bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry
Authors: WISNOSKEY Brian J. , SINKINS William G. , SCHILLING William P.
Published: June 2003

To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca2+ signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf 9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf 9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca2+ concentration ([Ca2+]i), with EC50 values of 166 pM, 24.5 nM and 3.89 μM respectively. In the absence of extracellular Ca2+, both capsaicin and RTX caused an increase in [Ca2+]i with EC50 values of approx. 10 μM and 10 nM respectively. This TRPV1-induced release of Ca2+ from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin- and RTX-sensitive internal Ca2+ pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca2+ activates endogenous store-operated Ca2+ entry, the effect of 2-aminoethoxydiphenyl borate (2-APB) on Ba2+ influx was examined. 2-APB blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores, but fail to activate endogenous store-operated Ca2+ entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca2+ influx mechanisms, could play an important role in Ca2+ signalling within specific subcellular microdomains.
Protective effect of creatine against inhibition by methylglyoxal of mitochondrial respiration of cardiac cells
Authors: ROY Soumya Sinha , BISWAS Swati , RAY Manju , RAY Subhankar
Published: June 2003

Previous publications from our laboratory have shown that methylglyoxal inhibits mitochondrial respiration of malignant and cardiac cells, but it has no effect on mitochondrial respiration of other normal cells [Biswas, Ray, Misra, Dutta and Ray (1997) Biochem. J. 323, 343–348; Ray, Biswas and Ray (1997) Mol. Cell. Biochem. 171, 95–103]. However, this inhibitory effect of methylglyoxal is not significant in cardiac tissue slices. Moreover, post-mitochondrial supernatant (PMS) of cardiac cells could almost completely protect the mitochondrial respiration against the inhibitory effect of methylglyoxal. A systematic search indicated that creatine present in cardiac cells is responsible for this protective effect. Glutathione has also some protective effect. However, creatine phosphate, creatinine, urea, glutathione disulphide and β-mercaptoethanol have no protective effect. The inhibitory and protective effects of methylglyoxal and creatine respectively on cardiac mitochondrial respiration were studied with various concentrations of both methylglyoxal and creatine. Interestingly, neither creatine nor glutathione have any protective effect on the inhibition by methylglyoxal on the mitochondrial respiration of Ehrlich ascites carcinoma cells. The creatine and glutathione contents of several PMS, which were tested for the possible protective effect, were measured. The activities of two important enzymes, namely glyoxalase I and creatine kinase, which act upon glutathione plus methylglyoxal and creatine respectively, were also measured in different PMS. Whether mitochondrial creatine kinase had any role in the protective effect of creatine had also been investigated using 1-fluoro-2,4-dinitrobenzene, an inhibitor of creatine kinase. The differential effect of creatine on mitochondria of cardiac and malignant cells has been discussed with reference to the therapeutic potential of methylglyoxal.
Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells
Authors: LAMBERTINI Elisabetta , PENOLAZZI Letizia , GIORDANO Silvia , DEL SENNO Laura , PIVA Roberta
Published: June 2003

(O)estrogen receptor-α (ERα), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERα gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at −117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of γ-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PFa and PFb, that do not present binding sites for known transcription factors and that are involved in a strong DNA–protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERα gene expression in bone. These data provide evidence for different promoter usage of the ERα gene through the recruitment of tissue-specific transcription activators and co-regulators.
Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases
Authors: SMEDS Emanuel , HABUCHI Hiroko , DO Anh-Tri , HJERTSON Eva , GRUNDBERG Helena , KIMATA Koji , LINDAHL Ulf , KUSCHE-GULLBERG Marion
Published: June 2003

Glycosaminoglycan heparan sulphate interacts with a variety of proteins, such as growth factors, cytokines, enzymes and inhibitors and, thus, influences cellular functions, including adhesion, motility, differentiation and morphogenesis. The interactions generally involve saccharide domains in heparan sulphate chains, with precisely located O-sulphate groups. The 6-O-sulphate groups on glucosamine units, supposed to be involved in various interactions of functional importance, occur in different structural contexts. Three isoforms of the glucosaminyl 6-O-sulphotransferase (6-OST) have been cloned and characterized [H. Habuchi, M. Tanaka, O. Habuchi, K. Yoshida, H. Suzuki, K. Ban and K. Kimata (2000) J. Biol. Chem. 275, 2859–2868]. We have studied the substrate specificities of the recombinant enzymes using various O-desulphated poly- and oligo-saccharides as substrates, and using adenosine 3′-phosphate 5′-phospho[35S]sulphate as sulphate donor. All three enzymes catalyse 6-O-sulphation of both -GlcA-GlcNS- and -IdoA-GlcNS- (where GlcA represents d-glucuronic acid, NS the N-sulphate group and IdoA the l-iduronic acid) sequences, with preference for IdoA-containing targets, with or without 2-O-sulphate substituents. 6-OST1 showed relatively higher activity towards target sequences lacking 2-O-sulphate, e.g. the -GlcA-GlcNS- disaccharide unit. Sulphation of such non-O-sulphated acceptor sequences was generally favoured at low acceptor polysaccharide concentrations. Experiments using partially O-desulphated antithrombin-binding oligosaccharide as the acceptor revealed 6-O-sulphation of N-acetylated as well as 3-O-sulphated glucosamine residues with each of the three 6-OSTs. We conclude that the three 6-OSTs have qualitatively similar substrate specificities, with minor differences in target preference.
Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position −107, implicating the Sp1 transcription factor
Authors: DEAKIN Sara , LEVIEV Ilia , BRULHART-MEYNET Marie-Claude , JAMES Richard W.
Published: June 2003

Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P<0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (−107, −824 and −907), the −107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the −107 site (−107C) alone. No significant contribution was observed for the −824 and −907 sites. The coding-region Leu55→Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the −107 site. A molecular basis for the effect of the −107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (−107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(−107)T polymorphism for gene expression in vivo. Differences in the affinity of the −107C and −107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the −107 polymorphism.
Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma
Authors: FINI Carlo , TALAMO Fabio , CHERRI Silvia , COLI Marcello , FLORIDI Ardesio , FERRARA Lino , SCALONI Andrea
Published: June 2003

Ecto-5′-nucleotidase (ecto-5′-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5′-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln547 and/or Phe548. Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn403. Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5′-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.
Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA
Authors: McLEAN Kirsty J. , SCRUTTON Nigel S. , MUNRO Andrew W.
Published: June 2003

The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent Km for NADH=50.6±3.1 μM; NADPH=4.1±0.3 μM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 μM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964–1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate (kred)=25.4±0.7 s−1, apparent Kd=42.9±4.6 μM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is −235±5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (−274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005–3013].
Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering
Authors: CALVETE Juan J. , MORENO-MURCIANO M. Paz , THEAKSTON R. David G. , KISIEL Dariusz G. , MARCINKIEWICZ Cezary
Published: June 2003

We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin α5β1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the α4β1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both α5β1 and α4β1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (adisintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.
The random-coil ‘C’ fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors
Authors: HAARMANN Claudia S. , GREEN Daniel , CASAROTTO Marco G. , LAVER Derek R. , DULHUNTY Angela F.
Published: June 2003

The actions of peptide C, corresponding to 724Glu–Pro760 of the II–III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM–10 μM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 μM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5×105 s−1·M−1 was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9×103 s−1·M−1. The combined actions of peptide C and peptide A (II–III loop sequence 671Thr–Leu690) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation–contraction coupling or interactions between the proteins at rest.
Polymorphic glutathione S-transferase subunit 3 of rat liver exhibits different susceptibilities to carbon tetrachloride: differences in their interactions with heat-shock protein 90
Authors: MAYAMA Jun , KUMANO Takayuki , HAYAKARI Makoto , YAMAZAKI Takehiko , AIZAWA Shu , KUDO Toshihiro , TSUCHIDA Shigeki
Published: June 2003

Rat glutathione S-transferase (GST) subunit 3 gene has polymorphism, one type encoding Asn198-Cys199 (NC type) and another encoding Lys198-Ser199 (KS type). To examine whether the two types of GST 3-3 exhibit different susceptibilities to oxidative stress in vivo, rats were administered with CCl4, a hepatotoxin causing severe oxidative stress, and its effect on liver GST 3-3 was compared. Decrease in GST activities in liver due to CCl4 administration was more evident in NC type rats than in KS type rats, and most GST activities of KS type rats were confined to S-hexylglutathione–Sepharose, whereas those of NC type rats were not. Decreases in GST subunits 1 and 3 were more marked in NC type rats and glutathiolated NC type GST 3-3 was also detected. These results indicated that KS and NC type GST 3-3 of rat livers exhibited different susceptibilities to CCl4in vivo. A protein consisting of a subunit with molecular mass of 90 kDa was shown to bind to KS type GST 3-3 but not to NC type. This protein was identified as heat-shock protein (HSP) 90β by N-terminal amino acid sequencing and immunoblotting. A specific HSP90 inhibitor geldanamycin released their binding. There was no difference in the binding of apoptosis signal-regulating kinase 1 to GST 3-3 between NC and KS type rats. These findings suggest that HSP90 interacts with KS type GST 3-3 and thereby protects it from inactivation due to CCl4.
Cobalt activation of Escherichia coli 5'-nucleotidase is due to zinc ion displacement at only one of two metal-ion-binding sites
Authors: McMILLEN Lyle , BEACHAM Ifor R. , BURNS Dennis M.
Published: June 2003

Escherichia coli 5′-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co2+. Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E. coli 5′-nucleotidase (Asp41, His43, Asp84, His117, Glu118, His217 and His252) were selected for modification using site-directed mutagenesis of the cloned ushA gene. On the basis of comparative studies between the resultant mutant proteins and the wild-type enzyme, a model is proposed for E. coli 5′-nucleotidase in which a Co2+ ion may displace the Zn2+ ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn2+ ion already present. The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp84, Asn116, His217 and His252, leading to the observed increase in 5′-nucleotidase activity.
Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor
Authors: TARDIF Marianne , BROUCHON Laurence , RABIET Marie-Josèphe , BOULAY François
Published: June 2003

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott–Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1–321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active ‘open conformation’, the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.
Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands
Authors: PEDERSEN Katrine E. , EINHOLM Anja P. , CHRISTENSEN Anni , SCHACK Lotte , WIND Troels , KENNEY John M. , ANDREASEN Peter A.
Published: June 2003

Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop–sheet polymerization, in which the reactive centre loop of one molecule binds to β-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general.
Tyrosine phosphorylation of protein kinase CK2 by Src-related tyrosine kinases correlates with increased catalytic activity
Authors: DONELLA-DEANA Arianna , CESARO Luca , SARNO Stefania , RUZZENE Maria , BRUNATI Anna Maria , MARIN Oriano , VILK Greg , DOHERTY-KIRBY Amanda , LAJOIE Gilles , LITCHFIELD David W. , PINNA Lorenzo A.
Published: June 2003

Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (α and/or α´) and two regulatory β-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory β-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2α with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2α becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2α do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2α that had been phosphoradiolabelled by incubation with [32P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248–268 sequence, phosphorylated previously by c-Fgr at Tyr255in vitro. The identification of Tyr255 as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248–268 tryptic fragments from CK2α and by on-target phosphatase treatment. A CK2α mutant in which Tyr255 was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.
Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA
Authors: PATTON Jeffrey R. , PADGETT Richard W.
Published: June 2003

The formation of pseudouridine (Ψ) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Ψ were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNAVal (AAC), a putative Pus1p-modification site, was converted into Ψ in the wild-type worms, but the tRNAVal (AAC) from mutant worms lacked the modification. Ψ formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Ψ at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.
Daunorubicin-induced variations in gene transcription: commitment to proliferation arrest, senescence and apoptosis
Authors: MANSILLA Sylvia , PIÑA Benjamin , PORTUGAL José
Published: June 2003

We used a human cDNA macroarray containing various oncogenes and tumour suppressor genes to assess gene expression profiles in early-passage Jurkat T lymphocytes treated with clinically relevant concentrations of the antitumour antibiotic daunorubicin. Several oncogenes and tumour suppressor genes were either up- or down-regulated depending on the daunorubicin concentration used. The expression levels of some of these genes were confirmed by semi-quantitative reverse transcriptase–PCR. We also compared the changes in cell-cycle distribution and the apoptotic morphological characteristics of the cells treated with daunorubicin, using flow cytometry and fluorescence microscopy. Exposure to 182 nM daunorubicin (its IC75 in Jurkat T cells: where IC75 is the drug concentration that inhibits growth by 75%) resulted in cell-cycle arrest in G1 and almost immediate apoptosis. In contrast, decreasing the drug concentration to 91 nM (close to the IC50) caused G2 arrest and cell senescence-like growth arrest, whereas features of apoptosis and necrosis appeared only after longer incubation times. Gene expression profiles, cell-cycle distribution, the presence of DNA damage and the time-dependent response of Jurkat T cells to cell death were correlated clearly. The general behaviour of the genes suggests that cell-cycle arrest and cell death follow distinct pathways depending on drug concentration.
The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells
Authors: LIDELL Martin E. , JOHANSSON Malin E. V. , MÖRGELIN Matthias , ASKER Noomi , GUM James R. , KIM Young S. , HANSSON Gunnar C.
Published: June 2003

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse–chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.
Differential functional properties of calmodulin-dependent protein kinase IIgamma variants isolated from smooth muscle
Authors: GANGOPADHYAY Samudra S. , BARBER Amy L. , GALLANT Cynthia , GRABAREK Zenon , SMITH Janet L. , MORGAN Kathleen G.
Published: June 2003

Six variants of calmodulin-dependent protein kinase IIγ were isolated from a ferret-aorta smooth-muscle cDNA library. Variant G-2 is generated by a novel alternative polyadenylation, utilizing a site contained in an intron. The last 77 residues of the association domain are replaced with 99 residues of a unique sequence containing Src homology 3-domain-binding motifs, which alter catalytic activity. Variant C-2 has an eight-residue deletion in an ATP-binding motif and does not autophosphorylate Thr286, but does phosphorylate exogenous substrate. Two variants, B and J, autodephosphorylate. Four variants differing only in the variable domain have differing catalytic activities, despite identical sequences in the catalytic domains. Thus structural features determined by variable and association domains are important for the catalytic activity of calmodulin-dependent protein kinase II.
Molecular characterization of a male-specific glycosyl hydrolase, Lma-p72, secreted on to the abdominal surface of the Madeira cockroach Leucophaea maderae (Blaberidae, Oxyhaloinae)
Authors: CORNETTE Richard , FARINE Jean-Pierre , ABED-VIELLARD Dehbia , QUENNEDEY Brigitte , BROSSUT Rémy
Published: June 2003

The epicuticular surface protein Lma-p72 is specific to the abdominal secretions of Leucophaea maderae (Madeira cockroach) adult males. Natural Lma-p72 was purified and the complete cDNA sequence determined by reverse-transcription PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p72 was expressed in the tergal and sternal glands. Sequence alignment indicates that Lma-p72 is closely related to the family 1 glycosyl hydrolases (EC 3.2.1). Native Lma-p72 was proved to be active in the abdominal secretions and exhibit a β-galactosidase-like activity. However, weak specificity with respect to the C-4 configuration of the substrate was observed. Two main hypotheses were proposed concerning the function of this enzyme: Lma-p72 could hydrolyse oligosaccharides from the male abdominal secretions, making them more phagostimulatory for the female during the precopulatory behaviour. The protein could also cleave a pheromone–sugar conjugate to release the pheromonal compounds on to the cuticular surface. Such a sugar conjugate could be a transport form. Data from the first in vivo inhibition tests indicate that a glycosidase could be directly involved in the production process of some pheromonal compounds in L. maderae males.
A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction
Authors: VRZHESHCH Peter V. , BATANOVA Elena A. , MEVKH Alevtina T. , VARFOLOMEEV Sergei D. , GAZARYAN Irina G. , THORNELEY Roger N. F.
Published: June 2003

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector ‘kinetic connectivities’ between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H2O2. To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I–adrenaline complex.
Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells
Authors: LINDEMANN Ralph K. , BRAIG Melanie , HAUSER Craig A. , NORDHEIM Alfred , DITTMER Jürgen
Published: June 2003

Parathyroid hormone-related protein (PTHrP) promotes the metastatic potential and proliferation of breast cancer cells, and acts anti-apoptotically. In invasive MDA-MB-231 breast cancer cells, transforming growth factor β-regulated PTHrP synthesis is mediated by an Ets1/Smad3-dependent activation of the PTHrP P3 promoter. In the present study, we studied the regulation of PTHrP expression in non-invasive, Ets1-deficient and transforming growth factor β-resistant MCF-7 cells. We found PMA to be a strong stimulator of P3-dependent PTHrP expression in MCF-7 cells. Mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase 1 (MEK-1)/ERK1/2 inhibitor PD98059 interfered with this activity. Promoter studies revealed that the PMA effect depended on the Ets and stimulating protein-1 (Sp1)-binding sites. Of several Ets factors tested, Ets2, but not Ese-1, Elf-1 or Ets1, supported the PMA-dependent increase in promoter activity. PD98059 and a threonine to alanine mutation of the ERK1/2-responsive Ets2 phosphorylation site at position 72 inhibited the Ets2/PMA effect. Activated protein kinase C (PKC)ε could mimic PMA by stimulating the P3 promoter alone or in co-operation with Ets2 in an MEK-1/ERK1/2-dependent manner. Activated PKCα, although capable of co-operating with Ets2, failed to induce transcription from the P3 promoter on its own. The Ets2/PKCα synergistic effect was neither sensitive to PD98059 nor to Thr72/Ala72 mutation. PMA neither increased the expression of Sp1 nor modulated the transcriptional activity of Sp1. However, it induced the displacement of a yet unknown factor from the Sp1-binding site, which may result in Sp1 recruitment to the promoter. Our results suggest an ERK1/2-dependent Ets2/PKCε synergism to be involved in PTHrP expression in MCF-7 breast cancer cells.
Leucine-zipper-mediated homo- and hetero-dimerization of GIT family p95-ARF GTPase-activating protein, PIX-, paxillin-interacting proteins 1 and 2
Authors: PARIS Simona , LONGHI Renato , SANTAMBROGIO Paolo , de CURTIS Ivan
Published: June 2003

ADP-ribosylation factor GTPase-activating proteins (ARFGAPs) of the G-protein-coupled receptor kinase interactor 1/p95 paxillin kinase linker/p95-ARFGAP Pak-interacting exchange factor paxillin-binding protein (APP)-1 family are multidomain proteins, which interact functionally with both ARF and Rac GTPases. These proteins are involved in the dynamic reorganization of adhesion and the cytoskeleton during cell motility. Our previous work [Di Cesare, Paris, Albertinazzi, Dariozzi, Andersen, Mann, Longhi and de Curtis (2000) Nat. Cell Biol. 2, 521–530] has pointed out a role for p95-APP1 in the regulation of ARF6-mediated membrane recycling. These proteins include different domains, and are capable of interacting stably with proteins that are supposed to play a role in the regulation of actin dynamics and adhesion. They contain a coiled-coil region comprising a putative leucine zipper, predicted to be involved in dimerization. In the present study, we have investigated the possibility that these proteins form dimers. Our results show that p95-APP1 forms homodimers and may also form heterodimers with the other member of the family, p95 paxillin kinase linker/p95-APP2. Both homo- and heterodimerization are disrupted by mutation of two leucine residues in the coiled-coil region of p95-APP1. The N-terminal portion of p95-APP1, including the ARFGAP domain, three ankyrin repeats and the Pak-interacting exchange factor-binding region, are not required for dimerization. Evidence is presented for the existence of endogenous oligomeric complexes. The implication of dimerization/oligomerization in the functioning of these proteins is discussed.
The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb
Authors: MATRE Vilborg , HØVRING Per I. , FJELDHEIM Åse-Karine , HELGELAND Lars , ORVAIN Christophe , ANDERSSON Kristin B. , GAUTVIK Kaare M. , GABRIELSEN Odd S.
Published: June 2003

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb–Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.
Arachidonate release and prostaglandin production by group IVC phospholipase A2 (cytosolic phospholipase A2gamma)
Authors: MURAKAMI Makoto , MASUDA Seiko , KUDO Ichiro
Published: June 2003

While the role of the group IVA Ca2+-dependent cytosolic phospholipase A2α (cPLA2α) in arachidonic acid (AA) metabolism has been well documented, that of its paralogue, Ca2+-independent group IVC PLA2 (cPLA2γ), has remained uncertain. Here we show, using a transfection strategy, that cPLA2γ has the ability to increase the spontaneous and stimulus-induced release of cellular fatty acids. The AA released by cPLA2γ was metabolized further to prostaglandin E2 via cyclo-oxygenase-1 (COX-1) in the immediate response, and via COX-2 in the delayed response. Mutation of the putative catalytic-centre residue Ser82 abrogated the AA-releasing function of cPLA2γ both in vitro and in vivo. Confocal microscopy revealed that cPLA2γ was distributed in the perinuclear endoplasmic reticulum membranes. Mutating the C-terminal prenylation site of cPLA2γ abrogated its intracellular membrane localization and cellular AA-releasing function, without reducing its enzyme activity in vitro. Our results indicate that cPLA2γ is the second cPLA2 enzyme that contributes to cellular AA metabolism and phospholipid remodelling under appropriate conditions.
Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter
Authors: LI Hongyan , LI Fei , SUN Hongzhe , QIAN Zhong Ming
Published: June 2003

Divalent-metal transporter 1 (DMT1) is involved in the intestinal iron absorption and in iron transport in the transferrin cycle. It transports metal ions at low pH (≈5.5), but not at high pH (7.4), and the transport is a proton-coupled process. Previously it has been shown that transmembrane domain 4 (TM4) is crucial for the function of this protein. Here we provide the first direct experimental evidence for secondary-structural features and membrane insertions of a 24-residue peptide, corresponding to TM4 of DMT1 (DMTI-TM4), in various membrane-mimicking environments by the combined use of CD and NMR spectroscopies. The peptide mainly adopts an α-helical structure in trifluoroethanol, SDS and dodecylphosphocholine micelles, and dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol small unilamellar vesicles. It has been demonstrated from both Hα secondary shifts and nuclear-Overhauser-enhancement (NOE) connectivities that the peptide is well folded into an α-helix from Val8 to Lys23 in SDS micelles at pH 4.0, whereas the N-terminus is highly flexible. The α-helical content estimated from NMR data is in agreement with that extracted from CD simulations. The highest helicity was observed in the anionic phospholipids {1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]}, indicating that electrostatic attraction is important for peptide binding and insertion into the membranes. The secondary-structural transition of the peptide occurred at pH 4.3 in the 2,2,2-trifluoroethanol (TFE) water mixed solvent, whereas at a higher pH value (5.6) in SDS micelles, DMT1-TM4 exhibited a more stable structure in SDS micelles than that in TFE in terms of changing the pH and temperature. PAGE did not show high-molecular-mass aggregates in SDS micelles. The position of the peptide relative to SDS micelles was probed by the effects of 5- and 16-doxylstearic acids on the intensities of the peptide proton resonances. The results showed that the majority of the peptide is inserted into the hydrophobic interior of SDS micelles, whereas the C-terminal residues are surface-exposed. The ability of DMT1-TM4 to assume transmembrane features may be crucial for its biological function in vivo.
Characterization of the nutrient-sensing response unit in the human asparagine synthetase promoter
Authors: ZHONG Can , CHEN Chin , KILBERG Michael S.
Published: June 2003

Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis-elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides −68 to −60 and the NSRE-2 core sequence is within nucleotides −48 to −43. Through insertion or deletion of 5–10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.