bluuebunny/crossref_metadata_2025_split huggingface dataset:
Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells
Authors: Hadjiolova Krassimira V. , Hadjiolov Asen A. , Bachellerie Jean‐Pierre 
Published: March 2005

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long beenknown, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01–0.08 μg/ml) of actinomycin D caused within 1–2 h a 2–7‐fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full‐length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5′ external transcribed spacer), whereas the synthesis of full‐length transcripts was abolished.Removal of actinomycin D from the medium caused within 8–24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3–4‐kb transcripts to about 50–80‐fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full‐length transcripts was not restored even 24 h after removal of the drug.The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G+C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes. However, transcriptiondriven positive supercoiling of rDNA is enhanced by actinomycin D and above a critical transcript length it causes a blockage of transcription elongation and the accumulation of aborted rDNA transcripts.
The Molybdenum Nitrogenase from Wild‐type Xanthobacter autotrophicus Exhibits Properties Reminiscent of Alternative Nitrogenases
Authors: Schneider Klaus , Müller Achim , Krahn Eugen , Hagen Wilfred R. , Wassink Hans , Knüttel Karl‐Heinz 
Published: March 2005

In the presence of molybdate (1 μM) 2–3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen‐fixing hydrogen‐oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm).The maximal specific activity obtained was 145 nmol ethylene reduced ċ min−1· mg protein−1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate‐starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo‐independent nitrogenase does not exist. However, the molybdate‐containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases.The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non‐saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH‐dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S =3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.
Oligosaccharyl Transferase is a Constitutive Component of an Oligomeric Protein Complex from Pig Liver Endoplasmic Reticulum
Authors: Breuer Wilhelm , Bause Ernst 
Published: March 2005

Oligosaccharyl transferase (OST), an intrinsic component of the endoplasmic reticulum membrane, catalyses the N‐glycosylation of specific asparagine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/detergent extraction, concanavalin‐A precipitation, S‐Sepharose, MonoP and concanavalin‐A–Sepharose chromatographies. A highly purified OST preparation exerting catalytic activity, contained two protein subunits of 48 kDa and 66 kDa, from which the 66‐kDa species was identified by immunoblotting as ribophorin I. The function of ribophorin I in this dimeric protein complex is unknown. The high degree of similarity between its transmembrane region and a putative dolichol‐recognition consensus sequence suggests that ribophorin I could be involved in glycolipid binding and delivery. Several lines of evidence indicate that the catalyti‐cally active 48‐kDa/66‐kDa polypeptides are associated in the endoplasmic reticulum membrane with other proteins, including ribophorin II and a 40‐kDa glycoprotein. The implication of ribophorins I and II in the translocation machinery and their apparent association with the OST activity point to a close relationship between polypeptide synthesis, translocation and N‐glycosylation, both spacially and temporally.Kinetic studies with the MonoP‐purified Oligosaccharyl transferase showed that the enzyme transfers dolichyl‐diphosphate‐linked GlcNAc2 to synthetic tripeptides and hexapeptides, containing the Asn‐Xaa‐Thr motif, at a comparable rate. The glycosylation reaction was found to have a pH optimum close to 7 and to require divalent metal ions, with Mn2+ being most effective. Substitution of threonine in the N‐ glycosylation motif by serine impairs its function as an acceptor, measured by Vmax/Km, by approximately 17‐fold, consisting of a 7.3‐fold increase in Km and a 2.3‐fold decrease in Vmax. This indicates that the side chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its participation in the catalytic mechanism of transglycosylation. The Km values of peptide acceptors improved significantly when dolichyl‐phosphate‐bound oligosaccharides were used instead of lipid‐linked GlcNAc2 as the glycosyl donor. We conclude from this observation that the sugar residues on the outer branches of the glycolipid donor induce conformational changes in the active site of the Oligosaccharyl transferase, thus influencing the association constant of the peptide substrate.
13C‐NMR Spectroscopy of Keratan Sulphates
Authors: Huckerby Thomas N. , Brown Gavin M. , Nieduszynski Ian A. 
Published: March 2005

Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and the complete 13C chemical shift data are reported for the four tetrasaccharides which derived from the repeat region. They have the structures:Galβ(1–4)GlcNAc(6S)β(1–3) Galβ(1–4)GlcNAc(6S)‐ol, Gal(6S)β(1–4)GlcNAc(6S)β(1–3)Gal(1–4)GlcNAc(6S)‐ol, Galβ(1–4)GlcNAc(6S)β(1–3)Gal(6S)β(1–4)GlcNAc(6S)‐ol, and Gal(6S)β(1–4)GlcNAc(6S)(1–3)Gal(6S)β(1–4)GlcNAc(6S)‐ol, where GlcNAc(6S)‐ol represents N ‐acetyl‐glucosaminitol‐6‐O ‐sulphate. The value of these data for microstructural analysis of undegraded keratan sulphate samples using 13C‐NMR spectroscopy is discussed with respect to galactose sulphation levels and the block sulphation structure.
Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4
Authors: Cox Andrew D. , Taylor Catherine J. , Anderson Alistair J. , Perry Malcolm B. , Wilkinson Stephen G. 
Published: March 2005

Like several other strains of Burkholderia (Pseudomonas) cepacia, the reference strain for serogroup 04 in the French typing scheme [Heidt, A., Monteil, H. & Richard, C. (1983) J. Clin. Microbiol. 18, 738–740] produces a lipopolysaccharide containing two distinct polymers. Attempts to separate the polymers chromatographically were unsuccessful, but the periodate‐resistant major polymer could be isolated by application of the Smith degradation technique to the mixture. By means of chemical and NMR spectroscopic analyses, the following structure could be assigned to the repeating unit of the major polymer:→3)‐α‐d‐Galp ‐(1→3)‐β‐d‐Galp ‐(1→3)‐β‐d‐Galp NAc‐(1→.The following structure of the repeating unit of the minor polymer was established from similar studies of its degradation product, resulting from the oxidation of L‐rhamnose (Rha), and of the original mixture:→3)‐α‐d‐Galp NAc‐(1→3)‐β‐d‐Galp NAc‐(1→4)‐α‐L‐Rhap‐(1→.Individually, the polymers have recently been found in related strains of B. cepacia. The minor polymer was identified as the O‐antigen in serotype A of a Canadian typing scheme [Beynon, L. M. & Perry, M. B. (1993) Biochem. Cell Biol. 71, 417–420], and the major polymer in serotype C of a Japanese typing scheme [Paramonov, N. A., Shashkov, A. S., Knirel, Y. A., Soldatkina, M. A. & Zakharova, I. Y. (1994) Bioorg. Khim. 20, 984–993]. In the case of the O4 strain studied here, both polymers were produced under a variety of growth conditions.
Peroxisome Proliferators Differentially Regulate Long‐chain Acyl‐CoA Thioesterases in Rat Liver
Authors: Svensson L. Thomas , Wilcke Mona , Alexson Stefan E. H. 
Published: March 2005

We have investigated the effects of peroxisome proliferators on rat liver long‐chain acyl‐CoA thioesterase activities. Subcellular fractionations of liver homogenates from control, clofibrate‐ and di(2‐ethylhexyl)phthalate‐treated rats confirmed earlier studies which demonstrated that peroxisome‐proliferating drugs induce long‐chain acyl‐CoA thioesterase activity mainly in the mitochondrial and cytosolic fractions. The aim of the present study was to investigate whether the induced activities were due to increases in normally expressed enzymes, or due to induction of novel enzymes. To investigate whether structurally different peroxisome proliferators differentially induced thioesterase activities, we tested the effects of di(2‐ethylhexyl)phthalate (a plastisizer) and the hypolipidemic drug clofibrate. For this purpose, we established an analytical size exclusion chromatography method. Chromatography of solubilised mitochondrial matrix proteins showed that the activity in control mitochondria was mainly due to enzymes with molecular masses of about 50 kDa and 35 kDa. The activity in samples prepared from clofibrate‐ and di(2‐ethylhexyl)phthalate‐treated rats eluted as proteins of about 40 kDa and 110 kDa. Highly purified peroxisomes contained two peaks of activity, which were not induced, that corresponded to molecular masses of 40 kDa and 80 kDa. The 80‐kDa peak was shown to be due to dimerization by addition of glycerol. Chromatography of cytosolic fractions from control rat livers indicated the presence of long‐chain acyl‐CoA thioesterases with molecular masses of approximately 35 kDa and 125 kDa and a broad peak corresponding to a high‐molecular‐mass protein. The activity in cytosolic fractions from peroxisome‐proliferator‐treated rats eluted mainly as peaks corresponding to 40, 110 and 150 kDa. In addition, in the 110‐kDa peak, a different degree of induction and different chain‐length specificities were caused by clofibrate and di(2‐ethylhexyl)phthalate, suggesting that these peroxisome proliferators differentially regulate the cytosolic acyl‐CoA thioesterase activities. Western blot analysis showed that enzymes in the 40‐kDa peak of the peroxisomal and cytosolic fractions were structurally related, but not identical, to a 40‐kDa mitochondrial very‐long‐chain acyl‐CoA thioesterase.Our data show that the increased acyl‐CoA thioesterase activities in mitochondria and cytosol were mainly due to induction of acyl‐CoA thioesterases which are not, or only weakly, expressed under normal conditions.
The pH Dependence and Modification by Diethyl Pyrocarbonate of Isocitrate Lyase from Phycomyces blakesleeanus
Authors: Rúa Javier , Soler Joaquín , Busto Félix , De Arriaga Dolores 
Published: March 2005

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaXpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units.Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second‐order rate constant of 18.58M−1 s−1 (at pH 6.0 and 20°C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N‐carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O‐carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N‐carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data‐protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.
Transcriptional Activation of Psoriasis‐Associated Cytokeratin K17 by Interferon‐γ
Authors: Vogel Ulrich , Denecke Bernd , Troyanovsky Sergey M. , Leube Rudolf E. , Böttger Erik C. 
Published: March 2005

The acid cytokeratin K17 is inducible by interferon‐γ (IFN‐γ), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN‐γ in epithelial cells. The 5′‐flanking region of the K17 gene (positions −1762 to −13). cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN‐γ but not IFN‐α in transient transfection assays. Sequence analysis revealed three putative γ‐interferon activation sites (GAS). Band‐shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions −1528 to −1515) was found to bind GAF/STAT91 and to compete with tryptophanyl‐tRNA synthetase (IFP53/WRS)‐GAS for binding to GAF; in contrast, GAS1 (positions −183 to −171) and GAS2 (positions −290 to −277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5′‐flanking region (positions −1762 to −111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN‐γ.Surprisingly, transfection of a CAT‐reporter gene construct harbouring a promoter segment (positions –111 to +13) devoid of the GAS elements revealed enhanced CAT‐gene expression upon IFF‐γ treatment. The interaction of GAS1 with the interferon‐responsive promoter region in the physiological context remains to be clarified.
Cloning and Primary Structure of Murine 11β‐Hydroxysteroid Dehydrogenase/Microsomal Carbonyl Reductase
Authors: Oppermann Udo C. T. , Netter Karl J. , Maser Edmund 
Published: March 2005

Screening of a mouse liver λgt 11 cDNA library with a rat liver 11β‐hydroxysteroid dehydrogenase cDNA (11β‐HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11β‐hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11β‐hydroxysteroid dehydrogenase structures. Open‐reading‐frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short‐chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post‐translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11β‐dehydrogenation/11‐oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P. C. (1993) Mol. Endocrinol. 7, 154–160; Agarwal, A. K., Tusie‐Luna, M. T., Monder, C. & White, P. C. (1990) Mol. Endocrinol. 4, 1827–18321. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti‐(microsomal carbonyl reductase) serum and N‐terminal sequence analysis of the purified protein confirms the identity of microsomal 11β‐hydroxysteroid dehydrogenase with the previously described, microsomal‐bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol., 1805–1812], and points to an involvement of the 11β‐HSD1A isofom in the reductive phase‐I metabolism of xenobiotic compounds, besides its endocrinological functions.The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11β‐HSD primary structure.
13C‐NMR Studies of Corynebacterium melassecola Metabolic Pathways
Authors: Rollin Catherine , Morgant Vincent , Guyonvarch Armel , Guerquin‐Kern Jean‐Luc 
Published: March 2005

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55 %) and the pentose phosphate pathway (45 %).Absolute glutamate 13C enrichments resulting from the incorporation of [1‐13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenol pyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32 %) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs' cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs. cycle were determined to be 38 % and 62 %, respectively.
The Oxidation of Hemocyanin
Authors: Beltramini Mariano , Bubacco Luigi , Casella Luigi , Alzuet Gloria , Gullotti Michele , Salvato Benedetto 
Published: March 2005

The reaction that gives met‐hemocyanin from Octopus vulgaris oxy‐hemocyanin has been reinvesti‐gated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. R., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050–1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43–108, J. Wiley, New York].Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo‐dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and precedes the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met‐hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di‐μ‐hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met‐hemocyanin with low affinity (K= 30 M−1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K= 1500 M−1), where a second azide ligand also binds in the terminal mode (K= 20 M−1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.
The Use of Beef Bull Semen Reduced the Risk of Abortion inNeospora‐seropositive Dairy Cows
Authors: López‐Gatius F. , Santolaria P. , Yániz J. L. , Garbayo J. M. , Almería S. 
Published: March 2005

SummaryThere is an evidence that the epidemiology of neosporosis differs in dairy and beef cattle, such that beef cattle carry a lower risk of abortion. The aim of the present study was to establish whether artificial insemination using semen from beef bulls could reduce the risk of abortion in dairy cows seropositive for theNeospora caninumparasite. Our study was based on yearly serological screening for neosporosis and on the confirmation ofNeosporainfection in aborted fetuses in two high‐producing dairy herds with a mean 28% seroprevalence ofN. caninumantibodies. The study population comprised of 273 pregnancies in seropositive animals: 156 pregnancies monitored after insemination using Holstein–Friesian semen and 117 after insemination using beef bull semen. Abortion rates for these animals were 28.2% (77 of 273), 34.6% (54 of 156) and 19.7% (23 of 117). Logistic regression analysis indicated no significant effects of lactation number and previous abortion on the abortion rate. Based on the odds ratio, a 1‐unit increase in theNeosporaantibody titre yielded a 1.01‐fold increase in the abortion rate. The likelihood of abortion was two times higher for cows in one of the two herds and 2.8 times lower (one of 0.36) for pregnant cows inseminated with beef bull semen rather than Holstein–Friesian semen. Our results indicate that the use of beef bull semen can reduce the risk of abortion in dairy cows, and suggest that annual screening for neosporosis, specifically the antibody titre to the protozoan, could be an useful predictor of abortion risk in reproductive health programmes.