bluuebunny/crossref_metadata_2025_split huggingface dataset:
The majority of human glutathione peroxidase type 5 (GPX5) transcripts are incorrectly spliced: implications for the role of GPX5 in the male reproductive tract
Authors: HALL Len , WILLIAMS Karin , PERRY Anthony C. F. , FRAYNE Jan , JURY Jennifer A. 
Published: August 2015

An epididymis-specific, secretory glutathione peroxidase (GPX5) has been proposed previously to play a role in protecting mammalian sperm membranes from the deleterious effects of lipid peroxidation, which, if not contained, can lead to reduced fertilizing capacity. Here we report the cDNA cloning of human GPX5 and show that the majority of transcripts contain a 118 nt frame-shifting deletion, arising, most likely, from inappropriate excision of exon 3 during processing. Antisera raised against recombinant human GPX5 cross-reacted with rat and macaque (Macaca fascicularis) epididymal proteins of the size expected for full-length, active GPX5. However, no similar reactivity could be demonstrated in any of the human samples tested.
CD23 (Fc∊RII) release from cell membranes is mediated by a membrane-bound metalloprotease
Authors: MAROLEWSKI Ariane E. , BUCKLE Derek R. , CHRISTIE Gary , EARNSHAW David L. , FLAMBERG Pearl L. , MARSHALL Lisa A. , SMITH David G. , MAYER Ruth J. 
Published: August 2015

CD23 (low-affinity IgE receptor, FcεRII) is expressed as a Type II extracellular protein on a variety of cells such as B-cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in the regulation of IgE synthesis. Here we report that the release of CD23 from the cell surface is mediated by a metalloprotease. An assay utilizing purified CD23 and an neo-epitope antibody specific for one of the known cleavage products is described and used to demonstrate unambiguously the cleavage of CD23 by a distinct protease. Characterization of the mechanism of CD23 processing shows that the protease exists as an integral membrane protein with a functional molecular mass of approx. 63 kDa as determined by gel-filtration chromatography. The CD23-cleaving activity found in enriched plasma membranes from RPMI 8866 cells is inhibited by the metalloprotease inhibitors 1,10-phenanthroline and imidazole and by the matrix metalloprotease inhibitor batimastat, but not by inhibitors of cysteine proteases, serine proteases or acid proteases. The same or a similar activity that cleaves CD23 to the known 33 kDa fragment and is inhibited by batimastat is present in diverse cell types such as unstimulated fibroblasts and monocytic cell lines not expressing CD23, as well as in the Epstein–Barr virus-transformed B-cell line, RPMI 8866, which constitutively expresses CD23.
Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin
Authors: SCHILLING Donna , REID James D. , HUJER Andrea , MORGAN Dewey , DEMOLL Edward , BUMMER Paul , FENSTERMAKER Robert A. , KAETZEL David M. 
Published: August 2015

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF α-and β-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK → EEE), whereas an RKK → SSS mutant also exhibited a modest loss in affinity for β-receptors. Replacements with serine at either Arg160 (RKK → SKK) or at all three positions (RKK → SSS) had little effect on binding to α-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK → EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29–32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK → EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK → EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of β-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties.
Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass
Authors: FOLEY Vivienne , SHEEHAN David 
Published: August 2015

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.
Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae
Authors: MILLER Richard W. , EADY Robert R. , GORMAL Carol , FAIRHURST Shirley A. , SMITH Barry E. 
Published: August 2015

Investigation of the interaction of MgADP- and MgATP2- with the Fe protein of Klebsiella pneumoniae nitrogenase by 31P NMR showed that the adenine nucleotides are reversibly bound in slow exchange with free nucleotides. Dissociation of the MgADP-–Fe protein complex was slow enough to enable its isolation by gel filtration, thus permitting the assignment of resonances to bound nucleotides. Spectra of ADP bound to Kp2 were similar to spectra of ADP bound to the myosin motor domain. Oxidative inactivation of a Kp2–MgADP- complex with excess ferricyanide ion eliminated exchange between bound and free ADP, indicating that the intact iron sulphur cluster, located 20 Å from the binding sites, is required for the reversible binding of MgADP-. A change in conformation on controlled oxidation of Kp2 with indigocarmine increased the chemical shift of the β phosphate resonance of bound MgADP-. Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The 31P resonances of both the β and γ phosphates of bound MgATP2- indicated major changes in environment and labilization of both groups on binding to the Fe protein. Highly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bound nucleotides. Partial occupation of Kp2 MgATP2--binding sites (N = 1.9±0.2, Kd = 145 µM) in concentrated protein solutions was demonstrated by flow dialysis. Scatchard plots of data for bound and free ligand obtained after equilibration with Kp2 were linear and no co-operative interactions were detected. We conclude that MgADP- stabilizes the oxidized Fe protein conformer and this conformation in turn triggers the dissociation of the Fe protein from the MoFe protein in the rate-limiting step of the overall process of dinitrogen reduction.
MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage
Authors: WICKSTRÖM Claes , DAVIES Julia R. , ERIKSEN Gitte V. , VEERMAN Enno C. I. , CARLSTEDT Ingemar 
Published: August 2015

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B ‘glycoforms ’. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.
SH2-Bα is an insulin-receptor adapter protein and substrate that interacts with the activation loop of the insulin-receptor kinase
Authors: KOTANI Kei , WILDEN Peter , PILLAY Tahir S. 
Published: August 2015

We identified SH2-Bα as an insulin-receptor-binding protein based on interaction screening in yeast hybrid systems and co-precipitation in cells. SH2-Bα contains pleckstrin-homology (‘PH’) and Src homology 2 (SH2) domains and is closely related to APS (adapter protein with a PH domain and an SH2 domain) and lnk, adapter proteins first identified in lymphocytes. SH2-Bα is ubiquitously expressed and is present in rat epididymal adipose tissue, liver and skeletal muscle, physiological sites of insulin action. On SDS/PAGE, SH2-Bα migrates at a molecular mass of 98 kDa, although the predicted size of SH2-Bα is 79.6 kDa. Insulin causes an electrophoretic mobility shift. SH2-Bα can be immunoprecipitated using anti-(insulin receptor) antibody from insulin-stimulated cells. Anti-phosphotyrosine antibody or the growth factor receptor-binding protein 2 (Grb2) SH2 domain precipitate SH2-Bα after insulin stimulation, suggesting that SH2-Bα is tyrosine-phosphorylated and may be a substrate for the insulin receptor. The SH2-Bα SH2 domain did not interact with insulin-receptor substrate (IRS) proteins or epidermal-growth-factor receptor. Mutation of the juxtamembrane and C-terminus of the insulin receptor did not abolish the interaction with the SH2 domain. This was further confirmed using a panel of activation-loop single point mutants where mutation of Tyr1158, Tyr1162 and Tyr1163 abolished interaction. Thus SH2-Bα is a likely component in the insulin-signalling pathway and may function as an alternative signalling protein by interacting with the activation loop of the insulin-receptor cytoplasmic domain.
Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain
Authors: VENKATESWARLU Kanamarlapudi , GUNN-MOORE Frank , OATEY Paru B. , TAVARÉ Jeremy M. , CULLEN Peter J. 
Published: August 2015

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32.2±5.2 nM) and inositol phosphate specificity [Kd values for Ins(1,3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283±32, &gt;10000, &gt;10000 and &gt;10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP–GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 µM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.
The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin fibroblasts
Authors: LEPPIMÄKI Petra , KRONQVIST Robert , SLOTTE J. Peter 
Published: August 2015

Plasma membrane sphingomyelin (SM) is known to affect the cellular distribution of cholesterol. The aim of this work was to examine how SM homoeostasis in human skin fibroblasts is affected by alterations in the level of cholesterol in the cell. The cellular cholesterol level was decreased by exposing cells to 2-hydroxypropyl-β-cyclodextrin, and increased by exposing cells to cholesterol–methyl-β-cyclodextrin inclusion complexes. A lowering of the cellular unesterified cholesterol content by 20% was shown to increase the incorporation of [14C]palmitic acid into SM by 70%. Subsequently, the cellular SM mass was shown to be increased (24% increase after a 24 h period). Since l-cycloserine completely abolished the increased incorporation of [14C]palmitic acid into SM in cholesterol-depleted cells, we concluded that the de novo synthesis of the sphingosine backbone of SM was activated in cholesterol-depleted cells. This conclusion was further verified by performing a cell-free assay of serine C-palmitoyltransferase (SPT) in cholesterol-depleted cells, which showed that the activity of the enzyme was increased by 30% after cholesterol depletion. Most of the newly synthesized SM in cholesterol-depleted cells was susceptible to degradation by sphingomyelinase, indicating that it was transported efficiently to the cell surface. Loading of fibroblasts with cholesterol had essentially the opposite effects on SM homoeostasis to those of cholesterol depletion, i.e. 20–30% decreased incorporation of [14C]palmitic acid into SM and decreased activity of SPT. The results of this study show that cellular cholesterol levels have marked effects on the homoeostasis of SM.
Partial purification of heparanase activities in Chinese hamster ovary cells: evidence for multiple intracellular heparanases
Authors: BAME Karen J. , HASSALL Alan , SANDERSON Crystal , VENKATESAN Indumati , SUN Chao 
Published: August 2015

Heparanases are mammalian endoglycosidases that cleave heparan sulphate glycosaminoglycans from proteoglycan core proteins and degrade them into shorter chains. The enzymes have been proposed to act in a variety of cellular processes, including proteoglycan catabolism, remodelling of basement membranes and release of heparan sulphate-binding ligands from their extracellular storage sites. Additional functions for heparanases may be to generate short heparan sulphate chains that stabilize or activate other proteins. While heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for these activities. Our recent studies characterizing the short glycosaminoglycans produced in Chinese hamster ovary (CHO) cells suggested that multiple heparanases are necessary for the formation of the short heparan sulphate chains [Bame and Robson (1997) J. Biol. Chem. 272, 2245–2251]. We examined whether this is the case by purifying heparanase activity from CHO cell homogenates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were partially purified. All four activities cleave free glycosaminoglycans over a broad pH range of 3.5–6.0 or 6.5, suggesting that they act in the endosomal/lysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two of the activities the product size is pH-dependent. Three of the four activities degrade proteoglycans as well as the free glycosaminoglycan chain. Interestingly, all four enzymes generate short glycosaminoglycans with a sulphate-rich, modified domain at the non-reducing end of the newly formed chain. Since our previous studies showed that in CHO cells there is also a population of short heparan sulphates with a modified domain at the reducing end of the chain, this suggests that there may be another heparanase in CHO cells that was not purified. Alternatively, our findings suggest that the formation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend on the proportions of the different enzymes and the environment in which the chains are degraded.
Isolation and characterization of the rat huntingtin promoter
Authors: HOLZMANN Carsten , MÄUELER Winfried , PETERSOHN Dirk , SCHMIDT Thorsten , THIEL Gerald , EPPLEN Jörg T. , RIESS Olaf 
Published: August 2015

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)&gt;37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region. This region lacks a TATA and a CAAT box, is rich in GC content and has several consensus sequences for binding sites for SP1, PEA3, Sif and H2A. The stretch between nucleotides -56 and -206 relative to the first ATG is highly conserved between human and rodents and it harbours several potential binding sites for transcription factors. We analysed deletion mutants fused with the chloramphenicol acetyltransferase reporter gene in transfected, HD-expressing neuronal (NS20Y, NG108-15) and non-neuronal Chinese hamster ovary cell lines. Hence these cells should contain the required trans-acting factors necessary for HD gene expression. Partial deletion of the evolutionarily conserved part of the promoter significantly decreases the activity in both neuronal and non-neuronal cells, indicating that the core promoter activity is located between nucleotides -332 and -15. DNase I footprinting and electrophoretic mobility-shift assays were used to define the nucleotide positions and binding affinity of DNA–protein interactions.
Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith–Wiedemann syndrome and tumorigenesis
Authors: GAITANOU Maria , BUANNE Pasquale , PAPPA Christina , GEORGOPOULOU Niki , MAMALAKI Avgi , TIRONE Felice , MATSAS Rebecca 
Published: August 2015

Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith–Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.
Sequence data for the human and mouse BM88 cDNAs have been deposited in the EMBL/GenBank Nucleotide Sequence Databases under accession numbers AF235030 and AF243130, respectively.