bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of metallothionein and stomatin by interleukin‐6 and glucocorticoids in a human amniotic cell line
Authors: SNYERS Luc , CONTENT Jean 
Published: March 2005

Interleukin 6 (IL‐6) is an important mediator of various kinds of inflammatory and immune responses. The human amniotic cell line UAC has an increased number of IL‐6 receptors after treatment by glucocorticoids. To find a possible activity of IL‐6 on these cells, a cDNA library of IL‐6‐ and dexamethasone‐treated cells was screened with cDNA probes from both induced and non‐induced cells.Two cDNAs showed a differential hybridization signal. The first one corresponds to metallothionein, a group of small cysteine‐rich proteins thought to participate in the metabolism and storage of zinc and to protect cells against oxidative damage.A second cDNA corresponds to the recently cloned cDNA of band 7 integral membrane protein also called stomatin. In hereditary stomatocytosis, absence of this protein in erythrocyte membranes is associated with high Na+ and low K+ intracellular concentrations [Stewart, G. W., Hepworth‐Jones, B. E., Keen, J. N., Dash, B. C. J., Argent, A. C. & Casimir, C. M. (1992) Blood 79, 1593–1601].In UAC cells both metallothionein and stomatin are induced by dexamethasone and IL‐6 in a more than additive manner. Western blot analysis shows that stomatin protein is induced in a similar way as its mRNA.IL‐6 and dexamethasone induce a state of resistance against hydrogen peroxide toxicity in UAC cells. Metallothionein induction might be partly responsible for this cytoprotection against oxidative stress.
Mechanism of resistance to the antibiotic trichothecin in the producing fungi
Authors: IGLESIAS Maite , BALLESTA Juan P. G. 
Published: March 2005

Trichothecium roseum, an imperfecti fungus producer of the translation inhibitor trichothecin, is constitutively resistant to its product. Fusarium oxysporum, a fungi not described as a toxin producer, is sensitive to trichothecin but becomes resistant when grown in the presence of the drug. In both cases, the resistance occurs at the level of the ribosomes. In cell‐free polypeptide polymerization systems, trichothecin resistance is associated with the presence of 60S subunits from the resistant organisms. Resistant ribosomes can be prepared in vitro by incubating sensitive ribosomes, from either non‐induced F. oxysporum or Saccharomyces cerevisiae, with cell extracts from the resistant cells in the presence of S‐adenosylmethionine. An in‐vitro specific differential methylation is detected in the sensitive ribosomes but not in resistant particles using radioactive S‐adenosylmethionine.The results indicate for the first time the existence in eukaryotic organims of an antibiotic‐resistance mechanism involving a ribosomal methylation similar to that described previously in prokaryotic systems.
(S)‐2‐Amino‐1,3‐propanediol‐3‐phosphate‐carrying diradylglyceroglycolipids
Authors: FISCHER Werner , HARTMANN Rudolf , PETER‐KATALINIĆ Jasna , EGGE Heinz 
Published: March 2005

Two novel aminophosphoglycolipids (I, II) were isolated from Clostridium innocuum which constitute 51% (I) and 15% (II) of total polar membrane lipids. The structures, established by quantitative and methylation analyses, fast‐atom‐bombardment mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy, are (I) S‐2‐amino‐1,3‐propanediol‐3‐phospho‐6‐α‐d‐galactopyranosyl(1‐2)α‐d‐glucopyranosyl(1‐3)diradylglycerol and (II) an acylated derivative of (I) that carries an additional fatty acid ester on O6 of the glucosyl moiety. The stereochemical configuration of the 2‐amino‐1,3‐propanediol 3‐phosphate residue was elucidated by conversion to N‐acetylserine 3‐phosphate, with subsequent release and identification of l‐serine by HPLC. In addition to diacylglycerol species, both aminophosphoglycolipids contain 15–32% 1‐O‐(alk‐1‐enyl)‐2‐O‐acyl‐glycerol species in which C14, C16, and C18 vinyl ether are combined predominantly with unsaturated C16 and C18 fatty acid ester. Hydrogenation of the vinyl ether was required to desorb the alkyl, acylsubstituted species in fast‐atom‐bombardment mass spectrometry. Hydrogenation made it further possible to release the alkyl glycerols by acid hydrolysis and to locate the ether bond at O1 of the glycerol moiety.In contrast to the glycerophosphoglycolipids of other Gram‐positive bacteria, the aminophosphoglycolipids are metabolically not related to the lipoteichoic acid of C. innocuum and serve, therefore, exclusively as major membrane components. Their large abundance among membrane lipids suggests bilayer‐forming physicochemical properties.
Structural Studies of the O‐Antigenic Polysaccharides of Escherichia coli O3 and the Enteroaggregative Escherichia coli Strain 17‐2
Authors: Medina Ernesto C. , Widmalm Göran , Weintraub Andrej , Vial Pablo A. , Levine Myron M. , Lindberg Alf A. 
Published: March 2005

The polysaccharide part of the lipopolysaccharide isolated from an enteroaggregative Escherichia coli isolated from a young child with diarrhoea in Santiago, Chile (strain 17‐2), has been investigated. Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H‐NMR and 13C‐NMR spectroscopies revealed that the polysaccharide is composed of pentasaccharide repeating units. The structure of the repeating unit of E. coli strain 17‐2 O‐polysaccharide is:
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The structure of the O‐polysaccharide from E. coli O3 was shown to be identical to that of E. coli strain 17‐2 by sugar and methylation analyses and by 1H‐NMR and 13C‐NMR spectroscopies.
ADP‐Induced Changes in Ordering of Spin‐Labelled Myosin Heads in Muscle Fibres
Authors: Belágyi Joseph , Frey István , Pótó László 
Published: March 2005

Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate‐based spin label attached to the fast‐reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2–3 ± 0.1 μm static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels.The spetcra of isothiocyanate spin‐labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75° and 56°. In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a θ value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin‐labelled fibres, however, the effect of MgADP on the probe angular distribution was small.
Evidence fromIn vivoManipulations of Lipid Composition in Mutants that the Δ3‐Trans‐Hexadecenoic Acid‐Containing Phosphatidylglycerol is Involved in the Biogenesis of the Light‐Harvesting Chlorophylla/b‐Protein Complex ofChlamydomonas reinhardtii
Authors: Dubertret Guy , Mirshahi Ali , Mirshahi Massoud , Gerard‐Hirne Catherine , Trémolieres Antoine 
Published: March 2005

The phosphatidylglycerol containing the unusual Δ3‐transhexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light‐harvesting chlorophylla/b‐protein complex has been evidenced byin vivotargeting this lipid to photosynthetic membranes ofChlamydomonas reinhardtiimutants lacking this lipid. In themf1andmf2mutants, this deficiency results in (a) the absence of the oligomeric lightharvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77‐K fluorescence emission in the photosystem–2 region.We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem‐2 light‐harvesting complex; it contains less chlorophyllband some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack oftrans‐C16: 1‐phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light–harvesting complex (pigment and polypeptide composition, oligomerization) after liposome‐mediated,in vivoincorporation of this lipid into the photosynthetic membranes of themflmutant.Trans‐C16: 1‐phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem‐2 light‐harvesting chlorophylla/b‐protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.
Interaction of Plant Profilin with Mammalian Actin
Authors: Giehl Klaudia , Valenta Rudolf , Rothkegel Martin , Ronsiek Melanie , Mannherz Hans‐Georg , Jockusch Brigitte M. 
Published: March 2005

The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(l‐proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two‐dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus.In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration‐dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg2+‐sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography.Cross‐linking experiments with actin, birch profilin, the G‐actin binding peptide thymosin β4 and gelsolin segment 1, the N‐terminal fragment of an actin capping protein, showed that profilin competed with thymosin β4, but had no effect on segment 1 binding to actin.These data indicate that the actin‐binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.
Structure and Function of Mutant Arg44Lys of 4‐Hydroxybenzoate Hydroxylase
Authors: Eppink Michel H. M. , Schreuder Herman A. , Van Berkel Willem J. H. 
Published: March 2005

Arg44, located at the si‐face side of the flavin ring in 4–hydroxybenzoate hydroxylase, was changed to lysine by site‐specific mutagenesis. Crystals of [R44K]4‐hydroxybenzoate hydroxylase complexed with 4‐hydroxybenzoate diffract to 0.22‐nm resolution. The structure of [R44K]4‐hydroxybenzoate hydroxylase is identical to the wild‐type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The φ,ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4‐Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type.[R44K]4‐Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped‐flow kinetics showed that the effector role of 4‐hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge‐transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.
Hepatocyte‐Derived Interleukin‐6 and Tumor‐Necrosis Factor α Mediate the Lipopolysaccharide‐Induced Acute‐Phase Response and Nitric Oxide Release by Cultured Rat Hepatocytes
Authors: Saad Bashar , Frei Karl , Scholl Florence A. , Fontana Adriano , Maier Peter 
Published: March 2005

The regulation of acute‐phase protein production and nitric oxide (NO) release in lipopolysaccharide‐induced liver injury is thought to occur in response to monocytes/macrophages and Kupfrer‐cell‐derived cytokines. In this study, we used primary cultured rat hepatocytes maintained as a differentiated phenotype to investigate the direct effects of endotoxin (lipopolysaccharide) on the production of the acute‐phase proteins and on NO release.Lipopolysaccharide (10 (μg/ml) increased the production of α2‐macroglobulin 2.5‐fold compared to untreated cultures and decreased the production of albumin by 50%. The effect of lipopolysaccharide was mimicked by adding interleukin‐6 (IL‐6) and tumor‐necrosis factor α(TNF‐α), cytokines being induced by treatment of hepatocytes with lipopolysaccharide. Maximal TNF‐α (600 pg/ml) and IL‐6 (1800pg/ml) concentrations were observed 4 h and 6 h after lipopolysaccharide stimulation, respectively. The lipopolysaccharide‐induced acute‐phase protein response was blocked by anti‐(IL‐6) but not by anti‐(TNF‐α) IgG. The latter reduced the lipopolysaccharide‐induced IL‐6 production by 60%. Besides its effects on the acute‐phase proteins, endotoxin caused a significant increase in NO production in cultured rat hepatocytes. Unlike anti‐(IL‐6) IgG, anti‐(TNF‐α) IgG reduced the lipopolysaccharide‐induced NO production by 50% indicating that endotoxin‐induced NO production is partially mediated by TNF‐α but not by IL‐6. Precul‐ture with gadolinium chloride (GdCl3), an inhibitor of Kupffer cells, did not change the response of hepatocytes to lipopolysaccharide indicating that the observed findings are direct endotoxin effects on hepatocytes. The data demonstrate that by their production of TNF‐α and IL‐6 rat hepatocytes respond to lipopolysaccharide treatment with an IL‐6 mediated acute‐phase protein and a TNF‐a‐mediated NO production. These features have previously been attributed to monocytes/macrophages and Kupffer cells.
Use of Mathematical Models for Predicting the Metabolic Effect of Large‐Scale Enzyme Activity Alterations
Authors: Schuster Ronny , Holzhütter Hermann‐Georg 
Published: March 2005

There are numerous examples showing that the metabolism of cells can be severely impaired if the activity of only one of the participating enzymes undergoes large‐scale alterations, resulting, for example, from spontaneous mutations (inherited or aquired enzymopathies), the administration of toxic drugs or self‐inactivation of enzymes during cell aging. However, a quantitative relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction is very difficult to establish by experimental means. An alternative is to tackle this problem by mathematical modelling. Our approach is based on a comprehensive mathematical model of the energy and redox metabolism for human erythrocytes. We calculate stationary states of the cell metabolism, varying the activity of each of the participating enzymes by several orders of magnitude. The metabolic states are then evaluated in terms of a performance function which relates the metabolic variables to the overall functional fitness of the cell. The performance function for the erythrocyte takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione), and the osmotic state. Based on the behaviour of the performance function at varying enzyme activities, we estimate those ranges of enzyme activities, in which the metabolic alterations should be either tolerable, associated with non‐chronic or chronic diseases, or letal. For most enzymopathies, the experimental and clinical observations can be satisfactorily rationalized by the computational results. Moreover, a surprisingly high correlation is found between the range of the activity range where disease is predicted by the model and the observed number of diseased probands.Another objective of our study was to contribute to the theory of metabolic control. The well‐elaborated concept of the metabolic control theory is restricted to (infinitely) small activity alterations. In order to quantify the metabolic effect of finite (large‐scale) changes in the activity of an enzyme, we propose, as a control measure, the effective activity Ea, defined as the relative activity of an enzyme (with respect to the activity in a reference state) required to bring about a change in the stationary value of a metabolic variable by the (finite) factor α. We demonstrate that none of the existing extrapolation methods using the conventional control coefficient is capable to provide reliable predictions of the effective activities for all enzymes of erythrocyte metabolism.
Prolonged Half‐Life in the Circulation of a Chemical Conjugate Between a Pro‐Urokinase Derivative and Human Serum Albumin
Authors: Breton Jérôme , Pezzi Nieves , Molinari Antonio , Bonomini Luisella , Lansen Jacqueline , De Buitrago Gonzalo Gonzalez , Prieto Ignacio 
Published: March 2005

Pro‐urokinase is a natural plasminogen activator that displays a clot‐lysis activity through a fibrin‐dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue‐type plasminogen activator and two‐chain urokinase‐type plasminogen activator, pro‐urokinase has a very short half‐life in circulation.It has been described that conjugation of serum albumin with pro‐urokinase in plasma may occur that could protect this protein from degradation.In this study we describe the insertion of an extra cysteine residue in the N‐terminal end of des‐(C11–K135)‐pro‐urokinase (Δ125‐proUK), a pro‐urokinase deletion mutant lacking amino acids 11–135. We have expressed and purified the new mutein [H5K, S9C, N10T]des‐(C11‐K135)‐pro‐urokinase (Cys‐Δ125‐pro‐urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys‐Δ125‐pro‐urokinase.The purified conjugate obtained has a lower specific amidolytic activity (72000 U/mg) than unconjugated Cys‐Δ125‐pro‐urikinase (240000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of Δ125‐pro‐urokinase.We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection.The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half‐life in the circulation, with respect to pro‐urokinase and Δ125‐pro‐urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro‐urokinase.
Structural Aspects of Bile Acids Involved in the Regulation of Cholesterol 7α‐Hydroxylase and Sterol 27‐Hydroxylase
Authors: Twisk Jaap , Hoekman Marco F. M. , Muller Linda M. , Iida Takashi , Tamaru Tamaaki , Ijzerman Ad , Mager Willem H. , Princen Hans M. G. 
Published: March 2005

We have recently reported that coordinate down‐regulation of cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase by bile acids results in suppression of bile acid synthesis in cultured rat hepatocytes [Twisk, J., De Wit, E. & Princen, H. M. G. (1995) Biochem. J. 305, 505–511]. In the current study, we have assessed the effects of a large group of different bile acids, both naturally occurring and synthetic, on these two key enzymes, to elucidate structural features which render bile acids potent as regulators of bile acid synthesis.Addition of 50 μM deoxycholate or cholate, two relatively hydrophobic bile acids, to the culture medium of hepatocytes resulted in strong suppression of cholesterol 7α‐hydroxylase (suppression of 75% and 88%, respectively) and sterol 27‐hydroxylase activity (suppression of 76% and 72%, respectively). These effects were also reflected in the mRNA levels and the transcriptional activities of the two enzymes, showing a parallel suppression of both parameters in response to cholate (suppression of 78% and 43% for cholesterol 7α‐hydroxylase mRNA and transcription, respectively, and suppression of 76% and 42% for sterol 27‐hydroxylase mRNA and transcription, respectively). In contrast, no effects were observed with the two hydrophilic bile acids, β‐muricholate and ursocholate. Transient expression analysis in cultured rat hepatocytes, using a promoter‐reporter construct containing the proximal part of the cholesterol 7α‐hydroxylase promoter, demonstrated a reduction of transcriptional activity by cholate (reduction of 72%), but not by ursocholate.Assessment of the effects of 27 different bile acids, varying in the number, position and orientation (α/β) of hydroxyl groups on the steroid nucleus of the molecule, on cholesterol 7α‐hydroxylase mRNA showed only a moderate correlation with the hydrophobicity index of the bile acid involved (r= 0.61; P < 0.0001). Analysis of the three‐dimensional structure of a number of these bile acids suggests that hydroxyl groups situated in close proximity to each other within the molecule, creating a hydrophilic environment, as in the case of cholate, may be a prerequisite for a strong inhibitory potency. Deviation from this situation leads to a markedly lesser effect on suppression of cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase.