bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom
Authors: BRILLARD-BOURDET Michèle , NGUYÊN Vinh , FERRER-DI MARTINO Michèle , GAUTHIER Francis , MOREAU Thierry 
Published: August 2015

Cobra cystatin, a new cysteine-proteinase inhibitor of the cystatin superfamily, was isolated from the venom of the Taiwan cobra (Naja naja atra) by affinity chromatography on S-carboxymethylpapain–Sepharose and reverse-phase chromatography. The venom contained two forms of the inhibitor, one of 11870 Da and the other of 12095 Da, as determined by MS, and pI values of 6.2 and 6.1. Cobra cystatin strongly inhibits cysteine proteinases of the papain family, but not calpain. Papain, cathepsin L, cathepsin B and cathepsin S are inhibited with Ki values of 0.19, 0.1, 2.5 and 1.2 nM respectively. The amino acid sequence of cobra cystatin shows that it is a Type 2 cystatin. The amino acid sequence is 73% identical with that of the cystatin in African-puff-adder (Bitis arietans) venom, with which it shares a unique six-residue insertion in a region opposite the reactive inhibitory site. Cobra cystatin is 25–42% identical with other Type 2 cystatins, the most closely related being the recently described human cystatin M, which also has a similar five-residue insertion starting at position 76 (chicken cystatin numbering). A molecular phylogenetic tree of 16 representative members of Family 2 cystatins was constructed by parsimony analysis; it suggests that snake cystatins, together with Tachypleus tridentatus (Japanese horseshoe crab) cystatin and human cystatin M, form a new subfamily within cystatin Family 2.
Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways
Authors: TUDAN Christopher , JACKSON John K. , CHARLTON Lorin , PELECH Steven L. , SAHL Bill , BURT Helen M. 
Published: August 2015

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70S6K was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70S6K and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70S6K that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70S6K in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70S6K could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70S6K may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils.
Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid
Authors: MIRA-Y-LOPEZ Rafael , JARAMILLO Silvia , JING Yongkui 
Published: August 2015

We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5´-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100–200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300–500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5´-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA–CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA–CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription.
Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer
Authors: LEHTO Marty T. , SHAROM Frances J. 
Published: August 2015

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5´-nucleotidase (5´-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5´-nucleotidase all obeyed Michaelis–Menten kinetics, with a Km for 5´-AMP in the range 11–16 µM. The GPI anchor was removed from essentially all 5´-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5´-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5´-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5´-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5´-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria
Authors: JORCKE Dierk , ZIEGLER Mathias , HERRERO-YRAOLA Andrés , SCHWEIGER Manfred 
Published: August 2015

NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 µM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.
Isolation and characterization of the human aldehyde oxidase gene: conservation of intron/exon boundaries with the xanthine oxidoreductase gene indicates a common origin
Authors: TERAO Mineko , KUROSAKI Mami , DEMONTIS Silvia , ZANOTTA Stefania , GARATTINI Enrico 
Published: August 2015

Aldehyde oxidase (AO) is a molybdo-flavo enzyme involved in the metabolism of various endogenous and exogenous N-heterocyclic compounds of pharmacological and toxicological importance. The enzyme is the product of a gene which is implicated in the aetio-pathogenesis of familial recessive amyotrophic lateral sclerosis. Here, we report the cloning and structural characterization of the human AO gene. AO is a single copy gene approximately 85 kb long with 35 transcribed exons. The transcription-initiation site and the sequence of the 5´-flanking region, containing several putative regulatory elements, were determined. The 5´-flanking region contains a functional promoter, as assessed by appropriate reporter constructs in transient transfection experiments. Comparison of the AO gene structure shows conservation of the position and type of exon/intron junctions relative to those observed in the gene coding for another molybdo-flavoprotein, i.e. xanthine oxidoreductase (XOR). As the two genes code for proteins with a high level of amino acid identity, our results strongly suggest that the AO and XOR genetic loci arose as the consequence of a duplication event. Southern blot analysis conducted on genomic DNA from various animal species with specific cDNA probes indicates that the AO gene is less conserved than the XOR gene during evolution.
Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator
Authors: MACHEROUX Peter , HILL Susan , AUSTIN Sara , EYDMANN Trevor , JONES Tamera , KIM Sung-Oog , POOLE Robert , DIXON Ray 
Published: August 2015

Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly α-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.
Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome
Authors: STEFANELLI Claudio , BONAVITA Francesca , STANIC Ivana , PIGNATTI Carla , FARRUGGIA Giovanna , MASOTTI Lanfranco , GUARNIERI Carlo , CALDARERA Claudio M. 
Published: August 2015

Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.
Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase γ
Authors: SHIOKAWA Daisuke , TANUMA Sei-ichi 
Published: August 2015

An endonuclease named DNase γ has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675–681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase γ. A 1.6 kb mRNA coding for the DNase γ precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase γ mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase γ reveals that full-length DNase γ, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase γ, from which the N-terminal precursor has been removed, has the same properties as purified DNase γ: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase γ cDNA, exogenously introduced DNase γ is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase γ in DNA fragmentation and nuclear structural changes during apoptosis.
Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen
Authors: PASQUET Jean-Max , DACHARY-PRIGENT Jeanne , NURDEN Alan T. 
Published: August 2015

Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1,4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with µ-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an ‘in-gel ’ assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for µ-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.
Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene
Authors: STRACHAN Philip D. , FREER Andrew A. , FEWSON Charles A. 
Published: August 2015

A method was developed for the purification of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1–2 µM) and Vmax (13–19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH 9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH 7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a = 111.9 Å, b = 78.1 Å, c = 134.6 Å, β = 100 °. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1,2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AF043741.
Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.)
Authors: LACEY Dominic J. , WELLNER Nikolaus , BEAUDOIN Frederic , NAPIER Johnathan A. , SHEWRY Peter R. 
Published: August 2015

Oil bodies were isolated from mature seeds of sunflower (Helianthus annuus L.) and safflower (Carthamus tinctorius L.). Oil body preparations containing only oleosin proteins could be obtained from safflower seeds by salt-washing followed by centrifugation on discontinuous sucrose density gradients. However, it was necessary to treat sunflower oil bodies with urea to obtain preparations of similar purity. Incubation of the oil bodies with proteinases gave two fragments with molecular masses of 6 and 8 kDa which were protected from digestion. These fragments represented the hydrophobic domain of the oleosins, as determined by N-terminal sequencing. Intact and proteinase-treated oil bodies of both species were analysed by Fourier-transform infrared spectroscopy, as dry films and in aqueous medium, the spectra being compared with those obtained for pure oil samples in order to identify the bands resulting from the oleosin proteins and protected peptides. This investigation showed that the hydrophobic domain of the oleosins in intact oil bodies is predominantly α-helical in structure and that the conformation was not greatly affected by washing the oil bodies with urea during preparation.
Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes
Authors: CAPEILLÈRE-BLANDIN Chantal , MARTIN Christelle , GAGGERO Nicoletta , PASTA Piero , CARREA Giacomo , COLONNA Stefano 
Published: August 2015

The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 °C. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1–10 s-1 range and around 43±26 µM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (δ+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4–8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3–4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity.