bluuebunny/crossref_metadata_2025 huggingface dataset:
The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin αIIbβ3
Authors: DERRICK Jerry M. , TAYLOR Donald B. , LOUDON Robert G. , GARTNER T. Kent
Published: August 2015

A novel peptide (designed to bind to αIIbβ3) caused platelet aggregation and aggregation-independent secretion of the contents of α-granules in an αIIbβ3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, αIIbβ3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized αIIbβ3. The agonist peptide apparently activates αIIbβ3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding.
Evidence for the existence of a pristanoyl-CoA oxidase gene in man
Authors: VANHOOREN Johannes C. T. , MARYNEN Peter , MANNAERTS Guy P. , VAN VELDHOVEN Paul P.
Published: August 2015

In the rat, 2-methyl branched fatty acids and the bile acid intermediates di- and tri-hydroxycoprostanic acids are desaturated by pristanoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase respectively. In the human, these compounds are oxidized by a single enzyme, branched-chain acyl-CoA oxidase, which according to its amino acid sequence is the human homologue of rat trihydroxycoprostanoyl-CoA oxidase. Pristanoyl-CoA oxidase is apparently absent from human tissues as indicated by immunoblot analysis [Van Veldhoven, Van Rompuy, Fransen, de Béthune and Mannaerts (1994) Eur. J. Biochem. 222, 795–801] and Northern-blot analysis [Vanhooren, Fransen, de Béthune, Baumgart, Baes, Torrekens, Van Leuven, Mannaerts and Van Veldhoven (1996) Eur. J. Biochem. 239, 302–309] of human tissues. In this paper we present evidence, however, that at least the gene for pristanoyl-CoA oxidase is present in the human. A human liver cDNA encoding a protein of 700 amino acids, showing 75% amino acid identity with rat pristanoyl-CoA oxidase and harbouring a peroxisomal C-terminal-targeting signal (SKL), was isolated. Bacterial expression of the cDNA resulted in a fusion protein that was cross-reactive with antibodies directed against rat pristanoyl-CoA oxidase and the C-terminal SKL sequence. Screening of a genomic library with the isolated cDNA as a probe resulted in a genomic clone in which four introns were localized. By means of fluorescence in situhybridization the gene for human pristanoyl-CoA oxidase was mapped at chromosome position 4p15.3. We conclude that a gene for pristanoyl-CoA oxidase is present in the human genome. The gene appears to be expressed to such a low extent in liver that its mRNA cannot be detected by routine Northern-blot analysis and that its product remains undetected by standard immunoblotting or by enzyme activity measurements. We speculate that the gene may be expressed under special (e.g. certain developmental stages) conditions or in certain specialized tissues not examined thus far.
Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster α1B-adrenoceptor by ligands that act as inverse agonists
Authors: LEE Tae Weon , COTECCHIA Susanna , MILLIGAN Graeme
Published: August 2015

The α1-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM α1B-adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as α1-adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type α1B-adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM α1B-adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM α1B-adrenoceptor-expressing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of α1-antagonists were shown to possess the characteristics of inverse agonists of the CAM α1B-adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.
Parallel changes in nuclear and cytosolic calcium in mouse pancreatic β-cells
Authors: BROWN Graham R. , KÖHLER Martin , BERGGREN Per-Olof
Published: August 2015

In the neuroendocrine pancreatic β-cell, elevations in intracellular Ca2+ lead to insulin secretion and the initiation of gene transcription. However, the relationship between cytosolic and nuclear Ca2+ in these cells is unknown. The Ca2+ permeability of the nuclear membrane would therefore determine if Ca2+ could play a direct role in Ca2+-dependent nuclear processes. Using confocal fluorescence microscopy with the ratiometric Ca2+ indicator indo-1 and carefully correcting for compartmentalized indicator, we now demonstrate that there is no difference between the nuclear Ca2+ concentration and the cytosolic Ca2+ concentration ([Ca2+]c) in the resting β-cell. Slow Ca2+ oscillations induced by glucose, fast oscillations induced by glucagon-like peptide-1 and responses to potassium and carbachol all indicate that changes in cytosolic Ca2+ are reflected within the nucleus. We conclude that there are no restrictions on Ca2+ entry into the nucleus of the pancreatic β-cell subsequent to increases in [Ca2+]c. This implies that any signal involved in increasing [Ca2+]c, and thereby insulin release, may also promote nuclear Ca2+-induced gene transcription.
Synthetic histatin analogues with broad-spectrum antimicrobial activity
Authors: HELMERHORST Eva J. , HOF Wim VAN 'T , VEERMAN Enno C. I. , SIMOONS-SMIT Ina , AMERONGEN Arie V. NIEUW
Published: August 2015

Histatins are salivary histidine-rich cationic peptides, ranging from 7 to 38 amino acid residues in length, that exert a potent killing effect in vitro on Candida albicans. Starting from the C-terminal fungicidal domain of histatin 5 (residues 11–24, called dh-5) a number of substitution analogues were chemically synthesized to study the effect of amphipathicity of the peptide in helix conformation on candidacidal activity. Single substitutions in dh-5 at several positions did not have any effect on fungicidal activity. However, multi-site substituted analogues (dhvar1 and dhvar2) exhibited a 6-fold increased activity over dh-5. In addition, dhvar1 and dhvar2 inhibited the growth of the second most common yeast found in clinical isolates, Torulopsis glabrata, of oral- and non-oral pathogens such as Prevotella intermedia and Streptococcus mutans, and of a methicillin-resistant Staphylococcus aureus. In their broad-spectrum activity, dhvar1 and dhvar2 were comparable to magainins (PGLa and magainin 2), antimicrobial peptides of amphibian origin. Both the fungicidal and the haemolytic activities of dhvar1, dhvar2 and magainins increased at decreasing ionic strength.
Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase
Authors: WEBER Jason D. , RABEN Daniel M. , PHILLIPS Polly J. , BALDASSARE Joseph J.
Published: August 2015

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulated mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induced G1 phase progression. ERK1 activation was biphasic and was sustained throughout the G1 phase of the cell cycle. PDGF induced cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) was correlated with growth arrest. In contrast, growth was unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, resulted in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1–cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreased ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression was observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also resulted in G1 growth arrest. These data provide evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression.
Immortalization and characterization of a cell line exhibiting a severe multiple sulphatase deficiency phenotype
Authors: NELSON Kathy , BIELICKI Julie , ANSON Donald S.
Published: August 2015

Multiple sulphatase deficiency (MSD) is a rare genetic defect that causes a simultaneous deficiency of all known sulphatases. All available evidence suggests that the deficient gene product is normally responsible for the post-translational modification of a conserved cysteine residue to 2-amino-3-oxopropionic acid and that this modification is necessary for sulphatase activity. MSD often has an enzymically mild phenotype, with significant levels of residual sulphatase activity being detectable. Here we identify an MSD cell line in which the residual activity of the sulphatases assayed was generally very low. To characterize the phenotype of this cell line further, immortalized lines were established after transformation with simian virus 40 (SV40) T antigen. Immortalized cell lines representing normal and MSD phenotypes were then transduced with a retroviral vector carrying the gene encoding human N-acetylgalactosamine-4-sulphatase. Analysis of N-acetylgalactosamine-4-sulphatase protein synthesis and enzyme activity showed that transduced cell lines expressed large amounts of enzyme and that the specific activity of this enzyme was approx. 0.5–1.5% of normal, confirming that this cell line defines a severe phenotype for MSD. N-Acetylgalactosamine-4-sulphatase purified from a transduced MSD cell line seemed normal on denaturing PAGE. Kinetic analysis of the purified enzyme suggests that the residual activity is due to small amounts of normal enzyme rather than unmodified enzyme with low levels of residual activity. These cell lines and the availability of large amounts of inactive N-acetylgalactosamine-4-sulphatase from MSD cells should facilitate the further study of this disorder.
Interaction of nitric oxide with non-haem iron sites of Escherichia coli bacterioferritin: reduction of nitric oxide to nitrous oxide and oxidation of iron(II) to iron(III)
Authors: LE BRUN Nick E. , ANDREWS Simon C. , MOORE Geoffrey R. , THOMSON Andrew J.
Published: August 2015

The bacterioferritin (BFR) of Escherichia coliconsists of 24 identical subunits, each containing a dinuclear metal-binding site consisting of two histidines and four carboxylic acid residues. Earlier studies showed that the characterization of iron binding to BFR could be aided by EPR analysis of iron–nitrosyl species resulting from the addition of NO to the protein [Le Brun, Cheesman, Andrews, Harrison, Guest, Moore and Thomson (1993) FEBS Lett. 323, 261–266]. We now report data from gas chromatographic head space analysis combined with EPR spectroscopy to show that NO is not an inert probe: iron(II)–BFR catalyses the reduction of NO to N2O, resulting in oxidation of iron(II) at the dinuclear centre and the subsequent detection of mononuclear iron(III). In the presence of excess reductant (sodium ascorbate), iron(II)–BFR also catalyses the reduction of NO to N2O, giving rise to three mononuclear iron–nitrosyl species which are detectable by EPR. One of these, a dinitrosyl–iron complex of S = ½, present at a maximum of one per subunit, is shown by EPR studies of site-directed variants of BFR not to be located at the dinuclear centre. This is consistent with a proposal that the diferric form of the centre is unstable and breaks down to form mononuclear iron species.
A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins
Authors: BRITTAIN Thomas , HOFMANN Oliver M. , WATMOUGH Nicholas J. , GREENWOOD Colin , WEBER Roy E.
Published: August 2015

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the α2ϵ2 and ζ2ϵ2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.
Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes
Authors: KATAEVA Irina , GUGLIELMI Gérard , BÉGUIN Pierre
Published: August 2015

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD–CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.
Hydrolysis of somatostatin by human tissue kallikrein after the amino acid pair Phe-Phe
Authors: PIMENTA Daniel C. , JULIANO Maria A. , JULIANO Luiz
Published: August 2015

Somatostatin-(1–14) was hydrolysed by human tissue kallikrein at the Phe7-Trp8 bond, after a Phe-Phe pair of amino acids, with similar kinetic parameters to those described for human high- and low-molecular-mass kininogens. Substance P and human insulin, which also contain a Phe-Phe pair in their sequences, were both resistant. More details of this hydrolytic specificity of human tissue kallikrein were obtained by synthesizing and assaying internally quenched fluorescent peptides containing the sequence of somatostatin-(1–14), as well as the reactive-centre loop of human kallikrein-binding protein (kallistatin). We also observed that human tissue kallikrein hydrolysed growth hormone-releasing hormone at the Arg11-Lys12 bond, although this peptide contains in its structure a pair of leucines (Leu22-Leu23), in contrast with the Phe-Phe pair in somatostatin. We have also demonstrated the susceptibility to human tissue kallikrein of some chromogenic peptides with the general structure X-Phe-Phe-p-nitroanilide and of d-Pro-Phe-Phe-4-methylcoumaryl-7-amide.
Common-type acylphosphatase: steady-state kinetics and leaving-group dependence
Authors: PAOLI Paolo , CIRRI Paolo , CAMICI Lucia , MANAO Giampaolo , CAPPUGI Gianni , MONETI Gloriano , PIERACCINI Giuseppe , CAMICI Guido , RAMPONI Giampietro
Published: August 2015

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 °C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme–substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O–inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. All these findings, as well as the strong dependence of kcat upon the leaving group pKa, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.
Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro
Authors: LEGGETT S. David , CANDIDO M. E. Peter
Published: August 2015

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.
Aging of di-isopropyl-phosphorylated human butyrylcholinesterase
Authors: MASSON Patrick , FORTIER Pierre-Louis , ALBARET Christine , FROMENT Marie-Thérèse , BARTELS F. Cynthia , LOCKRIDGE Oksana
Published: August 2015

Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed ‘aging’, involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 °C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (). The was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased 60-fold. Mutation W82A increased 13-fold. Mutation D70G increased 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the ω loop (Cys-65-Cys-92) in the mutant enzyme.
Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3
Authors: RIETVELD E. G. Luc , HOLTHUIZEN P. Elly , SUSSENBACH S. John
Published: August 2015

Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3.
Allosteric communication in mammalian muscle aldolase
Authors: SYGUSCH Jurgen , BEAUDRY Danielle
Published: August 2015

Mixed disulphide formation in the presence of oxidized glutathione reversibly inactivates rabbit skeletal muscle aldolase. Inactivation is allosteric, preferentially modifying Cys-72 on the surface of the aldolase homotetramer distant from active-site locations and subunit interfaces. Ion-exchange chromatography fractionates partly inactivated aldolase into three distinct enzymic species: unmodified enzyme, inactive fully modified enzyme corresponding to one thiol reacted per subunit, and inactive singly modified enzyme in which only one thiol has reacted. Acid-precipitable enzymic intermediates formed in the presence of substrate, D-fructose 1,6-bisphosphate, and product, dihydroxyacetone phosphate, indicates that active site binding is unaffected upon modification. The absence of enamine carbanion formation in the presence of substrate but not product is consistent with mixed disulphide formation's blocking -C-C- cleavage and/or subsequent D-glyceraldehyde 3-phosphate release. Inactivation upon single subunit modification and substrate protection against modification denotes that the blocked step is associated with a long-range conformational transition involving highly co-operative subunit behaviour.
Intracellular coupling of bikunin and the heavy chain of rat pre-α-inhibitor in COS-1 cells
Authors: BLOM M. Anna , THUVESON Maria , FRIES Erik
Published: August 2015

Pre-α-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-α-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.
Characterization of homocysteine metabolism in the rat kidney
Authors: HOUSE D. James , BROSNAN E. Margaret , BROSNAN T. John
Published: August 2015

Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine β-synthase and cystathionine γ-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine β-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine γ-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.
Rapid induction of apoptosis by deregulated uptake of polyamine analogues
Authors: HU Rei-Huang , PEGG E. Anthony
Published: August 2015

Treatment of Chinese hamster ovary cells with α-difluoromethylornithine for 3 days, followed by exposure to cycloheximide, led to an unregulated, rapid and massive accumulation of polyamine analogues. This accumulation led to cell death by apoptosis within a few hours. Clear evidence of DNA fragmentation was seen in response to both N-terminally ethylated polyamines and to polyamines containing methyl groups on the terminal carbon atoms. Programmed cell death was induced within 2-4 h of exposure to 1 μM or higher concentrations of N1,N11-bis(ethyl)norspermine. The presence of cycloheximide increased the uptake of the polyamine analogues and therefore led to cell death at lower analogue concentrations, but it was not essential for the induction of apoptosis, since similar effects were seen when the protein synthesis inhibitor was omitted and the concentration of N1,N11-bis(ethyl)norspermine was increased to 5 μM or more The induction of apoptosis was blocked both by the addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or by the addition of the polyamine oxidase inhibitor N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine (MDL 72,527). These experiments provide evidence to support the concepts that: (1) polyamines or their oxidation products may be initiators of programmed cell death; (2) regulation of polyamine biosynthesis and uptake prevents the accumulation of toxic levels of polyamines; and (3) the anti-neoplastic effects of bis(ethyl) polyamine analogues may be due to the induction of apoptosis in sensitive tumour cells.
Bradykinin stimulates cAMP synthesis via mitogen-activated protein kinase-dependent regulation of cytosolic phospholipase A2 and prostaglandin E2 release in airway smooth muscle
Authors: PYNE J. Nigel , TOLAN Deborah , PYNE Susan
Published: August 2015

Bradykinin stimulates cAMP synthesis in cultured airway smooth muscle (ASM) cells. This occurs via a pathway that involves: (1) the protein kinase C (PKC)-dependent activation of mitogen-activated protein kinase (MAPK); (2) the MAPK-dependent phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) and (3) the utilization of cPLA2-derived arachidonate by the cyclo-oxygenase pathway to produce prostaglandin E2 (PGE2). PGE2 is released and binds to cell surface receptors to stimulate intracellular cAMP synthesis. The signalling pathway was confirmed by the use of PD098059 [the inhibitor of MAPK kinase-1 (MEK-1) activation], AACOCF3 (an inhibitor of cPLA2) and indomethacin (an inhibitor of cyclo-oxygenase), which all reduced bradykinin-stimulated cAMP synthesis. Bradykinin also elicits the inhibition of approx. 60% of the total cAMP phosphodiesterase activity in the cell [Stevens, Pyne, Grady and Pyne (1994) Biochem. J. 297, 233-239]. This is likely to decrease the rate of cAMP degradation markedly and therefore to potentiate PGE2-stimulated cAMP synthesis. Acute treatment of ASM cells with PMA (a direct activator of PKC) also stimulated the MAPK-dependent phosphorylation of cPLA2. However, in contrast with bradykinin, PMA did not stimulate arachidonate release, suggesting that additional signals (e.g. Ca2+ ions) are required for phosphorylation by MAPK to activate cPLA2. PMA was also without effect on PGE2 release and cAMP synthesis. Evidence that PKC can also directly regulate adenylate cyclase was obtained by using cells pretreated with cholera toxin. Under these conditions, PMA stimulated cAMP synthesis independently of arachidonate metabolites. Furthermore the combined treatment of cells with PMA (to activate PKC) and PGE2 (to activate Gs) stimulated synergistic cAMP synthesis. This might be due to the presence of the type 2 adenylate cyclase, which is synergistically activated by Gs and PKC.
Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation
Authors: PACKHAM Graham , BRIMMELL Matthew , CLEVELAND L. John
Published: August 2015

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5ʹ sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Suprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.
Covalent complex of microperoxidase with a 21-residue synthetic peptide as a maquette for low-molecular-mass redox proteins
Authors: IPPOLITI Rodolfo , PICCIAU Alessandra , SANTUCCI Roberto , ANTONINI Giovanni , BRUNORI Maurizio , RANGHINO Graziella
Published: August 2015

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an ‘open’ and a ‘closed’ structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron (‘open’ conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH (‘closed’ conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.
Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe
Authors: FELSCHOW M. Donna , MI Zenghui , STANEK J. , FREI J. , PORTER W. Carl
Published: August 2015

Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 μM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the identified pbps may be associated with that function.
Temperature-sensitive mRNA degradation is an early event in hepatocyte de-differentiation
Authors: WANG Xiu-Jun , HODGKINSON P. Conrad , WRIGHT C. Matthew , PAINE J. Alan
Published: August 2015

The isolation and culture of metabolically active hepatocytes by proteolytic digestion of the extracellular matrix of the liver results in the transcriptional silencing of liver-specific genes encoding cytochromes P-450 (CYP) and albumin together with an induction of cellular RNase activity. The levels of albumin mRNA are maintained in cultured hepatocytes at similar levels to that present in the intact liver for at least 24 h, whereas the major constitutively expressed CYP2C11 mRNA is rapidly degraded. Hepatocytes heat-shocked at 40 °C during the isolation procedure (which results in an induction of heat-shock protein mRNA species) blocks the increase in RNase activity and abrogates the loss of CYP2C11 mRNA for at least 4 h. Cycloheximide-dependent inhibition of protein synthesis blocks the temperature-dependent induction of heat-shock proteins without affecting the protection afforded to CYP2C11 mRNA, indicating that CYP2C11 mRNA levels are not directly dependent on heat-shock protein induction and suggesting that the induction of RNase activity might be responsible for the specific loss of CYP2C11 mRNA in hepatocytes isolated at 37 °C. Differential rates of degradation of CYP2C11 transcribed in vitro and of albumin mRNA are observed in the presence of cellular extracts from cultured hepatocytes isolated at 37 °C (which have maximally induced levels of cellular RNase activity) but not in comparable extracts from cultured hepatocytes isolated at 40 °C, supporting the hypothesis that an RNase activity is induced in culture that specifically degrades CYP2C11 mRNA but not albumin mRNA. These results suggest that an early event in hepatocyte de-differentiation involves the induction of RNase activity in addition to transcriptional silencing of liver-specific genes and that the induced RNase activity demonstrates specificity within liver-specific gene products.
Regulation of the serine-base exchange enzyme system by CD4: effects of monoclonal antibodies, jacalin, interleukin 16 and the HIV membrane protein gp120
Authors: DUMAURIER Marie-Jeanne , PELASSY Claudette , BREITTMAYER Jean-Philippe , AUSSEL Claude
Published: August 2015

Phosphatidylserine (PtdSer) is synthesized by an exchange of the polar head group of phospholipids for a serine residue. The enzyme responsible for this reaction, the serine-base exchange enzyme system (serine-BEES) is inhibited during lymphocyte activation. We show here that triggering the CD4 cell surface molecule in several CD4+ T-cell lines regulates the serine-BEES activity, thus resulting in marked changes in PtdSer synthesis. CD4 ligands able to generate an activating signal in T-cells such as the lectin jacalin, down-regulate the synthesis of PtdSer. In contrast, monoclonal antibodies (mAbs) directed against the CD4 molecule, such as IOT4 and IOT4a, which have previously been described as generating an inhibitory signal to T-cells, induced an up-regulation of the serine-BEES and impaired CD3-induced inhibition of PtdSer synthesis. Similarly, the HIV-gp120 envelope glycoprotein, in both soluble and cross-linked forms, induces an increase in PtdSer synthesis. The protein tyrosine kinase p56lck participates in the regulation of serine-BEES activity because the effect of CD4 mAbs was additive to that of amino-hydroxyflavone, an inhibitor of p56lck. Also, CD4 mAbs were inactive in J Cam 1.6 cells or when the CD3 signals were bypassed by using thapsigargin. These results demonstrate that the CD4 surface molecule can transmit both activating and inhibiting intracellular signals depending on the CD4 ligand used. We suggest that PtdSer synthesis would be one of the intracellular signals that could explain the opposite effects of different CD4 ligands on T-cells.
Kinetic and chemical mechanisms for the effects of univalent cations on the spectral properties of aromatic amine dehydrogenase
Authors: ZHU Zhenyu , DAVIDSON L. Victor
Published: August 2015

Univalent cations and pH influence the UV-visible absorption spectrum of the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH). Little spectral perturbation was observed when pH was varied in the absence of univalent cations. The addition of alkali metal univalent cations (K+, Na+, Li+, Rb+ or Cs+) to oxidized AADH caused significant changes in its absorption spectrum. The apparent Kd for each cation, determined from titrations of the spectral perturbation, decreased with increasing pH. Transient kinetic studies involving rapid mixing of AADH with cations and pH jump revealed that the rate of the cation-induced spectral changes initially decreased with increasing cation concentration to a minimum value, then increased with increasing cation concentration. A kinetic model was developed to fit these data, determine the true pH-independent Kd values for K+ and Na+, and explain the pH-dependence of the apparent Kd. A chemical reaction mechanism, based on the kinetic data, is presented in which the metallic univalent cation facilitates the chemical modification of the TTQ prosthetic group to form an hydroxide adduct which gives rise to the spectral change. Addition of NH4+/NH3 to AADH caused changes in the absorption spectrum which were very different form those caused by addition of the metallic univalent cations. The kinetics of the reaction induced by addition of NH4+/NH3 were also different, being simple saturation kinetics. Another reaction mechanism is proposed for the NH4+/NH3-induced spectral change that involves nucleophilic addition of the unprotonated NH3 to TTQ. The general relevance of these data and models to the physiological reactions of TTQ-dependent enzymes and to the roles of univalent cations in modulating enzyme activity are discussed.
Effect of long-term treatment of 3T3-L1 adipocytes with chlorate on the synthesis, glycosylation, intracellular transport and secretion of lipoprotein lipase
Authors: MASUNO Hiroshi , SAKAYAMA Kenshi , OKUDA Hiromichi
Published: August 2015

Lipoprotein lipase (LPL) is synthesized and glycosylated in the endoplasmic reticulum (ER), transported through the Golgi to the cell surface, and finally secreted. To examine the role of heparan sulphate proteoglycans (HSPG) in the synthesis, activity, intracellular transport and secretion of LPL, 3T3-L1 adipocytes were cultured for 7 days in the presence of 20 mM chlorate, an inhibitor of sulphation of HSPG. Treatment of cells with 20 mM chlorate for 7 days caused a 55% decrease in LPL activity in the intracellular compartment and a 79% decrease in the cell-surface compartment. The synthetic rate of LPL in chlorate-treated cells was identical with that in control cells as determined by biosynthetic labelling. The study with endoglycosidase H (endo H) showed that the treatment with chlorate increased the proportion of LPL subunits which were totally endo H-sensitive. The study with a heparin-Sepharose column showed that 3T3-L1 adipocytes contained three forms of LPL. The first form, accounting for 35% of the LPL, did not bind to the heparin-Sepharose column and had little or no activity; the second form, accounting for 32%, bound to the column and was eluted with 0.4-0.75 M NaCl but had no activity; the third form, accounting for 33%, bound to the column and was eluted with 0.8-1.2 M NaCl and had activity. In chlorate-treated cells, the first form accounted for 66% of the LPL, the second form 15% and the third form 19%. When cells were incubated for 1 h with brefeldin A, which translocates Golgi proteins to the ER [J. Lippincott-Schwartz, L. C. Yuan, J. S. Banifacino and R. D. Klausner (1989) Cell 56, 801-813; J. Lippincott-Schwartz, J. Glickman, J. E. Donaldson, J. Robbins, T. E. Kreis, K. B. Seamon, M. P. Sheetz and R. D. Klausner (1991) J. Cell Biol. 112, 567-577], the chlorate-induced decrease in cellular LPL activity was restored. These findings indicate that LPL synthesized in chlorate-treated cells can be processed to be fully active, but chlorate-treated cells are unable to transport LPL to the Golgi and accumulate inactive LPL with a lower affinity for heparin in the ER. The treatment with chlorate decreased the proportion of LPL subunits that were endo H-resistant, indicating that the processing of oligosaccharide chains of LPL in the trans-Golgi was impaired in chlorate-treated cells. The amount of 35S-labelled LPL secreted by chlorate-treated cells was identical with that secreted by control cells, whereas the level of LPL activity in the medium of chlorate-treated cells was 25% of that in the medium of control cells, indicating that most of the LPL secreted by chlorate-treated cells was inactive.