bluuebunny/crossref_metadata_2025 huggingface dataset:
A potent and highly selective peptide substrate for protein kinase C assay
Authors: TOOMIK Reet , EK Pia
Published: August 2015

Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49±0.13 μM and Vmax of 10.0±0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts.
Rapid decrease in cellular sodium and chloride content during cold incubation of cultured liver endothelial cells and hepatocytes
Authors: GIZEWSKI Elke R. , RAUEN Ursula , KIRSCH Michael , REUTERS Irith , DIEDERICHS Herbert , GROOT Herbert de
Published: August 2015

Hypothermia, as used for organ preservation in transplantation medicine, is generally supposed to lead to an intracellular accumulation of sodium, and subsequently of chloride, via inhibition of the Na+/K+-ATPase. However, on studying the cellular sodium concentration of cultured liver endothelial cells using fluorescence microscopy, we found a 55% decrease in the cellular sodium concentration after 30 min of cold incubation in University of Wisconsin (UW) solution. To confirm this surprising result, we set up a capillary electrophoresis method that allowed us to determine the cellular contents of inorganic cations and of inorganic anions. Using this method we measured a decrease in the cellular sodium content from 104±11 to 55±4 nmol/mg of protein, accompanied by a decrease in the chloride content from 71±9 to 25±5 nmol/mg of protein, after 30 min of cold incubation in UW solution. When the endothelial cells were incubated in cold Krebs–Henseleit buffer or in cold cell culture medium instead of UW solution, similar early decreases in cellular sodium and chloride contents were observed, thus excluding the possibility of the decreases being dependent on the preservation solution used. Furthermore, experiments with cultured rat hepatocytes yielded a similar decrease in sodium content during initiation of cold incubation in UW solution, so the decrease does not appear to be cell-specific either. These results suggest that, contrary to current opinion, sodium efflux predominates over sodium influx during the early phase of cold incubation of cells.
Glutathione transferase mimics: micellar catalysis of an enzymic reaction
Authors: LINDKVIST Björn , WEINANDER Rolf , ENGMAN Lars , KOETSE Marc , ENGBERTS Jan B. F. N. , MORGENSTERN Ralf
Published: August 2015

Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as α,β-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium-co-dodecylmethyldiallylammon ium) bromide (86/14) ≫cetyltrimethylammonium bromide > zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) > zwittergent 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) ≈ N,N-dimethyl-laurylamine N-oxide > N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to α,β-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding.
DNA polymerase β: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics
Authors: KRAYNOV Vadim S. , WERNEBURG Brian G. , ZHONG Xuejun , LEE Hui , AHN Jinwoo , TSAI Ming-Daw
Published: August 2015

DNA polymerase β (pol β) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd (app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol β, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol β. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol β. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol β. Since the chemical step may not be rate-limiting for wild-type pol β, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol β displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.
Glutathione S-transferase Yc cDNA from Syrian hamster kidney
Authors: MAGGOUTA Frideriki , LI Sara A. , LI Jonathan J. , NORRIS James S.
Published: August 2015

A cDNA encoding alpha-class glutathione S-transferase Yc (GSTYc) has been isolated from a Syrian hamster kidney library, and its nucleotide sequence (968 bp) has been determined. Analysis of the deduced amino acid sequence revealed a high level of identity between Syrian hamster GSTYc, rat GST Yc1 and Yc2 and mouse GSTYc. Northern-blot experiments demonstrated that Syrian hamster GSTYc expression is tissue-specific. A GSTYc mRNA of approx. 1 kb is expressed in liver, kidney, vas deferens and epididymis. Expression of the GSTYc transcript was not detected in testis or uterus.
Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration
Authors: BILLINGSLEY Melvin L. , KINCAID Randall L.
Published: August 2015

This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal–membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation.
Allosteric equilibrium model explains steady-state coupling of β-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes
Authors: UGUR Özlem , ONARAN H. Ongun
Published: August 2015

We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated β-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of β-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we attempt to unify the equilibrium and collision coupling approaches to provide a consistent theoretical basis for the G-protein-mediated β-adrenergic receptor signalling in turkey erythrocyte membranes.
Isolated rat hepatocytes differentially bind and internalize bovine lactoferrin N- and C-lobes
Authors: SITARAM Maheshwari P. , McABEE Douglas D.
Published: August 2015

Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) and its bound iron in a Ca2+-dependent manner. In this study, we determined if one or both halves of Lf (N- and C-lobes) were responsible for the interaction of Lf with hepatocytes. We isolated three tryptic fragments of bovine Lf. Cleavage at Arg284–Ser285 generated two fragments: N-terminal pp36 that contained 80% of Lf N-lobe and C-terminal pp51. A second cleavage at Arg338–Ala339 generated a smaller fragment (pp44) that contained all of the C-lobe with no N-lobe elements. Hepatocytes bound Lf and pp51 in a Ca2+-dependent manner with the same affinity (Kd approx. 75 nM) and to nearly identical extents (approx. 106 sites per cell). Lf and pp51 competed with each other for binding to cells over a similar titration range. Hepatocytes internalized Lf at a faster rate than pp51 (kin=0.28 and 0.19 min-1 respectively), but cells degraded pp51 at approx. twice the rate of native Lf. pp44 competed with 125I-labelled Lf for binding to Ca2+-dependent binding sites on hepatocytes as well as native Lf or pp51. In contrast, hepatocytes bound pp36 (Kd = 90 nM, ⩽ 5×106 sites per cell) but did not internalize or degrade it appreciably. Moreover, pp36 binding to cells was not Ca2+-dependent, and pp36 competed poorly with native Lf and pp51 for binding to cells. We conclude from these findings that the Lf determinants responsible for binding to the Ca2+-dependent receptor on hepatocytes is present within the C-lobe of Lf.
Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells
Authors: CROOK Richard B. , CHANG Alan T.
Published: August 2015

Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 μM stimulated cGMP formation 8.2(±1.2)-fold, 4.8(±0.6)-fold and 87.3(±12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD = 0.30±0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced > 95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30–38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4α phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 μM PMA was 30.6(±4.0)% with 500 nM CNP, but 83.4(±8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(±5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.
Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet
Authors: ADAMS Sean H. , ALHO Clarice S. , ASINS Guillermina , HEGARDT Fausto G. , MARRERO Pedro F.
Published: August 2015

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2–3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling–weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.
Network organization of cell metabolism: monosaccharide interconversion
Authors: NUÑO Juan C. , SÁNCHEZ-VALDENEBRO Ignaciol , PÉREZ-IRATXETA Carolina , MELÉNDEZ-HEVIA Enrique , MONTERO Francisco
Published: August 2015

The structural properties of carbohydrate metabolism are being studied. The present contribution focuses mainly on those processes involving the transfer of carbon fragments among sugars. It is shown how enzymic activities fix the way the system self-organizes stoichiometrically at the steady state. It is proven that there exists a specific correspondence between the set of all possible enzymic activities, the activity set, and the set of stoichiometrically compatible flux distributions through the pathway. On the one hand, there are enzymic activities that do not allow a stoichiometrically feasible coupling at the steady state of the reactions involved in the conversion. On the other hand, there are enzymic activities that are related to one or more flux distributions at the steady state (i.e. with one or several rate vectors respectively). For this latter group, it can be demonstrated that the structure of the system depends on other non-structural factors, such as boundary constraints and the kinetic parameters. As a consequence, it is suggested that this kind of metabolic process must be viewed as a complex reaction network instead of a sequential number of steps. Some implications of these derivations are illustrated for the particular conversion CO2 →C3. General remarks are also discussed within the framework of network models of cell metabolism.
Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells
Authors: MacKENZIE Simon , FLEMING Iona , HOUSLAY Miles D. , ANDERSON Neil G. , KILGOUR Elaine
Published: August 2015

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-α, -γ, -δ, -ϵ and -ζ. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-δ and the partial depletion of PKC-α but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-δ and perhaps PKC-α are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-δ anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-δ anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-δ and -ϵ anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-δ and -ϵ isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-δ.
Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cγ1
Authors: BALDASSARE Joseph J. , HENDERSON Patricia A. , TARVER Alan , FISHER Gary J.
Published: August 2015

We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cγ1 (PLCγ1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCγ1 were immunoprecipitated as a complex by a polyclonal antibody to PLCγ1. The association of gelsolin and actin was specific for PLCγ1 because immunoprecipitates of PLCs β2, β3, γ2 and δ1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCγ1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCγ1. After stimulation with thrombin, PLCγ1 activity in immunoprecipitates was increased 2–3-fold. This elevation in PLCγ1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCγ1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCγ1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCγ1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCγ1 from gelsolin and actin, and the stimulation of PLCγ1 activity.
Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145→Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy
Authors: FEDERWISCH Matthias , HASSIEPEN Ulrich , BENDER Kirsten , DEWOR Manfred , RAJEWSKY Manfred F. , WOLLMER Axel
Published: August 2015

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 °C but remained monomeric at 4 °C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8.4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The α-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its α-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.
Chemoattractants induce tyrosine phosphorylation of ERK2 in Dictyostelium discoideum by diverse signalling pathways
Authors: KOSAKA Chiya , PEARS Catherine J.
Published: August 2015

Two homologues of mitogen-activated protein kinases have been identified in Dictyostelium discoideum (ERK1 and EKR2). We here demonstrate transient tyrosine phosphorylation of ERK2 in response to the chemoattractants cAMP and folic acid that correlates with activity. To investigate the signalling pathways, we studied the response in strains with altered cAMP-dependent protein kinase (PKA) status. The degree of cAMP-induced ERK2 tyrosine phosphorylation was increased in cells overexpressing PKA activity but no such increase was observed in the response to folic acid. Our observations suggest that cAMP-induced ERK2 tyrosine phosphorylation is positively modulated by a PKA-regulated step which is not involved in the response to folic acid, suggesting the presence of diverse signalling pathways leading to ERK2 activation.
Heterologous expression of rab4 reduces glucose transport and GLUT4 abundance at the cell surface in oocytes
Authors: MORA Silvia , MONDEN Ingrid , ZORZANO Antonio , KELLER Konrad
Published: August 2015

To evaluate the role of the small rab GTP-binding proteins in glucose transporter trafficking, we have heterologously co-expressed rab4 or rab5 and GLUT4 or GLUT1 glucose transporters in Xenopus oocytes. Co-injection of rab4 and GLUT4 cRNAs resulted in a dose-dependent decrease in glucose transport; this effect was specific for rab4, since co-injection of an inactive rab4 mutant or rab5 cRNA did not have any effect on glucose transport. The effect of rab4 was selective for GLUT4, since no effect was detected in GLUT1-expressing oocytes. The inhibitory effect of rab4 on GLUT4-induced glucose transport was not the result of a change in overall cellular levels of GLUT4 glucose transporters. However, rab4 expression caused a marked decrease in the abundance of GLUT4 transporters present at the cell surface. Finally, rab4 and inhibitors of PtdIns 3-kinase showed additive effects in decreasing glucose transport in GLUT4-expressing oocytes. We conclude that rab4 plays an important role in the regulation of the intracellular GLUT4 trafficking pathway, by contributing to the intracellular retention of GLUT4 through a PtdIns 3-kinase-independent mechanism.
Differential functional significance of AP-1 binding sites in the promoter of the gene encoding mouse tissue inhibitor of metalloproteinases-3
Authors: KIM Hyungtae , PENNIE William D. , SUN Yi , COLBURN Nancy H.
Published: August 2015

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular-matrix-associated protein that suppresses tumorigenicity or invasion in several model systems. We have identified, by in vitro footprinting, six AP-1 (activator protein-1) or AP-1-like binding sites in the mouse TIMP-3 promoter that bind purified c-Jun homodimers. Electrophoretic mobility shift assays revealed that the non-consensus fifth AP-1 binding site (AP-720; nt -720 to -714) had the strongest binding activity for recombinant c-Jun protein, and that the fourth binding site (AP-763; nt -763 to -754) and AP-720 showed strong binding activity for cellular nuclear proteins. Antibody supershift and blocking experiments suggest that AP-720, but not AP-763, binds authentic AP-1 components. Transient transfection reporter assays of deletion constructs showed that the region spanning AP-720 has the highest transcriptional activity, and that sequences 5′ to this region (nt -2846 to -747) may contain negative regulatory elements. The deletion construct containing about 500 nt 5′ to the transcriptional start, but no AP-1 sites, showed lower but significant activity, suggesting both AP-1-dependent and -independent regulation of the mouse TIMP-3 promoter. Mutational inactivation of AP-720 abolished the activity increment that distinguished the reporter construct containing both AP-720 and sixth AP-1 binding site (AP-617; nt -617 to -611) from that containing only AP-617. In summary, we report here that both AP-1 and non-AP-1 elements contribute to activity, with the non-consensus AP-1 site at -720 showing the greatest functional significance among the AP-1 sites.
Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-γ and aldolase C
Authors: BULLIARD Catherine , ZURBRIGGEN Rinaldo , TORNARE Joelle , FATY Mahamadou , DASTOOR Zubin , DREYER Jean-Luc
Published: August 2015

NADH–dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-γ. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.
Evidence for glutathione involvement in platelet-derived growth-factor-mediated signal transduction
Authors: RIGACCI Stefania , IANTOMASI Teresa , MARRACCINI Patrizia , BERTI Andrea , VINCENZINI Maria Teresa , RAMPONI Giampietro
Published: August 2015

Recent studies show that glutathione, while being involved in the well-known physiological processes of amino acid transport and detoxification, can also play a part in cell proliferation events. Cell treatment with l-buthionine sulphoximine, which causes glutathione depletion, is accompanied by a decrease in cell proliferation. At present no precise relationship between this thiol and any critical intermediate of the mitogenic cascade has been proved. In this study, conducted on NIH/3T3 murine fibroblasts, we demonstrate a strict correlation between glutathione levels and platelet-derived growth-factor-receptor activation in response to stimulation and cell proliferation. The receptor autophosphorylation is severely impaired at low glutathione cellular levels. The interaction of glutathione with this growth-factor receptor in vivo, while being rather specific, is complex and may involve both cytosolic and extracellular receptor domains.
Expression of human cholesterol 7α-hydroxylase in atherosclerosis-susceptible mice via adenovirus infection
Authors: MOORE Gina L. , DREVON Christian A. , MACHLEDER Dietrich , TRAWICK John D. , McCLELLAND Alan , ROY Soumitra , LYONS Russette , JAMBOU Robert , DAVIS Roger A.
Published: August 2015

Adenovirus is a vector for the delivery of genes mainly to the liver. Short-term (~3 days) studies using adenovirus transfection have provided valuable insights into how genes can complement normal and pathological phenotypes. When atherosclerosis-susceptible C57BL/6 mice were infected with an adenovirus vector containing the human 7α-hydroxylase cDNA (AV17h1) and fed on a chow diet, human 7α-hydroxylase mRNA and enzyme activity doubled compared with that in mice infected with an adenovirus vector (AV1Null) alone. In AV17h1-infected mice fed on a high fat cholic acid (HFCA) diet, mRNA expression and activity of both the endogenous and adenovirus (human) 7α-hydroxylase were repressed. AV17h1-infected mice fed on a HFCA diet and killed at mid-light had increased 7α-hydroxylase activity and mRNA compared with mice killed at mid-dark. Since expression of AV17h1 is driven by a constitutive Rous sarcoma virus promoter, the repression of human 7α-hydroxylase by the HFCA diet was unexpected. In spite of this post-transcriptional repression by the HFCA diet, AV17h1-infected mice expressed the human 7α-hydroxylase mRNA, causing its enzyme activity to be 3-fold greater than in AV1Null-infected mice. In AV17h1-infected mice, the 7α-hydroxylase enzyme activity varied as a linear function of human mRNA abundance. In conclusion, the accumulation of apolipoprotein B-containing lipoproteins in plasma of C57BL/6 mice fed on the HFCA diet was not reduced by longer-term (2 weeks) 7α-hydroxylase expression, probably because of its diminished expression caused by the diet and hepatic inflammation from the adenovirus infection. These results may suggest that adenovirus is effective in promoting longer-term (2 weeks) expression of 7α-hydroxylase.
A conserved GATA motif in a tissue-specific DNase I hypersensitive site of the cardiac α-myosin heavy chain gene
Authors: HUANG Weei-Yuarn , LIEW Choong-Chin
Published: August 2015

Transgenic analysis has indicated that far upstream regulatory elements of the cardiac α-myosin heavy chain (MyHC) gene are required for appropriate transgene expression [Subramaniam, Gulick, Neumann, Knotts and Robbins (1993) J. Biol. Chem. 268, 4331–4336]. In an attempt to identify these as-yet-undefined regulatory elements, we mapped the DNase I hypersensitive sites (DHSs) in the 4 kb upstream region of the hamster cardiac α-MyHC gene. When using nuclei isolated from late-gestational and adult heart ventricles, a strong DHS was identified in the -1.9 kb region (α-1.9 kb site). It cannot be detected in kidney, liver or cardiofibroblast nuclei. Within this site, we found a conserved GATA-motif that interacts specifically with GATA-binding factors in nuclear extracts of cardiomyocytes at various developmental stages. These data provide further evidence to support the role of GATA factors in the regulation of cardiac α-MyHC gene expression.
Molecular cloning of up-regulated cytoskeletal genes from regenerating skeletal muscle: potential role of myocyte enhancer factor 2 proteins in the activation of muscle-regeneration-associated genes
Authors: AKKILA Waleed M. , CHAMBERS Rebecca L. , ORNATSKY Olga I. , McDERMOTT John C.
Published: August 2015

A subtractive hybridization and cloning strategy was used to identify genes that are up-regulated in regenerating compared with normal skeletal muscle. The gastrocnemius muscle of CD1 mice was injected with a myotoxic agent (BaCl2). A cDNA library was constructed from the regenerating muscle, and was screened with subtracted probes enriched in genes up-regulated during regeneration. Cofilin and vimentin cDNA clones were isolated. Both cofilin and vimentin were demonstrated to be overexpressed in regenerating compared with non-regenerating muscle (17-fold and 19-fold induction respectively). Cofilin and vimentin mRNAs also exhibited an increased expression in C2C12 myoblasts and a decreased expression in differentiated myotubes. Analysis of the regeneration-induced vimentin enhancer/promoter region revealed a consensus binding site for the myocyte enhancer factor 2 (MEF2) transcription factors. Electrophoretic mobility-shift assays and in vivo reporter assays revealed that MEF2 DNA-binding activity and transcriptional activation are increased in regenerating skeletal muscle, indicating that they may play a role in the activation of muscle genes during regeneration. These data suggest that both cofilin (an actin-regulatory protein) and vimentin (an intermediate filament) may be key components of the cytoskeletal reorganization that mediates muscle cell development and adult skeletal-muscle repair.
The study of cell-death proteins in the outer mitochondrial membrane by chemical cross-linking
Authors: ALI Sohail T. , COGGINS John R. , JACOBS Howard T.
Published: August 2015

Chemical cross-linking was used to study the interactions of the anti-cell-death protein Bcl2 with other proteins in the outer mitochondrial membrane. Cross-linking of mitochondrial surface proteins produced a large Bcl2-containing complex (> 200 kDa), and a Bcl2-derived peptide was shown to cross-link specifically with a mitochondrial protein identified by immunoblotting as Raf-1 kinase.
Ca2+ and protein kinase C-dependent mechanisms involved in gastrin-induced Shc/Grb2 complex formation and P44-mitogen-activated protein kinase activation
Authors: DAULHAC Laurence , KOWALSKI-CHAUVEL Aline , PRADAYROL Lucien , VAYSSE Nicole , SEVA Catherine
Published: August 2015

The proliferative effects of gastrin on normal and neoplastic gastro-intestinal tissues have been shown to be mediated by the gastrin/CCKB (G/CCKB) G-protein-coupled receptors. We have recently reported that gastrin stimulates the tyrosine phosphorylation of Shc proteins and their subsequent association with the Grb2/Sos complex, leading to mitogen-activated protein kinase (MAPK) activation, a pathway known to play an important role in cell proliferation. We undertook the present study to characterize the signalling pathways used by this receptor to mediate the activation of the Shc/Grb2 complex. Since G/CCKB receptor occupancy leads to the activation of the phospholipase C (PLC)/protein kinase C (PKC) pathway, we examined whether PKC stimulation and Ca2+ mobilization contribute to the phosphorylation of Shc proteins and their association with Grb2 in response to gastrin. Our results indicate that Shc proteins are tyrosine phosphorylated and associate with Grb2 in response to phorbol esters, suggesting that activation of PKC is a potential signalling pathway leading to activation of the Shc/Grb2 complex. Inhibition of PKC by GF109203X completely blocked the effect of PMA on Shc tyrosine phosphorylation and its subsequent association with Grb2, but had a partial inhibitory effect on the response to gastrin. Depletion of the intracellular Ca2+ pools by treatment with thapsigargin blocked the increase in intracellular free calcium concentration induced by gastrin and diminished the ability of the peptide to stimulate Shc phosphorylation and recruitment of Grb2. In addition, removal of extracellular Ca2+ partially inhibited the effect of gastrin on Shc phosphorylation as well as its association with Grb2, indicating that the effects of gastrin are also mediated by Ca2+-dependent mechanisms. Furthermore, we show that blockage of the two major early signals generated by activation of PLC, which induced the activation of the Shc/Grb2 complex, also blocked gastrin-induced MAPK activation.
Asymmetry adjacent to the collagen-like domain in rat liver mannose-binding protein
Authors: WALLIS Russell , DRICKAMER Kurt
Published: August 2015

Rat liver mannose-binding protein (MBP-C) is the smallest known member of the collectin family of animal lectins, many of which are involved in defence against microbial pathogens. It consists of an N-terminal collagen-like domain linked to C-terminal carbohydrate-recognition domains. MBP-C, overproduced in Chinese-hamster ovary cells, is post-translationally modified and processed in a manner similar to the native lectin. Analytical ultracentrifugation experiments indicate that MBP-C is trimeric, with a weight-averaged molecular mass of approx. 77 kDa. The rate of sedimentation of MBP-C and its mobility on gel filtration suggest a highly elongated molecule. Anomalous behaviour on gel filtration due to this extended conformation may explain previous suggestions that MBP-C forms a higher oligomer. The polypeptide chains of the MBP-C trimer are linked by disulphide bonds between two cysteine residues at the N-terminal junction of the collagen-like domain. Analysis of an N-terminal tryptic fragment reveals that the disulphide bonding in MBP-C is heterogeneous and asymmetrical. These results indicate that assembly of MBP-C oligomers probably proceeds in a C- to N-terminal direction: trimerization at the C-terminus is followed by assembly of the collagenous domain and finally formation of N-terminal disulphide bonds. The relatively simple organization of MBP-C provides a template for understanding larger, more complex collectins.
Activation of sea-urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux
Authors: van DORSTEN Ferdi A. , WYSS Markus , WALLIMANN Theo , NICOLAY Klaas
Published: August 2015

The kinetics of the creatine kinase (CK) reaction were studied in suspensions of quiescent and active, intact sea-urchin spermatozoa in artificial seawater, using 31P-NMR magnetization transfer. In inactive sperm, no CK-mediated exchange flux was detected, whereas in activated motile sperm, the forward pseudo-first-order rate constant was 0.13±0.04 s-1 at 10 °C, corresponding to a steady-state CK flux of 3.1±0.5 mM·s-1. Intracellular pH shifted from 6.6±0.1 to 7.6±0.1 upon activation. The phosphocreatine (PCr)/ATP and PCr/Pi ratios were only marginally reduced in activated sperm, whereas the estimated cytosolic free ADP concentration increased remarkably from 9 μM in quiescent, to 114 μM in activated spermatozoa. The elevation of CK flux upon sperm activation is discussed in the light of the proposition that in sea-urchin spermatozoa, which are fuelled entirely by oxidative phosphorylation, high-energy phosphate transport is mediated by a ‘CK/PCr shuttle‘.
Glutathione conjugation of chlorambucil: measurement and modulation by plant polyphenols
Authors: ZHANG Kai , WONG Kim Ping
Published: August 2015

Chlorambucil (CMB), an anticancer drug, was cytotoxic at concentrations of 5–20 μM to human colon adenocarcinoma cells. It inhibited [14C]thymidine uptake in a dose-dependent manner. Both effects were potentiated by simultaneous exposure of the cells to 10 μM plant polyphenols. In an attempt to explain the possible mechanism of action of the polyphenols in relation to these observations, an HPLC-radiometric method was developed to measure the conjugation of CMB with glutathione in these cells and to monitor the export of monochloromonoglutathionyl CMB (MG-CMB), its main glutathione conjugate. At micromolar concentrations, five polyphenols, namely quercetin, butein, tannic acid, 2′-hydroxychalcone and morin, inhibited the efflux of CMB significantly; an inhibition of 40% was observed with 10 μM quercetin. The glutathione S-transferase (GST) activity of the cancer cells, measured with 1-chloro-2,4-dinitrobenzene, was also inhibited by the polyphenols. Their combined action on GST and on the efflux of MG-CMB conjugate could provide an enhanced positive modulation of sensitivity of the tumour cells to CMB.
Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2
Authors: SUSZTÁK Katalin , MÓCSAI Attila , LIGETI Erzsébet , KAPUS András
Published: August 2015

The potential role of cytosolic phospholipase A2 (cPLA2) in the regulation of the electrogenic arachidonic acid (AA)-activatable H+ translocator of neutrophils was investigated. (1) The trifluoromethyl ketone analogue of arachidonate (AACOCF3), a newly developed selective blocker of cPLA2, inhibited both the N-formylmethionyl-leucylphenylalanine (fMLP)- and the phorbol-ester-induced rheogenic H+ efflux (K0.5 ≈ 5 μM) and abrogated the stimulus-triggered release of AA from these cells. The drug failed to reduce the fMLP-evoked Ca2+ signal or protein tyrosine phosphorylation and did not affect the activity of protein kinase C. By using the patch-clamp technique we verified that the agent did not interfere with the voltage- and the pH-dependent activation of the H+ conductance of the peritoneal macrophages and therefore is not a direct blocker of the H+ channel itself. AACOCF3, however, slightly decreased the AA-induced stimulation of the H+ currents. We conclude that AA, liberated by the agonist-induced stimulation of cPLA2, is a direct activator of H+ conductance. (2) AACOCF3 did not inhibit superoxide generation, indicating that activation of cPLA2 may not be a prerequisite for turning on NADPH oxidase. (3) Since neither acid generation by the oxidase, nor the basal or stimulated Na+/H+ exchange (the predominant acid-eliminating mechanism) were influenced by the drug, we could use AACOCF3 to address whether the H+ channel in fact opens and plays any physiological role during activation of neutrophils. Stimulus-induced cytosolic alkalinization was smaller, whereas depolarization became larger, in the presence of AACOCF3. Stimulated H+ conductance therefore does contribute to intracellular pH (pHi) homoeostasis and membrane potential changes of intact neutrophils.
Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus
Authors: OHKURA Naoki , OKUHARA Hiroaki , INOUE Seiji , IKEDA Kiyoshi , HAYASHI Kyozo
Published: August 2015

Three distinct types of phospholipase A2 (PLA2) inhibitory proteins (PLIα, PLIβ, and PLIγ) were isolated from the blood plasma of the Chinese mamushi, Agkistrodonblomhoffiisiniticus. PLIα is an inhibitor that we have already purified and whose amino acid sequence we have already determined [Ohkura, Inoue, Ikeda and Hayashi (1993) J. Biochem. (Tokyo) 113, 413–419]. It inhibited selectively the group-II acidic PLA2s from Crotalidae venom. PLIβ was a 160-kDa glycoprotein having a trimeric structure composed of 50-kDa subunits. The amino acid sequence of the first 30 amino acids of the N-terminal part of the 50-kDa subunit was determined and found to have no significant homology to that of known proteins. PLIβ was a selective inhibitor against the group-II basic PLA2s from Crotalidae venom. Some amino acid residues located in or close to the interfacial binding surface of the group-II basic PLA2s were suggested to be involved in selective binding to PLIβ. PLIγ was a 100-kDa glycoprotein containing 25-kDa and 20-kDa subunits and inhibited all of the PLA2s investigated equally, including Elapidae venom PLA2s (group I), Crotalidae and Viperidae venom PLA2s (group II) and honey-bee PLA2 (group III). From the N-terminal sequences of the two subunits, PLIγ was found to be the same type of PLI that had been purified from Thailand cobra plasma.