2008 04:00:00 +0100 1595521 http://www.medworm.com/index.php?rid=1595522&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080233 In the present studies, we examined the role of phospholipase C (PLC)δ1 in the regulation of cellular proliferation. We demonstrate that RNAi-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCε, induces a proliferation defect in Rat 1 and NIH3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approximately 60 percent inhibition of 3H-thymidine incorporation. Analysis of the cell cycle with bromodeoxyuridine (BrdU) / propidium iodide labeled fluorescence-activated cell sorting (FACS) demonstrated an accumulation of cells in G0/G1 and a corresponding decrease in S phase. Further examination of the cell cycle after synchronization by serum starvation demonstrated normal movement t... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595522 Fri, 27 Jun 2008 04:00:00 +0100 1595522 http://www.medworm.com/index.php?rid=1595523&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080973 No studies have been performed on mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that significantly impact malaria parasite transmission in endemic regions. Here, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells (ASE line) from Anopheles stephensi, a major vector of malaria in India, Southeast Asia and parts of the Middle East. ASE cell mitochondria shared many features in common with mammalian muscle mitochondria, despite the fact that these cells have a larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays a major role in NADH oxidation in ASE cell... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595523 Thu, 26 Jun 2008 04:00:00 +0100 1595523 http://www.medworm.com/index.php?rid=1595524&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071652 In conclusion, we provide evidence that in MCs the uPA/uPAR system regulates survival/apoptosis processes in a stimulus-specific fashion via mitochondria-dependent mechanism and that BAD protein serves as a downstream molecule. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595524 Thu, 19 Jun 2008 04:00:00 +0100 1595524 http://www.medworm.com/index.php?rid=1595525&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20070960 Hydrogen peroxide (H2O2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischemia-reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H2O2 inhibits sodium chloride absorption and activates chloride secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl-/OH- (HCO3-). The present studies were, therefore, undertaken to examine the direct effects of H2O2 on OH- gradient-driven DIDS sensitive 36Cl- uptake utilizing post-confluent transformed human intestinal epithelial cell line Caco2. Our results demonstrate that H2O2 (1 mM, 1h) significantly inhibi... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595525 Wed, 18 Jun 2008 04:00:00 +0100 1595525 http://www.medworm.com/index.php?rid=1595527&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080795 Rhoptries are secretory organelles involved in the virulence of the human pathogen Toxoplasma gondii. We have used high performance liquid chromatography and capillary gas-liquid chromatography to isolate and quantify lipids from whole Toxoplasma cells and their purified rhoptries. This comparative lipidomic analysis revealed an enrichment of cholesterol, sphingomyelin and, most of all, saturated fatty acids in the rhoptries. These lipids are known, when present in membranes, to be contributing to their rigidity and, interestingly, fluorescence anisotropy measurements confirmed that rhoptry-derived membranes have a lower fluidity than membranes from whole T. gondii cells. Moreover, while rhoptries were initially thought to be highly enriched in cholesterol, we demonstrated it is present in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595527 Mon, 16 Jun 2008 04:00:00 +0100 1595527 http://www.medworm.com/index.php?rid=1595526&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080463 RACK1 is an abundant scaffolding protein, which binds active PKCβII increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity of the mRNA exit channel. We tested the hypothesis that PKCβII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. Here we find that specific inhibition of PKCβI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR targets. These data suggest that PKCβII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. By far-Western, we found that PKCβII binds ribosomes in vitro. Coimmunoprecip... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595526 Mon, 16 Jun 2008 04:00:00 +0100 1595526 http://www.medworm.com/index.php?rid=1595528&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080881 Cellular synthesis of peptide hormones requires prohormone convertases (PCs) for the endoproteolysis of prohormones. Antral G-cells synthesize most gastrin and express PC1/3, 2 and 5/6 in rat and man. But the cleavage sites in progastrin for each PC have not been determined. Therefore, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3 null-mice and compared the results to those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridisation of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that also mouse G... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595528 Fri, 13 Jun 2008 04:00:00 +0100 1595528 http://www.medworm.com/index.php?rid=1595529&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080948 RKTG (Raf-1 Kinase Trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with the N-terminus facing cytosol, distinct from classical G protein coupled receptors. We analyzed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized in the cytosolic side. Two stretches of short amino acid sequences at the membrane proximal N- and C-termini (amino acids 61-71 and 299-303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three amino acid l... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595529 Wed, 11 Jun 2008 04:00:00 +0100 1595529 http://www.medworm.com/index.php?rid=1595531&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080149 The small molecule inhibitor Exo2 has been reported to completely disrupt the Golgi apparatus and to stimulate Golgi-endoplasmic reticulum (ER) fusion in mammalian cells, akin to the well-characterised fungal toxin brefeldin A (BFA). It has also been reported that Exo2 does not affect the integrity of the trans-Golgi network (TGN), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2 and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in both HeLa and BS-C-1 cells. However Exo2, unlike BFA, does not induce the tubulation and merging of TGN and endosomal compartments. Furthermore... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595531 Wed, 04 Jun 2008 04:00:00 +0100 1595531 http://www.medworm.com/index.php?rid=1595530&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080662 Prenylation (or Geranylgeranylation, GG) of Rab GTPases is catalysed by Rab Geranylgeranyl Transferase (RGGT) and requires Rab Escort Protein (REP). In the classical pathway, REP associates first with unprenylated Rab which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. Here we show that REP mutants (REP1F282L and REP1F282L/V290F) defective in RGGT binding are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP acts as a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595530 Wed, 04 Jun 2008 04:00:00 +0100 1595530 http://www.medworm.com/index.php?rid=1595533&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080313 Hemoglobin (Hb) based-blood substitutes represent a class of therapeutics designed to correct oxygen deficit in conditions of anemia and traumatic blood loss. The influences of these agents on hypoxia-inducible factor (HIF-1α) target genes involved in adaptation to hypoxia have not been studied. In the study presented rats underwent 80% exchange transfusion (ET) with either hetastarch (HS) or a polymerized Hb (Oxyglobin®) (OG). HS induced dramatic erythropoietin (EPO) gene transcription reaching a maximum at 4 hours post ET. In contrast, OG suppressed EPO transcription until approximately 24 hours post ET. Large plasma EPO levels that were observed post ET with HS were significantly blunted in animals transfused with OG. OG unlike HS induced a sharp increase in heme oxygenase... BJ Cell journals http://www.medworm.com/rss/comments.php?id=1595533 Fri, 23 May 2008 04:00:00 +0100 1595533