bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that the inhibition of cartilage proteoglycan breakdown by mannosamine is not mediated via inhibition of glycosylphosphatidylinositol anchor formation
Authors: BRYSON Heather , BUTTLE David J. , KOZACI L. Didem , JOHNATTY Rachel N. , BUNNING Rowena A. D.
Published: August 2015

The effect of mannosamine, an inhibitor of glycosylphosphatidylinositol (GPI) anchor formation, on chondrocyte-mediated cartilage proteoglycan breakdown was investigated using cartilage explant cultures. Mannosamine inhibited interleukin 1α-, tumour necrosis factor α- and retinoic acid-stimulated proteoglycan release from bovine nasal and articular cartilage, and retinoic acid-stimulated proteoglycan release from human cartilage. Its effects on two GPI-anchored proteins [the urokinase receptor, which binds urokinase-type plasminogen activator (uPA) to cell surfaces, and alkaline phosphatase] were also studied using bovine chondrocytes. Enzyme histochemistry and zymography demonstrated cell-associated uPA-like serine proteinase activity and PA activity respectively which was not reduced by treatment of chondrocytes with mannosamine at concentrations effective at inhibiting cartilage proteoglycan breakdown. Similarly, the activity of cell-associated alkaline phosphatase was not reduced, except at mannosamine concentrations much higher than those used to inhibit proteoglycan breakdown. These results demonstrate that inhibition of proteoglycan breakdown by mannosamine is too potent to be explained by an effect on GPI-anchor formation.
Analysis of tissue inhibitor of metalloproteinases-2 effect on pro-matrix metalloproteinase-2 activation by membrane-type 1 matrix metalloproteinase using baculovirus/insect-cell expression system
Authors: JO Yihyung , YEON Jungheum , KIM Hwa-Jung , LEE Seung-Taek
Published: August 2015

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14) is known to activate pro-matrix metalloproteinase-2 (pro-MMP-2; progelatinase A) on the cell surface. To analyse the tissue inhibitor of metalloproteinases-2 (TIMP-2) effect on activation of pro-MMP-2 by MT1-MMP, we have expressed the full-size MT1-MMP (fMT1-MMP) and a transmembrane (TM)-domain-deleted soluble MT1-MMP (sMT1-MMP) in the baculovirus/Sf9 (Spodoptera frugiperda 9) insect-cell system, where neither endogenous gelatinolytic MMPs nor TIMP-2 are expressed. Both fMT1-MMP and sMT1-MMP expressed in the expression system were found not to contain the pro-domain and were able to activate the TIMP-2-free pro-MMP-2. Both in the insect cells and in vitro, activation of pro-MMP-2 by fMT1-MMP was enhanced at low concentrations of TIMP-2 and inhibited by its higher concentrations. The maximal enhancing effect was detected at 0.05 molar fraction of TIMP-2/fMT1-MMP. In contrast, activation of pro-MMP-2 by sMT1-MMP was dose-dependently inhibited by TIMP-2. These results demonstrate that the TM domain of MT1-MMP is not required for the ability to activate pro-MMP-2, but is required for the enhancing effect of TIMP-2 on pro-MMP-2 activation by recruiting pro-MMP-2 to the MT1-MMP-TIMP-2 complex as a cell-surface pro-MMP-2 receptor. Moreover, our data strongly suggest that the pro-domain of MT1-MMP is not required for the TIMP-2-mediated enhancing effect on pro-MMP-2 activation. In addition, the pro-MMP-2 in the MT1-MMP-TIMP-2-pro-MMP-2 ternary complex was not activated without external activator, but readily by addition of sMT1-MMP. This result demonstrates that MT1-MMP free of TIMP-2 would be the enzyme responsible for activation of the pro-MMP-2 in the ternary complex under physiological conditions.
Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase
Authors: FAMULSKI Konrad S. , LIUZZI Michel , BASHIR Saber , MIRZAYANS Razmik , PATERSON Malcolm C.
Published: August 2015

A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein ‘prefers#x2019; a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5ʹ-OH and 3ʹ-PO4 termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 °C or greater, and is neither stimulated by Mg2+ or Ca2+ nor inhibited by Zn2+. AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.
A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family
Authors: TUNG Hui-shan , GUSS Bengt , HELLMAN Ulf , PERSSON Lena , RUBIN Kristofer , RYDÉN Cecilia
Published: August 2015

Staphylococcus aureus bacteria, isolated from bone and joint infections, specifically interact with bone sialoprotein (BSP), a glycoprotein of bone and dentine extracellular matrix, via a cell-surface protein of Mr 97000 [Yacoub, Lindahl, Rubin, Wendel, Heinegård and Rydén, (1994) Eur. J. Biochem. 222, 919-925]. Amino acid sequences of seven trypsin fragments from the 97000-Mr BSP-binding protein were determined. A gene encoding a protein encompassing all seven peptide sequences was identified from chromosomal DNA isolated from S. aureus strain O24. This gene encodes a protein with 1171 amino acids, called BSP-binding protein (Bbp), which displays similarity to recently described proteins of the Sdr family from S. aureus. SdrC, SdrD and SdrE encode putative cell-surface proteins with no described ligand specificity. Bbp also shows similarity to a fibrinogen-binding protein from S. epidermidis called Fbe. A serine-aspartic acid repeat sequence was found close to the cell-wall-anchoring Leu-Pro-Xaa-Thr-Gly sequence in the C-terminal end of the protein. Escherichia coli cells were transformed with an expression vector containing a major part of the bbp gene fused to the gene for glutathione S-transferase. The affinity-purified fusion protein bound radiolabelled native BSP, and inhibited the binding of radiolabelled BSP to staphylococcal cells. Serum from patients suffering from bone and joint infection contained antibodies that reacted with the fusion protein of the BSP-binding protein, indicating that the protein is expressed during an infection and is immunogenic. The S. aureus Bbp protein may be important in the localization of bacteria to bone tissue, and thus might be of relevance in the pathogenicity of osteomyelitis.
Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin
Authors: ANDBERG Martina , WETTERHOLM Anders , MEDINA Juan F. , HAEGGSTRÖM Jesper Z.
Published: August 2015

Leukotriene A4 hydrolase is a bifunctional Zn2+-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B4. From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC50 values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A4 hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.
Isolation and characterization of glycosylphosphatidylinositol-anchored, mucin-like surface glycoproteins from bloodstream forms of the freshwater-fish parasite Trypanosoma carassii
Authors: LISCHKE Antje , KLEIN Christian , STIERHOF York-Dieter , HEMPEL Michaela , MEHLERT Angela , ALMEIDA Igor C. , FERGUSON Michael A. J. , OVERATH Peter
Published: August 2015

Wild and farmed freshwater fishes are widely and heavily parasitized by the haemoflagellate Trypanosoma carassii. In contrast, common carp, a natural host, can effectively control experimental infections by the production of specific anti-parasite antibodies. In this study we have identified and partially characterized mucin-like glycoproteins which are expressed in high abundance [(6.0±1.7)×106 molecules·cell-1] at the surface of the bloodstream trypomastigote stage of the parasite. The polypeptide backbone of these glycoproteins is dominated by threonine, glycine, serine, alanine, valine and proline residues, and is modified at its C-terminus by a glycosylphosphatidylinositol membrane anchor. On average, each polypeptide carries carbohydrate chains composed of about 200 monosaccharide units (galactose, N-acetylglucosamine, xylose, sialic acid, fucose, mannose and arabinose), which are most probably O-linked to hydroxy amino acids. The mucin-like molecules are the target of the fish's humoral immune response, but do not undergo antigenic variation akin to that observed for the variant surface glycoprotein in salivarian trypanosomes. The results are discussed with reference to the differences between natural and experimental infections, and in relation to the recently delineated molecular phylogeny of trypanosomes.
Signalling via ADP-ribosylation factor 6 lies downstream of phosphatidylinositide 3-kinase
Authors: VENKATESWARLU Kanamarlapudi , CULLEN Peter J.
Published: August 2015

ADP-ribosylation factor (ARF) 6 regulates plasma membrane trafficking and cortical actin formation by cycling between inactive GDP and active GTP-bound conformations. Here we show that agonist stimulation of phosphatidylinositide 3-kinase (PI 3-kinase) activates a pathway that leads to ARF6 activation. We also describe experiments that propose a central role in this pathway for the PI 3-kinase-dependent plasma membrane recruitment of the cytohesin-1 family of PtdIns(3,4,5)P3-binding ARF-exchange factors.
Platelet-derived growth factor (PDGF)-induced activation of signal transducer and activator of transcription (Stat) 5 is mediated by PDGF β-receptor and is not dependent on c-Src, Fyn, Jak1 or Jak2 kinases
Authors: PAUKKU Kirsi , VALGEIRSDÓTTIR Sigrídur , SAHARINEN Pipsa , BERGMAN Mathias , HELDIN Carl-Henrik , SILVENNOINEN Olli
Published: August 2015

Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF β-receptor (PDGF β-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF β-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-β-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr579 and Tyr581 in PDGF β-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src-/- and Fyn-/- mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF β-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.
Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis
Authors: WAY Gemma , O'LUANAIGH Niamh , COCKCROFT Shamshad
Published: August 2015

The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcϵR1, with antigen. We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses. When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In addition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP2) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP2 in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPα) to increase PIP2 levels. Exocytosis, restored by either ARF1 or PITPα, was inhibited when PIP2 levels were depleted by phospholipase C∆1. We conclude that the function of ARF1 and PITPα is to increase the local synthesis of PIP2, the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment.
Regulation of the juvenile hormone esterase gene by a composite core promoter
Authors: JONES Grace , CHU Yan Xia , SCHELLING Douglas , JONES Davy
Published: August 2015

Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5ʹ region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5ʹ-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the ‘downstream promoter element’ described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.
The ShBle resistance determinant from Streptoalloteichus hindustanus is expressed in Haloferax volcanii and confers resistance to bleomycin
Authors: NUTTALL Stewart D. , DEUTSCHEL Simone E. , IRVING Robert A. , SERRANO-GOMICIA Juan A. , DYALL-SMITH Michael L.
Published: August 2015

We have designed a gene cassette for expression of the bleomycin-resistance protein from Streptoalloteichus hindustanus (ShBle) in the extremely halophilic archaeon Haloferax volcanii, and shown that transformed haloarchaea are resistant to bleomycin. Recombinant ShBle was purified by a one-step affinity-chromatography procedure as a correctly folded, dimeric protein. ShBle thus provides a useful haloarchaeal selectable marker and represents the first non-halophilic and soluble heterologous protein to be expressed in the Haloarchaea.
Some polyphenols inhibit the formation of pentyl radical and octanoic acid radical in the reaction mixture of linoleic acid hydroperoxide with ferrous ions
Authors: IWAHASHI Hideo
Published: August 2015

Effects of some polyphenols and their related compounds (chlorogenic acid, caffeic acid, quinic acid, ferulic acid, gallic acid, D-(+)-catechin, D-(-)-catechin, 4-hydroxy-3-methoxybenzoic acid, salicylic acid, L-dopa, dopamine, L-adrenaline, L-noradrenaline, o-dihydroxybenzene, m-dihydroxybenzene, and p-dihydroxybenzene) on the formation of 13-hydroperoxide octadecadienoic (13-HPODE) acid-derived radicals (pentyl radical and octanoic acid radical) were examined. The ESR spin trapping showed that chlorogenic acid, caffeic acid, gallic acid, D-(+)-catechin, D-(-)-catechin, L-dopa, dopamine, L-adrenaline, L-noradrenaline, and o-dihydroxybenzene inhibited the overall formation of 13-HPODE acid-derived radicals in the reaction mixture of 13-HPODE with ferrous ions. The ESR peak heights of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/13-HPODE-derived radical adducts decreased to 46±4% (chlorogenic acid), 54±2% (caffeic acid), 49±2% (gallic acid), 55±1% [D-(+)-catechin], 60±3% [D-(-)-catechin], 42±1% (L-dopa), 30±2% (dopamine), 49±2% (L-adrenaline), 24±2% (L-noradrenaline), and 54±5% (o-dihydroxybenzene) of the control, respectively. The high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatography-electron spin resonance-mass spectrometries (HPLC-ESR-MS) showed that caffeic acid inhibited the formation of octanoic acid radical and pentyl radical to 42±2% and 52±7% of the control, respectively. On the other hand, the polyphenols and their related compounds had few inhibitory effects on the radical formation in the presence of EDTA. Visible absorbance measurement revealed that all the polyphenols exhibiting the inhibitory effect chelate ferrous ions. Above results indicated that the chelation of ferrous ion is essential to the inhibitory effects of the polyphenols.
Target site search and effective inhibition of leukaemic cell growth by a covalently closed multiple anti-sense oligonucleotide to c-myb
Authors: MOON Ik-Jae , LEE Youngik , KWAK Chun-Sik , LEE Je-Ho , CHOI Kyusam , SCHREIBER Alan D. , PARK Jong-Gu
Published: August 2015

Systematic secondary structure simulation of a target mRNA sequence is shown to be effective for locating a good anti-sense target site. Multiple selected anti-sense sequences were placed in a single molecule. The anti-sense oligonucleotide (oligo) was covalently closed to avoid exonuclease activities and was designated CMAS (covalently closed multiple anti-sense)-oligo. CMAS-oligo was found to be stable, largely preserving its structural integrity after 24 h of incubation in the presence of either exonuclease III or serum. When human c-myb mRNA was targeted by the c-myb CMAS-oligo, expression of the gene was completely abolished. Further, tumour cell growth was inhibited by 82±3% as determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and by 90±1% by [3H]thymidine incorporation. When a leukaemic cell line K562 was treated with CMAS-oligo, colony formation on soft agarose was also decreased by 93%. In contrast, treatment with a scrambled control oligo did not significantly inhibit leukaemic cell growth. These results suggest that a rational target site search is possible for an anti-sense oligo and that CMAS-oligo can be employed as an effective anti-sense agent with enhanced stability.
Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase
Authors: PALAPARTI Anuradha , LI Yuan , ANAND-SRIVASTAVA Madhu B.
Published: August 2015

Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP4-23 [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANP4-23-NH2]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP4-23. In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP4-23-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 μM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not inhibited by antisense-oligonucleotide treatment. Our results demonstrate conclusively that the elimination of ANP-C receptor by antisense oligonucleotide attenuates ANP-induced inhibition of adenylyl cyclase and provide evidence that antisense oligonucleotide of the ANP-C receptor may serve as a useful pharmacological tool to elucidate the physiological functions of the ANP-C receptor.
Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils
Authors: SMITH Gerry A. , DIXON Henry B. F. , KIRSCHENLOHR Heide L. , GRACE Andrew A. , METCALFE James C. , VANDENBERG Jamie I.
Published: August 2015

The measurement of cardiac Ca2+ transients using spectroscopic Ca2+ indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca2+ buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca2+ dissociation rate constants and which would account for the observed effects of 19F-NMR indicators on the cardiac Ca2+ transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca2+ dependency of myofibril activation in cardiac myocytes is mediated by a single Ca2+-binding site on troponin C. A model based on 1:1 Ca2+ binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca2+ binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca2+ binding, giving 58 and 2.0 μM respectively. In addition to reproducing our experimental data on the concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i), the resulting Ca2+ and ATP affinities given by fitting of the model also provided good predictions of the Ca2+ dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca2+ mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca2+ indicators. The new model was used to estimate the extent of perturbation of the Ca2+ transient caused by different concentrations of indicators. As little as 10 μM of a Ca2+ indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca2+]i and 30 μM will cause approx. 50% reduction in the peak-systolic [Ca2+]i in a heart paced at 1.0 Hz.
Thyroxine regulation of monolysocardiolipin acyltransferase activity in rat heart
Authors: MUTTER Thomas , DOLINSKY Vernon W. , MA Brian J. , TAYLOR William A. , HATCH Grant M.
Published: August 2015

Treatment of rats with thyroxine has been shown to elevate the biosynthesis and content of cardiolipin in the heart [Cao, Cheng, Angel and Hatch (1995) Biochim. Biophys. Acta 1256, 241-244]. Treatment with thyroxine resulted in a 1.8-fold increase (P < 0.025) in [1-14C]linoleate and a 1.7-fold increase (P < 0.025) in [1-14C]oleate incorporated into cardiolipin in perfused hearts, compared with controls. The mechanism for the elevation in incorporation of unsaturated fatty acids into cardiolipin was a 1.6-fold (P < 0.025) increase in mitochondrial monolysocardiolipin acyltransferase activity. The results demonstrate that the acylation of cardiac monolysocardiolipin is regulated by thyroid hormone. Thus an elevation in cardiolipin biosynthesis is accompanied by an elevation in monolysocardiolipin acyltransferase activity to maintain the appropriate molecular species composition of cardiolipin in the cardiac mitochondrial membrane. We postulate that monolysocardiolipin acyltransferase might be a rate-limiting enzyme for the molecular remodelling of cardiolipin in the heart.
Continuous exposure to high concentrations of nitric oxide leads to persistent inhibition of oxygen consumption by J774 cells as well as extraction of oxygen by the extracellular medium
Authors: ORSI Antonia , BELTRÁN Belén , CLEMENTI Emilio , HALLÉN Katarina , FEELISCH Martin , MONCADA Salvador
Published: August 2015

Nitric oxide (NO) plays a key role in many physiological and pathophysiological events, including the control of cell respiration. Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of NO by cells. We have exposed the murine macrophage cell line J774 to high concentrations of NO, such as are generated in some pathological conditions, and determined their effect on oxygen consumption. We observed a persistent inhibition of respiration which was due to a redox-dependent, progressive inhibition of complex I activity. No other enzyme of the respiratory chain was inhibited in this way. At the same time, we detected a paradoxical removal of oxygen by the extracellular medium. This removal was due to a chemical interaction between dissolved oxygen and NO-related species released from cells exposed to NO. A similar removal of oxygen by the cell supernatant also occurred following activation of cells with cytokines and bacterial products. Thus, the amounts of NO generated during pathological conditions may contribute to tissue hypoxia both by inhibiting cell respiration and by promoting removal of oxygen from the extracellular medium.
Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells
Authors: GARDNER Robert C. , ASSINDER Stephen J. , CHRISTIE Gary , MASON Grant G. F. , MARKWELL Roger , WADSWORTH Harry , MCLAUGHLIN Mark , KING Ron , CHABOT-FLETCHER Marie C. , BRETON John J. , ALLSOP David , RIVETT A. Jennifer
Published: August 2015

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The Ki values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3×Ki). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFĸB activation with IC50 values comparable to their Ki values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
Vitamin C protects against and reverses specific hypochlorous acid- and chloramine-dependent modifications of low-density lipoprotein
Authors: CARR Anitra C. , TIJERINA Terry , FREI Balz
Published: August 2015

Activated phagocytes produce the highly reactive oxidant hypochlorous acid (HOCl) via the myeloperoxidase-catalysed reaction of hydrogen peroxide with chloride ions. HOCl reacts readily with a number of susceptible targets on apolipoprotein B-100 of low-density lipoprotein (LDL), resulting in uncontrolled uptake of HOCl-modified LDL by macrophages. We have investigated the efects of vitamin C (ascorbate), an effective water-soluble antioxidant, on the HOCl- and chloramine-dependent modification of LDL. Co-incubation of vitamin C (25-200 μM) with LDL resulted in concentration-dependent protection against HOCl (25-200 μM)-mediated oxidation of tryptophan and lysine residues, formation of chloramines and increases in the relative electrophoretic mobility of LDL. Vitamin C also partially protected against oxidation of cysteine residues by HOCl, and fully protected against oxidation of these residues by the low-molecular-mass chloramines, Nα-acetyl-lysine chloramine and taurine chloramine, and to a lesser extent monochloramine (each at 25-200 μM). Further, we found that HOCl (25-200 μM)-dependent formation of chloramines on apolipoprotein B-100 was fully reversed by 200 μM vitamin C; however, the loss of lysine residues and increase in relative electrophoretic mobility of LDL were only partially reversed, and the loss of tryptophan and cysteine residues was not reversed. Time-course experiments showed that the reversal by vitamin C of HOCl-dependent modifications became less efficient as the LDL was incubated for up to 4 h at 37 °C. These data show that vitamin C not only protects against, but also reverses, specific HOCl- and chloramine-dependent modifications of LDL. As HOCl-mediated LDL modifications have been strongly implicated in the pathogenesis of atherosclerosis, our data indicate that vitamin C could contribute to the anti-atherogenic defence against HOCl.
Rabaptin4, a novel effector of the small GTPase rab4a, is recruited to perinuclear recycling vesicles
Authors: NAGELKERKEN Bas , VAN ANKEN Eelco , VAN RAAK Marcel , GEREZ Lisya , MOHRMANN Karin , VAN UDEN Nathalie , HOLTHUIZEN Jessica , PELKMANS Lucas , VAN DER SLUIJS Peter
Published: August 2015

The small GTPase rab4a is associated with early endocytic compartments and regulates receptor recycling from early endosomes. To understand how rab4a mediates its function, we searched for proteins which associate with this GTPase and regulate its activity in endocytic transport. Here we identified rabaptin4, a novel effector molecule of rab4a. Rabaptin4 is homologous with rabaptin5 and contains a C-terminal deletion with respect to rabaptin5. Rabaptin4 preferentially interacts with rab4a-GTP and to a lesser extent with rab5aGTP. We identified a rab4a-binding domain in the N-terminal region of rabaptin4, and two binding sites for rab5, including a novel N-terminal rab5a-binding site. Rabaptin4 is a cytosolic protein that inhibits the intrinsic GTP hydrolysis rate of rab4a and is recruited by rab4a-GTP to recycling endosomes enriched in cellubrevin and internalized indocarbocyanine-3 (Cy3)-labelled transferrin. We propose that rabaptin4 assists in the docking of transport vesicles en route from early endosomes to recycling endosomes.
Molecular decipherment of Rho effector pathways regulating tight-junction permeability
Authors: FUJITA Hirotada , KATOH Hironori , HASEGAWA Hiroshi , YASUI Hidekazu , AOKI Junko , YAMAGUCHI Yoshiaki , NEGISHI Manabu
Published: August 2015

We reported recently that the activation of RhoA induced an increase in transepithelial electrical resistance (TER). To clarify effectors of Rho for this RhoA-induced regulation of tight-junction permeability, we introduced two effector-loop mutants of constitutively active RhoAV14, RhoAV14/L40 and RhoAV14/C42, into Mardin-Darby canine kidney cells in an isopropyl β-D-thiogalactoside-inducible expression system. RhoAV14 and the two effector-loop mutants interacted in vitro with the Rho-binding domain of Rho-associated kinase, ROKα. Next we examined two parameters of Rho functions, stress-fibre formation and TER elevation, induced by RhoAV14. Stress-fibre formation was induced by RhoAV14/C42 but not by RhoAV14/L40. On the other hand, TER elevation was induced by neither RhoAV14/L40 nor RhoAV14/C42. RhoA-associated kinase inhibitor, Y-27632, inhibited both stress-fibre formation and TER elevation induced by RhoAV14. These results demonstrated that RhoA-induced regulation of tight-junction permeability is mediated by Rho-associated kinase and at least one other unidentified effector, the coupling to RhoA being disrupted by mutation at position 40 or 42 in the effector loop.
Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties
Authors: LIZANO Sergio , ANGULO Yamileth , LOMONTE Bruno , FOX Jay W. , LAMBEAU Gérard , LAZDUNSKI Michel , María GUTIÉRREZ; José
Published: August 2015

Myotoxic phospholipases A2 (PLA2s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA2 (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI = 4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA2 and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to γ-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with α-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA2 receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.
Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain
Authors: FALANY Charles N. , XIE Xiaowei , WANG Jin , FERRER Jorge , FALANY Josie L.
Published: August 2015

The sulphotransferase (SULT) gene family is involved with the conjugation of many small drugs, xenobiotics and endogenous compounds. In this report, we describe the cloning and expression of novel cDNAs from human and rat brain, which are structurally related to the SULTs. These cDNAs have been termed ‘brain sulphotransferase-like’ (BR-STL), because of their similarity to the SULTs and their selective expression in brain tissue. The proteins encoded by the human and rat BR-STL cDNAs (hBR-STL-1 and rBR-STL cDNA respectively), denoted as hBR-STL and rBR-STL, are 98% identical and 99% similar in sequence. The hBR-STL-1 cDNA contains an 852-nt open reading frame encoding a 284-amino-acid protein with a calculated molecular mass of 33083 Da. Northern-blot analyses of RNA isolated from eight human tissues indicate that the hBR-STL message is selectively expressed in brain. Characterization of different brain regions showed that message levels were highest in cortical brain regions. rBR-STL message levels were relatively low in brains of 1-day-old male and female rats, but increased to adult levels in RNA from 7-day-old rats, and remained at that level in adult animals. The hBR-STL and rBR-STL cDNAs were expressed in both Escherichia coli and Sf9 insect cells in the presence or absence of an N-terminal histidine-affinity tag (His-tag). Polyclonal antibodies were raised in chickens against purified His-tagged hBR-STL, and were used to detect the presence of rBR-STL in adult male and female rat brain cytosol. The high degree of sequence conservation, and the selective localization of the BR-STL message in brain, suggest an important function in the central nervous system.
PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters
Authors: HASEGAWA Tadao , TAKEUCHI Akihide , MIYAISHI Osamu , XIAO Hengyi , MAO Jalin , ISOBE Ken-ichi
Published: August 2015

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proα2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proα2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.
Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes
Authors: PANI Giovanni , COLAVITTI Renata , BORRELLO Silvia , GALEOTTI Tommaso
Published: August 2015

Molecular events mediating the T-lymphocyte response to lectins are still incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H2O2 have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid ‘burst’ of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.
Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution
Authors: MATSUSHIMA Norio , HAYASHI Nobuhiro , JINBO Yuji , IZUMI Yoshinobu
Published: August 2015

Small-angle X-ray scattering (SAXS), which determines the radius of gyration, Rg, and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca2+ in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca2+-saturated CaM molecule induces structural changes from a ‘dumb-bell’ shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca2+-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca2+-saturated CaM molecule. Even in the absence of Ca2+, the conformational changes of CaM occur on TFP binding, giving a slightly smaller Rg than Ca2+-free CaM alone.
Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras
Authors: HENRIKSSON Maria L , ROSQVIST Roland , TELEPNEV Maxim , WOLF-WATZ Hans , HALLBERG Bengt
Published: August 2015

We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.
Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor
Authors: FOWLES Lindsay F. , STACEY Katryn J. , MARKS Denese , HAMILTON John A. , HUME David A.
Published: August 2015

Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter.