bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Interaction Between Plasminogen and Staphylokinase
Authors: Lijnen H. Roger , De Cock Frans , Van Hoef Berthe , Schlott Bernhard , Collen Désiré 
Published: March 2005

Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka=ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real‐time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu‐plasminogen was 0.93×108M‐1 as compared to 2.0×108M‐1 and 1.6×108M‐1, respectively, for the binding to [S741A]recombinant plasminogen or Lys‐[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active‐site blocked plasmin occurred with Ka, values of 4.0×108M‐1 and 8.4×108M‐‐1, respectively, whereas active‐site blocked LMM‐plasmin (a plasmin derivative lacking kringles 1– 4) and the plasmin B‐chain bound with Ka values of 1.0×108M‐1 and 0.49×108M‐1, respectively. Lysine‐binding site I (a plasminogen derivative consisting of kringles 1–3) and lysine‐binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2×105M‐1 and 2.9×105M‐1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin‐SakSTAR complex to streptokinase and binding of the plasmin‐streptokinase complex to SakSTAR, were, respectively, 44‐fold and 30‐fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site‐specific mutagenesis of Met26 to arginine or alanine) were 10–20‐fold lower than that of native staphylokinase.These results indicate that: (a) the affinity of staphylokinase for Glu‐plasminogen and Lysplasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1– 4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high‐affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.
Modulation of Activator Protein‐1 (AP‐1) Transcription Factor and Protein Kinase C by Hydrogen Peroxide and d‐α‐Tocopherol in Vascular Smooth Muscle Cells
Authors: Stäuble Barbara , Boscoboinik Daniel , Tasinato Andrea , Azzi Angelo 
Published: March 2005

The effects of hydrogen peroxide, d‐α‐tocopherol and of d‐β‐tocopherol on proliferation, protein kinase C and activator protein‐1 (AP‐1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by d‐α‐tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, d‐α‐tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol‐myristate‐acetate‐stimulated AP‐1 binding to DNA but stimulated it if protein kinase C was down‐regulated or inhibited. d‐α‐Tocopherol promoted AP‐1 activation in quiescent cells but prevented its activation by phorbol myristate acetate.None of the described effects of d‐α‐tocopherol were shared by d‐β‐tocopherol, suggesting a non‐antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and d‐α‐tocopherol affect more than one element in the cell signal‐transduction cascade.
Reactions of Prostaglandin H Synthase with Monosubstituted Hydrazines and Diazenes
Authors: Mahy Jean‐Pierre , Gaspard Sarra , Delaforge Marcel , Mansuy Daniel 
Published: March 2005

The reaction of p‐chlorophenylhydrazine with prostaglandin H synthase (PGHS) Fe(III) under aerobic conditions leads to a partial destruction of the heme and to a new complex absorbing at 436 nm. This complex is also obtained by reaction of p‐chlorophenyldiazene (pClPhN = NH) with PGHS Fe(III) under anaerobic conditions and by oxidation of the PGHS Fe(II)(pClPhN = NH) diazene complex by Fe(CN)6K3. The similarity between those reactions and those of arylhydrazines and aryldiazenes with other hemoproteins such as cytochrome P450 and hemoglobin and myoglobin, as well as the similarities between the spectroscopic and chemical properties of this complex and those of the σ‐aryl complexes of other hemoproteins such as hemoglobin and myoglobin, strongly suggested a PGHS Fe(III)‐pClPh structure for this complex. It was completely established after the extraction of its heme, by butan‐2‐one at 0°C under neutral or acidic conditions, which led to the σ‐aryl PGHS‐Fe(III)‐pClPh complex and to N‐phenylprotoporphyrin IX, respectively. A mechanism is proposed for the formation of the PGHS Fe(III) pClPh complex; it includes the reduction of PGHS Fe(III) into PGHS Fe(II) with formation of the diazene pClPhN = NH. This diazene can bind to PGHS Fe(II) or be oxidized with formation of pClPh free radicals. These radicals can react with PGHS Fe(II) to form the PGHS Fe(III)‐pClPh complex or with the protein, or may initiate free radical oxidations which could lead to destruction of the heme or of the protein.Other alkylhydrazines or arylhydrazines also react with PGHS Fe(III) under aerobic conditions with the formation of PGHS Fe(III)‐R or aryl (Ar) complexes and heme destruction. Alkylhydrazines such as methylhydrazine, which lead to very reactive alkyl radicals, lead to very low amounts of PGHS Fe(III)‐R complex and high amounts of heme destruction, whereas arylhydrazines bearing electron‐withdrawing substituents such as 3,4‐dichlorophenylhydrazine, which lead to stabilized aryl radicals, lead to a high amounts of PGHS Fe(III)‐Ar complex and low amounts of heme destruction.Some information concerning the structure of the active site of PGHS has been drawn from those studies as follows: (a) the formation of σ‐aryl PGHS Fe(III)‐Ar and PGHS Fe(II)(ArN = NH) complexes bearing bulky ligands on the iron is in agreement with a large hydrophobic active site with an easily accessible iron atom and, (b) a comparison of the UV‐visible characteristics of PGHS Fe(III)‐R or Ar complexes with those of other hemoprotein‐Fe(III)‐R or hemoprotein‐Fe(III)‐Ar complexes is in agreement with a histidine as the axial proximal ligand of the iron of PGHS.Finally, the formation of iron(III)‐R or iron (III)‐Ar complexes and the free radical oxidative degradation of the heme could both explain why monosubstituted hydrazines are powerful inhibitors of PGHS and very bad reducing cosubstrates of the peroxidase function of PGHS.
Autoregulation of Actin Synthesis in Hepatocytes by Transcriptional and Posttranscriptional Mechanisms
Authors: Reuner Karl Heinrich , Wiederhold Matthias , Dunker Petra , Just Ingo , Bohle Rainer Maria , Kröger Manfred , Katz Norbert 
Published: March 2005

Treatment of rat hepatocytes with the filamentous‐actin‐stabilizing toxin phalloidin decreased the amount of globular actin by 77% in the cytosol and by 80% in the nucleus within 12 h. Simultaneously, actin mRNA was specifically increased by 230%. The de‐novo synthesis of actin mRNA, as measured by nuclear run‐on transcription, was enhanced by 250%. Treatment of cells with actinomycin D blocked the increase of actin mRNA. The apparent half‐life of actin mRNA was not significantly altered during treatment with phalloidin.In contrast, the globular‐actin‐stabilizing botulinum C2 toxin increased the amount of cytosolic globular actin by 50% within 12 h. Simultaneously, the actin mRNA level was decreased by 62%. However, de‐novo synthesis of actin mRNA was not impaired. The apparent half‐life of actin mRNA was decreased by approximately 60% during treatment with C2 toxin.The data strongly suggest an autoregulatory control of actin synthesis on the basis of the globular/filamentous actin ratio in rat hepatocytes at the transcriptional as well as at the posttranscriptional levels.
Inversion of Lipase Stereospecificity for Fluorogenic Alkyldiacyl Glycerols
Authors: Zandonella Gerhild , Haalck Lutz , Spener Fritz , Faber Kurt , Paltauf Fritz , Hermetter Albin 
Published: March 2005

We synthesized enantiomeric 1‐O‐alkyl‐2,3‐diacyl‐sn‐glycerol and 3‐O‐alkyl‐1,2‐diacyl‐sn‐glycerol containing pyrene as a fluorescent reporter and the trinitrophenylamino residue as a fluorescence quencher; both reporter groups were covalently bound to the ω end of the acyl chains at positions sn‐2 and sn‐3(1), respectively. The fluorescence of the intact substrate molecules was very low. Chemical or enzymic release of the fatty acyl chains lead to fluorescence dequenching. The rate of lipolysis could be measured from the time‐dependent increase in fluorescence intensity. We used the respective substrates for the continuous determination of activity and stereopreference of four different microbial lipases from Chromobacterium viscosum, Candida rugosa, Pseudomonas sp., Rhizopus arrhizus, as well as cutinase from Fusarium solani and lipoprotein lipase from bovine milk. The stereopreference of the lipases depended, in general, on how the substrate was solubilized in the reaction medium. All lipases under investigation preferentially hydrolysed the sn‐1 acyl ester bond, if the lipid analog was dispersed in albumin‐containing Tris/HCl buffer in the absence of detergent or organic solvent.In mixtures of 1:1 (by vol.) water/ethanol, the enzymes showed higher activity toward the sn‐3 acyl ester bond, except for lipoprotein lipase which preferred the sn‐1 acyl isomer under all conditions tested. Different stereopreferences were observed with the different lipases if the substrate was solubilized by amphiphiles (micelles of N‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonate). C. rugosa lipase and F. solani cutinase showed high stereopreference for the sn‐3 acyl ester, whereas Pseudomonas sp. lipase and C. viscosum lipase hydrolysed both enantiomers at similar rates.From spectroscopic studies, it can be inferred that the conformation of the fluorescent lipids is probably similar in water, mixtures of water and organic solvents, and in micelles. The possible effects of reaction conditions on substrate accessibility and enzyme conformation on stereoselectivity of the respective lipases are discussed.
Cytochrome‐c Oxidase in Developing Rat Heart Enzymic Properties and Amino‐terminal Sequences Suggest Identity of the Fetal Heart and the Adult Liver Isoform
Authors: Schägger Hermann , Noack Heiko , Halangk Walter , Brandt Ulrich , Von Jagow Gebhard 
Published: March 2005

Perinatal development of cytochrome‐c oxidase (complex IV) and ubiquinol–cytochrome‐c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densito‐metrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223–231].A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.
The Ontogeny of Apolipoprotein Expression in Rat Liver
Authors: Hall Graham P. , Redgrave Trevor G. , Yeoh George C. T. 
Published: March 2005

The pattern of apolipoprotein (apo) A‐I, A‐IV and E expression in developing rat liver was established by determining steady‐state levels of the respective mRNAs. Apo A‐I and A‐IV altered in a coordinate fashion; the transcripts were detected from day 13 of gestation, whereas apo E was first detected on day 19 of gestation. Apo A‐I and A‐IV mRNA levels increased with developmental age until day 19, then declined until birth, after which they increased. In contrast, apo E mRNA levels progressively increased from day‐13 gestation until 3 days postnatal at which time it reached adult levels.In cultured hepatocytes established from immature (15‐day gestation) and near‐term (19‐day gestation) fetuses the difference in regulation between apo A‐I and A‐IV and apo E was also observed. In 3‐day‐old fetal hepatocyte cultures established from 19‐day gestation rats, dexamethasone, insulin, thyroxine and glucagon each substantially increased levels of apo A‐I and A‐IV mRNA but markedly decreased apo E mRNA. Thus fetal and adult hepatocytes respond similarly to the hormones tested with respect to apolipoprotein expression. Unexpectedly, 15‐day gestation hepatocytes expressed apo E in culture, even without hormone supplementation. The discrepancy between in vivo and in vitro data suggests that, in the fetus, apo E expression may be suppressed by high levels of circulating steroid, insulin and thyroxine and that establishment of the hepatocytes in culture removes the inhibition, thereby inducing apo E expression in these immature cells. The data are also consistent with the view that the same group of hormones may be responsible for regulating levels of apo A‐I and A‐IV in the perinatal period. Both apolipoproteins progressively increase as the fetus reaches term at a time when these hormones which induce their expression are also increasing.
Hybrid Bacillus amyloliquefaciens X Bacillus licheniformisα‐Amylases
Authors: Conrad Birgit , Hoang Viet , Polley Andreas , Hofemeister Jürgen 
Published: March 2005

A series of 33 single and mosaic hybrid α‐amylases was constructed from the genes amyBA or amyLI, encoding the α‐amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus licheniformis (AmyLI). The hybrid proteins, consisting of the entire α‐amylase sequence with a variable portion of AmyBA or AmyLI origin, were characterized in order to find enzymes with new properties (thermostability, temperature and pH optima, and substrate specificity), and to localize the amino acid sequence regions responsible for the changes.The thermostability of the AmyBA/AmyLI (AL‐type) hybrid proteins correlated with the position and the length of the hybrid sequence. The hybrid enzymes fell into six groups retaining, in comparison to AmyBA, a certain value of the extra‐thermostability of AmyLI or becoming more thermolabile than AmyBA. Four regions are proposed to contain thermostability determinants (TSDs). They map between amino acid residues 34–76,112–142,174–179 and 263–276 of the respective hybrid enzymes, indicating the dominance of the N‐terminal half of AmyLI for these hybrid enzymes' resistance against irreversible inactivation. Two (TSD3 and TSD4) coincide with regions I and II that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chem. 264, 18933–18938]. The temperature dependence of activity of the AL‐type hybrid α‐amylases was compared at pH 6.4 and pH 7.6 and the hybrid enzymes of one thermostability group were found to have similar temperature responses.A hybrid region between residues 34–76 is demonstrated to correlate with the α‐amylases' substrate specificity, i.e. either hydrolysis or accumulation of maltohexaose. This region was therefore named the G6G5 region. The exchange of internal sequences between residues 17–201 of AmyBA by the AmyLI counterpart in ALA‐type mosaic hybrid α‐amylases, with one exception (ALA99–429), unexpectedly destabilized the respective ALA‐type hybrids. Two of these hybrid α‐amylases (ALA17–151 and ALA76–151) were less thermostable than AmyBA, while others (ALA112–151, ALA112–201) were enzymically inactive. These data support specific roles of the predicted Al‐B domain portion between residues 17–201 of those Bacillusα‐amylases probably for correct folding and enzymic activity.
Chemical Structures of the Core Region of Campylobacter Jejuni O:3 Lipopolysaccharide and an Associated Polysaccharide
Authors: Aspinall Gerald O. , Lynch Christina M. , Pang Henrianna , Shaver Robert T. , Moran Anthony P. 
Published: March 2005

The complete structure for the core oligosaccharide region of the water‐insoluble low‐Mr lipopolysaccharide of Campylobacter jejuni serotype O:3 from phenol/water extraction of bacterial cells was assigned through studies on derivatives of the liberated oligosaccharide. Structure determinations were performed using 1H‐NMR and 31P‐NMR spectroscopies, methylation analysis supported by fast‐atom‐bombardment mass spectrometry, and Smith degradation experiments. It was concluded that the complete chains in the core oligosaccharide had the following structure in which a proportion of the terminal residues were phosphorylated:
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From a similar series of experiments, it was concluded that an associated polysaccharide, which was isolated from the water phase of the phenol/water extracts, had the following repeating unit in which a proportion of the previously unknown L‐glycero ‐D‐ido ‐heptose (L‐α‐D‐ido ‐Hep) residues were present as 3‐hydroxypropanoyl esters, and were not covalently linked to the lipopolysaccharide:
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A Novel Approach to Introduce Site‐Directed Specific Cross‐Links Within RNA‐Protein Complexes
Authors: Zenkova Marina , Ehresmann Chantal , Caillet Joël , Springer Mathias , Karpova Galina , Ehresmann Bernard , Romby Pascale 
Published: March 2005

We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The first step involves site‐directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3′ or 5′ phosphate, a 4‐[‐N ‐(2‐chloroethyl)‐N ‐methylamino]benzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4‐dinitro‐5‐fluorophenylazide) is condensed to the modified nucleotide.This method was used to induce specific cross‐links between Escherichia coli threonyl‐tRNA synthetase and the leader region of threonyl‐tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl‐tRNA synthetase can be cross‐linked to the enzyme, depending on the nucleotide modified.
Binding of a Fluorescent Nucleotide Analog to Hsc70
Authors: Churchich Jorge E. 
Published: March 2005

2′‐Deoxy‐3′‐anthraniloyladenosine‐5‐triphosphate(Ant‐dATP) was used as an environmentally sensitive probe of the nucleotide‐binding site of the molecular chaperone Hsc70.When coordinated to the lanthanide ion Tb3+, Ant‐dATP is not hydrolyzed by Hsc70. The lanthanide ion acts as strong competitive inhibitor with respect to Mg2+ (Ki= 0.1 μM). Tb · Ant‐dATP recognizes the nucleotide site of Hsc70 as revealed by an increase in the emission anisotropy from 0.03 to 0.21 and by a change in the fluorescence‐decay time from 2.52 ns to 3.75 ns. Sensitized luminescence arising from resonance energy transfer from the anthraniloyl group to Tb3+ is substantially enhanced in the presence of Hsc70.Binding of a 20‐amino‐acid‐residue peptide (Rnase‐S peptide) to Hsc70 causes a blue shift in the fluorescence spectrum of Ant‐dATP and enhances Tb3+ luminescence upon excitation at 330 nm. It is postulated that binding of the peptide to the COOH‐terminal domain of Hsc70 initiates domain movement and the structural changes might extend to the nucleotide‐binding site.
Manganese Peroxidase from Phanerochaete chrysosporium
Authors: Selvaggini Carlo , Salmona Mario , De Gioia Luca 
Published: March 2005

A detailed three‐dimensional model of manganese peroxidase was constructed using lignine peroxidase as the structural scaffold. This is the only protein in the peroxidase family except for cytochrome c peroxidase for which a resolved crystal structure is available.The model was built using the following procedure: (a) structurally preserved regions were derived from similar regions in the sequence alignment of the two proteins; (b) non‐similar regions were modelled by searching a set of resolved protein structures for fragments which fitted in geometrically and choosing the best fitting fragment. Side chains were constructed by calculating rotamer‐rotamer interaction energies and minimizing intramolecular energy. Model refinement was performed by molecular mechanics calculation.The quality of the model was assessed on the basis of the propensity of the amino acids to be inserted into regular secondary‐structure elements and to be exposed to solvent.All the lignine peroxidase regions not used for model construction because of the lack of similarity, except the helix fragment Leu261‐Phe269, correspond to external loops, suggesting reliable modelling.The manganese peroxidase model structure was analyzed in detail and several functionally relevant structural features were predicted, the most important being: (a) the very close structural similarity between lignine and manganese peroxidase active sites, suggesting a similar mode of hydrogen peroxide activation; (b) the substitution of polar residues for the hydrophobic amino acids exposed at the edge of the channel involved in substrate recognition in lignine peroxidase, suggesting that manganese peroxidase does not directly bind aromatic substrates; (c) the location of residues potentially able to bind Mn2+, spatially positioned on the side of the 3‐CH3 heme edge.
Anti‐La Monoclonal Antibodies Recognizing Epitopes Within the RNA‐Binding Domain of the La Protein Show Differential Capacities to Immunoprecipitate RNA‐Associated La Protein
Authors: Pruijn Ger J. M. , Thijssen José P. H. , Smith Paul R. , Williams David G. , Van Venrooij Walther J. 
Published: March 2005

The La (SS‐B) autoimmune antigen is an RNA‐binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti‐La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre‐tRNA, pre‐5S rRNA, hY RNAs, pre‐U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7‐2 RNA.Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross‐competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3.Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA‐binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts.