bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, characterization and sequence analysis of Omp50,a new porin isolated from Campylobacter jejuni
Authors: BOLLA Jean Michel , DÉ Emmanuelle , DOREZ Aurélien , PAGÈS Jean Marie
Published: August 2015

A novel pore-forming protein identified in Campylobacter was purified by ion-exchange chromatography and named Omp50 according to both its molecular mass and its outer membrane localization. We observed a pore-forming ability of Omp50 after re-incorporation into artificial membranes. The protein induced cation-selective channels with major conductance values of 50Ő60pS in 1M NaCl. N-terminal sequencing allowed us to identify the predicted coding sequence Cj1170c from the Campylobacterjejuni genome database as the corresponding gene in the NCTC 11168 genome sequence. The gene, designated omp50, consists of a 1425bp open reading frame encoding a deduced 453-amino acid protein with a calculated pI of 5.81 and a molecular mass of 51169.2Da. The protein possessed a 20-amino acid leader sequence. No significant similarity was found between Omp50 and porin protein sequences already determined. Moreover, the protein showed only weak sequence identity with the major outer-membrane protein (MOMP) of Campylobacter, correlating with the absence of antigenic cross-reactivity between these two proteins. Omp50 is expressed in C. jejuni and Campylobacter lari but not in Campylobacter coli. The gene, however, was detected in all three species by PCR. According to its conformation and functional properties, the protein would belong to the family of outer-membrane monomeric porins.
In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression
Authors: SRIRAM Bandi , BANERJEA Akhil C.
Published: August 2015

Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10Ő23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg2+ only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.
Expression and regulation of pyruvate dehydrogenase kinase isoforms in the developing rat heart and in adulthood: role of thyroid hormone status and lipid supply
Authors: SUGDEN Mary C. , LANGDOWN Maria L. , HARRIS Robert A. , HOLNESS Mark J.
Published: August 2015

Activation of the pyruvate dehydrogenase (PDH) complex (PDHC) promotes glucose disposal, whereas inactivation conserves glucose. The PDH kinases (PDHKs) regulate glucose oxidation through inhibitory phosphorylation of PDHC. The adult rat heart contains three PDHK isoforms PDHK1, PDHK2 and PDHK4. Using Western-blot analysis, with specific antibodies raised against individual recombinant PDHK1, PDHK2 and PDHK4, the present study investigated PDHK isoform expression in the developing rat heart and adulthood. We identified clear differences in the patterns of protein expression of each of these PDHK isoforms during the first 3 weeks of post-natal development, with most marked up-regulation of isoforms PDHK1 and PDHK4. Distinctions between the three cardiac PDHK isoforms were also demonstrated with respect to post-neonatal maturational up-regulation; with greatest up-regulation of PDHK1 and least up-regulation of PDHK4 from the post-neonatal period until maturity. The study also examined the role of thyroid hormone status and lipid supply on PDHK isoform expression. We observed marked selective increases in the amount of PDHK4 protein present relative to total cardiac protein in both hyperthyroidism and high-fat feeding. Overall, our data identify PDHK isoform PDHK1 as being of more potential regulatory importance for glucose oxidation in the adult compared with the neonatal heart, and cardiac PDHK4 as a PDHK isoform whose expression is specifically responsive to changes in lipid supply, suggesting that its up-regulation during early post-natal life may be the perinatal switch to use fatty acids as the energy source. We also identify regulation of pyruvate sensitivity of cardiac PDHK as a physiological variable, a change in which requires factors in addition to a change in lipid supply.
Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor
Authors: VITALE Gaetano , BERNARDI Lorenzo , NAPOLITANI Giorgio , MOCK Michèle , MONTECUCCO Cesare
Published: August 2015

The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn2+-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific proteinŐprotein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1´ are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.
Specific induction of RGS16 (regulator of G-protein signalling 16) mRNA by protein kinase C in CEM leukaemia cells is mediated via tumour necrosis factor α in a calcium-sensitive manner
Authors: FONG Chee Wai , ZHANG Yi , NEO Soek Ying , LIN Sheng-Cai
Published: August 2015

The RGS (regulator of G-protein signalling) proteins are GTPase-activating proteins for activated Gα subunits. We investigated the effects of protein kinase C (PKC) on RGS proteins in various T cell lines by treating them with PMA. mRNA levels of both RGS16 and tumour necrosis factor α (TNFα) were found to be up-regulated in CEM leukaemia cells in a PKC-dependent manner. Mezerein, a non-phorbol-ester activator of PKC, also elevated RGS16 and TNFα mRNA levels, while the specific PKC inhibitor Go6983 abrogated their expression. In view of the slower kinetics of PMA-induced RGS16 expression and the tight correlation between TNFα and RGS16 mRNA induction among the cell lines studied, we suggest that activation of PKC up-regulates RGS16 via TNFα. Indeed, addition of recombinant TNFα to CEM cells rapidly stimulated RGS16 mRNA expression independently of PKC. Furthermore, mobilization of calcium by A23187 and thapsigargin blocked the TNFα-mediated induction of RGS16, which was reversed by EGTA and by the immunosuppressants FK506 and cyclosporin A, suggesting that the calcineurin/NF-AT (nuclear factor of activated T cells) pathway may repress the up-regulation process. Our results demonstrate for the first time that activation of PKC induces RGS16 expression via TNFα in a calcium-sensitive manner, thereby implicating RGS16 in the regulation of T cell responses to inflammation.
Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions
Authors: MURATA Naoya , SATO Koichi , KON Junko , TOMURA Hideaki , YANAGITA Masayo , KUWABARA Atsushi , UI Michio , OKAJIMA Fumikazu
Published: August 2015

The concentration of sphingosine 1-phosphate (S1P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol phosphate response to plasma or serum mediated by Edg-3, one of the S1P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S1P in these samples. The inositol phosphate response to exogenous S1P was markedly attenuated in the presence of charcoal-treated low-S1P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S1P and was associated with the trapping of exogenous S1P; the inositol phosphate response to P2-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S1P than lipoprotein-deficient serum. Consistent with this observation, we detected a 15Ő100-fold higher amount of S1P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S1P is distributed in this fraction. These results suggest that the tight binding of S1P to the components of serum or plasma, including lipoproteins, may interfere with the S1P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.
Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization
Authors: VAN DE LOO Jan-Willem H. P. , TEUCHERT Meike , PAULI Ilse , PLETS Evelyn , DE VEN Wim J. M.VAN , CREEMERS John W. M.
Published: August 2015

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t1/2 ≈ 50min) than that of the LPC polypeptide backbone (t1/2 ≈ 3h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.
Conversion of a synthetic fructosamine into its 3-phospho derivative in human erythrocytes
Authors: DELPIERRE Ghislain , VANSTAPEL Florent , STROOBANT Vincent , VAN SCHAFTINGEN Emile
Published: August 2015

Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent Km of 30mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284, 363Ő366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent Ki of 100µM. 31P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent Km (≈ 100µM) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.
Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology
Authors: CÈBE Régis , GEISER Martin
Published: August 2015

The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host. After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced. In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular domain of the phage minor coat protein III. The new adapter was expressed in Escherichia coli and refolded from inclusion bodies. The two different domains joined within the chimaera were found to be biologically functional. We also demonstrated that non-covalent interactions between a non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity. To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule. In our model systems we show that the size of either the ligand or the target is a limiting factor for the technology.
Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates
Authors: LEE Chang Hoon , PARK Myung Hee
Published: August 2015

Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [3H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N1-guanyl-1,7-diamino[3H]heptane (GC7), but not with [14C]putrescine or [14C]spermine. Although [3H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC7 and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be ≈ 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.
Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus
Authors: BUTLER Clive S. , CHARNOCK John M. , GARNER C. David , THOMSON Andrew J. , FERGUSON Stuart J. , BERKS Ben C. , RICHARDSON David J.
Published: August 2015

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4FeŐ4S] cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated. Results are presented that show NAP is sensitive to SCN- (thiocyanate) inhibition, with SCN- acting as a competitive inhibitor of nitrate (Ki ≈ 4.0mM). The formation of a novel EPR Mo(V) species with an elevated gav value (gav ∼ 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN-. Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three MoŐS ligands at 2.35Å (1 Å = 0.1nm) and a MoŐO ligand at 2.14 Å. The addition of SCN- to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 Å) Mo(IV) species with four MoŐS ligands at 2.34 Å. Taken together, the competitive nature of SCN- inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR gav value following redox cycling in the presence of SCN- and the increase in sulphur co-ordination of Mo(IV) upon SCN- binding, provide strong evidence for the direct binding of SCN- via a sulphur atom to Mo.
Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis
Authors: CAMPOS Narciso , RODRÍGUEZ-CONCEPCIÓN Manuel , SAURET-GÜETO Susanna , GALLEGO Francesca , LOIS Luisa-María , BORONAT Albert
Published: August 2015

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.
The role of c-Myb in the up-regulation of methionine adenosyltransferase 2A expression in activated Jurkat cells
Authors: ZENG Zhaohui , YANG Heping , HUANG Zong-Zhi , CHEN Changjin , WANG Jiaohong , LU Shelly C.
Published: August 2015

Methionine adenosyltransferase (MAT) is a critical cellular enzyme which catalyses the formation of S-adenosylmethionine (SAM), the principal methyl donor. In mammals, two different genes, MAT1A and MAT2A, encode liver-specific and non-liver-specific MATs, respectively. SAM level increases during T-lymphocyte activation and is required for proliferation. A major mechanism for the increase in SAM level is increased MAT2A transcription. In the current work we examined the molecular mechanism of increased MAT2A expression in activated Jurkat cells. Treatment of Jurkat cells with interleukin-2 (IL-2), PMA or PMA plus phytohaemagglutinin (PHA) resulted in a 2-fold increase in MAT2A mRNA levels and a 2-fold increase in luciferase activity driven by the transfected human MAT2A promoter construct -571/+60 but not -270/+60. The region -571 to -270 of the human MAT2A contains a c-Myb consensus binding site. c-Myb is known to be induced during T-lymphocyte activation and its mRNA level was increased after treatment of Jurkat cells with IL-2, PMA or PMA plus PHA. Increased nuclear binding to the MAT2A c-Myb site was confirmed on electrophoretic mobility-shift and supershift analyses. Mutation of the MAT2A c-Myb site abolished the stimulatory effect of these agents on c-Myb nuclear binding and MAT2A promoter activities. Overexpression of c-Myb increased MAT2A promoter activity by 2-fold. Dexamethasone, a known inhibitor of lymphocyte activation, blocked the effect of these agents on MAT2A expression by preventing the increase in c-Myb expression.
Negative cross-talk between interleukin-3 and interleukin-11 is mediated by suppressor of cytokine signalling-3 (SOCS-3)
Authors: MAGRANGEAS Florence , BOISTEAU Olivier , DENIS Sébastien , JACQUES Yannick , MINVIELLE Stéphane
Published: August 2015

Previous studies have shown that addition of interleukin-3 (IL-3) abrogated the B-cell potential of primary colonies supported by IL-11, erythropoietin, IL-7 and steel factor. However, the mechanism by which IL-3 exerts its inhibitory role is not understood. Using a variant of the mouse pro-B cell line Ba/F3 which expresses both IL-3 and IL-11 receptors, we showed that pretreatment of these cells with IL-3 before stimulation by IL-11 suppressed the tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3). This inhibition occurred within 30min and required the synthesis of a negative regulator. The onset of IL-3-dependent inhibition was correlated temporally with the appearance of SOCS-3 (suppressor of cytokine signalling-3) protein. In addition, overexpression of SOCS-3 in the pro-B cell line effectively blocked STAT3 activation induced by IL-11. These findings establish that a cytokine (IL-3) that has been shown to modulate its own signal of activation is also able to down-regulate signalling activated by a different cytokine (IL-11). This cross-talk involves activation of the JAK (Janus kinase)/STAT signalling pathway, but not mitogen-activated protein kinase pathways, and is mediated, at least in part, by SOCS-3.
Fatty-acyl-CoA thioesters inhibit recruitment of steroid receptor co-activator 1 to α and γ isoforms of peroxisome-proliferator-activated receptors by competing with agonists
Authors: MURAKAMI Koji , IDE Tomohiro , NAKAZAWA Tomoko , OKAZAKI Takashi , MOCHIZUKI Toshiro , KADOWAKI Takashi
Published: August 2015

Peroxisome-proliferator-activated receptors (PPARs) α and γ are ligand-dependent transcription factors that are key regulators of lipid and carbohydrate homoeostasis. Fatty acids bind to the ligand-binding domains (LBDs) of PPARα and PPARγ and activate these receptors. To clarify whether fatty-acyl-CoAs interact directly with the LBDs of PPARα and PPARγ, we performed a competition binding assay with radiolabelled KRP-297, a known dual agonist for these receptors. We show here that fatty-acyl-CoAs bind directly to PPARα and PPARγ. Interestingly, fatty-acyl-CoAs, unlike fatty acids, failed to recruit steroid receptor co-activator 1 (SRC-1), on the basis of conformational changes in the LBDs of PPARα and PPARγ. Moreover, fatty-acyl-CoAs also markedly inhibited agonist-induced recruitment of SRC-1. These findings demonstrate that fatty-acyl-CoAs have a novel function in the signalling pathways of PPARα and PPARγ.
Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis
Authors: WU Guoyao , HAYNES Tony E. , LI Hui , YAN Wene , MEININGER Cynthia J.
Published: August 2015

L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24h in the presence of 0, 0.1, 0.5 or 2mM D-glucosamine, or of 0.2 or 2mM L-glutamine with or without 20µM 6-diazo-5-oxo-L-norleucine (DON) or with 100µM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20µM) and azaserine (100µM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca2+ levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.
Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells
Authors: LIN Hong , CHEN Catheryne , CHEN Ben D.-M.
Published: August 2015

In this study we investigated the underlying mechanisms that confer resistance on mature macrophages with the use of macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDM). In the presence of M-CSF, immature precursor cells were induced to undergo proliferation and differentiation into mature macrophages in vitro with cell morphology similar to that of tissue macrophages by day 7Ő10. Immunoblot analyses showed that bone marrow precursors express appreciable levels of caspase-3 and caspase-9 but no or very low levels of c-fms (M-CSF receptor) and the apoptosis regulators X-linked inhibitor of apoptosis protein (XIAP), c-IAP-1, Bcl-2 and Bax. The differentiation of BMDM is associated with a steady and gradual increase in the levels of c-fms, XIAP, c-IAP-1, Bcl-2 and Bax, reaching maximal levels by day 7. However, the levels of caspase-3 and caspase-9 stayed essentially unchanged even after prolonged incubation (more than 10 days) with M-CSF. Unlike bone marrow precursor cells, mature BMDM (day 7Ő10) were resistant to apoptosis induced by M-CSF depletion, which includes the activation of caspase-3 and caspase-9 and the degradation of XIAP, Bcl-2 and Bax proteins in the process. Treatment of day 7 BMDM with XIAP anti-sense oligonucleotides (oligos), but not sense oligos, partly abolished their resistance to apoptosis. By using a gel-shift assay and a specific nuclear factor κB (NF-κB) inhibitor, we demonstrated that NF-κB activity is responsible for the up-regulation of XIAP in M-CSF-treated macrophages. In addition, treatment of starved macrophages with M-CSF induced a rapid phosphorylation of Akt kinase before the activation of NF-κB. Our results showed that XIAP is one of the anti-apoptotic regulators that confer resistance on mature macrophages by M-CSF.
5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)
Authors: GIONO Luciana E. , VARONE Cecilia L. , CÁNEPA Eduardo T.
Published: August 2015

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.
Cross-talk between steroid-receptor-mediated and cell-membrane-receptor-mediated signalling pathways results in the in vivo modulation of c-Met and ornithine decarboxylase gene expression in mouse kidney
Authors: DUDKOWSKA Magdalena , STACHURSKA Agnieszka , CHMURZYŃSKA Wanda , GRZELAKOWSKA-SZTABERT Barbara , MANTEUFFEL-CYMBOROWSKA Małgorzata
Published: August 2015

The cross-talk in vivo between two signalling pathways activated by testosterone via intracellular androgen receptor, and induced by damage to renal tubules evoked by anti-folate [N10-propargyl-5,8-dideazafolic acid (CB 3717)] or folate is reported. We show that CB 3717/folate induces the expression of the hepatocyte growth factor (HGF)/c-Met signalling system in injured kidneys in which a significant, but transient, elevation of the HGF mRNA level occurs. It is followed by a severalfold increase in the c-Met transmembrane receptor message that persists for up to 24h. The c-Met expression is also positively controlled by testosterone, which induces a significant increase in its mRNA level that is abolished by an anti-androgen, casodex. However, when testosterone and anti-folate/folate are administered sequentially, a substantial (3.5Ő4.0-fold) decrease in the increase of c-Met expression caused by CB 3717/folate alone occurs. Similarly, testosterone-induced ornithine decarboxylase (ODC) mRNA level and activity are decreased 2.8Ő7.7-fold when the androgen is applied together with CB 3717. Antagonism between these pathways is also visible under physiological conditions in the kidneys of male mice in which, owing to elevated endogenous testosterone levels, neither the ODC activity nor the mRNA level is induced by anti-folate/folate, whereas the c-Met message response to these drugs is significantly decreased. Our results document a substantial negative regulation of c-Met and ODC gene expression as a result of the cross-talk between testosterone-activated and HGF-activated pathways and suggest a sex-differentiated response to injury of mouse kidneys.
Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones
Authors: FRANKLIN Trevor J. , MORRIS William P. , EDWARDS Philip N. , LARGE Michael S. , STEPHENSON Robert
Published: August 2015

Examples of a novel series of phenanthrolinones are shown to be potent competitive inhibitors of avian prolyl 4-hydroxylase, and of collagen hydroxylation, in embryonic chick tendon cells and human foreskin fibroblasts in vitro and in the oestradiol-stimulated rat uterus in vivo. Two compounds, Compound 1 (1,4-dihydrophenanthrolin-4-one-3-carboxylic acid) and Compound 5 [8-(N-butyl-N-ethylcarbamoyl)-1,4-dihydrophenathrolin-4-one-3-carboxylic acid], with comparable potencies in vivo, were chosen to investigate the effect of the inhibition of the hydroxylation of newly synthesized uterine collagen on the turnover of this protein in vivo. Inhibition of hydroxylation by more than 50% for approx. 8h following single oral doses of the compounds was associated with significant losses of radiolabelled proline and 4-hydroxyproline from collagen during this period. Progressive hydroxylation of collagen over 48h, as the inhibitory action of the compounds declined, was accompanied by a decreased loss of radiolabel from the uterine collagen. Earlier reports indicated that underhydroxylated collagen, accumulating within the endoplasmic reticulum in cells where prolyl 4-hydroxylase is inactivated, is slowly degraded, but is then rapidly hydroxylated and secreted when the activity of prolyl 4-hydroxylase is restored. Taken with the present results, this suggests that the potential use of inhibitors of prolyl 4-hydroxylase to control excessive collagen deposition in pathological fibrosis may be limited by the need to maintain continuous inhibition of collagen hydroxylation so as to facilitate intracellular degradation of the accumulated protein.
Overexpression of the human 2-oxoglutarate carrier lowers mitochondrial membrane potential in HEK-293 cells: contrast with the unique cold-induced mitochondrial carrier CGI-69
Authors: YU Xing Xian , LEWIN David A. , ZHONG Alan , BRUSH Jennifer , SCHOW Peter W. , SHERWOOD Steven W. , PAN Guohua , ADAMS Sean H.
Published: August 2015

Using differential mRNA expression analysis, a previously uncharacterized gene was found to be up-regulated 2-fold in brown adipose tissue (BAT) of mice exposed to cold (4°C) for 48h. Contig and homology analysis revealed that the gene represents the murine orthologue to a sequence from a public database encoding a putative human protein (CGI-69). The presence of mitochondrial carrier domains in the human protein, its transmembrane topology and cold-induction of the mouse CGI-69 gene in BAT prompted an analysis of the idea that CGI-69 may represent a new uncoupling protein (UCP) functional homologue. However, transfection of human CGI-69 isoforms in HEK-293 cells yielded no change in mitochondrial membrane potential (∆Ψm), despite localization of FLAG-tagged CGI-69 to mitochondria of MCF7 cells. Surprisingly, overexpression of the human 2-oxoglutarate carrier (OGC) protein (originally designed as a negative control) sparked a significant drop in ∆Ψm, possibly signalling a previously unappreciated uncoupling activity for the OGC.
Interaction of the α subunit of Na,K-ATPase with cofilin
Authors: LEE Kyunglim , JUNG Jaehoon , KIM Miyoung , GUIDOTTI Guido
Published: August 2015

The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the α subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive 86Rb+ uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.
mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2
Authors: YU Zhenbao , MAOUI Meryem , WU Liangtang , BANVILLE Denis , SHEN Shi-Hsiang
Published: August 2015

The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily. By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E. The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes. The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction. This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor. When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell–cell interactions. In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9. The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33. Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.
Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle
Authors: MEGÍAS Alicia , MARTÍNEZ-SENAC M. Mar , DELGADO Jerónimo , SABORIDO Ana
Published: August 2015

Transverse tubule (T-tubule) ecto-ATPase from chicken skeletal muscle is an integral membrane glycoprotein that seems to exist as a homodimer and exhibits unusual properties. Treatment of T-tubule membranes with concanavalin A (Con A) did not significantly affect the thermal variation of the fluorescence anisotropy of vesicles labelled with 1,6-diphenyl-1,3,5-hexatriene or trimethylammonium-1,6-diphenyl-1,3,5-hexatriene. Cross-linking of membrane components with glutaraldehyde elicited effects on ecto-ATPase activity very similar to those of Con A treatment: a severalfold increase in activity, a decrease in Triton X-100 sensitivity and a requirement to be present before ATP to exert its action. In addition, glutaraldehyde and Con A normalized the temperature dependence and the kinetic behaviour of the enzyme. Membrane-perturbing agents (detergents, alcohols and cholesterol oxidase), with the sole exception of digitonin, caused a marked decrease in ecto-ATPase activity; the prior presence of Con A prevented this inhibition, whereas when the lectin was added after the membrane perturbing agent, recovery of the activity was not always possible. The addition of nucleotides before Con A led to a suppression of ecto-ATPase stimulation; it occurred when the nucleotide was hydrolysed (ATP or UTP) and when it was not (adenosine 5′-[β,γ-imido]triphosphate) and even in the presence of 3mM Pi. A model is proposed for the complex regulatory mechanisms of chicken T-tubule ecto-ATPase that involves the occurrence of two different catalytic states in an equilibrium modulated by lectins and cross-linking agents, by the structure of the membrane and by the presence of ligands for a regulatory site.
Nicotinic acid–adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells
Authors: YUSUFI Ahad N.-K. , CHENG Jingfei , THOMPSON Michael A. , CHINI Eduardo N. , GRANDE Joseph P.
Published: August 2015

Nicotinic acid–adenine dinucleotide phosphate (NAADP), a molecule derived from β-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not β-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.
Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells
Authors: BAKOWSKI Daniel , PAREKH Anant B.
Published: August 2015

Whole-cell patch-clamp experiments were performed to examine the mechanism underlying the inability of intracellular Ins(1,4,5)P3 to activate the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL)-1 cells under conditions of weak cytoplasmic Ca2+ buffering. Dialysis with Ins(1,4,5)P3 in weak Ca2+ buffer did not activate any macroscopic ICRAC even after precautions had been taken to minimize the extent of Ca2+ entry during the experiment. Following intracellular dialysis with Ins(1,4,5)P3 for > 150s in weak buffer, external application of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin activated ICRAC, and the current developed much more quickly than when thapsigargin was applied in the absence of Ins(1,4,5)P3. This indicates that the Ins(1,4,5)P3 receptors had not inactivated much over this timecourse. When external Ca2+ was replaced by Ba2+, Ins(1,4,5)P3 still failed to generate any detectable ICRAC even though Ba2+ permeates CRAC channels and is not taken up into the intracellular Ca2+ stores. In strong Ca2+ buffer, ICRAC could be activated by muscarinic-receptor stimulation, provided protein kinase C (PKC) was blocked. In weak buffer, however, as with Ins(1,4,5)P3, stimulation of these receptors with carbachol did not activate ICRAC even after inhibition of PKC. The inability of Ins(1,4,5)P3 to activate macroscopic ICRAC in weak Ca2+ buffer was not altered by inhibition of Ca2+-dependent phosphorylation/dephosphorylation reactions. Our results suggest that the inability of Ins(1,4,5)P3 to activate ICRAC under conditions of weak intracellular Ca2+ buffering is not due to strong inactivation of the Ins(1,4,5)P3 receptors. Instead, a futile Ca2+ cycle across the stores seems to be occurring and SERCA pumps resequester sufficient Ca2+ to ensure that the threshold for activation of macroscopic ICRAC has not been exceeded.
Role of calcium ions in the structure and function of thedi-isopropylfluorophosphatase from Loligo vulgaris
Authors: HARTLEIB Judith , GESCHWINDNER Stefan , SCHARFF Eileen I. , RÜTERJANS Heinz
Published: August 2015

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca2+-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-({2-[bis(carboxymethyl)amino]-5-methylphenoxy}-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840nM in the absence and the presence of 150mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3nM. Stopped-flow experiments have shown that the dissociation of bound Ca2+ occurs in two phases, with rates of approx. 1.1 and 0.026s-1 corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca2+ dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca2+ from DFPase. The Ca2+ dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca2+ dissociation and the denaturation results we conclude that the higher-affinity Ca2+ site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.
Phosphorylation and desensitization of α1d-adrenergic receptors
Authors: GARCÍA-SÁINZ J. Adolfo , VÁZQUEZ-CUEVAS Francisco G. , ROMERO-ÁVILA María Teresa
Published: August 2015

In rat-1 fibroblasts stably expressing rat α1d-adrenoceptors, noradrenaline and PMA markedly decreased α1d-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [35S]guanosine 5′-[γ-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified α1d-adrenoceptors as a broad band of 70–80kDa. α1d-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC50 75nM), rapid (maximum at 1min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC50 25nM), slightly slower (maximum at 5min) and stable for at least 60min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of cross-talk of α1d-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce α1d-adrenoceptor phosphorylation. In summary, it is shown for the first time here that α1d-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions.