bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNA cloning and characterization of human Δ5-desaturase involved in the biosynthesis of arachidonic acid
Authors: LEONARD Amanda E. , KELDER Bruce , BOBIK Emil G. , CHUANG Lu-Te , PARKER-BARNES Jennifer M. , THURMOND Jennifer M. , KROEGER Paul E. , KOPCHICK John J. , HUANG Yung-Sheng , MUKERJI Pradip
Published: August 2015

Two human expressed sequence tag (EST) cDNA sequences with identity with ∆5- and ∆6-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq® database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3ʹ EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a ∆5-desaturase, as determined by the conversion of dihomo-γ-linolenic acid (C20:3,n-6) into arachidonic acid (C20:4,n-6). The human ∆5-desaturase contained a predicted N-terminal cytochrome b5-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.
Identification of the catalytic residues of the first family of β(1–3)glucanosyltransferases identified in fungi
Authors: MOUYNA Isabelle , MONOD Michel , FONTAINE Thierry , HENRISSAT Bernard , LÉCHENNE Barbara , LATGÉ Jean-Paul
Published: August 2015

A new family of glycosylphosphatidylinositol-anchored β(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.
Involvement of NRAMP1 from Arabidopsis thaliana in iron transport
Authors: CURIE Catherine , ALONSO Jose M. , JEAN Marie LE , ECKER Joseph R. , BRIAT Jean-François
Published: August 2015

Nramp genes code for a widely distributed class of proteins involved in a variety of processes, ranging from the control of susceptibility to bacterial infection in mammalian cells and taste behaviour in Drosophila to manganese uptake in yeast. Some of the NRAMP proteins in mammals and in yeast are capable of transporting metal ions, including iron. In plants, iron transport was shown to require a reduction/Fe(II) transport system. In Arabidopsis thaliana this process involves the IRT1 and Fro2 genes. Here we report the sequence of five NRAMP proteins from A. thaliana. Sequence comparison suggests that there are two classes of NRAMP proteins in plants: A. thaliana (At) NRAMP1 and Oriza sativa (Os) NRAMP1 and 3 (two rice isologues) represent one class, and AtNRAMP2-5 and OsNRAMP2 the other. AtNramp1 and OsNramp1 are able to complement the fet3fet4 yeast mutant defective both in low- and high-affinity iron transports, whereas AtNramp2 and OsNramp2 fail to do so. In addition, AtNramp1 transcript, but not AtNramp2 transcript, accumulates in response to iron deficiency in roots but not in leaves. Finally, overexpression of AtNramp1 in transgenic A. thaliana plants leads to an increase in plant resistance to toxic iron concentration. Taken together, these results demonstrate that AtNramp1 participates in the control of iron homoeostasis in plants.
Alanine mutagenesis of high-mobility-group-protein-1 box B (HMG1-B)
Authors: TAUDTE Susann , XIN Hong , KALLENBACH Neville R.
Published: August 2015

We have generated a set of alanine-scanning substitutions in high-mobility-group protein 1 box B (HMG1-B; the second domain of the HMG1 nuclear protein from the rat) in order to explore the influence of specific surface side chains on its function and folding. Guanidine hydrochloride and thermal unfolding studies have been carried out to investigate the effect of substituted residues on the folding pathway. Binding to four-way junction and linear-duplex DNA has been assayed to determine which residues play an important role in DNA binding. We have identified several mutants that are more stable or bind more tightly to the junction than the wild-type, including the particular phenylalanine side chain that is thought to intercalate into the DNA. Thus the interaction between HMG1-B and branched DNA substrates should exhibit differences from present models based on the structure of the complexes that have been solved to date.
Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions
Authors: FAIK Ahmed , DESVEAUX Darrell , MACLACHLAN Gordon
Published: August 2015

Polypeptide assemblies cross-linked by S-S bonds (molecular mass > 200 kDa) and single polypeptides folded with internal S-S cross-links (< 41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [14C]Gal or [3H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[14C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[14C]Gal into intact nasturtium membrane vesicles and the incorporation of [14C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.
Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus
Authors: BISWAS Indranil , VIJAYVARGIA Ravi
Published: August 2015

ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is not very clear. We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution. Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP. ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein. Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA. Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes. The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair.
pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of αIIbβ3 integrin in human platelets
Authors: GIURIATO Sylvie , BODIN Stéphane , ERNEUX Christophe , WOSCHOLSKI Rüdiger , PLANTAVID Monique , CHAP Hugues , PAYRASTRE Bernard
Published: August 2015

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4,5)P4-phosphatase and a PtdIns(3,4,5)P3-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60c-src is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.
Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties
Authors: MCABEE Douglas D. , JIANG Xin , WALSH Kevin B.
Published: August 2015

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca2+ and receptor acylation. Binding of 125I-lactoferrin and 125I-ASOR to isolated rat hepatocytes at 4 °C decreased sharply at pH < 6, following similar titration curves. Binding of 125I-lactoferrin and 125I-ASOR to hepatocytes was Ca2+-dependent. Binding increased progressively at ≥ 300 μM CaCl2, in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of 125I-lactoferrin and 125I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of 125I-ASOR binding as well as the complete loss of specific 125I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca2+, and also requires the receptor subunits to be acylated.
Inhibition by long-chain acyl-CoAs of glucose 6-phosphate metabolism in plastids isolated from developing embryos of oilseed rape (Brassica napus L.)
Authors: JOHNSON Philip E. , FOX Simon R. , HILLS Matthew J. , RAWSTHORNE Stephen
Published: August 2015

The effects of long-chain acyl-CoA (lcACoA) esters (both added exogenously and synthesized de novo) and acyl-CoA binding protein (ACBP) on plastidial glucose 6-phosphate (Glc6P) and pyruvate metabolism were examined using isolated plastids. The binding of lcACoA esters by ACBP stimulated the utilization of Glc6P for fatty acid synthesis, starch synthesis and reductant supply via the oxidative pentose phosphate (OPP) pathway. Stimulation occurred at low (1-10 μM) concentrations of ACBP. Pyruvate-dependent fatty acid synthesis was not directly affected by ACBP. However, addition of ACBP did increase the Glc6P-dependent stimulation of pyruvate utilization mediated through the OPP pathway. On the basis of these experiments, we conclude that lcACoA esters may inhibit Glc6P uptake into plastids, and that this inhibition is relieved by ACBP. We also suggest that utilization of other substrates for fatty acid synthesis may be affected by lcACoA/ACBP via their effects on the OPP pathway.
Regulation of ATP-induced calcium release in COS-7 cells by calcineurin
Authors: BANDYOPADHYAY Arun , SHIN Dong-Wook , KIM Do Han
Published: August 2015

Experiments were conducted to examine the role of calcineurin in regulating Ca2+ fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 μM) of ATP triggered intracellular Ca2+ release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca2+ release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnA∆CaMAI) inhibited the ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i), in both the presence and the absence of extracellular Ca2+. These results suggest that the constitutively active calcineurin prevented Ca2+ release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca2+ release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnA∆CaMAI and calcineurin B. On the addition of 10 μM CsA, the inhibited phosphorylation of Ins(1,4,5)P3R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca2+ release through Ins(1,4,5)P3R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.
Comparative aspects of the diffusion of norfloxacin, cefepime and spermine through the F porin channel of Enterobacter cloacae
Authors: CHEVALIER Jacqueline , MALLÉA Monique , PAGÈS Jean-Marie
Published: August 2015

In Enterobacteriaceae, the permeability of the outer membrane to hydrophilic antibiotics is associated with the presence of pore-forming proteins. We tested the diffusion of the fluoroquinolone norfloxacin in four Enterobacter cloacae strains: a clinical isolate and three derivatives variously producing or lacking the D and F porins. We analysed the entry of norfloxacin into E. cloacae cells in the presence of either the polyamine spermine or the recently developed cefepime, which are known to penetrate through the Escherichia coli OmpF porin. Uptake of the fluoroquinolone was decreased in both cases; the initial rate of penetration decreased as more spermine blocked the channel. Our results indicate that, like β-lactam molecules, fluoroquinolones translocate through the outer membrane via the F porin and that cefepime and norfloxacin entries are polyamine-sensitive. This suggests that the closure of the F porin channel by polyamines might modulate the susceptibility of E. cloacae to both fluoroquinolone and cephalosporin antibiotics.
Novel induction of α-lactalbumin-mediated lacdiNAc-R expression in vivo
Authors: DO Su-Il , LEE Ki-Young , KIM Hee-Nam
Published: August 2015

α-Lactalbumin (α-LA) is a regulatory protein by which the mammalian β1,4-galactosyltransferase (β1,4-galT) is induced to utilize glucose as an acceptor instead of N-acetylglucosamine (GlcNAc) during lactose synthesis in mammary gland. α-LA can also modulate β1,4-galT to utilize UDP-N-acetylgalactosamine (UDP-GalNAc) as a donor towards GlcNAc acceptor substrate with high efficiency in vitro [Do, Do and Cummings (1995) J. Biol. Chem. 270, 18447-18451]. In the present study we transfected cDNA encoding bovine α-LA into Lec8 cells and examined whether nucleotide sugar switching of UDP-galactose (UDP-Gal) into UDP-GalNAc occurred in vivo and whether the neo-glycosylation of GalNAcβ1,4GlcNAc-R structure was synthesized in α-LA-stable transfectants. Our studies demonstrate that the stable expression of α-LA in Lec8 cells induces the formation of GalNAcβ1,4GlcNAc-R in vivo through the nucleotide sugar switching of β1,4-galT.
CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers
Authors: LURIN Claire , GÜCLÜ Josette , CHENICLET Catherine , CARDE Jean-Pierre , BARBIER-BRYGOO Hélène , MAUREL Christophe
Published: August 2015

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel (‘IMAC’) described previously in animal and plant mitochondria.
Positional- and stereo-selectivity of fatty acid oxygenation catalysed by mouse (12S)-lipoxygenase isoenzymes
Authors: BÜRGER Friederike , KRIEG Peter , MARKS Friedrich , FÜRSTENBERGER Gerhard
Published: August 2015

A quantitative stereochemical analysis of the products generated by recombinant mouse (12S)-lipoxygenase isoenzymes was performed with arachidonic acid and linoleic acid as substrates. The leucocyte-type (12S)-lipoxygenase generated, in addition to 12-hydroxyeicosatetraenoic acid (12-HETE) as the main product, 15- and 8-HETE from arachidonic acid and 13- and 9-hydroxyoctadecadienoic acid (13- and 9-HODE) from linoleic acid. The platelet-type enzyme oxygenated arachidonic acid to 12- and 8-HETE and linoleic acid to 13- and 9-HODE, whereas the epidermis-type (12S)-lipoxygenase reaction was essentially mono-specific with arachidonic acid but oxygenated linoleic acid to both 13- and 9-HODE. 12-HETE and 13-HODE were almost exclusively the S enantiomers. 8-HETE was the R enantiomer as a side-product of the platelet-type (12S)-lipoxygenase reaction but the S enantiomer as a side-product of the leucocyte-type reaction. 9-HODE was generated as the R enantiomer by the platelet-type and the epidermis-type isoenzymes and as the S enantiomer by the leucocyte-type (12S)-lipoxygenase. On the basis of published models of lipoxygenase-substrate interaction, the stereochemistry of the products generated by the platelet- and epidermis-type (12S)-lipoxygenases is in agreement with a fixed ‘tail-to-head’ orientation of the substrate fatty acid in the binding pocket of these enzymes, whereas that of the reaction products of the leucocyte-type (12S)-lipoxygenase can be explained only when the inverse orientation of the substrate or a rotational isomerism along the longitudinal axis of the substrate is allowed. Both the product spectra generated and the sensitivity towards the 12-lipoxygenase selective inhibitors N-benzyl-N-hydroxy-4-phenylpentanamide and cinnamyl-3,4-dihydroxy-α-cyanocinnamate indicated the platelet-type and the epidermis-type isoenzymes to be biochemically more related to each other than to the leucocyte-type (12S)-lipoxygenase.
Lipidation of apolipoprotein E influences its isoform-specific interaction with Alzheimer's amyloid β peptides
Authors: TOKUDA Takahiko , CALERO Miguel , MATSUBARA Etsuro , VIDAL Ruben , KUMAR Asok , PERMANNE Bruno , ZLOKOVIC Berislav , SMITH Jonathan D. , LADU Mary Jo , ROSTAGNO Agueda , FRANGIONE Blas , GHISO Jorge
Published: August 2015

The inheritance of the apolipoprotein E (apoE) ϵ4 allele is a prevailing risk factor for sporadic and familial Alzheimer's disease (AD). ApoE isoforms bind directly to Alzheimer's amyloid β (Aβ) peptides both in vitro and in vivo. Recent studies suggest that association of apoE with lipids may modulate its interaction with Aβ. We examined the binding of lipid-associated and delipidated apoE3 and apoE4 isoforms to Aβ utilizing a solid-phase binding assay and estimated the dissociation constants for the interaction of various apoE and Aβ species. Using native apoE isoforms from stably transfected RAW 264 and human embryonic kidney 293 cells, apoE3 had greater affinity than apoE4 for both Aβ1-40 and Aβ1-42. Delipidation of apoE decreased its affinity for Aβ peptides by 5-10-fold and abolished the isoform-specificity. Conversely, incorporation of apoE isoforms produced by baculovirus-infected Sf9 cells into reconstituted human high-density-lipoprotein lipoparticles restored the affinity values for Aβ peptides and resulted in preferential binding of apoE3. The data demonstrate that native lipid-associated apoE3 binds to Aβ peptides with 2-3-fold higher affinity than lipid-associated apoE4. Since the isoforms' binding efficiency correlate inversely with the risk of developing late-onset AD, the results suggest a possible involvement of apoE3 in the clearance or routing out of Aβ from the central nervous system as one of the mechanisms underlying the pathology of the disease.
Interaction of Bacillus subtilis CsaA with SecA and precursor proteins
Authors: MÜLLER Jörg P. , OZEGOWSKI Jörg , VETTERMANN Stefan , SWAVING Jelto , VAN WELY Karel H. M. , DRIESSEN Arnold J. M.
Published: August 2015

CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.
The m3 muscarinic acetylcholine receptor is coupled to mitogen-activated protein kinase via protein kinase C and epidermal growth factor receptor kinase
Authors: SLACK Barbara E.
Published: August 2015

The acetylcholine analogue carbachol rapidly activated mitogen-activated protein kinase (MAPK), and caused tyrosine phosphorylation of the adapter protein p52 Shc and the epidermalgrowth factor (EGF) receptor, in human embryonic kidney cells stably expressing m3 muscarinic receptors. The protein kinase C (PKC) inhibitor GF109203X caused a significant partial inhibition of m3 receptor-mediated activation of MAPK. The PKC-independent MAPK activity elicited by carbachol in the presence of GF109203X was reproducibly abolished by AG1478, an inhibitor of EGF-receptor tyrosine kinase activity, and by the Src tyrosine kinase inhibitor PP1. In a subset of these experiments, GF109203X concomitantly increased carbachol-induced tyrosine phosphorylation of p52 Shc and the EGF receptor. In co-stimulation experiments, carbachol and EGF activated MAPK in a non-additive fashion; moreover, EGF-induced association of Shc with the phosphorylated EGF receptor was inhibited by carbachol. This effect of carbachol was blocked by GF109203X. The results indicate that MAPK activation by m3 receptor stimulation is regulated by two pathways; one dependent on PKC, and the other mediated via the EGF receptor and Src. Moreover, the EGF-receptor-dependent pathway may be subject to negative-feedback regulation via m3 receptor-coupled activation of PKC.
Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes
Authors: WUHRER Manfred , RICKHOFF Sandra , DENNIS Roger D. , LOCHNIT Günter , SOBOSLAY Peter T. , BAUMEISTER Stefan , GEYER Rudolf
Published: August 2015

Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(β1-3)Man(β1-4)Glc(1-1)ceramide, GalNAc(β1-4)[phosphocholine-6]GlcNAc(β1-3)Man(β1-4)Glc(1-1)ceramide and Gal(α1-3)GalNAc(β1-4)[phosphocholine-6]GlcNAc(β1-3)Man(β1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C22h:0), tricosanoic (C23h:0) and tetracosanoic (C24h:0) acids, and C17 sphingosine (Cd17:1) (where h is hydroxylated and d is dihydroxylated).
Expression and protein-binding studies of the EEN gene family, new interacting partners for dynamin, synaptojanin and huntingtin proteins
Authors: SO Chi Wai , SHAM Mai Har , CHEW Sze Lun , CHEUNG Ngai , SO Cary K. C. , CHUNG Sookja K. , CALDAS Carlos , WIEDEMANN M. Leanne , CHAN Li Chong
Published: August 2015

EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin, as well as with the huntingtin protein. In the present study, we show that the expression of the EEN gene-family members is differentially regulated. Multiple-spliced variants were identified for EEN-B2. In the brain, EEN-B1 and EEN-B2 mRNA are preferentially expressed in the cerebellar Purkinje and granule cells, dentate gyrus cells, hippocampal pyramidal neurons and cerebral granule cells. The expression patterns of EEN-B1 and EEN-B2 mRNA in the brain overlap with those of dynamin-I/III, synaptojanin-I and huntingtin, whereas the ubiquitous expression of EEN is consistent with that of dynamin-II. In testes, members of the EEN family are co-expressed with testis-type dynamin and huntingtin in Sertoli cells and germ cells respectively. Our results on the overlapping expression patterns are consistent with the proposed interaction of EEN family members with dynamin, synaptojanin and huntingtin protein in vivo. Although all three EEN family members bind to dynamin and synaptojanin, EEN-B1 has the highest affinity for binding, followed by EEN and EEN-B2. We also demonstrate that amphiphysin, a major synaptojanin-binding protein in brain, can compete with the EEN family for binding to synaptojanin and dynamin. We propose that recruitment of the EEN family by dynamin/synaptojanin to clathrin-coated pits can be regulated by amphiphysin.
Bcl-2 accelerates retinoic acid-induced growth arrest and recovery in human gastric cancer cells
Authors: CHOU Hua-Kang , CHEN Show-Li , HSU Chung-Te , CHAO You-Chen , TSAO Yeou-Ping
Published: August 2015

The role of Bcl-2 as an anti-apoptotic protein has been well documented. In the present work, we present evidence that Bcl-2 may also be involved in cell growth regulation. SC-M1 is an unique cell line which responds to retinoic acid (RA) treatment with reversible growth arrest [Shyu, Jiang, Huang, Chang, Wu, Roffler and Yeh (1995) Eur. J. Cancer 31, 237-243]. In this study, when treated with RA, SC-M1/Bcl2 cells, which were generated by transfecting SC-M1 cells with bcl-2 DNA, were growth-arrested two days earlier than SC-M1/neo cells, which were generated by transfecting SC-M1 cells with vector DNA. This indicates that Bcl-2 accelerates RA-induced growth arrest. In addition to the accelerated growth arrest, RA-treated SC-M1/Bcl2 cells also recovered from growth arrest two days faster than SC-M1/neo cells after the removal of RA. Previously, we had identified the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) as a mediator of RA-induced growth arrest [Tsao, Li, Kuo, Liu and Chen (1996) Biochem. J. 317, 707-711]. In a search for the mechanism by which Bcl-2 affects growth regulation, we found that p21 gene expression was more prominent in SC-M1/Bcl2 cells than in SC-M1/neo cells in the presence of RA, but when RA was removed, p21 gene expression levels in SC-M1/Bcl2 cells were also reduced earlier than in SC-M1/neo cells. The present report is the first to show that Bcl-2 accelerates not only growth arrest but also recovery from growth arrest. Moreover, the close correlation between the effect of Bcl-2 on both RA-induced growth arrest and RA-induced p21 gene expression suggests the possibility that Bcl-2 affects cell growth through the mechanism of p21.
Auto-catalysed progression of aneuploidy explains the Hayflick limit of cultured cells, carcinogen-induced tumours in mice, and the age distribution of human cancer
Authors: RASNICK David
Published: August 2015

Evidence continues to accumulate that aneuploidy, an imbalance in the number of chromosomes, is responsible for the characteristic phenotypes of cancer, including the abnormal cellular size and morphology of cancer cells, the appearance of tumour-associated antigens, as well as the high levels of membrane-bound and secreted proteins responsible for invasiveness and loss of contact inhibition. Aneuploidy has also been demonstrated to be the self-perpetuating source of the karyotypic instability of cancer cells. Here it is shown that the auto-catalysed progression of aneuploidy explains the kinetics of the finite lifetime of diploid cells in culture, the time course of the appearance of papillomas and carcinomas in benzo[a]pyrene-treated mice, and the age-dependence of human cancers. Modelling studies indicate that the ease of spontaneous transformation of mouse cells in culture may be due to a chaotic progression of aneuploidy. Conversely, the strong preference towards senescence and resistance to transformation of human cells in culture may be the result of a non-chaotic progression of aneuploidy. Finally, a method is proposed for quantifying the aneuploidogenic potencies of carcinogens.
G-protein coupling of μ-opioid receptors (OP3): elevated basal signalling activity
Authors: BURFORD Neil T. , WANG Danxin , SADÉE Wolfgang
Published: August 2015

To determine μ-opioid receptor (OP3) signalling activity, guanosine 5ʹ-[γ-[35S]thio]triphosphate (GTP[35S]) binding to G-proteins was measured in the membranes of human embryonic kidney cells (HEK-293) transfected with μ-opioid receptor (HEK-μ). GTP[35S] binding to HEK-μ membranes was significantly elevated compared with HEK-293 control membranes (without OP3), and this was abolished by pertussis-toxin pretreatment. The irreversible antagonist β-chlornaltrexamine (β-CNA) dose-dependently decreased elevated basal G-protein coupling of HEK-μ to control levels in cells devoid of OP3. This characterizes β-CNA as an inverse OP3 agonist. Immunoprecipitation of solubilized G-proteins with Gi3α antisera demonstrated that basal GTP[35S] binding to Gi3α was also substantially elevated in HEK-μ membranes over the control, whereas Gi3α protein levels were unchanged. Basal GTP[35S] binding to Gi1α/Gi2α and Goα was also increased twofold in HEK-μ membranes over the control. Morphine further increased coupling to each of these Gα proteins with similar potency, but not to Gq/11α or Gsα. These results indicate that the wild-type OP3 can couple constitutively to endogenously expressed Gi3α, Gi1α/Gi2α and Goα subunits of G-proteins in HEK-293 cells.
A new method for the selection of protein interactions in mammalian cells
Authors: ROJO-NIERSBACH Eileen , MORLEY Debra , HECK Stephanie , LEHMING Norbert
Published: August 2015

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor ĸB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1α, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT- (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.
Possible interference between tissue-non-specific alkaline phosphatase with an Arg54→Cys substitution and a counterpart with an Asp277→Ala substitution found in a compound heterozygote associated with severe hypophosphatasia
Authors: FUKUSHI-IRIÉ M. , ITO M. , AMAYA Y. , AMIZUKA N. , OZAWA H. , OMURA S. , IKEHARA Y. , ODA K.
Published: August 2015

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg54 → Cys (R54C) or an Asp277 → Ala (D277A) substitution was found in a patient with hypophosphatasia [Henthorn, Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9924-9928]. To examine effects of these missense mutations on properties of TNSALP, the TNSALP mutants were expressed ectopically in COS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-β-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP (R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP (D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A) was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP (R54C) interferes with folding and assembly of TNSALP (D277A) in trans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.
Modulation of the smooth-muscle L-type Ca2+ channel α1 subunit (α1C-b) by the β2a subunit: a peptide which inhibits binding of β to the I–II linker of α1 induces functional uncoupling
Authors: HOHAUS Annette , POTESER Michael , ROMANIN Christoph , KLUGBAUER Norbert , HOFMANN Franz , MORANO Ingo , HAASE Hannelore , GROSCHNER Klaus
Published: August 2015

Modulation of the smooth-muscle Ca2+ channel α1C-b subunit by the auxiliary β2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the α1-β interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of α1C (AID-peptide). Ca2+ channels derived by co-expression of α1C-b and β2a subunits exhibited an about 3-fold higher open probability (Po) than α1C-b channels. High-Po gating of α1C-b·β2a channels was associated with the occurrence of long-lasting channel openings [mean open time (τ) > 10 ms] which were rarely observed in α1C-b channels. Modulation of fast gating by the β2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to β2 subunits of native Ca2+ channels. Binding of the β2 protein to immobilized AID-peptide was specifically inhibited (Ki of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 μM) to the cytoplasmic side of inside-out patches induced a substantial reduction of Po of α1C-b·β2a channels. The scrambled control peptide failed to affect α1C-b·β2a channels, and the AID-peptide (10 μM) did not modify α1C-b channel function in the absence of expressed β2a subunit. Our results demonstrate that the β2a subunit controls fast gating of α1C-b channels, and suggest the α1-β interaction domain in the cytoplasmic I-II linker of α1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of α1-β interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the α1-β interaction.
Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature
Authors: KOMATSU Hiroaki , BOUMA Barend , WIRTZ Karel W. A. , TARASCHI Theodore F. , JANES Nathan
Published: August 2015

Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol. The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-α in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles. Acute exposure to pharmacological levels of ethanol enhanced the function of PITP. Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes. Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer. Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces. The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane. Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles. Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids. The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action. In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid.
Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase
Authors: MONTALVETTI Andrea , PE±A-DÍAZ Javier , HURTADO Ramón , Miguel RUIZ-PÉREZ Luis , GONZÁLEZ-PACANOWSKA Dolores
Published: August 2015

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining Km values for the overall reaction of 40.3±5.8 μM for (R,S)-HMG-CoA and 81.4±5.3 μM for NADPH; Vmax was 33.55±1.8 units·mg-1. Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.
Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities
Authors: PAOLI Paolo , CAMICI Guido , MANAO Giampaolo , GIANNONI Elisa , RAMPONI Giampietro
Published: August 2015

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the γ-phosphate group of nucleoside triphosphates, and then attacks the β-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In constrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated kcat values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg2+ ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 °C, in the presence of millimolar Mg2+ concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.
Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket
Authors: SANTUCCI Roberto , BONGIOVANNI Cristiana , MARINI Stefano , CONTE Rebecca DEL , TIEN Ming , BANCI Lucia , COLETTA Massimo
Published: August 2015

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an ‘alkaline’ and an ‘acid’ transition), which indicates that (at least) two protonating groups change their pKb values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His173, in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N∆ of the proximal imidazole group is redox-linked and that it is crucial in regulating the ‘alkaline’ transition. On the other hand, none of the proximal mutants alters the ‘acid’ transition, suggesting that it is modulated by groups located in a different portion of the protein.