bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Active Streptolysin O in Escherichia coli as a Maltose‐Binding‐Protein‐Streptolysin‐O Fusion Protein
Authors: Weller Ulrich , Müller Lutz , Messner Martina , Palmer Michael , Valeva Angela , Tranum‐Jensen Jørgen , Agrawal Pushpa , Biermann Christine , Döbereiner Andreas , Kehoe Michael A. , Bhakdi Sucharit
Published: August 2004

Streptolysin O (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore‐forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose‐binding protein (MBP) gene and SLO were constructed, using the vectors pMAL‐p2 and pMAL‐c2. MBP‐SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP‐SLO fusion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N‐terminus was separated from the MBP carrier by cleavage with factor Xa. Cleavage with plasmin or trypsin also yielded active, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP‐SLO was also hemolytic and cytotoxic to human fibroblasts and keratinocytes. The MBP‐SLO fusion protein displayed equal activities to SLO. Sucrose density gradient analyses showed that the fusion protein assembled into polymers, and no difference in structure was discerned compared with polymers formed by native SLO. These studies show that the N‐terminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N‐terminus of the molecule is probably not inserted into the bilayer. In addition, they provide a simple means for producing mutants for structure/function studies and highly purified SLO for use as a permeabilising reagent in cell biology research.
Paramagnetic NMR Analysis of the Seven‐Iron Ferredoxin from the Hyperthermoacidophilic Archaeon Desulfurolobus ambivalens Reveals Structural Similarity to other Dicluster Ferredoxins
Authors: Bentrop Detlef , Bertini Ivano , Luchinat Claudio , Mendes Joaquim , Piccioli Mario , Teixeira Miguel
Published: August 2004

The seven‐iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens, has been investigated by one‐dimensional and two‐dimensional 1H‐NMR in its oxidized and dithionite‐reduced states. All iron atoms of both the three‐iron and the four‐iron cluster are bound to cysteine residues whose hyperfine‐shifted resonances were characterized. The pattern of these resonances is similar to those from three‐iron, four‐iron and eight‐iron ferredoxins previously described in the literature, but the four‐iron cluster has a shift pattern different from that in other seven‐iron proteins. A second set of hyperfine‐shifted resonances clearly indicates sample heterogeneity, which possibly involves the four‐iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence‐specific assignment of the hyperfine‐shifted resonances. The Cα‐Cβ‐S‐Fe dihedral angles of the cysteine residues coordinating the four‐iron cluster could be estimated, and the electronic structure of the three‐iron cluster is discussed.
Conformational Features of a Synthetic Cyclic Peptide Corresponding to the Complete V3 Loop of the RF HIV‐1 Strain in Water and Water/Trifluoroethanol Solutions
Authors: Vranken Wim F. , Budesinsky Milos , Martins José C. , Fant Franky , Boulez Kris , Gras‐Masse Hélène , Borremans Frans A. M.
Published: August 2004

The disulfide‐bridge‐closed cyclic peptide corresponding to the whole V3 loop of the RF HIV‐1 strain was examined by proton two‐dimensional NMR spectroscopy in water and water/trifluoroethanol solutions. Although most of the peptide is conformationally averaged in water, the NOE data support a β‐turn conformation for the central conservative GPGR region and the presence of nascent helix. Upon addition of trifluoroethanol, helix formation in the C‐terminal part becomes apparent. This is confirmed by CD data. NOEs indicative of multiple and transient β‐turns around the Asn6 glycosylation site and NOEs fitting X‐ray data on a linear V3 peptide–Fab complex also emerge. The C‐terminal helix is shown to have amphipathic character and might thus assist in the infection process.
Sequence Differences between Human Muscle and Liver cDNAs for UDPglucose Pyrophosphorylase and Kinetic Properties of the Recombinant Enzymes Expressed in Escherichia Coli
Authors: Duggleby Ronald G. , Chao Yu Chyi , Huang Joyce G. , Peng Hwei‐Ling , Chang Hwan‐You
Published: August 2004

UDP‐Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP‐Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5′ region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.‐L. & Chang, H.‐Y. (1993) FEBS Lett. 329, 153–158] and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis‐Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis‐Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration.
Glutathione Reductase and Glutamate Dehydrogenase of Plasmodium Falciparum, The Causative Agent of Tropical Malaria
Authors: Krauth‐Siegel R. Luise , Müller Joachim G. , Lottspeich Friedrich , Schirmer R. Heiner
Published: August 2004

The use of glutathione reductase inhibitors in chemotherapy is the raison d'être for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N‐terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20–34 of human glutathione reductase, represents the first β strand and the diphosphate‐fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N‐termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P. falcipurum glutathione reductase as a drug target are discussed.
Metabolism of 5‐Chlorosubstituted Muconolactones
Authors: Prucha Matthias , Wray Victor , Pieper Dietmar Helmut
Published: August 2004

The stereochemistry of the four stereoforms of 5‐chloro‐3‐methylmuconolactones could be deduced from NMR and stability data, and from the comparison with authentic (4R, 5S)‐5‐chloromuconolactone. Muconolactone isomerase of Alcaligenes eutrophus JMP 134 was shown to catalyze syn ‐elimination of hydrogen chloride from (4R, 5S)‐5‐chloro‐3‐methylmuconolactone, (4R, 5S)‐5‐chloro‐3‐methyl‐muconolactone and (4R, 5S)‐5‐chloromuconolactone to form 3‐methyl‐cis ‐dienelactone, 3‐methyl‐trans ‐dienelactone and a 3:1 mixture of cis‐ and trans‐dienelactone, respectively. 3‐methyl‐trans‐dienelactone was a substrate of pJP4‐encoded dienelactone hydrolase of A. eutrophus JMP 134, whereas 3‐methyl‐cis‐dienelactone transformation was negligible indicating a restricted substrate specificity of this enzyme. Both substrates were transformed into 3‐methylmaleylacetate which in turn was a substrate for maleylacetate reductase. This compound was shown to possess a cyclic structure (4‐hydroxy‐3‐methylmuconolactone) under acidic conditions.
Analyses of Cis ‐Acting and Trans ‐Acting Elements that are Crucial to Sustain Pregnancy‐Specific Glycoprotein Gene Expression in Different Cell Types
Authors: Koritschoner Nicolás P. , Panzetta‐Dutaiu Graciela M. , Bocco José L. , Dumur Catherine I. , Flury Alfredo , Patrito Luis C.
Published: August 2004

Pregnancy‐specific β1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidi‐form mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA‐protein interactions and the basal activities of the TATA‐box‐less PSG5 promoter were determined in different tissues and cell types. In DNAse‐I protection assays, DNA‐binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides –150 to –124, overlapping the farthest 5′ upstream cap site and resembling an initiator‐like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27‐bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5′ACCCAT3′ by 5′GATATC3′ within the 27‐bp motif revealed that this sequence is fundamental for the formation of the specific DNA‐protein complexes. We show in transient transfection experiments performed in HeLa, COS‐7 and JEG‐3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type.

Moreover, this mutation disrupted the formation of the specific DNA‐protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleopro‐teins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1‐TRIM) and HM, respectively, showing that the DNA‐binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis ‐acting and trans ‐acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.
Purification and Characterization of Human Topoisomerase I Mutants
Authors: Jensen Anne Dam , Svejstrup Jesper Q.
Published: August 2004

A system for rapid purification and characterization of eukaryotic topoisomerase‐I mutants has been developed. The system utilizes six‐histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel‐affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates.

Relaxation‐deficient mutants were obtained by site‐directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50‐fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu was comparable to that of wild‐type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo‐dUTP‐substituted DNA oligonucleotides carrying a topoi‐somerase‐I‐binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non‐covalently.
Surface Location and Orientation of the Lantibiotic Nisin Bound to Membrane‐Mimicking Micelles of Dodecylphosphocholine and of Sodium Dodecylsulphate
Authors: Van Den Hooven Henno W. , Spronk Chris A. E. M. , Van De Kamp Mart , Konings Ruud N. H. , Hilbers Cornelis W. , Van De Ven Frank J. M.
Published: August 2004

The interaction of nisin, a membrane‐interacting cationic polypeptide, with membrane‐mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was studied. Direct contacts have been established through the observation of NOEs between nisin and micelle protons. Spin‐labeled DOXYL‐stearic acids were incorporated into the two micellar systems. From the paramagnetic broadening effects induced in the 1H‐NMR spectrum of nisin it is concluded that the molecule is localized at the surface of the micelles. The interactions of nisin with zwitterionic and with anionic micelles resemble each other as do the nisin conformations [van den Hooven, H. W., Doeland, C. C. M., van de Kamp, M., Konings, R. N. H., Hilbers, C. W. & van de Ven, F. J. M. (1995) Eur. J. Biochem. 235, 382–393]. The hydrophobic residues are immersed into the micelles and oriented towards the center, whereas the more polar or charged residues have an outward orientation. The micellar systems are considered to model the first step in the mechanism of antimicrobial action of nisin, this step is the binding of nisin to the cytoplasmic membrane of target bacteria. Detailed information on this initial binding step is obtained. Hydrophobic and electrostatic interactions appear to be involved in the nisin‐micelle contacts. It is suggested that subtilin, a lantibiotic structurally related to nisin, has a comparable membrane interaction surface.
1H‐NMR‐Derived Secondary Structure and the Overall Fold of the Potent Anti‐Mammal and Anti‐Insect Toxin III from the Scorpion Leiurus quinquestriatus quinquestriatus
Authors: Landon Céline , Cornet Bruno , Bonmatin Jean‐Marc , Kopeyan Charles , Rochat Hervé , Vovelle Françoise , Ptak Marius
Published: August 2004

We describe the secondary structure and the overall fold of toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two‐dimensional‐1H‐NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long‐chain toxin category and is highly toxic to both mammals and insects. The overall fold was determined on the basis of 1208 inter‐proton‐distance restraints derived from NOE measurements and 90 Ψ, Φ dihedral‐angle restraints derived from NOE connectivities and 3JNH‐αH coupling constants using the HABAS program. This fold, which mainly consists of an α‐helix packed against a small antiparallel three‐stranded β‐sheet, and of several turns and loops, is similar to that of other long‐chain scorpion toxins. Aromatic and non‐polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this toxin. The central core of toxin III is compared with the cysteine‐stabilized αβ motif (an α‐helix connected to a β‐sheet through two disulfide bridges) found in insect defensins and plant thionins. Defensins and thionins are small proteins (≈40–50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine‐stabilized αβ motif is a common core to a number of small proteins from different origins and having different activities.
The Structure of the Semenogelin Gene Locus
Authors: Lundwall Åke
Published: August 2004

The sequence of 15.7 kb from the human semenogclin gene locus has been determined. Together with previously published sequences, this gives the structure of a 28‐kb region encompassing the two semenogelin genes. The two transcription units are separated by 11 616 bp intergenic DNA comprising more than 40% repetitive DNA sequences, predominantly located to a 4‐kb block of L1 and Alu repeats. Two more blocks of L1 sequences are present in the DNA flanking the genes, so that approximately 20% of the completed sequence consists of long interspersed repeated sequences, so called LINES. A comparison of the SgI gene and the SgII gene suggests that they evolved by the duplication of an approximately 8 kb DNA segment about 61 million years ago, probitbly by a mechanism involving recombination between L1 elements.
The Crystal Structure of Transthyretin from Chicken
Authors: Sunde Margaret , Richardson Samantha J. , Chang Linus , Pettersson Tom M. , Schreiber Gerhard , Blake Colin C. F.
Published: August 2004

The crystal structure of chicken transthyretin has been solved at 290‐pm resolution by molecular‐replacement techniques. Transthyretin is the protein component of the amyloid fibrils found in patients suffering from either familial amyloidotic polyneuropathy or senile systemic amyloidosis. Familial amyloidotic polyneuropathy is an autosomal dominant hereditary type of amyloidosis which involves transthyretin with either one or two amino acid substitutions. The three‐dimensional structure of chicken transthyretin was determined in order to compare a non‐amyloidogenic, species‐variant transthyretin with wild‐type and mutant transthyretin molecules. Of the 31 chicken‐to‐human residue differences, 9 occur at positions which in human transthyretin give rise to amyloidogenic variants although none corresponds to the appropriate side‐chain substitutions. The model of chicken transthyretin has been refined to an R ‐factor of 19.9%. The overall fold of the protein is that of an all‐β protein. Compared with wild‐type human transthyretin the avian transthyretin shows quite large differences in the region known to be involved in binding to retinol‐binding protein, it has a much shorter helical component than the human protein and some of the monomer‐monomer interactions are different.
A Factor Derived from Polymorphonuclear Leukocytes Enhances Interleukin‐1‐Induced Synovial Cell Collagenase and Prostaglandin E2 Production in Rats
Authors: Hashida Ryoichi , Kuwada Manabu , Chiba Ken‐ichi , Horizoe Tatsuo , Shirota Hiroshi , Nagai Yutaka
Published: August 2004

We found that short‐term culture medium and homogenate of casein‐induced rat peritoneal polymerphonuclear leukocytes (PMN) markedly induced collagenase and prostaglandin E2 (PGE2) production by normal rat synovial cells and these effects were abrogated by anti‐(rat interleukin‐1α) (IL‐1α) polyclonal antibodies. However, collagenase activity and PGE2 induced by recombinant rat IL‐1α were less than those induced by rat PMN culture medium. It was also proved by radioimmunoassay that rat PMN culture medium contains a relatively small amount of IL‐1α. The introduction of IL‐1α‐deleted PMN culture medium and recombinant rat IL‐1α together into the synovial cell culture system revealed that IL‐1α‐deleted PMN culture medium has a significant enhancing activity on IL‐1α‐induced synovial cell collagenase and PGE2 production. This new factor, which was shown to be a negatively charged protein of about 80 kDa, may have important roles in connective tissue destruction and chronic inflammation in diseases such as rheumatoid arthritis.
Relationship between Type II Phosphatidylinositol 4‐kinase Activity and Protein Tyrosine Phosphorylation in Membranes from Normal and Sickle Red Cells
Authors: De Neef René‐Serge , Hardy‐Dessources Marie‐Dominique , Giraud Françoise
Published: August 2004

To assess the origin of the previously reported higher type II phosphatidylinositol 4‐kinase (PtdIns 4‐kinase) activity of sickle‐red‐cell membranes [Rhoda‐Hardy‐Dessources, M. D., de Neef, R. S., Mérault, G. & Giraud, F. (1993) Biochim. Biophys. Acta 1181, 90–96], we have investigated the possible involvement of protein kinase C and tyrosine kinases in the regulation of the lipid kinase activity. Both protein kinase activities were found to be markedly higher in membranes from the pathological cells. When isolated normal‐red‐cell or sickle‐red‐cell membranes were assayed, phosphatidylinositol phosphorylation activity was not significantly modified after phorbol ester modulation of protein kinase C. In contrast, stimulation (with sodium orthovanadate) or inhibiton (by tyrphostin) of tyrosine phosphorylation led, respectively, to increased or decreased PtdIns 4‐kinase activity in membranes from both cell types. Moreover, immunoprecipitations of membrane extracts from normal and sickle red cells with anti‐PtdIns 4‐kinase antibody 4C5G, followed by immunoblotting with an anti‐phosphotyrosine Ig, revealed a 56‐kDa band migrating with PtdIns 4‐kinase activity. Taken together, these findings indicate that PtdIns 4‐kinase in red blood cells is a phosphotyrosine‐containing protein and could be regulated by a mechanism involving tyrosine phosphorylation, and the increase in PtdIns 4‐kinase activity of sickle‐red‐cell membranes is at least in part mediated by their high intrinsic tyrosine kinase activity.
Purification, Metal Cofactor, N‐Terminal Sequence and Subunit Composition of a 5‐Aminolevulinic Acid Dehydratase from the Unicellular Green Alga Scenedesmus obliquus, Mutant C‐2A′
Authors: Stolz Mirjam , Dörnemann Dieter
Published: August 2004

5‐Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C‐2A′, starting with serial affinity chromatography according to Wang et al. [Wang, W.‐Y., Gough, S. P. & Kannangara, C. G. (1981) Carlsberg Res. Commun. 46, 243–257], followed by separation on DEAE‐Cellulose DE 52, TSKgel Toyopearl HW‐55 and FPLC on Mono Q. The enzyme was purified 117‐fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 μkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N‐terminus could be determined, showing a moderate similarity to 5‐aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282±5 kDa. 42±1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six‐membered ring. Additionally, there is some evidence that two of the rings form a sandwich‐like complex.
Anti‐Peptide Immunoglobulins from Rabbit and Chicken Eggs Recognise Recombinant Human Dihydroorotate Dehydrogenase and a 44‐kDa Protein from Rat Liver Mitochondria
Authors: Knecht Wolfgang , Köhler Rolf , Minét Michèle , Löffler Monika
Published: August 2004

Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate. 312‐bp and 983‐bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32. The identity of the PCR products was verified by dideoxynucleotide sequencing. Transformation of Escherichia coli strain M15 resulted in expression of 13‐kDa and 36‐kDa proteins with an affinity tag consisting of six consecutive histidine residues; these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+‐chelating resin. In immunoblotting analyses, the fusion proteins were recognised by polyclonal avian and mammalian anti‐peptide immunoglobulins. These were generated against synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDNA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabbits and laying hens were immunized with these peptides without conjugation to a carrier protein. Comparison of the anti‐peptide immunogobulins produced from egg yolk and rabbit serum demonstrated that avian anti‐(dihydroorotate dehydrogenase) immunoglobulins may be considered a superior alternative to the mammalian equivalent; antibodies from both sources were applicable for all immunochemical purposes. Here, these antibodies were applied for identification of a 44‐kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrogenase activity.
Regulation of Expression of the Rat Serine Protease Inhibitor 2.3 Gene by Glucocorticoids and Interleukin‐6
Authors: Simar‐Blanchet Anne‐Emmanuelle , Paul Conception , Mercier Louis , Le Cam Alphonse
Published: August 2004

The rat serine protease inhibitor 2.3 gene (spi 2.3) is almost completely silent in normal animals and is transiently expressed during acute inflammation. It encodes a potential anti‐elastase which is likely to play a major physiological role for the host defense. Two well‐known inflammatory mediators, glucocorticoids and interleukin‐6 (IL‐6) activate the spi 2.3 promoter and increase steady‐state levels of mRNA in cultured hepatocytes. GC activation is mediated by a single glucocorticoid‐response element which seems to act autonomously. A unique array of four functional IL‐6‐response sites was identified in the spi 2.3 promoter. Three of them (C‐II–IV) bear structural identity to the CCAAT/enhancer‐binding‐protein‐binding site consensus sequence, whereas the forth closely resembles the consensus κB nuclear factor recognition motif. The C‐IV element, which is the most active, contains the motif 5′‐CTGGGA and binds the IL‐6‐inducible acute‐phase response factor present in liver nuclear extracts from inflamed rats. Both basal and IL‐6‐dependent activities of each individual cytokine‐response element tested separately are strongly down regulated by a recently identified regulatory sequence [Le Cam, A. & Legraverend, C. (1995) Eur. J. Biochem. 231, 620–6271, located in the 3′ untranslated region of the spi 2.3 gene. However, this repressor element does not significantly affect overall IL‐6‐dependent spi 2.3 promoter activity. This suggests that, in the context of the active gene in vivo, all four IL‐6‐response sites, which are largely redundant, cooperate to overcome the strong repressive effect of the 3′ untranslated region silencer and are needed to bring about a maximal TL‐6 response.

These data reveal a novel type of regulation of an acute‐phase gene involving different classes of IL‐6‐response elements controlled by a repressor and acting in conjunction with a glucocorticoid‐response element.
The Gly74→Ser and Ser3→Ala Mutations in Rhodobacter sphaeroides Y Thioredoxin
Authors: Pille Sabine , Assemat Karine , Breton Annick M. , Clément‐Métral Jenny D.
Published: August 2004

In this study, we report the effects of two different substitutions in Rhodobacter sphaeroides thioredoxin on two regions of the protein: the N‐terminus end and the hydrophobic area implicated in protein/protein interactions. We have produced by site‐directed mutagenesis R. sphaeroides thioredoxin single and double mutants in which the glycine residue at position 74 is changed to a serine and the serine: it position 3 is changed to an alanine; the three mutant proteins have been purified. The two substitutions are not equivalent. Substitution of serine by alanine increased the pI from 5.2 to 6.1; this pI value was the same in the double‐mutated protein, which demonstrates the presence of a local conformational change. In vivo studies showed that the Gly74→Ser substitution completely prevented phage T3/7 growth whereas the Ser3→Ala substitution had no effect. This finding was corroborated by the large decrease (100‐fold) of polymerase activity for the double mutant in the in vitro measurement of phage T7 DNA polymerase activity with the corresponding pure proteins. Although marginal(within a factor of two), the effects of the two substitutions on the catalytic activities of the thioredoxin reductase reaction confirmed their difference. Substitution of serine by alanine had no effect on the Km and resulted in an improvement in the catalytic efficiency. In contrast, the second substitution increased the Km value, without improving the catalytic efficiency. The following can be concluded (a) glyciiie74 of R. sphaeroides thioredoxin has a direct role in the binding of T7 gene 5 protein and the hydrophobic area of thioredoxin: (b) the N‐terminus plays a role in maintaining the conformational integrity of the active site; (c) the flexibility of Gly74 in the hydrophobic region involved in protein/protein interaction is the operative factor in the case of the activity of thioredoxin in the T7 DNA polymerase.
Purification and Characterisation of A Plasmin‐Sensitive Surface Protein of Staphylococcus aureus
Authors: Hildén Pekka , Savolainen Katri , Tyynelä Jaana , Vuento Matti , Kuusela Pentti
Published: August 2004

Certain methicillin‐resistant Staphylococcus aureus strains contain a 230‐kDa cell‐wall protein which is not present on the surface of other staphylococci. The presence of this 230‐kDa protein is associated with a negative test result in commercial assays designed to detect fibrinogen‐binding proteins and/or protein A on the staphylococcal surface. We have purified and partially characterised the 230‐kDa protein from a lysostaphin digest of a non‐agglutinating methicillin‐resistant S. aureus strain. Partial amino acid sequence data obtained from the purified protein did not reveal any significant similarities to known proteins which indicates that the protein is novel. The 230‐kDa protein was very sensitive to proteolysis; soluble plasmin, or plasmin formed on the bacterial‐cell surface, rapidly degraded the 230‐kDa protein to a 175‐kDa form. The finding that the 230‐kDa protein bound to lectins allowed its purification by affinity chromatography on immobilised wheat germ agglutinin. Furthermore, the degradation of the 230‐kDa protein was associated with an increased adherence of non‐agglutinating methicillin‐resistant S. aureus cells to solid‐phase fibronectin, fibrinogen or IgG.
Bradykinin Induces Tyrosine Phosphorylation in Human Foreskin Fibroblasts and 293 Cells Transfected with Rat B2 Kinin Receptor
Authors: Tippmer Stefanie , Bossenmaier Birgit , Häring Hans
Published: August 2004

The intracellular effects of bradykinin are mediated through the recently cloned B2 kinin receptor which belongs to the superfamily of receptors with seven transmembrane domains. The molecular events which transduce the bradykinin signal on the post‐receptor level are not understood in detail. We studied whether in human foreskin fibroblasts bradykinin treatment induces tyrosine phosphorylation of cellular proteins. Using phosphotyrosine antibodies we detected a bradykinin‐dependent phosphorylation of a group of proteins of about 130 kDa and an additional signal around 70 kDa after starvation of cells. The effect evoked by 10 nM bradykinin was rapid (2 min) and it was partially reduced by the B2‐kinin‐receptor antagonist Hoe 140 which was shown to be a weak inducer of tyrosine phosphorylation. The bradykinin‐mediated tyrosine phosphorylation events were reproduced in human embryonal kidney 293 fibroblasts which were transiently transfected with the rat B2 kinin receptor, but they were not observed in untransfected 293 control cells. These data suggest that the B2 kinin‐receptor subtype is involved. Upon fractionation of cells the 130‐kDa protein group was recovered both in the membrane and the cytosolic protein fraction. To assess the specificity of this bradykinin effect we stimulated human foreskin fibroblasts with epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), insulin‐like growth factor (IGF‐I) and insulin. While IGF‐I, insulin and EGF were almost ineffective, PDGF stimulated the tyrosine phosphorylation of 130‐kDa bands with a similar pattern to that produced by bradykinin. Immunoprecipitation experiments with specific antibodies against potential candidate proteins in the molecular‐mass range around 130 kDa revealed positive results for the focal adhesion kinase FAK and the p130 Src substrate while negative results were obtained for the GTPase‐activating protein GAP, the phospholipase C‐γ1, the Janus kinase JAK‐1 and vinculin. The data suggest that the tyrosine phosphorylation of FAK and the p130 Src substrate might be involved in the B2‐kinin‐receptor signalling cascade.
Translational Control of Poly(A)‐Binding Protein Expression
Authors: Bag Jnanankur , Wu Jun
Published: August 2004

Poly(A)‐binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5′ untranslated region (UTR) of PABP mRNA has several adenine‐rich regions which may serve as the PABP‐binding sites to control its translation by a feed‐back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell‐free system. Results of our studies show that removal of the putative PABP‐binding sites from the 5′ UTR of this mRNA enhances its translation in the rabbit reticulocyte cell‐free system. Furthermore, in vitro translation of the full‐length PABP mRNA was inhibited by addition of purified PABP to the cell‐free system. In contrast, translation of truncated mRNA lacking the putative PABP‐binding sites at the 5′ UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5′ UTR of PABP mRNA using ultraviolet‐mediated covalent cross‐linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5′ UTR of its full‐length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5′ UTR. The adenine‐rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell‐free system under conditions of mRNA translation, the polysomal and non‐translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA‐protein (mRNP) complexes following ultraviolet‐induced cross‐linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed‐back mechanism.
Molecular Sequencing and Modeling of Neobellieria bullata Trypsin
Authors: Borovsky Dov , Janssen Ine , Vanden Broeck Jozef , Huybrechts Roger , Verhaert Peter , De Bondt Hendrik L. , Bylemans Dany , De Loof Arnold
Published: August 2004

Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reverse transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3′ end of the gene (300 bp) was amplified by means of the rapid‐amplification‐of‐cDNA‐ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three‐dimensional models of Neobellieria trypsin and Drosophila trypsin were built and compared. Both models contain two domains of β‐barrel sheets as was shown by means of X‐ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Serl82 whereas Aspl76 sits at the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugarfed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin‐modulating oostatic factor (TMOF) at 10−9M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.
Transient Expression of a Mitochondrial Precursor Protein
Authors: Huckriede Anke , Heikema Astrid , Wilschut Jan , Agsteribbe Etienne
Published: August 2004

In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit‐9–H2folate‐reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that it was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature‐sized protein was folded into a protease‐resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit‐9–H2folate‐reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV‐mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders of organelle protein import.
Structure of the Human Gene Encoding the Phosphorylase Kinase β Subunit (PHKB)
Authors: Wüllrich‐Schmoll Andrea , Kilimann Manfred W.
Published: August 2004

We have determined the cDNA sequence and the gene structure of the human phosphorylase kinase β subunit (PHKB). With 95% amino acid sequence identity, the predicted primary structure is highly similar to that of the rabbit β subunit. At least 140 kilonucleotides in length, the gene is large and consists of 33 exons. Exons 26 and 27 are two homologous, mutually exclusively spliced exons in the middle of the gene, and exon 2 is a facultatively utilized cassette exon encoding an alternative N‐terminus of the β subunit. The previous assignment of the PHKB gene to chromosome 16 is confirmed by the successful screening of a chromosome 16‐specific genomic library. Plaque hybridization at reduced stringency led to the isolation of two processed pseudogenes, PHKBP1 and PHKBP2, but of no other PHKB ‐related sequences.
One Molecule of Molybdopterin Guanine Dinucleotide is Associated with Each Subunit of the Heterodimeric Mo‐Fe‐S Protein Transhydroxylase of Pelobacter acidigallici as Determined by SDS/PAGE and Mass Spectrometry
Authors: Reichenbecher Wolfram , Rüdiger Angelika , Kroneck Peter M. H. , Schink Bernhard
Published: August 2004

The molybdenum‐containing iron‐sulfur protein 1,2,3,5‐tetrahydroxybenzene:1,2,3‐trihydroxyben‐zene hydroxyltransferase (transhydroxylase) of Pelobacter acidigallici was investigated by various techniques including mass spectrometry and electron paramagnetic resonance. Mass spectrometry confirmed that the 133‐kDa protein is a heterodimer consisting of an α subunit (100.4 kDa) and a β subunit (31.3 kDa). The presence of a molybdenum cofactor was documented by fluorimetric analysis of the oxidized form A of molybdopterin. The enzyme contained 1.55 ± 0.14 mol pterin and 0.92 ± 0.25 mol molybdenum/mol enzyme (133 kDa). Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethyl)‐molybdopterin. Upon acid hydrolysis, 1.4 mol 5′GMP/mol enzyme (133 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide. The α and β subunits were separated by preparative gel electrophoresis. Both subunit fractions were free of molybdenum but contained equal amounts of a fluorescent form of the molybdenum cofactor. Mass spectrometry at various pH values revealed that an acid‐labile cofactor was released from the large subunit and also from the small subunit. At X‐band, 5–25 K, transhydroxylase (as isolated) showed minor EPR resonances with apparent g values around 4.3, 2.03 and, depending on the preparation, a further signal at g of approximately 1.98. This signal was still detectable above 70 K and was attributed to a Mo(V) center. Upon addition of dithionite, a complex set of intense resonances appeared in the region g 2.08–1.88. From their temperature dependence, three distinct sites could be identified: the Fe‐S center I with gx,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable <20 K); the Fe‐S center II with gx,y,z at approximately 1.872, 1.955 and 2.051 (gav 1.959, detectable >20K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable >70 K).
Effects of Overexpression of the Transferrin Receptor on the Rates of Transferrin Recycling and Uptake of Non‐Transferrin‐Bound Iron
Authors: Callus Bernard A. , Iacopetta Barry J. , Kühn Lukas C. , Morgan Evan H.
Published: August 2004

The possibilities that the recycling of the transferrin receptor is a rate‐limiting step in the efflux of endocytosed transferrin, and that the receptor functions as a trans‐membrane Fe transporter were investigated in untransfected Ltk‐ cells and in cells transfected with different levels of DNA for wild‐type, mutant and chimeric human transferrin receptors. The uptake of transferrin‐bound Fe and non‐transferrin‐bound Fe(II), and the surface binding, endocytosis and recycling of transferrin were measured. In cells that expressed increasing numbers of surface transferrin receptors, the rate of Fe uptake increased at a slower rate than the number of receptors. By measurement of the rates of endocytosis and recycling of transferrin it was shown that this effect was not due to a deficiency of endocytosis, but to a slower rate of recycling as the receptor numbers increased. Hence, a restricted recycling rate of the transferrin receptor appeared to be responsible for the slower rate of Fe uptake by cells with high receptor numbers, presumably because one or more cytosolic components required for recycling were in limited supply. The rate of uptake of non‐transferrin‐bound Fe(II) was not influenced by the number of transferrin receptors present on the surface of the cells even though this varied more than 20‐fold between the different cell lines. Hence, this investigation does not support the hypothesis that the receptors play a direct role in the transport of Fe(II) across cell membranes, as has been proposed previously [Singer, S. J. (1989) Biol. Cell 65, 1–5].
Synthesis and Solution Structure of the Antimicrobial Peptide Protegrin‐1
Authors: Aumelas André , Mangoni Mattéo , Roumestand Christian , Chiche Laurent , Despaux Ernest , Grassy Gérard , Calas Bernard , Chavanieu Alain
Published: August 2004

Protegrins are members of a family of five Cys‐rich, cationic antimicrobial peptides recently isolated from porcine cells. We have synthesised an 18‐amino‐acid peptide that corresponds to protegrin‐1. After Cys oxidation, the peptide has bactericidal activity against gram‐positive and gram‐negative bacteria, similar to that described for the natural peptide. The solution structure of protegrin‐1 was investigated by means of 1H‐NMR spectroscopy in water and in (CD3)2SO, with distance‐geometry and simulated‐annealing calculations. The C6‐C15 and C8‐C13 disulfide pattern was determined on the basis of NMR‐derived constraints. These two parallel disulfide bridges stabilised a β‐sheet structure which comprised two anti‐parallel strands (residues 5–9 and 12–16) linked by a distorted β‐turn (residues 9–12). The N‐terminus and C‐terminus were essentially disordered. The distribution of hydrophobic and hydrophilic residues at the peptide surface was found to be a structural feature shared with tachyplesin‐1, a related peptide which displays cytolytic activity, and, to a lesser extent, with mammalian defensins. These findings led us to assume that the distribution pattern could be required for the cytolytic activity of these peptides.