bluuebunny/crossref_metadata_2025 huggingface dataset:
Inconstant Association between 27‐kDa Heat‐Shock Protein (Hsp27) Content and Doxorubicin Resistance in Human Colon Cancer Cells
Authors: Garrido Carmen , Mehlen Patrick , Fromentin Annie , Hammann Arlette , Assem Mahfoud , Arrigo André‐Patrick , Chauffert Bruno
Published: August 2004

To investigate the role of the small 27‐kDa heat‐shock protein (Hsp27) in the intrinsic resistance of colon cancer cells to doxorubicin, we modified Hsp27 expression either genetically by transfection or pharmacologically by cisplatin treatment. HT‐29 cells were transfected with a full‐length Hsp27 construct in the sense or antisense orientation. We found a good correlation between cell survival after doxorubicin treatment and Hsp27 content. A similar correlation was found for the thermoresistance of the Hsp27‐transfected cells. In contrast, the sensitivity of the different transfected cells to 5‐fluorouracil was not modified. cis ‐Platinum(II)diammine dichloride (cisplatin) treatment of HT‐29 or Caco2 cells dramatically increased their Hsp27 mRNA and protein content. Accordingly, the cells became thermoresistant. Contrary to what has been previously assumed, however, cell resistance to doxorubicin was reduced. Our data suggest that the decreased resistance of the cells to doxorubicin may be due to a concomitant increase of topoisomerase II expression, the main target of anthracyclines. In conclusion, although Hsp27 seems to participate in the natural resistance of colon cancer cells to anthracyclines, its increase after cisplatin treatment is not associated with a decreased cytotoxicity to doxorubicin.
Efficient Transfer of Regulated Genes in Adipocytes and Hepatoma Cells by the Combination of Liposomes and Replication‐Deficient Adenovirus
Authors: Meunier‐Durmort Claire , Ferry Nicolas , Hainque Bernard , Delattre Jacques , Forest Claude
Published: August 2004

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3‐F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2‐CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3‐F442A adipocytes and FAO hepatoma cells with the HpofectAMINE/pSV2‐CAT complex in the presence of replication‐deficient recombinant type‐5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50‐fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing ‐2100 to +69 bp of the phosphoeno/pyruvate carboxykinase gene fused to the CAT gene (pPL1‐CAT). This construct was shown previously to be cAMP‐responsive after calcium‐phosphate‐mediated transfection of adipocytes and hepatoma cells. 3T3‐F442A or FAO cells in which Ppl1‐CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2–3‐fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.
Evidence That Anaerobic Oxidation of Toluene in the Denitrifying Bacterium Thauera aromatica is Initiated by Formation of Benzylsuccinate from Toluene and Fumarate
Authors: Biegert Thomas , Fuchs Georg , Heider Johann
Published: August 2004

Toluene is degraded anoxically to CO2 by the denitrifying bacterium Thauera aromatica. Toluene first becomes oxidized to benzoyl‐CoA by O2‐independent reactions. Benzoyl‐CoA is then reduced to non‐aromatic products by benzoyl‐CoA reductase. We set out to study the reactions employed for the initial activation of toluene and its oxidation to the level of benzoate. Evidence is provided for a novel way of toluene degradation based on experiments with cell‐free extracts and with whole toluene‐grown cells: Cell‐free extracts oxidized [14C]toluene to [14C]benzoyl‐CoA via several radioactive intermediates. This reaction was strictly dependent on the presence of fumarate, coenzyme A and nitrate as electron acceptor; acetyl‐CoA and ATP were not necessary for the reaction. The first product formed in vitro was benzylsuccinate; (2H8)toluene was converted to (2H7)benzylsuccinate. Formation of benzylsuccinate from toluene was independent of coenzyme A and nitrate, but it required the presence of fumarate. Other tricarboxylic acid cycle intermediates were converted to fumarate in cell extracts and therefore could partially substitute for fumarate. [14C]Benzylsuccinate was oxidized further to [14C]benzoyl‐CoA and [14C]benzoate in cell extracts if coenzyme A and nitrate were present. No benzyl alcohol and benzaldehyde and no phenylpropionate could be detected as intermediates. In isotope trapping experiments with cell suspensions, two intermediates from [14C]toluene were detected, benzoate and benzylsuccinate. This corroborates the sequence of reactions deduced from in vitro experiments. A hypothetical degradation pathway for the anaerobic oxidation of toluene to benzoyl‐CoA via an initial addition of fumarate to the methyl group of toluene and following β‐oxidation of the benzylsuccinate formed is suggested.
Alcaligenes eutrophus Possesses a Second Pyruvate Dehydrogenase (E1)
Authors: Hein Silke , Steinbüchel Alexander
Published: August 2004

Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase complex [Hein, S. & Steinbüchel, A. (1994) J. Bacteriol. 176, 4394–4408], were identified on two nonrelated A. eutrophus chromosomal Bam HI fragments by using a pdhA ‐specific DNA probe. These data indicated that A. eutrophus possesses, beside PdhA, two additional distinct pyruvate dehydrogenases (E1). A 6.8‐kbp genomic Bam HI fragment of A. eutrophus was cloned, and sequence analysis of a 3.896‐kbp region revealed the structural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (E1), which was not clustered with structural genes for other components of 2‐oxo acid dehydrogenase complexes. The A. eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E1 components of the pyruvate dehydrogenase complexes of A. eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, which are also composed of only one type of subunit. Heterologous expression of pdhE in the aceEF deletion mutant E. coli YYC202 was demonstrated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy. These complementation studies indicated that the E1 component of the A. eutrophus pyruvate dehydrogenase complex can be replaced by a functionally active pdhE gene product.
Cloning and Expression of Cytosolic Phospholipase A2 (cPLA2) and a Naturally Occurring Variant
Authors: Gordon Robert D. , Leighton Ian A. , Campbell David G. , Cohen Philip , Creaney Ann , Wilton David C. , Masters David J. , Ritchie Graham A. F. , Mott Richard , Taylor Ian W. F. , Bundell Kenneth R. , Douglas Lynne , Morten John , Needham Maurice
Published: August 2004

Full‐length cytosolic phopholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2 which lacks residues Val473–Ala749 but has a C‐terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full‐length cPLA2 was active in cPLA2 and lysophospholipase assays while DcPLA2 was inactive in both assays. cPLA2 was phosphorylated stoichiometrically by p42 mitogen–activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5‐fold increase in specific enzyme activity which was reversed by dephosphorylation.
Characterization of DNA Ligase from the Fungus Coprinus cinereus
Authors: Matsuda Shimako , Sakaguchi Kengo , Tsukada Kinji , Teraoka Hirobumi
Published: August 2004

DNA ligase was highly purified from the fungus Coprinus cinereus at the meiotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP‐labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16‐amino‐acid sequence in human DNA ligase I, which is conserved in C‐terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited‐degradation products of the 88‐kDa polypeptide during the isolation and purification procedures. Km values for ATP and (dT)20 hybridized with (dA)n were 1.5 μM and 90 nM, respectively. This enzyme was capable of joining (dT)20· (rA)n and (rA)12–18· (dT)n as well as (dT)20· (dA)n, and able to ligate blunt‐ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular mass, immuno‐reactivity, Km value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C. cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.
1H, 13C‐NMR and X‐ray Absorption Studies of Copper(I) Glutathione Complexes
Authors: Corazza Alessandra , Harvey Ian , Sadler Peter J.
Published: August 2004

The tripeptide glutathione (γ‐L‐Glu‐L‐Cys‐Cly, GSH) is an important intracellular reducing agent for Cu(II) and complexation agent for Cu(I). We have studied the complexation of Cu(I) to GSH in aqueous solution at a range of pH values and Cu(I):GSH molar ratios by 1H‐NMR and 13C‐NMR spectroscopy and X‐ray absorption spectroscopy. The NMR data are consistent with formation of a complex with approximate 1:1 stoichiometry [Cu(SG)] as the major species with only thiolate sulfur of GSH binding to Cu(I). The rate of exchange of GSH with GS‐Cu was determined to be 13 s‐1 at 283 K, pH 6.8. X‐ray absorption spectroscopic measurements showed that Cu(I) is coordinated to 3.1 ± 0.3 sulfur atoms at approximately 0.222 nm in solutions (and solids) containing GSH: Cu in 1:1 and 2:1 mol ratios. The possible structures of polymeric Cu(I)‐glutathione complexes are discussed. The high thermodynamic stability of Cu(I)‐S bonds in Cu(I)‐glutathione complexes coupled with their kinetic lability may provide efficient and specific pathways for the transport of copper in cells.
The Solution Structure of the Synthetic Circular Peptide
Authors: Rietman B. H. , Folkers P. J. M. , Folmer R. H. A. , Tesser G. I. , Hilbers C. W.
Published: August 2004

The cyclic disulfide peptide was prepared to obtain a constrained analogue of residues 17–27 of the DNA‐binding loop of the gene‐V‐encoded ssDNA‐binding protein of filamentous bacteriophage M13. Amino acid sequences very similar to that of this β‐loop have been found in various phage‐encoded ssDNA‐binding proteins, and it has been proposed that such a loop may occur as a common motif in this class of proteins. The conformation, in aqueous solution, of the synthetic gene‐V‐protein binding‐loop analogue has been investigated by means of two‐dimensional‐1H‐NMR techniques. Subsequent structure calculations show that the molecule forms a β‐loop that includes a turn formed by three residues. This structure, very unusually for a cyclic disulfide peptide, is highly similar to that of the analogous part of the binding loop of the native protein. Comparison with experiments on other cyclic disulfide peptides indicates that the formation, of the β‐sheet (β‐hairpin) secondary structure is essentially governed by the amino acid composition of the 11‐residue sequence. The disulfide bridge in the 11‐residue sequence is essential for conformational stability, as indicated by the finding that the open peptide analogue that encompasses residues Ser17–Ser27 does not adopt a detectable secondary structure in water. The bridge replaces the role of the loop formed by residues 49–58 in the protein, which act as a scaffold to hold the N‐terminal and C‐terminal ends of the DNA‐binding loop together.
Participation of Two Ser‐Ser‐Phe‐Tyr Repeats in Interleukin‐6 (IL‐6)‐Binding Sites of the Human IL‐6 Receptor
Authors: Kalai Michael , Montero‐Julian Felix A. , Grötzinger Joachim , Wollmer Axel , Morelle Danielle , Brochier Jean , Rose‐John Stefan , Heinrich Peter C. , Brailly Hervé , Content Jean
Published: August 2004

The α‐subunit of interleukin‐6 (IL‐6) receptor is a member of the hematopoietin receptor family. The alignment of its amino acid sequence with those of other members of this family (human somatotropin receptor/murine IL‐3 receptor β and human IL‐2 receptor β) has suggested that amino acids included in two SSFY repeats found in each of its hematopoietin receptor domains, contribute to the binding of the ligand. The involvement of these amino acids in IL‐6 binding and signal transduction was studied by site‐directed mutagenesis and molecular modelling. We present a computer‐derived three‐dimensional model of the IL‐6/IL‐6 receptor complex based on the structure of the human somatotropin/human somatotropin receptor complex. This model allowed the location of distinct regions important for IL‐6 and gp130 binding. We show that some of the residues included in the SSFY repeats located in our IL‐6 receptor model in the loops between β‐strands E and F of domain‐I and B' and C', of domain‐II, participate in the formation of a major IL‐6‐binding site. These residues are necessary for IL‐6 and gp130 binding and for signal transduction. Using our IL‐6 receptor mutants we mapped the epitopes of four anti‐(IL‐6 receptor) neutralising monoclonal antibodies to these residues. Our results demonstrate that a generic hematopoietin receptor family structural module can be used for the study of both α and β receptor subunits belonging to this family.
Dissimilatory Sulfite Reductase Revisited
Authors: Marritt Sophie J. , Hagen Wilfred R.
Published: August 2004

Assimilatory sulfite reductase contains a sirohaem that is very weakly coupled to a [4Fe‐4S] cubane, i.e. five iron atoms in total. Dissimilatory sulfite reductase is a complex system with 20 Fe atoms/α2β2γ2 hexamer. A recent revision of the purification procedure for the Desulfovibrio vulgaris dissimilatory enzyme has afforded a preparation of only 10 Fe atoms hexamer, this has led to the conclusion that the topology of prosthetic groups parallels that of the assimilatory system [Wolfe, B. M., Lui, S. M. & Cowan, J. A. (1994) Eur. J. Biochem. 223, 79–89]. The new purification procedure has been reproduced but the claimed molecular properties are not reproducible. The highly purified, active desulfoviridin contains 20, not 10, Fe atoms/molecule; the sirohaem is extensively demetallated, not metallated; and the S= 9/2 iron‐sulfur cluster is present, not absent.
Systematic Mutational Analysis of the Receptor‐Binding Region of the Human Urokinase‐Type Plasminogen Activator
Authors: Magdolen Viktor , Rettenberger Peter , Koppitz Marcus , Goretzkt Lothar , Kessler Horst , Weidle Ulrich H. , König Bernhard , Graeff Henner , Schmitt Manfred , Wilhelm Olaf
Published: August 2004

The amino‐terminal fragment of human uPA (ATF; amino acids 1–135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino‐terminal in‐frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell‐surface‐associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site‐directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20–30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA‐ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20–30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14–32 of ATF. Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR‐binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thrl8 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non‐reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR‐binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR‐binding activity. The results presented for the ATF mutants and uPA‐derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA‐derived peptide analogues as potential therapeutic agents to block tumor cell‐associated uPA/uPAR interaction.
Structure of the Gene Encoding the Sea Urchin Blastula Protease 10 (BP10), A Member of the Astacin Family of Zn2+–Metalloproteases
Authors: Lhomond Guy , Ghiglione Christian , Lepage Thierry , Gache Christian
Published: August 2004

Blastula protease 10 (BP10), a metalloprotease of the astacin family, is secreted at the blastula stage by the sea urchin embryo. The BP10 gene shows a precise temporal and spatial regulation during embryo‐genesis. It has been cloned from a sea urchin λ genomic library and the transcription unit has been entirely sequenced. It spans 6 kb and contains seven exons (2.8 kb) and six introns (3.2 kb). Sequence comparison and phylogeny analysis show that BP10 belongs to a sub‐family of modular proteins which all play a role during development. In the two cases where the exon/intron organization of the gene is known (BP10 and tolloid), the modular structure of the protein is not reflected at the gene level, which indicates that this sub‐family probably did not evolve by exon shuffling.
Cloning and Functional Characterization of the Amphibian Mesotocin Receptor, a Member of the Oxytocin/Vasopressin Receptor Superfamily
Authors: Akhundova Aida , Getmanova Elena , Gorbulev Valentin , Carnazzi Eric , Eggena Patrick , Fahrenholz Falk
Published: August 2004

Mesotocin is the oxytocin‐like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non‐mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non‐mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by Northern blot analysis and reverse‐transcriptase PCR. The results suggest a variety of functions for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.
The Anticoagulant Effect of the Human Secretory Phospholipase A2 on Blood Plasma and on a Cell‐Free System is Due to a Phospholipid‐Independent Mechanism of Action Involving the Inhibition of Factor Va
Authors: Mounier Carine , Franken Peet A. , Verheij Hubertus M. , Bon Cassian
Published: August 2004

Blood platelets play a central role in haemostasis by leading to plug formation and by increasing the efficiency of blood coagulation. We have previously shown that blood platelets contain a group II secretory phospholipase A2 (sPLA2 grII) which is released into the extracellular medium upon activation but is unable to stimulate blood platelets. We presently reported an investigation of the putative involvement of the human sPLA2 grII (hsPLA2 grII) in the coagulation process, both in the absence and in the presence of activated platelets. We show that this enzyme prolongs the recalcification time of blood plasma even in the presence of activated platelets.

The positive action of blood platelets on coagulation is correlated, at least in part, with the appearence at the cellular surface of procoagulant phospholipids which constitute a potential target for hsPLA2 grII. We therefore investigated the involvement of its enzymatic activity in the anticoagulant effect of this enzyme. We observed that the replacement of CaCl2 by SrCl2 to initiate the coagulation cascade did not suppress, but rather increased, the inhibitory action of hsPLA2 grII. Moreover, hsPLA2 grII hydrolyzed only a minor proportion of platelet phospholipids, and it did not affect plasma phospholipids. Taken together, these observations strongly suggest that the major action of hsPLA2 grII on blood coagulation does not involve the hydrolysis of phospholipids, in contrast with the strong anticoagulant effect of the group II venom phospholipase A2 from Crotalus durrissus terrificus.

We next studied which step of the coagulation cascade was affected by hsPLA2 grII. Using purified coagulation factors, we demonstrated that hsPLA2 grII strongly inhibited the prothrombinase activity. This inhibitory effect was independent of the presence of phospholipids but required factor Va, leading to the hypothesis that hsPLA2 grII inhibited this factor. Further, the anticoagulant effect of hsPLA2 grII was observed on normal and factor‐X‐deficient plasma, but not on factor‐V‐deficient plasma.

In conclusion, the anticoagulant action of hsPLA2 grII is based on a nonenzymatic mechanism of action involving the inhibition of factor Va.
A Klebsiella pneumoniae Strain that Shares a Type‐Specific Antigen with Shigella flexneri Serotype 6
Authors: Ansaruzzaman Mohammed , Albert M. John , Holme Tord , Jansson Per‐Erik , Rahman M. Mahbubur , Widmalm Göran
Published: August 2004

A strain of Klebsiella pneumoniae was found as the only isolate with pathogenic potential from the stool of a two‐year old patient with diarrhoea. A strong serological cross‐reactivity with Shigella flexneri serotype 6 was demonstrated. The cross‐reacting antigens were shown to reside in the cell wall lipopolysaccharide. Studies of the pathogenic potential of the Klebsiella strain showed low level of invasion of HEp‐2 cells. However, tests for adherence to HEp‐2 cells as well as tests for toxin production were negative. The strain had several small plasmids and was multidrug resistant. These data do not form a sufficient basis for estimating the pathogenic potential of the organism. No K antigen was detected. The structure of the O‐antigenic polysaccharide from the K. pneumoniae strain was investigated using methylation analysis, NMR spectroscopy, and Smith degradation as the principal methods. The O‐antigenic polysaccharide has the following pentasaccharide repeating unit:
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This structure is not identical to any of the previously described O‐antigens of K. pneumoniae. The strong serological cross‐reactivity with the Shigella flexneri serotype 6 O‐antigen can most likely be attributed to the structural element α‐l‐Rhap ‐(1→)‐α‐l‐Rhap present in the O‐polysaccharide repeating unit of this serotype. Antiserum raised against the K. pneumoniae strain also agglutinated S. dysenteriae serotype 1 and strains of all different serotypes of S. flexneri. The Shigella strains contain the structural element α‐l‐Rhap ‐(1→3)‐α‐l‐Rhap in their O‐antigen polysaccharides which may be responsible for the observed cross‐reactivity.
The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes
Authors: Belmouden Ahmed , Lederer Florence
Published: August 2004

Peroxisomal long‐chain 2‐hydroxy‐acid oxidase, an FMN‐dependent enzyme, catalyzes the oxidation of a variety of L‐2‐hydroxy acids into keto acids at the expense of oxygen. We recently reported the cloning and sequencing of its cDNA and the existence of a weakly expressed isozyme [Belmouden, A., Lê, K. H. D., Lederer, F. & Garchon, H. J. (1993) Eur. J. Biochem. 214, 17–25]. This isozyme, β2, differs from the major one in having a three–residue insertion,‐VRK‐, in loop 4 of the β8α8 barrel. In the crystal structures of homologous flavocytochrome b2 and glycolate oxidase, the corresponding region of loop 4 is disordered. We now report on the constitutive high‐level expression of isozymes β1 and β2 in Escherichia coli under control of the λpL promoter, and on the influence of the E. coli genetic background and the growth medium on the expression level. We describe the properties of isozyme β2 and compare them with those of pure isoform β1. The visible spectra of the purified enzymes differ in the position of the near‐ultraviolet band of the prosthetic group. pH titration studies indicate that the FMN ionizes at N3 at a lower pH than free flavin and that there is a small pKa difference between the isozymes. To our knowledge, the only other known case of a lowered pKa for the protein‐bound flavin is that of glycolate oxidase. In the CD spectra of the FMN region, a marked difference between isozymes in the 270–300‐nm region appears to be related to the pKa difference for the N3‐H bond. Kinetic parameters for a number of substrates and inhibitors are indistinguishable within the limits of experimental error, with the exception of values for kcat for mandelate (the most active substrate), Km for hydroxyhippurate (a new substrate), Ka for cinnamate and oxalate, and Ka for sulfite. The differences are no larger than twofold. The foregoing comparison between isozymes β1 and β2 shows that the naturally engineered insertion in loop 4 exerts some influence on the flavin spectral properties and the active‐site reactivity. Since the corresponding loop 4 regions in the three‐dimensional structures of flavocytochrome b2 and glycolate oxidase are 1.5–2.0 nm removed from the flavin, it would appear either that loop 4 has a very different conformation in hydroxy‐acid oxidase, or that it may interact with the active site due to mobility.
Sphingomyelin‐Derived Lipids Differentially Regulate the Extracellular Signal‐Regulated Kinase 2 (ERK‐2) and c‐Jun N‐Terminal Kinase (JNK) Signal Cascades in Airway Smooth Muscle
Authors: Pyne Susan , Chapman Joanna , Steele Leslie , Pyne Nigel J.
Published: August 2004

In ASM cells platelet‐derived growth factor stimulates rapid transient sphingosine phosphate formation, the activation of extracellular signal‐regulated kinase 2 (ERK‐2), the phosphorylation of p70S6K, and a ninefold increase in DNA synthesis. In contrast, this growth factor fails to activate c‐Jun N‐terminal kinase (JNK). Based upon these findings, we have tested whether the sphingomyelin‐derived sphingolipids play a role in growth factor signalling by assessing their effect on ERK‐2, JNK, and p70S6K.

We demonstrate that sphingosine phosphate induces the activation of ERK‐2, is ineffective against JNK, and fails to induce the phosphorylation of p70S6K. The latter may explain why it is a poor mitogen when added directly to ASM cells. In contrast, sphingosine and cell‐permeable ceramides elicit the prominent tyrosyl phosphorylation and activation of JNK, are poor stimulators of ERK‐2, and do not induce the phosphorylation of p70S6K. Therefore, the specificity of signalling through either ERK‐2 or JNK cascades may be determined by the rapid agonist‐dependent interconversion of these sphingomyelin‐derived lipids. This may also provide a dynamic mechanism that enables growth factors and cytokines to elicit pleiotropic cell responses, such as proliferation and cell survival. For instance, both ceramide and sphingosine will elicit growth arrest via activation of JNK, whereas sphingosine phosphate will potentiate growth‐factor‐stimulated DNA synthesis, a consequence of the activation of ERK‐2. Furthermore, under certain conditions, sphingosine and ceramide stimulate cAMP formation, a negative modulator of cell growth, whereas sphingosine phosphate depresses cAMP, thereby enhancing its own growth‐promoting properties.

From these studies, it is evident that sphingosine phosphate displays a signalling profile that is consistent with it mediating part of the action of platelet‐derived growth factor.
Molecular Characterization of a Saline‐Soluble Lectin from a Parasitic Fungus
Authors: Rosén Stefan , Kata Miroslaw , Persson Yvonne , Lipniunas Peter H. , Wikström Mats , Van Den Hondel Cees A. M. J. J. , Van Den Brink Johannes M. , Rask Lars , Hedén Lars‐Olof , Tunlid Anders
Published: August 2004

It has been proposed that the interactions between several parasitic and pathogenic fungi and their hosts are mediated by soluble lectins present in the fungus. We have cloned and analyzed a gene encoding such a lectin (AOL) from the nematophagous fungus Arthrobotrys oligospora (deuteromycete). The deduced primary structure of the AOL gene displayed an extensive similarity (identity 46.3%) to that of a gene encoding a lectin (ABL) recently isolated from the mushroom Agaricus bisporus (basidiomycete), but not to any other fungal, microbial, plant or animal lectins. The similarities between AOL and ABL were further demonstrated by the observation that an antibody specific for AOL cross‐reacted with ABL. Together with data showing that AOL has a binding specificity that is similar to that of ABL [Rosén, S., Bergström, J., Karlsson, K.‐A. & Tunlid, A. (1996) Eur. J. Biochem. 238, 830–837], these results indicate that AOL and ABL are members of a novel family of saline‐soluble lectins present in fungi. Southern blots indicated that there is only one AOL gene in the genome encoding a subunit (monomer) of the lectin. The primary structure of AOL did not show the presence of a typical N‐terminal signal sequence. Comparison of the deduced primary structure with the molecular mass of AOL as determined by electros‐pray mass spectrometry (16153 Da), indicated that AOL has an acetylated N‐terminal but no other post‐translational modifications, and that a minor isoform is formed by deamidation. Circular dichroism (CD) spectroscopy suggested that the secondary structure of AOL contains 34%β‐sheets, 21%α‐helix, and 45% turns and coils.
A Multispecific Saline‐Soluble Lectin from the Parasitic Fungus Arthrobotrys oligospora
Authors: Rosén Stefan , Bergström Jörgen , Karlsson Karl‐Anders , Tunlid Anders
Published: August 2004

Several fungi can express high levels of saline‐soluble and low‐molecular‐mass lectins that bind to glycoproteins such as fetuin and different mucins but not bind to any monosaccharides. In this paper, we report the binding specificities of such a lectin (designated AOL) isolated from the nematophagous fungus Arthrobotrys oligospora. The results show that AOL is a multispecific lectin that interacts with the following ligands: (a) Several sulfated glycoconjugates including sulfatide, dextran sulfate, and fucoidan. The specificity of this binding was indicated by experiments showing that none of the tested neutral‐ and sialic‐acid‐containing glycolipids, chondroitin sulfates B and C., heparin, and polyvinyl sulfate bound to AOL; (b) Phosphatidic acid and phospatidylglycerol, two out of several tested phospholipids. (c) N‐linked and O‐linked sugar chains bound to intact fetuin. The involvement of such sugar structures was demonstrated by analyzing the binding of AOL to chemically deglycosylated (trifluoromethanesulfonic acid) fetuin. Treating fetuin with O‐glycosidase and N‐glycosidase indicated that AOL bound to Galβ3‐GalNAcα‐Ser/Thr and to some N‐linked complex sugars, respectively. Further assays demonstrated that AOL could interact with several other glycoproteins containing O‐linked and/or N‐linked sugar chains. The observations that AOL did not bind to free N‐linked sugars isolated from fetuin, or to fetuin treated with trypsin or pronase, or to any of the tested neoglycoproteins and glycolipids with neutral‐or sialic‐acid‐containing sugars, indicated that the sugar chains need to be bound to an intact peptide backbone to interact with AOL. We have recently shown that the deduced primary structure of AOL has a high similarity to the sequence of a saline‐soluble lectin isolated from the mushroom Agaricus bisporus (ABL) (Rosén, S., Kata, M., Persson, Y., Lipniunas, P. H., Wikström, M., van den Hondel, C. A. M. J. J., van den Brink, J. M., Rask, L., Hedén L.‐O. and Tunlid, A., see companion paper). It is well known that ABL binds to Galβ3GalNAcα‐Ser/Thr, and in this paper we demonstrate that ABL binds to sulfatide, phosphatidic acid, phospatidylglycerol, and possibly also to the same N‐linked complex sugars as AOL. The above data indicate that AOL and ABL are members of a novel family of fungal lectins sharing similar primary structure and binding properties.
Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis
Authors: Kawachi Hiroyuki , Atomi Haruyuki , Ueda Mitsuyoshi , Tanaka Atsuo
Published: August 2004

A genomic DNA clone encoding carnitine acetyltransferases (EC 2.3.1.7), localized in two subcellular organelles, peroxisomes and mitochondria of an N‐alkane‐assimilating yeast Candida tropicalis, was isolated from the yeast λEMBL library using a carnitine acetyltransferase cDNA probe. Nucleotide sequence analysis disclosed that the open reading frame was 1881 bp, corresponding to 627 amino acids with a molecular mass of 70760 Da. Comparison of the predicted amino acid sequence of the C. tropicalis enzyme with that of Saccharomyces cerevisiae mitochondrial matrix carnitine acetyltransferase revealed 46.3% identity. It was noticeable that the C. tropicalis enzymes had amino acid sequences similar to both proposed mitochondrial and peroxisomal targeting signals. When the C. tropicalis gene was expressed in S. cerevisiae using its own 5′‐upstream region, a 12‐fold increase in activity was observed. Western blot analysis revealed the presence of two major proteins whose sizes corresponded to the peroxisomal and mitochondrial proteins detected in C. tropicalis. This suggested that peroxisomal and mitochondrial carnitine acetyltransferases were encoded by one gene, as suggested for the S. cerevisiae enzyme. Furthermore, we have separated and purified these enzymes from peroxisomes and mitochondria of C. tropicalis, and analyzed the amino‐terminal amino acid sequences of each. The amino‐terminal sequence of the mitochondrial enzyme suggested that a signal sequence had been cleaved during translocation into mitochondria. Concerning the peroxisomal enzyme, the evidence obtained indicated that in vivo the translation was initiated at the second methionine of the open reading frame.
Human and Plant Proliferating‐Cell Nuclear Antigen have a Highly Conserved Binding Site for the p53–Inducible Gene Product p21WAF1
Authors: Ball Kathryn L. , Lane David P.
Published: August 2004

The mechanism(s) whereby higher plants respond to environmental agents that damage their DNA, which leads to the arrest of cell division, is poorly understood. In mammalian cells, the tumour‐suppressor protein p53 plays a central role in a DNA‐damage‐induced cell‐cycle‐checkpoint pathway by induction of transcription of a set of gene products that have a direct role in a DNA‐damage‐induced cell‐cycle growth arrest. One such protein, p21WAF1, has been shown to be essential for radiation‐induced growth arrest. There appear to be at least two cellular targets of p21WAF1 during checkpoint control, the G1‐cyclin‐dependent kinases (CDK) and proliferating‐cell nuclear antigen (PCNA). The aim of the research reported here was to determine whether the interactions between the human growth inhibitor p21WAF1 and PCNA from plants and humans are conserved. If so, this would suggest that modulation of PCNA activity may play an important role in plant responses to DNA damage and would imply that functional homologue(s) of p21WAF1 exist in plants. We show that the p21WAF1–interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WAF1 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p21WAF1 peptide as an affinity matrix and showed that pea PCNA bound in a specific high‐affinity manner. This finding was used to develop a purification protocol that allowed PCNA from plant tissue to be purified to homogeneity. Pure pea PCNA forms a stable complex with full‐length human p21WAF1 and the specific amino acids of p21WAF1 required for the interaction have been identified. The critical residues were identical to those required for binding to human PCNA, which indicates that the interaction of human p21WAF1 with PCNA is highly conserved at each amino acid position between pea and human.
Crystallization and Crystal Packing of Recombinant 3 (or 17) β‐Hydroxysteroid Dehydrogenase from Comamonas testosteroni ATTC 11996
Authors: Benach Jordi , Knapp Stefan , Oppermann Udo C. T. , Hägglund Oskar , Jörnvall Hans , Ladenstein Rudolf
Published: August 2004

The enzyme 3 (or 17) β‐hydroxysteroid dehydrogenase from Comamonas testosteroni was crystallized. Crystals, of up to 0.6 mm in their longest dimension and suitable for a crystallographic analysis have been obtained by the vapour diffusion method. They belong to the orthorhombic lattice type and diffract to a maximum resolution of 0.23 nm. A final data set obtained by merging data from three crystals resulted in a completeness of 90% with an Rmerge of 6%. A molecular replacement search carried out by using 3α (or 20β)‐hydroxysteroid dehydrogenase from Streptomyces hydrogenans as a search model allowed us to assign I222 as the correct space group and to propose a model for the crystal packing, with one monomer per asymmetric unit. Thus, the whole unit cell contains two tetramers. The R‐factor after rigid body refinement is 48.1 %.
The C‐terminal Bisphosphorylated proenkephalin‐A‐(209–237)‐peptide from Adrenal Medullary Chromaffin Granules Possesses Antibacterial Activity
Authors: Goumon Yannick , Strub Jean‐Marc , Moniatte Marc , Nullans Gérard , Poteur Livia , Hubert Pierre , Van Dorsselaer Alain , Aunis Dominique , Metz‐Boutigue Marie‐Hélène
Published: August 2004

The chromaffin granules have been shown to be an excellent model to study the processing of proenkephalin‐A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin I and the occurrence of the C‐terminal chromogranin B‐derived peptide 614–626 which was shown to have antibacterial activity [Strub, J. M., Garcia‐Sablone, P., Looning, K., Taupenol, L., Hubert, P., Van Dorsselaer, A., Aunis, D. & Metz‐Boutigue, M. H. (1995) Eur. J. Biochem. 229, 356–368], We also observed that this new antibacterial activity present in chromaffin granules was associated with other endogenous protein‐derived fragments yet to be characterized. The present study reports the isolation and characterization of a peptide which possesses antibacterial activity and which corresponds to the C‐terminal 209–237 sequence of proenkephalin‐A. A detailed study using microsequencing and matrix‐assisted‐laser‐desorption time‐of‐flight mass spectrometry (MALD‐TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPLPSEEEGESYSKEVPEMEKRYGGFM) with post‐translational modifications. Endogenous bisphosphorylated proenkephalin‐A‐(209–237) was active on Micrococcus luteus and Bacillus megaterium killing bacteria in the 0.2–0.4 μM range but was inactive in similar conditions towards Escherichia coli. Enkelytin shares sequence and structural similarities with the antibacterial C‐terminal domain of diazepam‐binding inhibitor. According to this similarity, a prediction of secondary structure is proposed for enkelytin and discussed in relation to its biological activity.
Dissection of the Basic Subdomain of the c‐Jun Oncoprotein
Authors: Krebs Daniel , Dahmani Benamar , Monnot Monique , Mauffret Olivier , Troalen Frédéric , Fermandjian Serge
Published: August 2004

In a previous paper, we reported on the structural properties of a 35‐residue peptide corresponding to a modified basic subdomain (bSD) of the basic zipper protein c‐Jun (residues 252–281) as determined by combined use of 1H‐NMR, circular dichroism (CD) and Fourier transform infrared (FT‐IR) spectroscopies [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. (1995) Eur: J. Biochem. 231, 370–380]. The fragments NP and CP (the N‐terminal residues 1–19 and C‐terminal residues 16–35 of bSD, respectively) proved to be particularly useful for the assignment of the 1H‐NMR spectra of the full‐length bSD, which has been achieved completely in aqueous solution and partially in trifluoroethanol. Here, we report on The structural properties of NP and CP in aqueous solution and under varying H2O/trifluoroethanol conditions, again using 1H‐NMR, CD and FT‐IR experiments. Both CD and FT‐TR results established that the fragments are weakly structured in aqueous solution. Addition of trifluoroethanol to aqueous solutions of the peptides produced their stabilization into helix, following a profile sigmoidal for NP and nearly linear for CP. Quantitative NOES, secondary Ha chemical shifts, NH temperature coefficients and 3JαN coupling constants for the peptides in aqueous solutions provided indications for weak helix features (nascent helices) manifested within two sites (continuous dNN NOEs) in both NP and CP. For each peptide, an excellent agreement was observed between experiments and predictions with the AGADIR program fur the location of these nascent helices in the sequences. Trifluoroethanol provoked both the a‐helix stabilization within these sites and the α‐helix propagation to adjacent and no acid residues.

Finally, our results reflected the high flexibility and helix potential of the NP and CP fragments, these two properties seeming crucial for the accomodation of c‐Jun to its specific DNA targets. The results demonstrated also the fragmentation's benefits in dissecting a protein or a complex peptide into smaller fragments and analyzing their structure individually.
Dynamics of Parvalbumin Expression in Low‐Frequency‐Stimulated Fast‐Twitch Rat Muscle
Authors: Huber Bärbel , Pette Dirk
Published: August 2004

Similar to previous observations in rabbit muscle, chronic low‐frequency stimulation suppressed parvalbumin expression in fast‐twitch muscles of the rat. In extensor digitorum longus and tibialis anterior muscles, parvalbumin mRNA levels steeply declined with apparent half‐lives of approximately 26 h and 45 h, respectively. Measurements of parvalbumin synthesis indicated that the reduction in mRNA was immediately transmitted to the level of translation. Relative parvalbumin synthesis rates decayed with an apparent half‐life of approximately 60 h. Both the decrease in parvalbumin mRNA and synthesis considerably preceded the decay of parvalbumin protein. Although parvalbumin synthesis had approached zero in 14‐day‐stimulated muscles, parvalbumin content started to decrease only after some delay (28‐day‐stimulated muscles still contained 40–50% of their normal parvalbumin content). The lag time between fully suppressed synthesis and the onset of parvalbumin decay, as well as the stability of parvalbumin against tryptic cleavage in the presence of Ca2+ and Mg2+, indicated proteolysis as an important posttranslational control of parvalbumin levels. The decrease in parvalbumin mRNA followed a similar time course as that of the mRNA specific to the fast myosin heavy chain HCIIb. After complete suppression, parvalbumin mRNA reached control levels 4 days after cessation of stimulation, which demonstrates the complete reversibility of the stimulation‐induced parvalbumin suppression. These results show that a slow motoneuron‐like impulse pattern rapidly silences the parvalbumin gene, thus overriding fast‐fiber‐type‐specific programs of gene expression. Due to post‐transciptional regulation and the stability of parvalbumin, this high responsiveness of adult skeletal muscle to altered neuromuscular activity is more conspicuous at the mRNA level than at the protein level.
Redefining citizenship for the 21st century: from the National Welfare State to the UN Global Compact
Authors: Wagner Antonin
Published: August 2004

This article analyses the impact of globalisation on the changing role of citizenship as a state‐centred mechanism of societal integration. As more diverse forms of society emerged in the second half of the last century, national citizenship came under assault by identity‐based social groups from within. They function as integrative mechanisms for those members of society who diverge from the majority position and are committed to replace the nation‐state as the dominant integrative device. From without, vast movements of peoples across borders in search of jobs and refuge constitute an even more serious challenge to the traditional notion of citizenship. With reference to the current EU debates about immigration and the idea of a UN Global Compact, the article explores principles of societal integration that transcend the boundaries of national citizenship and involve a governance paradigm built on civil society and voluntary action.