bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase
Authors: COLLEN Desiré , VAN HOEF Berthe , SCHLOTT Bernhard , HARTMANN Manfred , GÜHRS Karl‐Heinz , LIJNEN H. Roger 
Published: March 2005

The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified plasminogens and α2‐antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent plasminogen activators (catalytic efficiencies, kcatKm, of 1.0 μM−1· s−1 and 0.3 μM−1· s−1, respectively). The complex with SK was insensitive to α2‐antiplasmin, which, however, rapidly inhibited the complex with STAR (second‐order rate constant, k1,app of 8 × 106 M−1· s−1). In a system composed of a 0.06‐ml 125I‐fibrin‐labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC50), with 120nM and 13 nM, respectively. Clot lysis with SK was non‐fibrin specific (residual fibrinogen <10%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine plasminogen reacted avidly with SK, but SK was rapidly degraded; it reacted rapidly and quantitatively with STAR to form a potent plasminogen‐activating complex (kcat/Km of 0.4 μM−1 s−1) which was sensitive to neutralization by α2‐antiplasmin (k1,app of 6 × 105 M−1· s−1). In a canine plasma milieu, SK was relatively potent (EC50 200 nM) and fibrin specific, whereas STAR was very potent (EC50 1.3 nM) but poorly fibrin specific. Baboon and rabbit plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous plasminogens (kcat/Km 0.02 μM−1· s−1) and reacted more slowly with α2‐antiplasmin (k1,app 5–10 × 105 M−1· s−1). Bovine plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of plasminogen and α2‐antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine plasminogen and α2‐antiplasmin.It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous plasminogen and by the rate of inhibition of these complexes by α2‐antiplasmin.
Large‐scale preparation of fully deuterated cell components
Authors: VANATALU Kalju , PAALME Toomas , VILU Raivo , BURKHARDT Nils , JÜNEMANN Ralf , MAY Roland , RÜHL Martin , WADZACK Jörg , NIERHAUS Knud H. 
Published: March 2005

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log‐phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, μ≤ 0.35 h−1, for acetate/succinate μ≤ 0.1 h−1) which avoids the accumulation of the substrate or of by‐products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source.The cells were first grown in fed‐batch to high cell densities (above 50 g wet cells/1) under conditions of substrate limitations. A steady‐flow fermentation followed keeping the growth rate at about μ of 0.1 h−1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.
Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis
Authors: MICHALON Pascal , COUTURIER Roger , BENDER Klaus , HECKER Hermann , MARION Christian 
Published: March 2005

The sensitive electric‐birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady‐state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times.In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1‐depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 μs. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA‐protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher‐order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1.The accessibility and structural role of histone‐like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilsin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked.The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by incrases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment.This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its inovolvement in nucleosome stabilization rather than higher‐order condensation.Therefore, in T. brucei brucei chromatin, the charcteristic properties a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N‐terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different. Since there is no binding with H1, it may be hypothesized that a (non‐histone) protein, or core protein domains, may play a part in chromatin compaction and gene expression regulation comparable to that of H1 in higher eukaryotes.
Complete sequence of human fast‐type and slow‐type muscle myosin‐binding‐protein C (MyBP‐C)
Authors: WEBER Franz E. , VAUGHAN Kevin T. , REINACH Fernando C. , FISCHMAN Donald A. 
Published: March 2005

Myosin‐binding‐protein C (MyBP‐C) is a myosin‐associated protein of unknown function found in the cross‐bridge‐bearing zone (C region) of A bands in striated muscle. Using a cDNA clone encoding the fast‐type isoform of chicken MyBP‐C, we screened a human fetal muscle cDNA library and isolated clones encoding the full‐length human fast‐type isoform of MyBP‐C. cDNA clones encoding the slow‐type isoform of human MyBP‐C, were also isolated and fully sequenced. Northern‐blot analysis demonstrated skeletal muscle‐specific expression of these gene products. Using human/hamster somatic‐cell hybrids, we were able to map the slow‐type MyBP‐C to human chromosome 12, and the fast‐type MyBP‐C to chromosome 19.The cDNA for human fast‐type MyBP‐C encodes a polypeptide of 1142 amino acids with an expected molecular mass of 128.1 kDa. Comparison of this cDNA with other members of the MyBP family reveals extensive primary‐sequence conservation. Each MyBP‐C contains seven immunoglobulin C2 motifs and three fibronectin type‐III repeats in the arrangement C2‐C2‐C2‐C2‐C2‐III‐III‐C2‐III‐C2. Regions of high identity shared by the chicken and the two human proteins are not restricted to the immunoglobulin and fibronectin motifs. Sequence comparison of all three proteins has allowed us to map a highly conserved region between the first and second C2 motifs, the only large spacer sequence present between motifs in these proteins.
Post‐translational modifications of theDictyostelium discoideumglycoprotein PsA
Authors: HAYNES Paul A. , GOOLEY Andrew A. , FERGUSON Michael A. J. , REDMOND John W. , WILLIAMS Keith L. 
Published: March 2005

Prespore‐specific antigen (PsA) is a cell‐surface glycoprotein isolated fromDictyostelium discoideum, which is post‐translationally modified by addition of carbohydrate to threonine residues of the carboxy‐terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane. The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined. The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono‐unsaturated fatty acid as the major component. The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol‐specific phospholipase‐C enzyme. The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Manα1‐2Manα1‐6Manα1‐4GlcNH2and Manα1‐2Manα1‐2Manα1‐6Manα1‐4GlcNH2. The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide. The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of theD. discoideumGPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes.Compositional analysis of the carbohydrate attached to threonine residues in the carboxy‐terminal peptide domain is also presented. The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.
The mannose‐specific lectins from ramsons (Allium ursinum L.) are encoded by three sets of genes
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J. 
Published: March 2005

Lectin cDNA clones encoding the two mannose‐binding lectins from ramsons (Allium ursinum L.) bulbs, AUAI and AUAII (AUA, Allium ursinum agglutinin), were isolated and characterized. Sequence comparison of the different cDNA clones isolated revealed three types of lectin clones called LECAUAG0, LECAUAG1 and LECAUAG2, which besides the obvious differences in their sequences also differ from each other in the number of potential glycosylation sites within the C‐terminal peptide of the lectin precursor. In vivo biosynthesis studies of the ramson lectins have shown that glycosylated lectin precursors occur in the organelle fraction of radioactively labeled ramson bulbs.Despite the similarities between the A. ursinum and the A. sativum (garlic) lectins at the protein level, molecular cloning of the two ramson lectins has shown that the lectin genes in A. ursinum are organized differently. Whereas in A. sativum the lectin polypeptides of the heterodimeric ASAI are encoded by one large precursor, those of the heterodimeric AUAI lectin are derived from two different precursors. These results are confirmed by Northern blot hybridization of A. ursinum RNA which, after hybridization with a labeled lectin cDNA, reveals only one band of 800 nucleotides in contrast to A. sativum RNA which yields two bands of 1400 and 800 nucleotides. Furthermore it is shown that the two mannose‐binding lectins are differentially expressed.
Heterogeneity of glycosylphosphatidylinositol‐anchored alkaline phosphatase of calf intestine
Authors: BUBLITZ Renate , ARMESTO Julio , HOFFMANN‐BLUME Eva , SCHULZE Margarete , RHODE Heidrun , HORN Anton , AULWURM Steffen , HANNAPPEL Ewald , FISCHER Werner 
Published: March 2005

A method is described for large‐scale purification of glycosylphosphatidylinositol‐anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components.No significant differences between native glycosylPtdIns‐anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids l‐leucine, l‐phenylalanine, and l‐tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively.Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl‐Sepharose chromatography. Fractions I corresponds to the anchorless enzyme, fractions II–V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns‐PLC or PLD action, almost 50% of fraction III is split by PtdIns‐PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively.Aggregates of glycosylPtdIns‐anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X‐100. By gel chromatography in the presence of Brij 35, fractions II–V form stable multiple aggregates of dimers and may bind different amounts of the detergent.These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns‐anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns‐PLC.The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns‐anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.
Activity and spectroscopic properties of bovine liver catalase in sodium bis(2‐ethylhexyl)sulfosuccinate/isooctane reverse micelles
Authors: HABER J. , MAŚALAKIEWICZ P. , RODAKIEWICZ‐NOWAK J. , WALDE P. 
Published: March 2005

Spectroscopic properties such as ultraviolet‐visible spectroscopy, circular dichroism, steady‐state fluorescence and the catalatic activity of bovine liver catalase (BLC) have been studied in reverse‐micelles microemulsions formed by sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in isooctane. Activity measurements were carried out with the highly water soluble hydrogen peroxide as substrate as a function of temperature and pH, at various concentrations of substrate, enzyme, AOT and at different wo values (wo= [H2O]/[AOT]).The folding of catalase in reverse micelles is not drastically altered, although there are changes in the local surrounding of the prosthetic group. Kinetic measurements have been carried out under first‐order conditions at low substrate concentration. They indicate that the measured substrate decomposition rate is limited by the exchange of substrate molecules between the substrate‐filled and the enzyme‐containing micelles. The specificity constant, ks=kcat/KM, as related to water pool concentrations, ks,wprm, was, at all wo values, considerably lower than ks measured in buffer (ksp). In contrast, overall values of ks in reverse micelles, ks,ovrm, were always 2–4‐times higher than ksb. This apparent superactivity can, however, be ascribed to concentration effects.No ‘bell‐curve’ to describe the wo dependence of catalase activity in reverse micelles was found, k or ks increasing monotonously up to wo= 50.
Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan
Authors: ILG Thomas , HARBECKE Dorothee , WIESE Martin , OVERATH Peter 
Published: March 2005

Leishmania promastigotes, the stage of the parasite characteristic for the sandfly vector, express an abundant glycoconjugate, called lipophosphoglycan, at their surface. Lipophosphoglycan consists of lysoalkyl‐sn‐glycerophosphoinositol linked to a phosphosaccharide core conserved in all species, which is connected to PO4‐6Galβ1,4Manα1 repeats with species‐specific substitutions at the Gal residue; the repeats are capped by conserved and species‐specific oligosaccharides. Most Leishmania species also secrete an acid phosphatase, which, in Leishmania mexicana, is a filamentous complex composed of a phosphorylated glycoprotein and non‐covalently associated proteo‐(high‐molecular‐mass)phosphoglycan.The secreted acid phosphatase complex was used as an antigen to derive a panel of monoclonal antibodies (mAbs). A total of 25 mAbs (17 novel and 8 previously described) were tested by different techniques for their specificity against lipophosphoglycan and secreted acid phosphatase from several Leishmania species. This comparison and the modification of the antigens by chemical or enzymic treatments allowed a classification of the mAbs into several groups.First, from 25 mAbs examined, 22 recognize lipophosphoglycan and the enzyme complex of L. mexicana; only three are specific for secreted acid phosphatase. Two of the latter group are also directed against carbohydrate structures, whereas the third mAb recognizes the 100‐kDa polypeptide of the complex. The secreted acid‐phosphatase‐specific class detects antigen in the flagellar pocket of promastigotes while all anti‐lipophosphoglycan mAbs bind to the cell surface.Second, all 15 anti‐lipophosphoglycan mAbs investigated in detail appear to be directed against the phosphosaccharide repeats or the cap structure rather than the phosphosaccharide core. Two mAbs recognize terminal cap‐structures containing Manα1,2Man residues. Four antibodies are specific for L. mexicana and are probably directed against PO4‐6[Glcβ1,3]Galβ1,4Manα1 repeats while six mAbs react with the unmodified repeats. Two antibodies specific for Leishmania major recognize Galβ1,3‐substituted repeats unique for lipophosphoglycan from this species.Analysis by immunoblotting indicates that the high‐molecular‐mass proteo‐phosphoglycan of L. mexicana secreted acid phosphatase carries epitopes for all anti‐lipophosphoglycan mAbs suggesting the presence of capped phosphosaccharide repeats while the enzymically active glycoprotein subunit is modified by caps but probably not by repeats. In the case of Leishmania donovani secreted acid phosphatase, the enzymically active polypeptide may be directly modified by repeats.The mAbs are used to characterize changes in lipophosphoglycan structure, which occur in culture during the transition of promastigotes from the logarithmic to the stationary growth phase. Furthermore, testing the mAbs against seven species demonstrates their potential for serotyping Leishmania.
The α‐mannosyl‐binding lectin from leaves of the orchid twayblade (Listera ovata)
Authors: SAITO Keiko , KOMAE Akiko , KAKUTA Mariko , VAN DAMME Els J. M. , PEUMANS Willy J. , GOLDSTEIN Irwin J. , MISAKI Akira 
Published: March 2005

The carbohydrate‐binding specificity of an α‐D‐mannose‐specific lectin isolated from leaves of the orchid twayblade (Listera ovata) was elucidated by quantitative precipitation of mannose‐containing polysaccharides and glycoproteins, hapten inhibition, and affinity chromatography on the immobilized lectin. L. ovata agglutinin (LOA) interacted with various α‐mannans and galactomannans of yeasts, fungi and bacteria, but not with α‐glucans, e.g., dextran and glycogen, as do mannose/glucose‐binding lectins. This lectin, LOA, appears to be highly specific for α1–3 mannosidic linkages. It reacted with a linear α1–3‐mannan (D. P. 15) and, surprisingly, even with a linear α1–3‐mannoheptasaccharide. The LOA/C. tropicalis mannan precipitation reaction was inhibited by α‐linked mannooligosaccharides, in the order, α1–3 > α1–6 > α1–2 linkages; α1–3 [Man]4 and [Man]5 were the best inhibitors among various mannooligosaccharides tested, having 7‐times greater potency than α1–3 [Man]2, and 18‐times that of methyl α‐mannoside. LOA/mannan interaction was also inhibited by periodate‐oxidized and reduced α1–3 [Man]5 which had an inhibitory potency similar to that of α1–3 [Man]3, confirming that LOA also recognizes the internal α1–3‐mannosidic linkages of carbohydrate chains.Complete resolution of mannan and glycogen from yeast cells, by affinity chromatography on an immobilized LOA column, and retention of several high‐mannose‐glycoproteins suggest this lectin to be a useful tool for purification and structural investigation of α‐mannosyl‐containing polysaccharides and glycoconjugates.
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: RENZ Paul , ENDRES Birgit , KURZ Birgit , MARQUART Jörg 
Published: March 2005

Eubacterium limosum transformed [2‐13C]5‐hydroxybenzimidazole not only into [2‐13C]5‐hy‐droxybenzimidazolylcobamide, but also into [2‐13C]5‐methoxy‐6‐methylbenzimidazolylcobamide and into [2‐13C]5,6‐dimethylbenzimidazolylcobamide (vitamin B12). [2‐13C]5‐Hydroxy‐6‐methyl‐benzimidazole was used by this bacterium to form [2‐13C]5‐hydroxy‐6‐methylbenzimidazolyl‐cobamide, [2‐13C]5‐methoxy‐6‐methylbenzimidazolylcobamide and [2‐13C]5,6‐dimethylbenzimid‐azolylcobamide. The 1H‐NMR spectrum of the 5,6‐dimethylbenzimidazole isolated from the 13Clabeled vitamin B12 preparations revealed that the externally added bases had been transformed into the vitamin B12 base almost without dilution of the label. This suggests that 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole are precursors of 5,6‐dimethylbenzimidazole.On the basis of these results, a hypothetical scheme for the biosynthesis of 5,6‐dimethylbenzimidazole via 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole is discussed. This scheme can also explain the formation of the other benzimidazole bases found in natural vitamin B12 analogs.