bluuebunny/crossref_metadata_2025 huggingface dataset:
The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine
Authors: BRÖER Angelika , WAGNER Carsten A. , LANG Florian , BRÖER Stefan
Published: August 2015

The cationic amino acid arginine, due to its positive charge, is usually accumulated in the cytosol. Nevertheless, arginine has to be released by a number of cell types, e.g. kidney cells, which supply other organs with this amino acid, or the endothelial cells of the blood–brain barrier which release arginine into the brain. Arginine release in mammalian cells can be mediated by two different transporters, y+LAT1 and y+LAT2. For insertion into the plasma membrane, these transporters have to be associated with the type-II membrane glycoprotein 4F2hc [Torrents, Estevez, Pineda, Fernandez, Lloberas, Shi, Zorzano and Palacin (1998) J. Biol. Chem. 273, 32437–32445]. The present study elucidates the function and distribution of y+LAT2. In contrast to y+LAT1, which is expressed mainly in kidney epithelial cells, lung and leucocytes, y+LAT2 has a wider tissue distribution, including brain, heart, testis, kidney, small intestine and parotis. When co-expressed with 4F2hc in Xenopus laevis oocytes, y+LAT2 mediated uptake of arginine, leucine and glutamine. Arginine uptake was inhibited strongly by lysine, glutamate, leucine, glutamine, methionine and histidine. Mutual inhibition was observed when leucine or glutamine was used as substrate. Inhibition of arginine uptake by neutral amino acids depended on the presence of Na+, which is a hallmark of y+LAT-type transporters. Although arginine transport was inhibited strongly by glutamate, this anionic amino acid was only weakly transported by 4F2hc/y+LAT2. Amino acid transport via 4F2hc/y+LAT2 followed an antiport mechanism similar to the other members of this new family. Only preloaded arginine could be released in exchange for extracellular amino acids, whereas marginal release of glutamine or leucine was observed under identical conditions. These results indicated that arginine has the highest affinity for the intracellular binding site and that arginine release may be the main physiological function of this transporter.
Responsiveness of the state of O-linked N-acetylglucosamine modification of nuclear pore protein p62 to the extracellular glucose concentration
Authors: HAN Innoc , OH Eok-Soo , KUDLOW Jeffrey E.
Published: August 2015

O-linked N-acetylglucosamine (O-GlcNAc) modification has been described in many proteins, including nuclear pore glycoproteins. In the present study we investigated the effect of extracellular glucose on the level of modification of nuclear pore protein p62 by O-GlcNAc. We found that exposure of cells to a high concentration of glucose caused an increased level of modification of p62 with O-GlcNAc, whereas the modification of other proteins did not change detectably. The increased O-GlcNAc modification of p62 in response to glucose required the metabolism of glucose to glucosamine. The exposure of the cells to glucosamine itself also caused increased O-GlcNAc modification, whereas mannosamine and galactosamine did not. Furthermore, changes in glucose concentrations within the physiological range induced the O-GlcNAc modification in p62 in rat aortic smooth-muscle cells, indicating that this modification of p62 might occur in an intact animal. These results imply that the ambient glucose concentration has an impact on the level of O-GlcNAc modification of proteins such as p62, and that functional changes in some of these proteins might ensue.
Tumour necrosis factor-α and interferon-γ synergistically activate the RANTES promoter through nuclear factor κB and interferon regulatory factor 1 (IRF-1) transcription factors
Authors: LEE Ahn Hwee , HONG Jeong-Ho , SEO Yeon-Soo
Published: August 2015

Inflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) synergistically activate expression of the RANTES (regulated upon activation, normal T-cell expressed and secreted) gene, which plays a crucial role in the chemoattraction of leukocytes during the inflammatory response. To understand at the molecular level the mechanism by which the two cytokines activate RANTES gene expression, we determined the requirement of cis-acting elements in the RANTES promoter and trans-acting factors. The murine RANTES promoter contained one putative interferon regulatory factor, IRF, and three putative nuclear factor κB (NF-κB) binding sites. Specific destruction of the IRF binding site and one of the three NF-κB binding sites abolished the inducibility of promoter activity by IFN-γ and TNF-α, respectively. In contrast, mutation of the other two putative NF-κB binding sites did not affect RANTES promoter activity significantly. In addition, the RANTES promoter was stimulated by co-transfection of plasmids that expressed either p65, an NF-κB family protein, or the IRF-1 transcription factor. RANTES promoters with mutations in the NF-κB or IRF binding sites were not stimulated by p65 or IRF-1 expression, respectively. In electrophoretic mobility-shift and immunologic assays, we showed that IRF-1 was induced after cells were treated with IFN-γ and that NF-κB was activated by TNF-α treatment. These results demonstrate that both NF-κB and IRF-1 transcription factors mediate the induction of RANTES expression via their cognate cis-acting elements when cells are stimulated by TNF-α and IFN-γ.
l-Leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the l-leucine-induced up-regulation of System A amino acid transport
Authors: PEYROLLIER Karine , HAJDUCH Eric , BLAIR Anne S. , HYDE Russell , HUNDAL Harinder S.
Published: August 2015

Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1h with L-leucine (2mM) led to a rapid (> 2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid (≈ 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by ≈ 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3, and this is likely to have important regulatory implications for translation initiation.
Disruption and overexpression of the Schizosaccharomyces pombe aph1 gene and the effects on intracellular diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A), ATP and ADP concentrations
Authors: INGRAM Stephen W. , BARNES Larry D.
Published: August 2015

Diadenosine oligophosphates are ubiquitous compounds that were discovered over 30 years ago. Diadenosine 5´,5″-P1,P4-tetraphosphate (Ap4A) is the most studied member of this family, and its function in yeast is unknown. To investigate possible functions, we changed the intracellular Ap4A concentration in Schizosaccharomyces pombe via disruption and overexpression of the aph1 gene, which encodes an Ap4A hydrolase (Aph1). S. pombe Aph1 is 52% identical with a human tumour suppressor protein, Fhit, in a core region of 109 amino acids. Disruption of aph1 resulted in an 85% decrease in Ap4A hydrolase activity and a 290-fold increase in the intracellular Ap4A concentration. The disruption and subsequent increase in intracellular Ap4A concentration had no significant effect on the growth of S. pombe. Overexpression of the S. pombe aph1 gene, resulting in 17- and 84-fold increases in Ap4A hydrolase activity above wild-type levels, resulted in 60 and 80% decreases respectively in the intracellular Ap4A concentration. This represents the first report of a decrease in the intracellular Ap4A concentration in response to overexpression of a degradative enzyme in any eukaryotic organism. We describe a new S. pombe expression plasmid, pPOX, which was used to achieve the largest increase in expression of aph1. Overexpression of aph1 at the highest level resulted in a 46% increase in generation time in comparison with the control strain. Neither overexpression nor disruption had any effect on the intracellular ATP or ADP concentrations. This is the first report of ADP and ATP concentrations in S. pombe. These data also indicate that Aph1 functions in vivo to degrade Ap4A, and that high-level overexpression of this enzyme reduces the growth rate.
A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein
Authors: VERSTEEG Henri H. , NIJHUIS Evert , VAN DEN BRINK Gijs R. , EVERTZEN Maaike , PYNAERT Gwenda N. , VAN DEVENTER Sander J. H. , COFFER Paul J. , PEPPELENBOSCH Maikel P.
Published: August 2015

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.
Biological effects of menadione photochemistry: effects of menadione on biological systems may not involve classical oxidant production
Authors: MCCORMICK Michael L. , DENNING Gerene M. , RESZKA Krzysztof J. , BILSKI Piotr , BUETTNER Garry R. , RASMUSSEN George T. , RAILSBACK Michelle A. , BRITIGAN Bradley E.
Published: August 2015

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca2+]i) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca2+]i response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca2+]i. Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca2+]i effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340nm) stimulates menadione-dependent formation of the DMPO/•OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca2+]i; and (c) light (maximum effect at 340nm) augments the deleterious effects of menadione on cell viability as determined by 51Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.
Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells
Authors: ISWANTINI Dyah , KANO Kenji , IKEDA Tokuji
Published: August 2015

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg2+. Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis–Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for d-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (kcat), which were determined to be 2.2×103 and 6.8±0.8×103s-1 (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (kf,PQQ) and with Mg2+ (kf,Mg) were determined to be 3.8±0.4×104 M-1·s-1 and 4.1±0.9M-1·s-1 respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg2+ were determined as the dissociation constants Kd,PQQ(Mg) and Kd,Mg to be 1.0±0.1nM and 0.14±0.01mM respectively. The dissociation constants for Ca2+ were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg2+ in solution. EDTA was effective to remove Mg2+ from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.
Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities
Authors: DOWLER Simon , CURRIE Richard A. , CAMPBELL David G. , DEAK Maria , KULAR Gursant , DOWNES C. Peter , ALESSI Dario R.
Published: August 2015

The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P3. The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P3. Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P3, but instead possess unexpected and novel phosphoinositide-binding specificitiesin vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P2 [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P2 (centaurin-β2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s).
Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells
Authors: TÖRNQUIST Kid , VAINIO Petri J. , BJÖRKLUND Sonja , TITIEVSKY Alexey , DUGUÉ Benoit , TUOMINEN Raimo K.
Published: August 2015

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca2+]i regulate several important functions, including the production of H2O2 (hydrogen peroxide). As H2O2 is of crucial importance for the production of thyroid hormones, we investigated the effects of H2O2 on [Ca2+]i in thyroid FRTL-5 cells. H2O2 itself did not modulate basal [Ca2+]i. However, H2O2 attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H2O2 was abrogated by the reducing agent β-mercaptoethanol. H2O2 also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H2O2 was, at least in part, mediated by activation of protein kinase C (PKC), as H2O2 enhanced the binding of [3H]phorbol 12,13-dibutyrate. H2O2 also stimulated the translocation of the isoenzyme PKCε from the cytosolic fraction to the particulate fraction. Furthermore, H2O2 did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H2O2 depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H2O2-evoked inhibition of calcium entry. In addition, H2O2 enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent β-mercaptoethanol. In conclusion H2O2 attenuates an increase in [Ca2+]i. As H2O2 is produced in thyroid cells in a calcium-dependent manner, our results suggest that H2O2 may participate in the regulation of [Ca2+]i in these cells via a negative-feedback mechanism involving activation of PKC.
A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp)
Authors: KONG Wei , SHIOTA Susumu , SHI Yixin , NAKAYAMA Hiroaki , NAKAYAMA Koji
Published: August 2015

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo.

The sequence data presented have been deposited in the GenBank/EMBL/DDBJ nucleotide sequence databases under the accession number AB037598.
The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1
Authors: SANDY John D. , THOMPSON Vivian , DOEGE Kurt , VERSCHAREN Christie
Published: August 2015

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val1–Glu1459 and Val1–Glu1274. These species were subsequently cleaved at the Glu373 site in the interglobular domain to form the terminal products, Val1–Glu373, Ala374–Glu1274 and Ala374–Glu1459. It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes insitu is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566–18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.
Characterization of the human liver fructose-1,6-bisphosphatase gene promoter
Authors: HERZOG Birger , WALTNER-LAW Mary , SCOTT Donald K. , ESCHRICH Klaus , GRANNER Daryl K.
Published: August 2015

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi. Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2,6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor κB (NF-κB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-κB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.
Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space
Authors: HAN Derick , WILLIAMS Everett , CADENAS Enrique
Published: August 2015

It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the intermembrane space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the intermembrane space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium trioxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the intermembrane space. The generation of superoxide anion towards the intermembrane space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.
Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects
Authors: WIESER Wolfgang , KRUMSCHNABEL Gerhard
Published: August 2015

The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells [Buttgereit and Brand (1995) Biochem. J. 312, 163-167]. Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived. We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na+/K+-ATPase, measuring their activity directly by [3H]leucine incorporation and Rb+-flux respectively. We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively). On the other hand, Na+/K+-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species. We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na+/K+-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na+/K+-ATPase of the two fish species. A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used. It was found that at 15mM cycloheximide [three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited] total oxygen consumption was suppressed by 71-75%, whereas the measured rate of [3H]leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx. 10% of the total oxygen consumption. On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium. Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a ‘hierarchy’ of ATP-consumers may reflect the lifestyle and physiology of the species studied.
Subcellular localization and adaptive up-regulation of the System A (SAT2) amino acid transporter in skeletal-muscle cells and adipocytes
Authors: HYDE Russell , CHRISTIE Graham R. , LITHERLAND Gary J. , HAJDUCH Eric , TAYLOR Peter M. , HUNDAL Harinder S.
Published: August 2015

The recently cloned amino acid transporter SAT2 is ubiquitously expressed and confers Na+-dependent transport of short-chain neutral amino acids, characteristics of the functionally defined System A transporter. Here we report the presence of SAT2 mRNA and protein in both skeletal muscle and adipocytes, and the characterization of polyclonal antibodies directed against this transporter. SAT2 protein was present in both plasma-membrane and internal-membrane fractions derived from rat skeletal muscle and adipose tissue, L6 myotubes and 3T3-L1 adipocytes, having a localization similar to that of the glucose transporter GLUT4. Moreover, consistent with the adaptive up-regulation of System A activity following chronic amino acid deprivation, a time-dependent increase in SAT2 protein abundance was observed in amino-acid-deprived L6 myotubes and 3T3-L1 adipocytes. These studies provide the first evidence regarding the subcellular distribution and adaptive up-regulation of SAT2 protein and the characterization of molecular probes for this physiologically important transporter, the function of which is altered in several disease states.
Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint
Authors: SÄÄMÄNEN Anna-Marja K. , SALMINEN Heli J. , RANTAKOKKO A. Juho , HEINEGÅRD Dick , VUORIO Eero I.
Published: August 2015

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7kb long cDNA contains an open reading frame of 1131nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.
The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bɛ at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase
Authors: WOODS Yvonne L. , COHEN Philip , BECKER Walter , JAKES Ross , GOEDERT Michel , WANG Xuemin , PROUD Christopher G.
Published: August 2015

The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the ε-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bε), which is inhibited by the GSK3-catalysed phosphorylation of Ser535. There is evidence that GSK3 is only able to phosphorylate eIF2Bε at Ser535 if Ser539 is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser539 have so far been identified. Here we show that Ser539 of eIF2Bε, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other ‘proline-directed’ protein kinases tested. We also establish that phosphorylation of Ser539 permits GSK3 to phosphorylate Ser535in vitro and that eIF2Bε is highly phosphorylated at Ser539in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr212in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr212 primes tau for phosphorylation by GSK3 at Ser208in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.
Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells
Authors: LAMB Maria E. , DE WEERD Willem F. C. , LEEB-LUNDBERG L. M. Fredrik
Published: August 2015

In this study, we analysed the agonist-promoted trafficking of human B2 (B2R) and B1 (B1R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B2R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B2R using radioligand binding and by imaging of B2R–GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B1R was sequestered to a considerably lesser extent upon binding of des-Arg10-kallidin. B2R sequestration was rapid (half-life ∼ 5min) and reached a steady-state level that was significantly lower than that of BK sequestration. B2R sequestration was minimally inhibited by K44A dynamin (22.4±3.7%), and was insensitive to arrestin-(319–418), which are dominant-negative mutants of dynamin I and β-arrestin respectively. Furthermore, the B2R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the β2-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2±16.3%) and by arrestin-(319–418) (36.9±4.4%). Our results show that B2R is sequestered to a significantly greater extent than is B1R upon agonist treatment in HEK293 cells. Furthermore, B2R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and β-arrestin-independent manner and, at least in part, involves caveolae.
Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase
Authors: GEISZT Miklós , DAGHER Marie-Claire , MOLNÁR Gergely , HAVASI Andrea , FAURE Julien , PACLET Marie-Hélène , MOREL Francoise , LIGETI Erzsébet
Published: August 2015

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [γ-32P]GTP-bound Rac revealed dominant GAP activity related to a 50kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.
Molten-globule structure and membrane binding of the N-terminal protease-resistant domain (63–193) of the steroidogenic acute regulatory protein (StAR)
Authors: SONG Maengseok , SHAO Haiyan , MUJEEB Anwer , JAMES Thomas L. , MILLER Walter L.
Published: August 2015

The first step in steroidogenesis is the movement of cholesterol from the outer to inner mitochondrial membrane; this movement is facilitated by the steroidogenic acute regulatory protein (StAR). StAR has molten-globule properties at low pH and a protease-resistant N-terminal domain at pH4 and pH8 comprising residues 63–193. To explore the mechanism of action of StAR we investigated the structural properties of the bacterially expressed N-terminal domain (63–193 StAR) using CD, limited proteolysis and NMR. Far- and near-UV CD showed that the amount of secondary structure was greater at acidic than at neutral pH, but there was little tertiary structure at any pH. Unlike 63–193 StAR liberated from N-62 StAR by proteolysis, biosynthetic 63–193 StAR was no longer resistant to trypsin or proteinase K at pH7, or to pepsin at pH4. Addition of trifluoroethanol and SDS increased secondary structure at pH7, and dodecylphosphocholine and CHAPS increased secondary structure at pH2, pH4 and pH7. However, none of these conditions induced tertiary structure, as monitored by near-UV CD or NMR. Liposomes of phosphatidylcholine, phosphatidylserine and their mixture increased secondary structure of 63–193 StAR at pH7, as monitored by far-UV CD, and stable protein–liposome complexes were identified by gel-permeation chromatography. These results provide further evidence that the N-terminal domain of StAR is a molten globule, and provide evidence that this conformation facilitates the interaction of the N-terminal domain of StAR with membranes. We suggest that this interaction is the key to understanding the mechanism of StAR's action.
Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor
Authors: PETRAT Frank , WEISHEIT Daniela , LENSEN Martina , de GROOT Herbert , SUSTMANN Reiner , RAUEN Ursula
Published: August 2015

Mitochondrial chelatable ('redox-active') iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe2+. RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2±4.9μM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.
Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility
Authors: BURTON David , ABDULRAZZAK Hassan , KNOTT Adam , ELLIOTT Kathryn , REDWOOD Charles , WATKINS Hugh , MARSTON Steven , ASHLEY Chris
Published: August 2015

We investigated the effects of two mutations in human cardiac troponin I, Arg145 → Gly and Gly203 → Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg145 → Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10–30% [Gly145]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly203 → Ser mutation and troponin C produced less Ca2+-activated force (64±8% of wild-type) and demonstrated lower Ca2+ sensitivity [ΔpCa50 (log of the Ca2+ concentration that gave 50% of maximal activation) 0.25 unit (P < 0.05)] compared with wild-type troponin I, but thin filaments containing [Ser203]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly203 → Ser mutation, or that the disease does not result directly from any particular alteration in contractility.
Regulation of novel protein kinase C ε by phosphorylation
Authors: CENNI Vittoria , DÖPPLER Heike , SONNENBURG Erica D. , MARALDI Nadir , NEWTON Alexandra C. , TOKER Alex
Published: August 2015

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.
Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions
Authors: ARDERIU Gemma , DÍAZ-RICART Maribel , BUCKLEY Byron , ESCOLAR Ginés , ORDINAS Antonio
Published: August 2015

After a vessel wall injury, platelets adhere to the subendothelium following a sequence of events: arrest of single platelets on the surface, progression to platelet spreading and final aggregation. Primary arrest of circulating platelets on subendothelial components occurs through platelet glycoprotein (GP) Ib and collagen receptors; then platelets spread and aggregate through a GPIIb-IIIa-dependent mechanism. A series of strategies were applied to analyse the tyrosine-phosphorylation mechanisms occurring at the different stages of platelet adhesion on subendothelial components under flow conditions, with special attention to primary arrest. To evaluate spread platelets, samples were exposed to acetylsalicylic acid, which blocks aggregate formation. To study single platelets in contact, a monoclonal antibody specific for GPIIb-IIIa was used to prevent platelet spreading and further aggregation. This experimental situation was also investigated using blood from two patients with Glanzmann's thrombasthenia (i.e. lacking GPIIb-IIIa). Results demonstrated that blockade of both spreading and aggregation results in significant changes in the tyrosine-phosphorylation patterns. Arrest of single platelets on collagen-rich surfaces resulted in phosphorylation of p125, identified as focal adhesion kinase (FAK), the 80/85kDa doublet (cortactin), and p72, identified as Syk. Arrest of single platelets on von Willebrand factor as adhesive substrate showed that interaction through GPIb induces Syk phosphorylation, but not that of cortactin and FAK. Our data indicate that the initial arrest of platelets on subendothelial components involves Syk phosphorylation, which seems to be GPIb-dependent, and this is followed by activation and phosphorylation of cortactin and FAK. These processes seem to occur before GPIIb-IIIa becomes activated.