bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteophosphoglycans of Leishmania mexicana: Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes
Authors: GÖPFERT Ulrich , GOEHRING Nathan , KLEIN Christian , ILG Thomas
Published: August 2015

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressedppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.
Interferon-γ-dependent stimulation of human involucrin gene expression: STAT1 (signal transduction and activators of transcription 1) protein activates involucrin promoter activity
Authors: TAKAHASHI Hidetoshi , ASANO Kazuhiro , NAKAMURA Satoshi , ISHIDA-YAMAMOTO Akemi , IIZUKA Hajime
Published: August 2015

Involucrin is one of the precursor proteins of the cornified cell envelope of keratinocytes, and is expressed during the later stages of keratinocyte differentiation. Interferon-γ (IFN-γ), a pleiotropic cytokine with anti-proliferative and immunomodulatory activities, is also a potent inducer of squamous differentiation. Using cultured normal human keratinocytes (NHK cells) and simian virus 40-transformed human keratinocytes (SVHK cells), we investigated the effects of IFN-γ on involucrin gene expression. Expression of involucrin was increased by about 3-fold after treating NHK cells with IFN-γ (100 units/ml). Northern blot analyses revealed that IFN-γ increased the expression of involucrin mRNA. The fragment +42 to -2463 in the 5ʹ-flanking region of the human involucrin gene was subcloned into a luciferase reporter vector and the construct (p2463Luc) was transfected into SVHK cells. p2463Luc produced a 3-fold increase in luciferase activity after IFN-γ treatment. Sequence analysis detected two putative IFN-γ-responsive regions [G1 (positions -883 to -874) and G2 (-784 to -775)]. Deletion analyses of the p2463Luc vector revealed that the G1 region is critical for the IFN-γ-dependent up-regulation of the involucrin gene. Gel-shift analyses revealed that STAT1 (signal transduction and activators of transcription 1) protein bound to the G1 region and that involucrin promoter activity was augmented by transfection of a STAT1 expression vector in the presence of IFN-γ. In contrast, transfection of a STAT1 dominant-negative expression vector suppressed the IFN-γ-dependent up-regulation of involucrin promoter activity. These results indicate that IFN-γ stimulates expression of the human involucrin gene via the G1 (-883 to -874) region of the involucrin gene promoter.
Immunosuppressants FK506 and rapamycin have different effects on the biosynthesis of cytoplasmic actin during the early period of T cell activation
Authors: MIYAMOTO Suzanne , SAFER Brian
Published: August 2015

FK506 and rapamycin are immunosuppressants that interfere with T cell activation. FK506 inhibits early events of T cell activation such as the induction of cytokine transcription, whereas rapamycin inhibits later interleukin 2 signalling events. However, both reagents either directly or indirectly reduce protein synthesis. Therefore a kinetic study was conducted in human primary T lymphocytes examining increased synthesis of proteins stimulated by either ionomycin+phorbol myristate acetate (PMA) or PMA alone. Three patterns of protein expression were observed. Synthesis of one group of proteins had enhanced synthesis with FK506, but reduced synthesis with rapamycin. A second group had reduced synthesis with rapamycin and either no change or a slight reduction with FK506 and a third group had reduction with both FK506 and rapamycin. One major protein of the first group, p42, had a rapid increase in synthesis that decreased by 8 h. Its synthesis was strongly enhanced by FK506, but reduced by rapamycin after ionomycin+PMA stimulation. In contrast, this protein was strongly induced by PMA alone in these cells and not affected by FK506 treatment, but still reduced by rapamycin. p42 was identified as cytoplasmic actin. mRNA levels of both γ- and β-actin were found to be enhanced with FK506 treatment suggesting that regulation of actin was at a transcriptional or post-transcriptional level. Results with actinomycin D indicated that FK506 is regulating actin biosynthesis at the post-transcriptional level. Rapamycin, however, appeared to be operating at the level of translation.
Molecular cloning and characterization of a novel dual-specificity protein phosphatase possibly involved in spermatogenesis
Authors: NAKAMURA Koji , SHIMA Hiroshi , WATANABE Masahiko , HANEJI Tatsuji , KIKUCHI Kunimi
Published: August 2015

Dual-specificity protein phosphatases (DSPs) play roles in the regulation of mitogenic signal transduction for extracellular stimulation and the cell cycle. In the present study, we identified a novel DSP, termed TMDP (testis- and skeletal-muscle-specific DSP). Nucleotide sequence analysis of TMDP cDNA indicated that the open reading frame of 597 bp encodes a protein of 198 amino acid residues with a predicted molecular mass of 22.5 kDa. The deduced amino acid sequence contains a motif for a conserved catalytic domain of DSPs and shows highest similarity to human Vaccinia HI-related phosphatase (45.5% identity) but low homology to the mitogen-activated protein kinase phosphatase and CDC25 subfamilies of DSPs. Recombinant TMDP protein exhibited intrinsic phosphatase activity towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities in vitro. Northern-blot analysis revealed that TMDP is most abundantly expressed in the testis. The expression in the testis is characterized as follows: (i) TMDP mRNA first appeared 3 weeks after birth, corresponding to the time that meiosis begins; (ii) TMDP mRNA was abundant in fractionated spermatocytes and round spermatids; and (iii) hybridization in situ showed that the TMDP mRNA is localized in spermatocytes and/or spermatids in seminiferous tubules. These data demonstrate that TMDP is a novel DSP abundantly expressed in the testis and suggest that TMDP may be involved in the regulation of meiosis and/or differentiation of testicular germ cells during spermatogenesis.
Identification of an anti-mycobacterial domain in NK-lysin and granulysin
Authors: ANDREU David , CARRE±O Cristina , LINDE Charlotte , BOMAN Hans G. , ANDERSSON Mats
Published: August 2015

NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively. The solution structure of NK-lysin comprises five amphipathic α-helices. To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin. CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of α-helical peptides. The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin. One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive. Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them. Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity. These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.
Metabolism, mitochondrial uptake and toxicity of 2′,3′-dideoxycytidine
Authors: ROSSI Luigia , SERAFINI Sonja , SCHIAVANO Giuditta F. , CASABIANCA Anna , VALLANTI Giuliana , CHIARANTINI Laura , MAGNANI Mauro
Published: August 2015

2ʹ,3ʹ-Dideoxycytidine (ddCyd) is a prescription anti-retroviral drug that causes mitochondrial toxicity and peripheral neuropathy. ddCyd is actively phosphorylated by cytosolic deoxycytidine kinase and nucleoside (di)phosphate kinase to the 5ʹ-triphosphate derivative. However, 2ʹ,3ʹ-dideoxycytidine 5ʹ-diphosphocholine (ddCDP-choline) was also found in human cells incubated with ddCyd. In this paper we show that ddCDP-choline is produced from dideoxyCTP (ddCTP) and phosphocholine by phosphocholine cytidylyltransferase. dCTP and CTP appear to activate this synthesis in a concentration-dependent manner. Although ddCTP and ddCDP-choline can both enter the mitochondria, ddCDP-choline uptake is more efficient than ddCTP uptake. These data suggest that ddCDP- choline is the ddCyd metabolite that is probably responsible for mitochondrial toxicity. The uptake of ddCTP and ddCDP-choline by mitochondria is inhibited by 3.0 mM L-carnitine in the cell-free system investigated; when added to U937 cells grown in the presence of 0.25 μM ddCyd, 3.0 mM L-carnitine partially abrogated the mitochondrial toxicity of ddCyd.
The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo
Authors: GRAY Alexander , V AN DER KAAY Jeroen , DOWNES C. Peter
Published: August 2015

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P3 levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H2O2. The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2.
The proteasome activator 11 S REG (PA28) and Class I antigen presentation
Authors: RECHSTEINER Martin , REALINI Claudio , USTRELL Vicença
Published: August 2015

There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is ‘11 S REG’ [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or ‘PA28’ [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely α, β and γ. Recombinant REGα forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of α and β subunits. REGγ is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGα and REGβ, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG α and β subunits is induced by interferon-γ, and this has led to the prevalent hypothesis that REG α/β hetero-oligomers play an important role in Class I antigen presentation. In the present review we focus on the structural properties of REG molecules and on the evidence that REGα/β functions in the Class I immune response.
Stimulation of c-Src by prolactin is independent of Jak2
Authors: VARA Juan Ángel FRESNO , CARRETERO María Victoria , GERÓNIMO Haydée , BALLMER-HOFER Kurt , MARTÍN-PÉREZ Jorge
Published: August 2015

Interaction of prolactin (PRL) with its receptor (PRLR) leads to activation of Jak and Src family tyrosine kinases. The PRL/growth hormone/cytokine receptor family conserves a proline-rich sequence in the cytoplasmic juxtamembrane region (Box 1) required for association and subsequent activation of Jaks. In the present work, we studied the mechanisms underlying c-Src kinase activation by PRL and the role that Jak2 plays in this process. PRL addition to chicken embryo fibroblasts (CEF) expressing the rat PRLR long form resulted in activation of c-Src and Jak2 and in tyrosine phosphorylation of the receptor. Receptor phosphorylation was due to associated Jak2, since in cells expressing either a Box 1 mutated PRLR (PRLR4P-A), which is unable to interact with Jak2, or a kinase-domain-deleted Jak2 (Jak2∆k), PRL did not stimulate receptor phosphorylation. Interestingly, addition of PRL to cells expressing PRLR4P-A resulted in an activation of c-Src equivalent to that observed with the wild-type receptor. These findings indicate that PRL-mediated stimulation of c-Src was independent of Jak2 activation and of receptor phosphorylation. Our results suggest that PRL-activated Src could send signals to downstream cellular targets independently of Jak2.
Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 °C
Authors: SALHANY James M. , CORDES Karen A. , SLOAN Renee L.
Published: August 2015

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 °C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4,4ʹ-di-isothiocyanostilbene-2,2ʹ-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport Vmax by using the same resealed ghosts. Incubation of membranes at 37 °C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 °C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 °C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 °C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 °C.
Characterization of plasma unsaturated lysophosphatidylcholines in human and rat
Authors: CROSET Martine , BROSSARD Nicole , POLETTE Anne , LAGARDE Michel
Published: August 2015

Unsaturated lysophosphatidylcholines (lysoPtdCho) bound to albumin circulate in blood plasma and seem to be a novel transport system for carrying polyunsaturated fatty acids (PUFA) to tissues that are rich in these fatty acids, such as the brain. The potential of these lysoPtdCho as a significant source of PUFA for cells has been assessed by comparing their plasma concentration with that of unsaturated non-esterified fatty acids (NEFA) bound to albumin. In humans, the PUFA concentration was 25.9±3.1 nmol/ml for these lysoPtdCho, compared with 33.4±9.6 nmol/ml for NEFA; in rats the equivalent values are 14.2±0.6 and 13.1±1.1 nmol/ml respectively (means±S.E.M.). The lysoPtdCho arachidonic acid content was 2-fold (human) and 5-fold (rat) higher than that of NEFA. In human and rat plasma, unsaturated lysoPtdCho were associated mainly with albumin rather than lipoproteins. The rate and extent of the acyl group shift from the sn-2 to sn-1 position of these lysoPtdCho were studied by the incubation of 1-lyso,2-[14C]C18:2n-6-glycerophosphocholine (GPC) with plasma. The rapid isomerization of this lipid occurred at pH 7 (20% isomerization within 2 min) and was not prevented by its association with albumin. The position of the acyl group in the lysoPtdCho circulating in plasma was studied by collecting blood directly in organic solvents containing 1-lyso,2-[14C]C18:2n-6-GPC as a marker of isomerization that occurred during sampling and analysis. Approx. 50% of the PUFA was located at the sn-2 position, demonstrating that substantial concentrations of 2-acyl-lysoPtdCho are present in plasma and are available for tissue uptake, where they can be reacylated at the sn-1 position to form membrane phospholipids.
Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats
Authors: GRECO Sandrine , HUGUENY Irene , GEORGE Pascal , PERRIN Pascale , LOUISOT Pierre , BIOL Marie-Claire
Published: August 2015

Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an α-1,2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in α-1,2-fucosyltransferase level and of the decrease in α-L-fucosidase level after treatment with spermine, several α-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development.
Identification of structurally important domains of lipid phosphate phosphatase-1: implications for its sites of action
Authors: ZHANG Qiu-Xia , PILQUIL Carlos S. , DEWALD Jay , BERTHIAUME Luc G. , BRINDLEY David N.
Published: August 2015

Lipid phosphate phosphatase-1 (LPP-1) dephosphorylates exogenous lysophosphatidate and thereby regulates the activation of lysophosphatidate receptors and cell division. Mutation of seven amino acids in three conserved domains of mouse LPP-1 abolished its activity. A glycosylation site was demonstrated between conserved Domains 1 and 2. LPP-1 is expressed in the plasma membrane, and the present results demonstrate the active site to be located on the outer surface.
Interaction of respiratory burst and uptake of dehydroascorbic acid in differentiated HL-60 cells
Authors: LAGGNER Hildegard , GOLDENBERG Hans
Published: August 2015

HL-60 cells differentiated with DMSO increased their rates of uptake of ascorbate when they were activated with PMA. The rates observed after this activation were essentially the same as those with dehydroascorbic acid as the original transport substrate. The effect of activation was sensitive to the antioxidant enzymes superoxide dismutase and catalase. When ascorbate was oxidized in situ by chemical or enzymic oxidation, the rates of uptake were similar to those after activation of the cells by phorbol ester; however, in the latter case the extracellular vitamin remained largely in the reduced form and there was very little loss by degradation, whereas after immediate oxidation no more reduced ascorbate could be found outside the cells after a few minutes and a significant part of the total vitamin was lost. The generation of superoxide by xanthine/xanthine oxidase stimulated the uptake of ascorbate much less than the activation by phorbol ester; H2O2 was even less effective. Stimulation of the uptake by phorbol ester was also insensitive to GSH, in contrast with stimulation by the chemical oxidation of ascorbate. Stimulation of ascorbate uptake by phorbol ester was sensitive to the respiratory-burst inhibitor diphenyliodonium as well as the protein kinase C inhibitor staurosporine, indicating the respiratory burst as the cause of stimulation. Activation of the cells by the phorbol ester also stimulated the uptake of dehydroascorbate as the original substrate, in a manner insensitive to antioxidants or inhibitors of the respiratory burst. In all cases the intracellular vitamin was completely in the reduced form. Kinetic characterization by the calculation of maximal velocities and apparent Km values and assaying for the dependence of uptake rates on the ionic milieu and for inhibition by glucose analogues and inhibitors of glucose transport revealed that after treatment with phorbol ester the uptake of total vitamin C in differentiated HL-60 cells was largely due to the low-affinity high-capacity glucose transporter. In contrast, in non-stimulated cells reduced ascorbate was taken up by the Na+-dependent high-affinity low-capacity ascorbate transporter. This change was probably due to the oxidation of ascorbate and, simultaneously, the recruitment of additional transporter molecules to the cell surface.
The platelet-derived-growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells
Authors: GOPPELT-STRUEBE Margarete , FICKEL Stefanie , REISER Christian O. A.
Published: August 2015

In renal mesangial cells, activation of protein tyrosine kinase receptors may increase the activity of mitogen-activated protein (MAP) kinases and subsequently induce expression of prostaglandin G/H synthase-2 (PGHS-2, cyclo-oxygenase-2). As examples, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) were shown to transiently enhance p42/44 MAP kinase activity, which was an essential step in the induction of PGHS-2 mRNA and protein. Inhibitors of receptor kinase activities, tyrphostins AG1296 and AG1478, specifically inhibited the effects of PDGF and EGF respectively. Activation of p42/44 and p38 MAP kinases and PGHS-2 induction were also mediated by lysophosphatidic acid (LPA), which binds to pertussis-toxin-sensitive G-protein-coupled receptors. LPA stimulation was inhibited by AG1296, but not AG1478, indicating involvement of the PDGF receptor kinase in LPA-mediated signalling. This was confirmed by pertussis-toxin-sensitive tyrosine phosphorylation of the PDGF receptor by LPA, whereas no phosphorylation of the EGF receptor was detected. For comparison, 5-hydroxytryptamine (‘serotonin’)-mediated signalling was only partially inhibited by AG1296, and also not affected by AG1478. A strong basal AG1296-sensitive tyrosine phosphorylation of the PDGF receptor and a set of other proteins was observed, which by itself was not sufficient to induce p42/44 MAP kinase activation, but played an essential role not only in LPA- but also in phorbol ester-mediated activation. Taken together, the PDGF receptor, but not the EGF receptor, is involved in LPA-mediated MAP kinase activation and PGHS-2 induction in primary mesangial cells, where both protein kinase receptors are present and functionally active.
Modulation of tau phosphorylation and intracellular localization by cellular stress
Authors: JENKINS Scott M. , ZINNERMAN Marcus , GARNER Craig , JOHNSON Gail V. W.
Published: August 2015

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.
Effect of cellular interaction on glycolytic oscillations in yeast: a theoretical investigation
Authors: WOLF Jana , HEINRICH Reinhart
Published: August 2015

On the basis of a detailed model of yeast glycolysis, the effect of intercellular coupling on the oscillatory dynamics is analysed theoretically. The model includes the main steps of anaerobic glycolysis, and the production of ethanol and glycerol. Transmembrane diffusion of acetaldehyde is included, since it has been hypothesized that this substance mediates the interaction. Depending on the kinetic parameters, the single-cell model shows both stationary and oscillatory behaviour. This agrees with experimental data with respect to metabolite concentrations and phase shifts. The inclusion of intercellular coupling leads to a variety of dynamical modes, such as synchronous oscillations, and different kinds of asynchronous behaviour. These oscillations can co-exist, leading to bi- and tri-rhythmicity. The corresponding parameter regions have been identified by a bifurcation analysis. The oscillatory dynamics of synchronized cell populations are investigated by calculating the phase responses to acetaldehyde pulses. Simulations are performed with respect to the synchronization of two subpopulations that are oscillating out of phase before mixing. The effect of the various processes on synchronization is characterized quantitatively. While continuous exchange of acetaldehyde might synchronize the oscillations for appropriate sets of parameter values, the calculated synchronization time is longer than that observed experimentally. It is concluded either that, in addition to the transmembrane exchange of acetaldehyde, other processes may contribute to intercellular coupling, or that intracellular regulatory feedback plays a role in the acceleration of the synchronization.
Cytoplasmic STAT proteins associate prior to activation
Authors: HAAN Serge , KORTYLEWSKI Marcin , BEHRMANN Iris , MÜLLER-ESTERL Werner , HEINRICH Peter C. , SCHAPER Fred
Published: August 2015

The commonly accepted model of STAT factor activation at the cytoplasmic part of the receptor assumes that signal transducers and activators of transcription (STATs) are recruited from a cytoplasmic pool of monomeric STAT proteins. Based on a previous observation that non-phosphorylated STAT3-Src homology 2 domains dimerize in vitro, we investigated whether the observed dimerization is of physiological relevance within the cellular context. We show that STAT1 and STAT3 are pre-associated in non-stimulated cells. Apparently, these complexes are not able to translocate into the nucleus. We provide evidence that the event of STAT activation is more complex than previously assumed.
Preliminary evidence for the existence of specific functional assemblies between enzymes of the β-oxidation pathway and the respiratory chain
Authors: PARKER Antony , ENGEL Paul C.
Published: August 2015

The electron-transferring flavoprotein (ETF) has been detected in two large soluble-protein complexes partially purified from sonicated porcine liver mitochondria. Size-exclusion chromatography and sucrose-density ultracentrifugation suggested molecular masses in the region of 390 to 420 kDa for the two complexes. Activities of medium-chain acyl-CoA dehydrogenase, sarcosine dehydrogenase and ETF:ubiquinone oxidoreductase were also detected. No evidence of oxidative-phosphorylation properties was obtained. Treatment with antimycin A inhibited the activity of both complexes. Pyridine haemochromogens, prepared from the partially purified species, show the presence of cytochrome proteins. The possible composition of these complexes and their relationship to the electron transport chain are discussed.
Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts
Authors: FROST Matthew T. , HALLIWELL Barry , MOORE Kevin P.
Published: August 2015

Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means±S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12±0.6 μg/ml and 14±0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7±1.7 μg/mg and 2.7±0.4 ng/mg respectively.
α-Crystallin assists the renaturation of glyceraldehyde-3-phosphate dehydrogenase
Authors: GANEA Elena , HARDING John J.
Published: August 2015

α-Crystallin, a major lens protein, has many of the properties of a molecular chaperone, but its ability to assist refolding of proteins has been less certain. In the present work it was shown that α-crystallin specifically increased the reactivation of guanidine-denatured glyceraldehyde-3-phosphate dehydrogenase with most of the activity being recovered. In the incubation mixture the recovered enzyme activity was partly free but mostly it appeared in a protective complex with α-crystallin. The aggregation of the denatured enzyme on dilution from the guanidine solution was prevented. Thus α-crystallin not only protects against aggregation and inactivation of enzymes during denaturation, but can also prevent aggregation and assist recovery of the native structure during renaturation.
Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits
Authors: FARR Tracy J. , CODDINGTON-LAWSON Sarah J. , SNYDER Peter M. , MCDONALD Fiona J.
Published: August 2015

The epithelial Na+ channel (ENaC) regulates Na+ absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na+-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na+-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na+-channel activity. Previously we showed that a proline-rich region of the α subunit of the Na+ channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na+-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the α, β and γ Na+-channel subunits but not to a mutated β subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na+-channel activity.
The recombinant N-terminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity
Authors: LIU Bing , RAYMENT Sean A. , GYURKO Csilla , OPPENHEIM F. G. , OFFNER Gwynneth D. , TROXLER Robert F.
Published: August 2015

MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys45 and Cys50) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD50 values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity.
Lipid metabolic changes caused by short-chain ceramides and the connection with apoptosis
Authors: ALLAN David
Published: August 2015

The effects of the short-chain ceramides D-erythro-N-acetylsphingosine (C2-ceramide), 6-[N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino]hexanoyl-D-erythro-sphingosine (NBD-ceramide) and N-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl]-D-erythro-sphingosine (DMB-ceramide) on the incorporation of [14C]acetate into baby-hamster kidney (BHK) fibroblasts have been examined. C2-ceramide at concentrations up to 20 μM caused an inhibition of synthesis of phosphatidylcholine (PtdCho), sphingolipids and cholesterol within 2 h. Similar effects in BHK cells were seen using other radioactive tracers ([3H]water, [3H]palmitate and [3H]choline) and using HL60 cells labelled with [14C]acetate. The inhibition of PtdCho synthesis corresponded to an accumulation of label in diacylglycerol and triacylglycerol, probably as a consequence of cytidylyltransferase blockade. With [3H]choline label, the decrease in sphingomyelin synthesis could be partly accounted for by accumulation of a slow-moving lipid, likely to be C2-sphingomyelin. NBD-ceramide also reduced sphingomyelin and cholesterol biosynthesis, but had much less effect on PtdCho and acylglycerols. In contrast, the only apparent effect of DMB-ceramide was to inhibit synthesis of sphingomyelin, with a reciprocal increase in DMB-sphingomyelin synthesis. However, all of these short-chain ceramides caused massive apoptosis after 18 h, whereas addition of N-acetyldihydrosphingosine or elevation of natural ceramide by treatment of cells with sphingomyelinase had little effect on lipid synthesis or apoptosis. The present findings suggest that the apoptotic effect of short-chain ceramides is sometimes associated with inhibition of cytidylyltransferase, but is more closely correlated with a competitive inhibition of normal sphingomyelin biosynthesis.
Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase
Authors: CHITAMBAR Christopher R. , WERELEY Janine P. , HEIMAN Thomas , ANTHOLINE William E. , O'BRIEN William J.
Published: August 2015

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in Vmax in DFe-T cells without a change in Km. Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.
A possible origin of differences between calorimetric and equilibrium estimates of stability parameters of proteins
Authors: SINHA Amita , YADAV Sushma , AHMAD Rizwan , AHMAD Faizan
Published: August 2015

To test the validity of thermodynamic parameters from the equilibrium method, we have studied the reversible heat-induced denaturations of lysozyme, ribonuclease A, cytochrome c and myoglobin at various pH values, using absorption spectral measurements. For each protein, if a linear temperature-dependence of the pre- and post-transition baselines is assumed for the analysis of the conformational-transition curve, the estimate of ∆H (the enthalpy change on denaturation at Tm, the midpoint of denaturation) is significantly less than ∆H, the value obtained by the calorimetric measurements. If the analysis of thermal-denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function, there exists an excellent agreement between ∆Hm values of all proteins obtained from equilibrium and calorimetric methods. The latter analysis is supported by the studies on model compounds, for measurements of absorption properties of tyrosine, tryptophan and haem as a function of temperature suggested that the temperature-dependencies of the optical properties are indeed non-linear. We have observed that for each protein the constant-pressure heat-capacity change on denaturation (∆Cp) determined from the plots of ∆H versus Tm is not only independent of the method of analysis of the transition curve, but it is also in excellent agreement with calorimetric ∆Cp. An important conclusion of this study is that for these proteins that exhibit two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.
14-3-3ζ interacts with the α-chain of human interleukin 9 receptor
Authors: SLIVA Daniel , GU Minyi , ZHU Yuan Xiao , CHEN Jun , TSAI Schickwann , DU Xiaoping , YANG Yu-Chung
Published: August 2015

Interleukin 9 (IL-9) exerts its pleiotropic effects through the IL-9 receptor (IL-9R) complex, which consists of the IL-9R α-chain, which determines the cytokine specificity, and the IL-2 receptor γ-chain. In the present study we used a modified yeast two-hybrid system to isolate cDNA species encoding proteins that interacted with the intracellular domain of the human IL-9R α-chain (hIL-9Rα). We have identified 14-3-3ζ as an hIL-9Rα-interacting protein. We also mapped residues 518-522 (Arg-Ser519-Trp-Thr521-Phe) in hIL-9Rα and helix I of 14-3-3ζ as being important for interaction. Moreover, peptide competition experi-ments suggested that interaction between hIL-9Rα and 14-3-3ζ requires the phosphorylation of Ser519 or Thr521. This is the first demonstration that 14-3-3 can interact with a non-tyrosine kinase receptor. The interaction between 14-3-3 and IL-9Rα but not IL-4Rα also suggests a potential role for 14-3-3 in determining cytokine specificity.
A pyruvate–proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle
Authors: VANDERHEYDEN Nicole , WONG Julian , DOCAMPO Roberto
Published: August 2015

Regulation of intracellular pH (pHi) and H+ efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pHi in bloodstream and procyclic trypomastigotes was 7.47±0.06 and 7.53±0.07 respectively. Differences in the mechanisms for the regulation of pHi were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pHi at neutral over a wide range of external pH values from 6 to 8, and in the absence of K+ or Na+. The H+-ATPase inhibitors N,Nʹ-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pHi and inhibited its recovery from acidification. The rate of H+ efflux in these forms was determined to be 62±6.5 nmol/min per mg of protein, and was substantially decreased by H+-ATPase inhibitors. The data support the presence of an H+-ATPase as the major regulator of pHi in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pHi and recovery from acidification were unaffected by H+-ATPase inhibitors, except for DCCD (100 μM). Their steady-state pHi was markedly decreased in glucose-free buffer or by ≥ 10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H+ efflux in bloodstream trypomastigotes was 534±38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499±34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4,4ʹ-di-isothiocyanatodihydrostilbene-2,2ʹ-disulphonic acid and α-cyanohydroxycinnamic acid. The pyruvate analogues β-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pHi, as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pHi supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pHi control mechanism in bloodstream forms. Inhibition by H+-ATPase inhibitors of residual H+ efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H+-ATPase(s) in control of pHi in bloodstream forms.