bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of glucokinase translocation in the mechanism by which resorcinol inhibits glycolysis in hepatocytes
Authors: AGIUS Loranne
Published: August 2015

Proglycosyn and resorcinol stimulate glycogen synthesis and inhibit glycolysis in hepatocytes. The former effect is attributed to inactivation of phosphorylase mediated by glucuronidated metabolites. This study investigated the mechanism by which resorcinol inhibits glycolysis. Resorcinol (150 μM) inhibited glycolysis in hepatocytes incubated with glucose (15–35 mM) but not with dihydroxyacetone (10 mM). The inhibition of glycolysis at elevated glucose concentration was associated with inhibition of glucose-induced dissociation of glucokinase and aldolase. The resorcinol concentration that caused half-maximal inhibition (20–43 μM) increased with increasing glucose concentration (15–35 mM). Resorcinol inhibited the translocation of glucokinase and the stimulation of detritiation of [2-3H]glucose and [3-3H]glucose caused by sorbitol (10–200 μM), but it potentiated the stimulation of glycogen synthesis. The inhibition of glycolysis by resorcinol could not be accounted for by diversion of substrate to glycogen. The glucose 6-phosphate content correlated with the free glucokinase activity. Resorcinol counteracted the increase in glucose 6-phosphate and fructose 2,6-bisphosphate caused by elevated glucose concentration or by sorbitol. The suppression of glucose 6-phosphate at high glucose concentration (15–35 mM) could be explained by the low activity of free glucokinase. However, the suppression at 5 mM glucose was due in part to an independent mechanism. The effect of resorcinol on glucokinase translocation was partly counteracted by galactosamine, which suppresses UDP-glucose and inhibits glucuronide formation, and was mimicked by phenol and p-nitrophenol but not by p-nitrophenylglucuronide. It is concluded that resorcinol inhibits glycolysis at elevated glucose concentration or when stimulated by sorbitol through increased glucokinase binding. The results indicate a link between glucuronidation and glucokinase translocation.
The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox
Authors: HENDERSON Lydia M. , THOMAS Stephen , BANTING George , CHAPPELL J. Brian
Published: August 2015

The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H+ ions through a H+ channel. gp91-phox, a protein component of the oxidase, has been shown previously to function as a H+ channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270, 5909–5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91-phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H+ fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H+ fluxes were indistinguishable from those observed in cells expressing full-length gp91-phox. Therefore the N-terminal 230 amino acids of gp91-phox contain all that is required to function as the NADPH oxidase-associated H+ channel.
Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28
Authors: PARRY Richard V. , OLIVE Daniel , WESTWICK John , SANSOM David M. , WARD Stephen G.
Published: August 2015

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite the lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327–329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.
From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase
Authors: JESPERSEN Hans M. , KJÆRSGÅRD Inger V. H. , ØSTERGAARD Lars , WELINDER Karen G.
Published: August 2015

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thalianaascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.
Induction of fatty acid synthase and S14 gene expression by glucose, xylitol and dihydroxyacetone in cultured rat hepatocytes is closely correlated with glucose 6-phosphate concentrations
Authors: MOURRIERAS Florence , FOUFELLE Fabienne , FORETZ Marc , MORIN Joëlle , BOUCHE Sandrine , FERRE Pascal
Published: August 2015

It is now well established that the transcription of several genes belonging to the glycolytic and lipogenic pathway is stimulated in the presence of a high glucose concentration in adipocytes and hepatocytes. We have previously proposed that glucose 6-phosphate could be the signal metabolite that transduces the glucose effect. This proposal has recently been challenged and both an intermediate of the pentose phosphate pathway, xylulose 5-phosphate, and metabolites of the later part of glycolysis (3-phosphoglycerate and phosphoenolpyruvate) have been proposed. To discriminate between these possibilities, we have measured concomitantly, in primary cultures of adult rat hepatocytes, the expression of the fatty acid synthase (FAS) and S14 genes and the concentration of glucose metabolites. We have used various substrates entering at different steps of the glycolytic pathway (glucose, dihydroxyacetone) and the pentose phosphate pathway (xylitol). When compared with 5 mM glucose, 25 mM glucose induces a marked increase in both S14 and FAS gene expression, detectable as early as 2 h and peaking at 6 h. Increasing concentrations (1–5 mM) of xylitol and dihydroxyacetone in the presence of 5 mM glucose are also able to induce S14 and FAS gene expression progressively. Among the various glucose metabolites measured, glucose 6-phosphate, in contrast with xylulose 5-phosphate and metabolites of the lower part of glycolysis, is the only one that shows a clear-cut parallelism between its concentration and the degree of S14 and FAS gene expression. We conclude that glucose 6-phosphate is the most likely signal metabolite for the glucose-induced transcription of this group of genes.
Chicken vigilin gene: a distinctive pattern of hypersensitive sites is characteristic for its transcriptional activity
Authors: GRÜNWELLER Arnold , PURSCHKE Werner G. , KÜGLER Sebastian , KRUSE Charli , MÜLLER Peter K.
Published: August 2015

Vigilin, a multidomain hn-ribonucleo-K-homologous protein, is part of a ribonucleoprotein complex with cognate tRNA and is found in both the nucleus and the cytoplasm. In an approach to identify genomic regions involved in regulation of the chicken vigilin gene, we carried out transfection studies with a reporter gene in suitable chicken cells. After including a distantly positioned 5′-sequence in the construct, we observed a 10.5-fold increase in luciferase (EC 1.13.12.7) expression compared with basal promoter activity. Accordingly, chromatin analysis of freshly isolated embryonic tendon fibroblasts with high levels of vigilin mRNA expression shows a DNase-I-hypersensitive site (DHS1) localized 2.2 kb upstream of the transcriptional start site. Similarly, phytohaemagglutinin-stimulated lymphocytes with a 4-fold elevated expression of vigilin mRNA compared with resting lymphocytes also exhibited this unique DHS, having switched from that found at 3.3 kb (DHS2) in resting lymphocytes. Furthermore, using gel-retardation experiments with DNA representing either DHS1 or DHS2, a specific interaction with chicken nuclear extracts was seen.
Interaction of platelet-derived growth factor with thrombospondin 1
Authors: HOGG Philip J. , HOTCHKISS Kylie A. , JIMÉNEZ Barbara M. , STATHAKIS Paul , CHESTERMAN Colin N.
Published: August 2015

Key factors that mediate vascular smooth muscle cell proliferation and migration are platelet-derived growth factor (PDGF) and thrombospondin 1 (TSP1). We now report that PDGFBB bound tightly and specifically to TSP1, that this interaction was markedly dependent on the disulphide bond arrangement in TSP1, and that binding of PDGFBB to TSP1 did not preclude PDGFBB from binding to its receptor on rat aortic vascular smooth-muscle cells. At physiological ionic strength and pH, PDGFBB bound to Ca2+-depleted TSP1 with a dissociation constant of 11±2 nM and to Ca2+-replete TSP1 with a dissociation constant of 32±5 nM. Binding was specific, as both soluble TSP1 and unlabelled PDGFBB competed for binding of iodinated PDGFBB to immobilized TSP1, whereas other platelet α-granule proteins did not compete. The tertiary structure of TSP1 is regulated by intramolecular disulphide interchange; we found that catalysis of disulphide interchange in TSP1 by protein disulphide isomerase ablated the binding of PDGFBB. The interaction of PDGFBB with TSP1 was weakened by increasing salt concentration and essentially ablated at 0.65 ionic strength; it was inhibited by heparin with a half-maximal effect at 20 i.u./ml, implying that the binding was mediated largely by ionic interactions. An anti TSP1 monoclonal antibody decreased the binding of iodinated PDGFBB to PDGF receptor on rat aortic vascular smooth-muscle cells by 37±2%, whereas platelet TSP1 non-competitively inhibited binding of iodinated PDGFBB. Uncomplexed PDGFBB bound to PDGF receptor with an affinity 5±2 times that of PDGFBB–TSP1 complexes. These results suggest that TSP1 might assist in the targeting of PDGF to its receptor on vascular smooth-muscle cells.
Role of ATP and Pi in the mechanism of insulin secretion in the mouse insulinoma βTC3 cell line
Authors: PAPAS Klearchos K. , LONG Robert C. , CONSTANTINIDIS Ioannis , SAMBANIS Athanassios
Published: August 2015

Understanding the biochemical events associated with glucose-stimulated insulin secretion by pancreatic β cells is of importance in gaining insight into both the pathophysiology of diabetes and the development of tissue-engineered bioartificial pancreatic substitutes. We have investigated the effects of glucose concentration on the bioenergetic status and on the metabolic and secretory functions exhibited by mouse insulinoma βTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cells entrapped in APA beads constitute a possible implantable bioartificial pancreas for the long-term treatment of insulin-dependent diabetes mellitus. Our results show that, in entrapped βTC3 cells, the oxygen consumption rate and the intracellular nucleotide triphosphate levels are unaffected by a step change in glucose concentration from 16 mM to 0 mM for 4.5 h and then back to 16 mM. The intracellular Pi level and the ammonia production rate were doubled, while insulin secretion was decreased 10-fold, upon switching from 16 mM to 0 mM glucose. The implications of these findings in the context of pancreatic β cell biochemistry and the mechanism of the ‘Fuel Hypothesis’ are discussed.
The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria: electrical nature and coupling of the exchange reaction with H+ translocation
Authors: INDIVERI Cesare , TONAZZI Annamaria , STIPANI Italo , PALMIERI Ferdinando
Published: August 2015

The mechanism and the electrical nature of ornithine/citrulline exchange has been investigated in proteoliposomes reconstituted with the ornithine/citrulline carrier purified from rat liver mitochondria. The stoichiometry of the exchanging substrates was close to 1:1. The exchange was not affected by inducing electrogenic flux of K+ with valinomycin. In contrast, the pH gradient generated by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient stimulated the ornithineout/citrullinein exchange, but not the ornithine/ornithine homoexchange. Experiments in which either the internal or the external pH was varied, while keeping constant the pH in the other compartment, indicated that maximal exchange rates are found at pH 6 in the compartment containing citrulline and at pH 8 in the compartment containing ornithine. Changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the exchanges ornithineout/citrullinein and citrullineout/ornithinein are accompanied by translocation of H+ in the same direction as citrulline. It is concluded that the mitochondrial ornithine/citrulline carrier catalyses an electroneutral exchange of ornithine+ for citrulline plus an H+. A reasonable model is one in which ornithine binds to a deprotonated carrier and citrulline to a protonated carrier and both substrate-carrier complexes are neutral. The physiological implications of this transport process are discussed.
Carboxymethylation of nuclear protein serine/threonine phosphatase X
Authors: KLOEKER Susanne , BRYANT C. Jeffrey , STRACK Stefan , COLBRAN J. Roger , WADZINSKI E. Brian
Published: August 2015

Specific rabbit polyclonal antibodies against peptides corresponding to the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to investigate the cellular and subcellular distribution of PP2A/C and PPX/C, as well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly high levels of PP2A/C and PPX/C in brain and testes respectively. In addition, immunoblots of subcellular fractions and immunocytochemical analyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treatment of nuclear extracts with alkali resulted in increased PPX/C immunoreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody against an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelectric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitated from COS cell extracts incubated with the methyl donor S-adenosyl-l-[methyl-3H]methionine. Thus the increase in immunoreactivity probably results from removal of a carboxymethyl group from PPX/C, as has been shown previously for PP2A/C [Favre, Zolnierowicz, Turowski and Hemmings (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indicate that the PPX catalytic subunit is a predominantly nuclear phosphatase and is methylated at its C-terminus.
Calnexin and calreticulin bind to enzymically active tissue-type plasminogen activator during biosynthesis and are not required for folding to the native conformation
Authors: ALLEN Simon , BULLEID J. Neil
Published: August 2015

The roles of the endoplasmic-reticulum lectins calnexin and calreticulin in the folding of tissue-type plasminogen activator (tPA) have been investigated using an in vitro translation system that reconstitutes these processes as they would occur in the intact cell. Using co-immunoprecipitation of newly synthesized tPA with antibodies to calnexin and calreticulin, it was demonstrated that the interaction of tPA with both lectins was dependent upon tPA glycosylation and glucosidase trimming. When tPA was synthesized in the presence of semi-permeabilized cells under conditions preventing complex formation with calnexin and calreticulin, the translation product had a specific plasminogenolytic activity identical with that when synthesized under conditions permitting interactions with both lectins. Furthermore, complexes of tPA bound to calnexin and calreticulin were shown to be enzymically active. These results demonstrate that calnexin and calreticulin can form a stable interaction with correctly folded tPA; however, such interactions are not required for the synthesis of enzymically active tPA.
Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris
Authors: MOREL Nathalie , MASSOULIÉ Jean
Published: August 2015

In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETΔ, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEΔ, reduced to the catalytic domain). We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast. The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P. pastoris can correctly process a mammalian C-terminal GPI-addition signal. Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity. In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide. We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide. Surprisingly, the highest yield was obtained with the endogenous signal peptide. For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides. The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P. pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.
Role of G-protein βγ subunits in the augmentation of P2Y2 (P2U) receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors
Authors: SELBIE A. Lisa , KING V. Natalie , DICKENSON M. John , HILL J. Stephen
Published: August 2015

Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (αβγ) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) α1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of Gβγ subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the β-adrenergic receptor kinase (βARK1 residues 495-689) and the transducin α subunit to associate with G-protein βγ subunit dimers and to act as Gβγ subunit scavengers. Transient expression of βARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both Gβγ subunit scavengers. Expression of βARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that Gβγ subunits, as inhibited by two different Gβγ scavengers, significantly contribute to the synergistic interaction between NPY Y1 Gi/o- and Gq/11-coupled receptor activity, and are required for the augmentation of IP production and AA release observed in this model cell system.
Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells
Authors: SHARMA K. Deepak , SMITH K. Terry , CROSSMAN Arthur , BRIMACOMBE S. John , FERGUSON A. J. Michael
Published: August 2015

De-N-acetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is the second step of glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent mannosylation of glucosaminyl-phosphatidylinositol (GlcN-PI) which leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. The substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells were studied with respect to the N-acyl groups (R) that could be removed from a series of GlcNR-PI substrates, where R = acetyl (Ac), propionyl (Pr), butyryl (Bu), isobutyryl (iBu), pentanoyl (Pen) or hexanoyl (Hex). The data show that the trypanosomal and HeLa enzymes had similar specificities and that the turnover of GlcNR-PIs by the trypanosomal enzyme was in the order GlcNAc-PI > GlcNPr-PI GlcNBu - PI ≈ GlcNiBu - PI ≈ GlcNPen - PI GlcNHex - PI. The trypanosome and HeLa de-N-acetylases were unable to de-N-acetylate mannosylated GlcNAc-PI intermediates, which explains why de-N-acetylation must precede mannosylation in the GPI biosynthetic pathway.
Molecular cloning, sequencing and functional study of the promoter region of the human α2C4-adrenergic receptor gene
Authors: SCHAAK Stéphane , DEVEDJIAN Jean-Christophe , CAYLA Cécile , SENDER Yolande , PARIS Hervé
Published: August 2015

Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5ʹ-flanking region, the open reading frame and 4 kb of the 3ʹ-flanking region of the α2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5ʹ to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5ʹ- and 3ʹ-ends of the α2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of α2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the α2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5ʹ-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5ʹ-non-coding region appeared to depend on the host cell. Removal of the 5ʹ-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5ʹ-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.
Mechanisms of mitogen-activated protein kinase activation by nicotine in small-cell lung carcinoma cells
Authors: CATTANEO G. Maria , D'ATRI Fabio , VICENTINI M. Lucia
Published: August 2015

We have previously reported that nicotine stimulates cell proliferation of three small-cell lung carcinoma (SCLC) cell lines by activating nicotinic receptors of the neuronal type. Here we report that, in the GLC-8 SCLC cell line, nicotine stimulates mitogen-activated protein (MAP) kinase activity in a concentration- and time-dependent manner (ED50 = 10 nM). The nicotine effect was antagonized by mecamylamine, an antagonist specific for neuronal nicotinic receptors. The absence of extracellular Ca2+, or pretreatment with pertussis toxin or the tyrosine kinase inhibitor genistein inhibited the action of nicotine on MAP kinase. Moreover, supernatants from nicotine-stimulated cells transferred to cells pretreated with mecamylamine were still capable of activating MAP kinase. On the other hand, the same supernatants transferred to cells pretreated with mecamylamine and pertussis toxin or genistein failed to activate MAP kinase. These findings suggest that nicotine elicits its stimulatory effect on MAP kinase in SCLC cells indirectly by inducing the production and/or release of a factor which then acts via a pertussis toxin- and tyrosine kinase-sensitive route.
Zeta, a novel class of glutathione transferases in a range of species from plants to humans
Authors: BOARD G. Philip , BAKER T. Rohan , CHELVANAYAGAM Gareth , JERMIIN S. Lars
Published: August 2015

Sequence alignment and phylogenetic analysis has identified a new subgroup of glutathione S-transferase (GST)-like proteins from a range of species extending from plants to humans. This group has been termed the Zeta class. An atomic model of the N-terminal domain suggests that the members of the Zeta class have a similar structure to that of other GSTs, binding glutathione in a similar orientation in the G site. Recombinant human GSTZ1-1 has been expressed in Escherichia coli and characterized. The protein is a dimer composed of 24.2 kDa subunits and has minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Although low in comparison with other GSTs, GSTZ1-1 has glutathione peroxidase activity with t-butyl and cumene hydroperoxides. The members of the Zeta class have been conserved over a long evolutionary period, suggesting that they might have a role in the metabolism of a compound that is common in many living cells.
Binding of urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex to the endocytosis receptors α2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor
Authors: RODENBURG W. Kees , KJØLLER Lars , PETERSEN H. Helle , ANDREASEN A. Peter
Published: August 2015

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors α2-macroglobulin receptor/low-density lipoprotein receptor-related protein (α2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA·PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified α2MR/LRP and VLDLR respectively. α2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA·PAI-1 complex to both α2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by α-helix D and β-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease·serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
Evidence for altered sensitivity of the nitric oxide/cGMP signalling cascade in insulin-resistant skeletal muscle
Authors: YOUNG E. Martin , LEIGHTON Brendan
Published: August 2015

Nitric oxide activates guanylate cyclase to form cGMP, comprising a signalling system that is believed to be a distinct mechanism for increasing glucose transport and metabolism in skeletal muscle. The effects of a selective cGMP phosphodiesterase inhibitor, zaprinast, on basal glucose utilization was investigated in incubated rat soleus muscle preparations isolated from both insulin-sensitive (lean Zucker; Fa/?) and insulin-resistant (obese Zucker; fa/fa) rats. Zaprinast at 27 μM significantly increased cGMP levels in incubated soleus muscle isolated from lean, but not obese, Zucker rats. Muscles were incubated with 14C-labelled glucose and various concentrations of zaprinast (3, 27 and 243 μM). Zaprinast (at 27 and 243 μM) significantly increased rates of net and 14C-labelled lactate release and of glycogen synthesis in lean Zucker rat soleus muscle; glucose oxidation was also increased by 27 μM zaprinast. In addition, regardless of concentration, the phosphodiesterase inhibitor failed to increase any aspect of 14C-labelled glucose utilization in soleus muscles isolated from obese Zucker rats. The maximal activity of nitric oxide synthase (NOS) was significantly decreased in insulin-resistant obese Zucker muscles. Thus the lack of effect of zaprinast in insulin-resistant skeletal muscle is consistent with decreased NOS activity. To test whether there is a defect in insulin-resistant skeletal muscle for endogenous activation of guanylate cyclase, soleus muscles were isolated from both insulin-sensitive and insulin-resistant Zucker rats and incubated with various concentrations of the NO donor sodium nitroprusside (SNP; 0.1, 1, 5 and 15 mM). SNP significantly increased rates of net and 14C-labelled lactate release, as well as glucose oxidation in muscles isolated from both insulin-sensitive and insulin-resistant rats. A decreased response to SNP was observed in the dose-dependent generation of cGMP within isolated soleus muscles from insulin-resistant rats. A possible link between impaired NO/cGMP signalling and abnormal glucose utilization by skeletal muscle is discussed.
Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase
Authors: NUMAZAWA Mitsuteru , YOSHIMURA Akiko , OSHIBE Mariko
Published: August 2015

To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6α- and 6β-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6α-alkyl derivatives with alkyl functions less than three carbons long, together with 6β-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6β-n-pentyl ADD and its n-heptyl analogue as well as 6β-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6α-alkyl androgens were more efficient substrates than the corresponding 6β isomers. The Vmax of 6α-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6α-methyl derivative are similar, it is implied that alignment of 6α-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series.
Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age-related changes
Authors: CHRETIEN Dominique , GALLEGO Jorge , BARRIENTOS Antoni , CASADEMONT Jordi , CARDELLACH Francesc , MUNNICH Arnold , RÖTIG Agnès , RUSTIN Pierre
Published: August 2015

It is now widely acknowledged that a large number of human diseases originate from respiratory-chain dysfunctions. Because the molecular bases of these diseases are still poorly known, a biochemical approach has to be used in the screening procedures for the diagnoses of these conditions. Assessment of respiratory-chain function in human samples faces several problems: (i) the small size of available samples, (ii) the determination of discriminating parameters, and (iii) the interfering factors, such as age and physical activity. The present study focuses on isolated mitochondria prepared from a minute amount (100-200 mg) of skeletal-muscle biopsies from 201 patients between 0 and 65 years. Whereas 42 patients presented an isolated complex (C)I, CII, CIII or CIV deficiency, no respiratory-chain dysfunction or indirect evidence for a mitochondrial disorder could be attested in 159 of these patients. In this reference group, there was little correlation between enzyme activities and age, whatever the age class considered, 0-3 or 0-65 years of age. However, a confident handling of data points was largely hampered by the marked scattering of enzyme activities measured in the reference population. Activity ratios between the various respiratory-chain complexes presenting a much reduced scattering may be considered as diagnostic tools. As to the effect of age, no correlation with any of the enzyme-activity ratios could be shown. Use of age-matched controls for the diagnosis of respiratory-chain disorders may therefore be avoided, enzyme-activity ratios being highly discriminating and age-independent parameters.
Dephosphorylation and deactivation of Ca2+/calmodulin-dependent protein kinase II in βTC3-cells is mediated by Mg2+- and okadaic-acid-sensitive protein phosphatases
Authors: EASOM A. Richard , TARPLEY L. Jim , FILLER R. Natalie , BHATT Harshika
Published: August 2015

The α-toxin-permeabilized βTC3 cell has been utilized as an experimental model for the identification of protein phosphatases responsible for the dephosphorylation and deactivation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in situ. In this model, the elevation of Ca2+ from 0.05 to 10 μM induced the near-total conversion of CaM kinase II into a Ca2+/calmodulin-independent (autonomous) form characteristic of autophosphorylated, activated enzyme. On the removal of Ca2+, the activation state of CaM Kinase II rapidly returned to prestimulated levels. This reversal was slowed, but not prevented, by the inhibitors of protein phosphatase-1 (PP-1) and PP-2A, okadaic acid and calyculin A, and by the selective chelation of Mg2+ by the addition of EDTA. Near-complete prevention of enzyme deactivation, however, was observed in the combined presence of both okadaic acid and EDTA. Under these conditions, CaM kinase II phosphatase was more sensitive to calyculin A relative to okadaic acid, characteristic of the involvement of PP-1. CaM kinase II deactivation was not affected by FK-506, eliminating the involvement of PP-2B in this process. These data suggest that CaM kinase II dephosphorylation and deactivation in the pancreatic β-cell is mediated by the combined action of an okadaic-acid-sensitive phosphatase and a Mg2+-dependent phosphatase, such as PP-2C.
One-electron oxidation pathway of thiols by peroxynitrite in biological fluids: bicarbonate and ascorbate promote the formation of albumin disulphide dimers in human blood plasma
Authors: SCORZA Giuseppe , MINETTI Maurizio
Published: August 2015

Recent studies have shown that peroxynitrite oxidizes thiol groups through competing one- and two-electron pathways. The two-electron pathway is mediated by the peroxynitrite anion and prevails quantitatively over the one-electron pathway, which is mediated by peroxynitrous acid or a reactive species derived from it. In CO2-containing fluids the oxidation of thiols might follow a different mechanism owing to the rapid formation of a different oxidant, the nitrosoperoxycarbonate anion (ONOOCO2-). Here we present evidence that in blood plasma peroxynitrite induces the formation of a disulphide cross-linked protein identified by immunological (anti-albumin antibodies) and biochemical criteria (peptide mapping) as a dimer of serum albumin. The albumin dimer did not form in plasma devoid of CO2 and its formation was enhanced by ascorbate. However, analysis of thiol groups showed that reconstituting dialysed plasma with NaHCO3 protected protein thiols against the oxidation mediated by peroxynitrite and that the simultaneouspresence of ascorbate provided further protection. Ascorbate alone did not protect thiol groups from peroxynitrite-mediated oxidation. ESR spin-trapping studies with N-t-butyl-α-phenylnitrone (PBN) revealed that peroxynitrite induced the formation of protein thiyl radicals and their intensity was markedly decreased by plasma dialysis and restored by reconstitution with NaHCO3. PBN completely inhibited the formation of albumin dimer. Moreover, the addition of iron-diethyldithiocarbamate to plasma demonstrated that peroxynitrite induced the formation of protein S-nitrosothiols and/or S-nitrothiols. Our results are consistent with the hypothesis that NaHCO3 favours the one-electron oxidation of thiols by peroxynitrite with formation of thiyl radicals, •NO2, and RSNOx. Thiyl radicals, in turn, are involved in chain reactions by which thiols are oxidized to disulphides.
Intercellular calcium signalling between chondrocytes and synovial cells in co-culture
Authors: D'ANDREA Paola , CALABRESE Alessandra , GRANDOLFO Micaela
Published: August 2015

Intercellular communication allows the co-ordination of cell metabolism between tissues as well as sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks that favour the intercellular exchange of nutrients and second messengers. Heterologous intercellular communication was studied in co-cultures of articular chondrocytes and HIG-82 synovial cells by measuring mechanically induced cytosolic changes in Ca2+ ion levels by digital fluorescence video imaging. In confluent co-cultures, mechanical stimulation induced intercellular Ca2+ waves that propagated to both cell types with similar kinetics. Intercellular wave spreading was inhibited by 18α-glycyrrhetinic acid and by treatments inhibiting the activation of purinoreceptors, suggesting that intercellular signalling between these two cell types occurs both through gap junctions and ATP-mediated paracrine stimulation. In rheumatoid arthritis the formation of the synovial pannus induces structural changes at the chondrosynovial junction, where chondrocyte and synovial cells come into close apposition: these results provide the first evidence for direct intercellular communication between these two cell types.
Analysis of the combined effect of two linear inhibitors on a single enzyme
Authors: MARTINEZ-IRUJO Juan J. , VILLAHERMOSA María L. , MERCAPIDE Javier , CABODEVILLA Jesús F. , SANTIAGO Esteban
Published: August 2015

Different methods for studying the concurrent effects of two linear inhibitors on a single enzyme have been published, including the fractional product of Webb, the Yonetani-Theorell plot or the method of Chou and Talalay. Recently the use of combination plots has also been advocated for this purpose. We have evaluated the applicability of these methods and found that most of them depend on assumptions about the mechanism of action of the inhibitors. If the mechanism of action is not completely understood, or if some assumptions about the mechanism are unfounded, the parameters obtained may be meaningless. Even if these assumptions are correct, the interaction can be advantageously measured using an alternative representation that does not require a knowledge of the inhibition constants and allows experimental data to be retrieved from the plot. In other cases it is the interpretation of the results rather than the validity of the method that is misleading. A common mistake is to take the exclusivity of the effects of two inhibitors as exclusivity of their binding. We show that this assumption is seldom justified. In any case, it is possible to decide whether the combination of two or more inhibitors is more effective than their individual use by means of isobolographic analysis, even when no information about their mechanism of action is available.
Hydrogen bonding changes of internal water molecules in rhodopsin during metarhodopsin I and metarhodopsin II formation
Authors: RATH Parshuram , DELANGE Frank , DEGRIP J. Willem , ROTHSCHILD J. Kenneth
Published: August 2015

Rhodopsin is a 7-helix, integral membrane protein found in the rod outer segments, which serves as the light receptor in vision. Light absorption by the retinylidene chromophore of rhodopsin triggers an 11-cis → all-trans isomerization, followed by a series of protein conformational changes, which culminate in the binding and activation of the G-protein transducin by the metarhodopsin II (Meta II) intermediate. Fourier transform IR difference spectroscopy has been used to investigate the structural changes that water, as well as other OH- and NH-containing groups, undergo during the formation of the metarhodopsin I (Meta I) and Meta II intermediates. Bands associated with the OH stretch modes of water are identified by characteristic downshifts upon substitution of H218O for H2O. Compared with earlier work, several negative bands associated with water molecules in unphotolysed rhodopsin were detected, which shift to lower frequencies upon formation of the Meta I and Meta II intermediates. These data indicate that at least one water molecule undergoes an increase in hydrogen bonding upon formation of the Meta I intermediate, while at least one other increases its hydrogen bonding during Meta II formation. Amino acid residue Asp-83, which undergoes a change in its hydrogen bonding during Meta II formation, does not appear to interact with any of the structurally active water molecules. Several NH and/or OH groups, which are inaccessible to hydrogen/deuterium exchange, also undergo alterations during Meta I and Meta II formation.
cDNA isolation of Alzheimer's amyloid precursor protein from cholinergic nerve terminals of the electric organ of the electric ray
Authors: IIJIMA Ko-ichi , LEE Dong-Seok , OKUTSU Jun-ichi , TOMITA Susumu , HIRASHIMA Naohide , KIRINO Yutaka , SUZUKI Toshiharu
Published: August 2015

Alzheimer's amyloid precursor protein (APP) is a transmembrane protein containing three phosphorylation sites in its cytoplasmic domain. In the present study, we isolated cDNA of APP from electric ray electric lobe (elAPP). This APP (elAPP699) consists of 699 amino acids, contains the β-amyloid domain and has 80.7% similarity with the human APP695 isoform. The cytoplasmic domain, including three phosphorylation sites, was completely conserved. In the nerve terminals of the cholinergic neuron from the electric ray electric organ, we found elAPP699 existed exclusively in the mature form. We found the phosphorylated form of mature elAPP699 in the nerve terminal as well as in cell body. Immature elAPP699 was not subject to phosphorylation. Our findings indicate that, in neurons, the phosphorylation of APP occurs after maturation.
Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function
Authors: HSIANG Chien-Yun , HO Tin-Yun , HSIANG Chin-Hui , CHANG Tien-Jye
Published: August 2015

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5ʹ-exonuclease and 3ʹ-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.