bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamic and kinetic analysis of the Escherichia coli thioredoxin-C′ fragment complementation system
Authors: GHOSHAL Alokesh K. , SWAMINATHAN Chittoor P. , THOMAS Celestine J. , SUROLIA Avadhesha , VARADARAJAN Raghavan
Published: August 2015

Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a long α-helix. The two fragments, 1-37 and 38-108, were purified and characterized by using CD and fluorescence spectroscopy. Both fragments lack structure at neutral pH and room temperature. The secondary and tertiary structural contents of the non-covalent complex formed on the mixing of the two peptide fragments are 47% and 35% of the intact protein respectively. The thermodynamics and kinetics of fragment association were characterized by titration calorimetry and stopped-flow fluorescence spectroscopy. Single phases were observed for both association and dissociation, with rate constants at 298 K of kon = 4971±160 M-1·s -1 and koff = 0.063±0.009 s-1 respectively. The ratio kon/koff was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process. The values for ∆Cp, ∆H0 and ∆G0 at 298 K for dissociation of the complex were 5.7 kJ·mol-1·K-1, 45.3 kJ·mol-1 and 29.8 kJ·mol-1 respectively. The value for ∆H0 was linearly dependent on temperature from 8-40 °C, suggesting that ∆Cp is independent of temperature. The values for ∆Cp and ∆G0 are very similar to the corresponding values for the unfolding of intact thioredoxin at 25 °C. However, both ∆H0 and ∆S are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.
Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells
Authors: GUTACKER Claudia , KLOCK Gerd , DIEL Patrick , KOCH-BRANDT Claudia
Published: August 2015

Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression.
Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events
Authors: COPPOLINO Marc G. , DEDHAR Shoukat
Published: August 2015

As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the α3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, αv immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i.e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the α3β1-mediated adhesion of PC-3 cells to collagen IV and the α2β1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.
Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin
Authors: KONDOH Masuo , USUI Takeo , NISHIKIORI Takaaki , MAYUMI Tadanori , OSADA Hiroyuki
Published: August 2015

We reported previously that pironetin and its derivatives were potent inhibitors of cell cycle progression at the M-phase and showed antitumour activity against a murine tumour cell line, P388 leukaemia, transplanted in mice. In this paper, we investigated the mechanism of action of pironetins in antitumour activity and cell cycle arrest at the M-phase. As reported previously for murine leukaemia P388 cells, pironetin showed antitumour activity in a dose-dependent manner in the human leukaemia cell line HL-60. Since DNA fragmentation was observed in both P388 and HL-60 cells, the antitumour activity of pironetin is thought to be due to the induction of apoptosis. Pironetin also induced the rapid phosphorylation of Bcl-2 before formation of the DNA ladder in HL-60 cells, as seen with several tubulin binders. These results suggest that the antitumour activity of pironetin is due to apoptosis caused by the phosphorylation of Bcl-2, and that pironetin targets the microtubules. Pironetin and demethylpironetin exhibited reversible disruption of the cellular microtubule network in normal rat fibroblast 3Y1 cells. However, epoxypironetin, which contains epoxide instead of the double bond of pironetin, showed only weak activity. Since the concentrations that inhibit cell cycle progression at the M-phase were the same as those for disruption of the microtubule network, it was suggested that the mitotic arrest induced by pironetin was the result of the loss of the mitotic spindle. These compounds also inhibited the microtubule-associated protein-induced and glutamate-induced tubulin assembly in vitro. Pironetin inhibited the binding of [3H]vinblastine, but not that of [3H]colchicine, to tubulin, and the Kd values revealed that the affinity of pironetin for tubulin is stronger than that of vinblastine. These results suggest that pironetins are novel antitumour agents which inhibit microtubule assembly.
Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney
Authors: MAULET Yves , LAMBERT Régis C. , MYKITA Serge , MOUTON Jerôme , PARTISANI Maria , BAILLY Yannick , BOMBARDE Guy , FELTZ Anne
Published: August 2015

ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.
Chemical cleavage of the overexpressed mitochondrial F1β precursor with CNBr: a new strategy to construct an import-competent preprotein
Authors: PAVLOV Pavel F. , MOBERG Per , ZHANG Xiao-Ping , GLASER Elzbieta
Published: August 2015

We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F1β precursor of the ATP synthase (N15pF1β). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N15pF1β contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N15pF1β was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc1 complex of the respiratory chain. Addition of N15pF1β at micromolar concentrations resulted in the inhibition of import of F1β precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc1 complex. N15pF1β conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F1β precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide.
Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine
Authors: HUANG Qihong , LAU Serrine S. , MONKS Terrence J.
Published: August 2015

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-L-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of α-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.
Osteocalcin binds tightly to the γ-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins
Authors: HOUBEN Roger J. T. J. , JIN Dayun , STAFFORD Darrel W. , PROOST Paul , EBBERINK Rob H. M. , VERMEER Cees , SOUTE Berry A. M.
Published: August 2015

Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme γ-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Kmfor peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 μM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 μM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to γ-glutamylcarboxylase at a site different from the propeptide-recognition site.
Polyunsaturated fatty acids inhibit fatty acid synthase and spot-14-protein gene expression in cultured rat hepatocytes by a peroxidative mechanism
Authors: FORETZ Marc , FOUFELLE Fabienne , FERRÉ Pascal
Published: August 2015

In vivo, polyunsaturated fatty acids (PUFA) inhibit the expression of hepatic genes related to the lipogenic process such as fatty acid synthase and spot-14-protein (S14) genes. In vitro studies have suggested that this was a direct transcriptional effect of PUFA. In hepatocytes, the inhibition of the lipogenic rate by PUFA is not specific, but is linked to a cytotoxic effect due to peroxidative mechanisms. We have investigated whether peroxidation could also explain the inhibitory effect of PUFA on gene expression. Rat hepatocytes were cultured for 24 h with mono-unsaturated or PUFA. PUFA inhibited the expression of fatty acid synthase and S14 genes, and this inhibition was directly related to the number of unsaturations. However, the β-actin and albumin mRNA concentrations were also affected by the most unsaturated fatty acids, suggesting a non-specific effect of PUFA on gene expression. Measurement of lactate dehydrogenase released into the medium indicated a cytotoxicity of PUFA. This was associated with their peroxidation as evaluated by the presence of thiobarbituric acid-reactive substances in the culture medium. The addition of high concentrations of antioxidants abolished lipid peroxidation and lactate dehydrogenase leakage and completely reversed the inhibitory effect of PUFA on gene expression. This suggests (i) that the results obtained previously in cultured hepatocytes in the presence of low concentrations of antioxidants must be interpretated cautiously and (ii) that in vivo, the inhibitory effect of PUFA on lipogenesis-related genes could be indirect through hormonal or metabolic changes or that their effect on gene expression is somehow linked to peroxidative mechanisms.
Characterization of trehalose phosphorylase from Schizophyllum commune
Authors: EIS Christian , NIDETZKY Bernd
Published: August 2015

During growth on D-glucose, the basidiomycete Schizophyllum commune produces an intracellular α,α-trehalose phosphorylase. Specific phosphorylase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the available D-glucose in the medium has been fully depleted. The variation with time of the concentrations of intracellular α,α-trehalose and Pi is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of α,α-trehalose (approx. 0.42 mmol/g dry cell) via phosphorolysis. The enzyme has been purified, 150-fold, to homogeneity in 55% yield and characterized. It is a monomeric 61 kDa protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of α,α-trehalose into α-D-glucose 1-phosphate and α-D-glucose in the absence of cofactors, with a catalytic-centre activity at 30 °C of 14 s-1. Double-reciprocal analysis of the initial velocities for trehalose phosphorolysis and synthesis yields intersecting patterns, and no exchange reaction occurs between α-D-glucose 1-phosphate and the phosphate analogue arsenate. Therefore trehalose phosphorylase operates by a ternary-complex, rather than a Ping-Pong, kinetic mechanism. The specificity constants (kcat/Km) of phosphate (6000 M-1˙s-1) and α-D-glucose 1-phosphate (3500 M-1˙s-1) compared with those of α,α-trehalose (161 M-1˙s-1) and D-glucose (260 M-1˙s-1), together with the inhibition by NaCl, which is competitive with respect to phosphate with a Ki of 67 mM, suggest an important role for ionic enzyme-phosphate interactions in the catalytic mechanism of trehalose phosphorylase. The isolated enzyme requires α,α-trehalose (0.1-0.3 M) for its conformational stability.
S-Adenosylmethionine attenuates the lipopolysaccharide-induced expression of the gene for tumour necrosis factor α
Authors: WATSON Walter H. , ZHAO Yanming , CHAWLA Rajender K.
Published: August 2015

Intracellular deficiency of S-adenosylmethionine (AdoMet) and elevated serum concentrations of tumour necrosis factor α (TNF) are hallmarks of toxin-induced liver injury. In these models, the administration of either exogenous AdoMet or antibody/soluble receptor for TNF attenuates the injury. We have demonstrated previously that the administration of exogenous AdoMet to AdoMet-deficient rats attenuated lipopolysaccharide (LPS)-induced liver injury and serum TNF concentrations. Here we report that AdoMet lowered the amount of TNF secreted by LPS-stimulated murine macrophage cells (RAW 264.7) in a dose-dependent manner. The inhibition of TNF release was correlated with changes in the steady-state TNF mRNA concentrations. Changes in TNF mRNA were not due to its altered stability and might have been due to an attenuation of the transcription rate of the TNF gene. The inhibition of TNF release in RAW cells was not mediated by GSH because treatment with AdoMet did not increase intracellular GSH. In addition, N-acetylcysteine, whereas it did increase GSH concentration, had no effect on LPS-stimulated TNF release in these cells. Exogenous AdoMet also attenuated LPS-induced serum TNF levels in normal rats sensitized with lead. Thus AdoMet administration might exert its hepatoprotective effects at least in part by its inhibitory effect on expression of the gene for TNF.
Sulphation heterogeneity in the trisaccharide (GalNAcSβ1,4GlcAβ1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate
Authors: WEST Leigh A. , ROUGHLEY Peter , NELSON Fred R. T. , PLAAS Anna H. K.
Published: August 2015

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(β1,4)GlcA(β1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 °C with 30 μ-units of endo-chondroitinase ABC per μg of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sβ1,4GlcAβ1,3GalNAc4S, GalNAc4Sβ1,4GlcAβ1,3GalNAc6S, GalNAc4,6Sβ1,4GlcAβ1,3GalNAc4S and GalNAc4,6Sβ1,4GlcAβ1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.
Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd
Authors: VERHAERT Raymond M. D. , DUIN Jan VAN , QUAX Wim J.
Published: August 2015

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (α subunit-internal peptide-β subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the β-subunit is covalently linked and the α-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.
Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases
Authors: ARIMITSU Eiji , AOKI Shinya , ISHIKURA Syuhei , NAKANISHI Kumiko , MATSUURA Kazuya , HARA Akira
Published: August 2015

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.
Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases
Authors: LEIPER James M. , MARIA Joanne SANTA , CHUBB Ann , MACALLISTER Raymond J. , CHARLES Ian G. , WHITLEY Guy St. J. , VALLANCE Patrick
Published: August 2015

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3.18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.
Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human α1A-adrenoceptors
Authors: COGÉ Francis , GUENIN Sophie-Pénélope , RENOUARD-TRY Anne , RIQUE Hervé , OUVRY Christine , FABRY Nelly , BEAUVERGER Philippe , NICOLAS Jean-Paul , GALIZZI Jean-Pierre , BOUTIN Jean A. , CANET Emmanuel
Published: August 2015

We have identified from human liver eight α1A-adrenoceptor (α1A-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these α1A-AR isoforms (α1A-1-AR, α1A-2a-AR and α1A-3a-AR) gave rise to receptors with seven transmembrane domains (7TMα1A-AR). The other five (α1A-2b-AR, α1A-2c-AR, α1A-3c-AR, α1A-5-AR and α1A-6-AR) led to truncated receptors lacking transmembrane domain VII (6TMα1A-AR). The 7TMα1A-AR isoforms transiently expressed in COS-7 cells bound [3H]prazosin with high affinity (Kd 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca2+ concentration, whereas the 6TMα1A-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged α1A-AR isoforms showed that the 7TMα1A-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMα1A-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated α1A-AR isoform inhibited [3H]prazosin binding and cell-surface trafficking of the co-expressed ‘original’ 7TMα1A-1-AR. However, there was no modification of either the [3H]prazosin-binding affinity or the pharmacological properties of α1A-1-AR. Immunoblotting experiments revealed that co-expression of the α1A-1-AR with 6TMα1A-AR isoforms did not impair α1A-1-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of α1A-AR.
Modification of luciferase to be a substrate for plant aspartic proteinase
Authors: AMIDON Wendy J. , PFEIL Joanne E. , GAL Susannah
Published: August 2015

The possibility of using firefly luciferase as a substrate for an aspartic proteinase was explored. Several amino acid modifications to the C-terminus of the luciferase were created on the basis of the known substrate of the Arabidopsis thaliana aspartic proteinase, pro-(barley lectin). One luciferase with the sequence Arg-Asp-Gly-Val-Phe-Ala-Ala instead of the native Arg-Glu-Ile-Leu-Ile-Lys-Ala at position -15 to -9 relative to the C-terminus of native luciferase was found to possess 17% of the original luciferase activity. When this modified luciferase was incubated with the aspartic proteinase, a specific loss in activity occurred that was not observed with the original luciferase. However, both enzymes seemed very sensitive to the acidic conditions required for aspartic proteinase activity. The other versions of luciferase with different numbers of pro-(barley lectin) amino acids were not active luciferases. This provided information on the structural requirements of the C-terminal portion of the protein for luciferase activity. The luciferase proteins were also monitored during the digestion by using Western blots and some were shown to be substrates for the aspartic proteinase. Contrary to what had been expected, the modified luciferase that incorporated the pro-(barley lectin) sequences was not simply cleaved at the engineered site but at additional positions in the protein. The Arabidopsis aspartic proteinase cleaved two other standard protein substrates at many sites, suggesting that this proteinase could have a role in the degradation of proteins in addition to processing propeptides in plants.
Molecular modelling indicates that the pathological conformations of prion proteins might be β-helical
Authors: DOWNING Donald T. , LAZO N. D.
Published: August 2015

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly α-helical and random-coil conformations, whereas the prion isoform is largely in the β conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the β-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The β-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases.
Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12
Authors: POTTER Laura C. , COLE Jeffrey A.
Published: August 2015

The seven nap genes at minute 47 on the Escherichia coli K-12 chromosome encode a functional nitrate reductase located in the periplasm. The molybdoprotein, NapA, is known to be essential for nitrate reduction. We now demonstrate that the two c-type cytochromes, the periplasmic NapB and the membrane-associated NapC, as well as a fourth polypeptide, NapD, are also essential for nitrate reduction in the periplasm by physiological substrates such as glycerol, formate and glucose. None of the three iron-sulphur proteins, NapF, NapG or NapH, are essential, irrespective of whether the bacteria are grown anaerobically in the presence of nitrate or fumarate as a terminal electron acceptor, or by glucose fermentation. Mutation of napD resulted in the total loss of Methyl Viologen-dependent nitrate reductase activity of the molybdoprotein, NapA, consistent with an earlier suggestion by others that NapD might be required for post-translational modification of NapA.
Haem oxygenase shows pro-oxidant activity in microsomal and cellular systems: implications for the release of low-molecular-mass iron
Authors: LAMB Nicholas J. , QUINLAN Gregory J. , MUMBY Sharon , EVANS Timothy W. , GUTTERIDGE John M. C.
Published: August 2015

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.
Transport activity of AE3 chloride/bicarbonate anion-exchange proteins and their regulation by intracellular pH
Authors: STERLING Deborah , CASEY Joseph R.
Published: August 2015

Plasma membrane Cl-/HCO3- anion-exchange (AE) proteins contribute to regulation of intracellular pH (pHi). We characterized the transport activity and regulation by pHi of full-length AE3 and the cardiac isoform, AE3c, both of which are expressed in the heart. AE3c is an N-terminal variant of AE3. We also characterized AE1, AE2 and a deletion construct (AE3tr) coding for the common region of AE3 and AE3c. AE proteins were expressed by transient transfection of HEK-293 cells, and transport activity was monitored by following changes of intracellular pH or intracellular chloride concentration associated with anion exchange. Transport activities, measured as proton flux (mM H+˙min-1), were as follows: AE1, 24; AE2, 32; full-length AE3, 9; AE3c, 4 and AE3tr, 4. The wide range of transport activities is not explained by variation of cell surface processing since approx. 30% of each isoform was expressed on the cell surface. pHi was clamped at a range of values from 6.0-9.0 to examine regulation of AE proteins by pHi. Whereas AE2 was steeply inhibited by acid pHi, AE1, AE3 and AE3c were essentially insensitive to changes of pHi. We conclude that AE3 and AE3c can contribute to pHi recovery after cellular-acid loading.
Cloning and characterization of two nuclear receptors from the filarial nematode Brugia pahangi
Authors: MOORE Joyce , DEVANEY Eileen
Published: August 2015

Nuclear receptors (NRs) encompass a superfamily of cytoplasmic/nuclear localized receptors that on ligand binding (or by phosphorylation) directly regulate the transcription of target genes. NRs are involved in many developmental processes, including moulting in insects and dauer larva formation in Caenorhabditis elegans. Here we report the isolation of two genes related to NRs from the filarial nematode Brugia pahangi. Bp-nhr-1 is a member of the NGF1-B subfamily of NRs and is expressed at very low levels in post-infective larval stage 3 (L3) after their transmission to the mammalian host. The second gene, Bp-nhr-2, is related to XR78E/F of Drosophila, a gene involved in the ecdysone response, over the region of its DNA-binding domain. cDNA and genomic clones have been isolated that correspond to Bp-nhr-2. The most striking feature of the encoded protein is that, although there is a DNA-binding domain similar to that of other NRs, the ligand-binding domain is absent. To investigate the pattern of transcription of Bp-nhr-2 in the filarial life cycle, semi-quantitative reverse-transcriptase-mediated PCR was performed; this analysis demonstrated that the gene is expressed in early stages after infection and in the adult and microfilariae, and is up-regulated just before the moult between L3 and L4 but is not expressed during the moult between L4 and adult. Antibodies raised against a peptide corresponding to the transactivation domain of Bp-nhr-2 demonstrate that the protein is expressed in microfilariae and adult samples and that another cross-reactive protein is expressed in these life-cycle stages.
Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2
Authors: BUSCHBECK Marcus , GHOMASHCHI Farideh , GELB Michael H. , WATSON Steve P. , BÖRSCH-HAUBOLD Angelika G.
Published: August 2015

Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H2O2 stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H2O2 and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H2O2, sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H2O2 and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H2O2 and sorbitol increased phosphorylation of cytosolic phospholipase A2 (cPLA2) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA2 by H2O2 occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA2 activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.
Phosphorylation of the rat pancreatic bile-salt-dependent lipase by casein kinase II is essential for secretion
Authors: PASQUALINI Eric , CAILLOL Nathalie , VALETTE Anne , LLOUBES Roland , VERINE Alain , LOMBARDO Dominique
Published: August 2015

Bile-salt-dependent lipase (BSDL, EC 3.1.1.-) is an enzyme expressed by the pancreatic acinar cells and secreted as a component of the pancreatic juice of all examined species. During its secretion route BSDL is associated with intracellular membranes. This association allows the complete glycosylation of the enzyme or participates in the inhibition of the enzyme activity, which can deleterious for the acinar pancreatic cell. Thereafter, the human BSDL is phosphorylated by a serine/threonine protein kinase and released from intracellular membranes. In the present study, we show that the rat pancreatic BSDL, expressed by AR4-2J cells used as a model, is phosphorylated by a protein kinase that is insensitive to inhibitors of protein kinases A, C or G and that the phosphorylation process is favoured by okadaic acid (an inhibitor of protein phosphatases 1 and 2A). However, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), which is a specific inhibitor of casein kinase II, abolishes the phosphorylation in vitro of BSDL within micro- somes of AR4-2J pancreatic cells. We showed further that the α-subunit of casein kinase II co-locates with BSDL within the lumenal compartment of the Golgi. Genistein, which perturbs the trans-Golgi network, also inhibits the phosphorylation of BSDL, suggesting that this post-translational modification of BSDL probably occurred within this cell compartment. The inhibition of the phosphorylation of BSDL by DRB also decreases the rate at which the enzyme is secreted. Under the same conditions, the rate of α-amylase secretion was not modified. These data strongly suggest that phosphorylation is a post-translational event, which appears to be essential for the secretion of BSDL.
A change of the metal-specific activity of a cambialistic superoxide dismutase from Porphyromonas gingivalis by a double mutation of Gln-70 to Gly and Ala-142 to Gln
Authors: HIRAOKA B. Yukihiro , YAMAKURA Fumiyuki , SUGIO Shigetoshi , NAKAYAMA Koji
Published: August 2015

Gln-70, which is located near the active-site metal, is conserved in aligned amino acid sequences of iron-containing superoxide dimutases (Fe-SODs) and cambialistic SOD from Porphyromonas gingivalis, but is complementarily substituted with Gln-142 in manganese-containing SODs (Mn-SODs). In order to clarify the contribution of this exchange of Gln to the metal-specific activity of P. gingivalis SOD, we have prepared a mutant of the enzyme with conversions of Gln-70 to Gly and Ala-142 to Gln. The ratio of the specific activities of Mn- to Fe-reconstituted P. gingivalis SOD increased from 1.4 in the wild-type to 3.5 in the mutant SODs. Furthermore, the visible absorption spectra of the Mn- and Fe-reconstituted mutant SODs more closely resembled that of Mn-specific SOD than that of the wild-type SOD. We conclude that a difference in configuration of the Gln residues of P. gingivalis SOD partially accounts for the metal-specific activity of the enzyme.
Phospholipase D: molecular and cell biology of a novel gene family
Authors: LISCOVITCH Mordechai , CZARNY Malgorzata , FIUCCI Giusy , TANG Xiaoqing
Published: August 2015

Interaction of extracellular-signal molecules with cell-surface receptors often activates a phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine and other phospholipids, generating phosphatidic acid. The activation of PLD is believed to play an important role in the regulation of cell function and cell fate. Multiple PLD activities were characterized in eukaryotic cells, and, more recently, several PLD genes have been cloned. A PLD gene superfamily, defined by a number of structural domains and sequence motifs, also includes phosphatidyltransferases and certain phosphodiesterases. Among the eukaryotic PLD genes are those from mammals, nematodes, fungi and plants. The present review focuses on the structure, localization, regulation and possible functions of cloned mammalian and yeast PLDs. In addition, an overview of plant PLD genes, and of several distinct PLD activities that have not yet been cloned, is provided. Emerging evidence from recent work employing new molecular tools indicates that different PLD isoforms are localized in distinct cellular organelles, where they are likely to serve diverse functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.
Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent
Authors: SAVINI Isabella , DUFLOT Sylvie , AVIGLIANO Luciana
Published: August 2015

Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.
Purification, characterization and gene cloning of two α-l-arabinofuranosidases from Streptomyces chartreusis GS901
Authors: MATSUO Noriki , KANEKO Satoshi , KUNO Atsushi , KOBAYASHI Hideyuki , KUSAKABE Isao
Published: August 2015

α-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. α-L-Arabinofuranosidase I hydrolysed all of the α-linkage types that normally occur between two α-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: α-(1 → 2) α-(1 → 3) α-(1 → 5). This enzyme cleaved the (1 → 3) linkages of the arabinosyl side-chains of methyl 3,5-di-O-α-L-arabinofuranosyl-α-L-arabinofuranoside in preference to the (1 → 5) linkages. α-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, α-L-Arabinofuranosidase II hydrolysed only (1 → 5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1 → 5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of α-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of α-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1,5-α-L-arabinofuranosidase, it represents a novel type of enzyme.