bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure elucidation of the O‐antigenic polysaccharide from the enteroaggregative Escherichia coli strain 62D1
Authors: Staaf Mikael , Urbina Felipe , Weintraub Andrej , Widmalm Göran 
Published: March 2003

The O‐antigen polysaccharide of the lipopolysaccharide from the enteroaggregative Escherichia coli strain 62D1 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy revealed the components of the repeating unit. Two‐dimensional NOESY and heteronuclear multiple‐bond correlation experiments were used to deduce the sequence. 1H and 13C NMR spectra indicate heterogeneity in the polysaccharide. Methylation analysis and 1H NMR spectra of native and Smith‐degraded material show that the majority (65%) of the repeating units has the following structure:Minor resonances in the NMR spectra are consistent with the presence of repeating units which lack the α‐d‐Galp terminal residue (35%).
Structure determination of the O‐antigenic polysaccharide from the enteroinvasive Escherichia coli O136
Authors: Staaf Mikael , Weintraub Andrej , Widmalm Göran 
Published: March 2003

The structure of the O‐antigen polysaccharide of the lipopolysaccharide from the enteroinvasive Escherichia coli O136 has been elucidated. The composition of the repeating unit was established by sugar and methylation analysis together with 1H and 13C NMR spectroscopy. Two‐dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple‐bond correlation experiments were used to deduce the sequence. The absolute configuration for the nonulosonic acid (NonA) could be determined using spin‐spin coupling constants, 13C chemical shifts and NOESY. The anomeric configuration of the NonA was determined via vicinal and geminal 13C,1H coupling constants. The structure of the repeating unit of the polysaccharide from E. coli O136 is as follows, in which β‐NonpA is 5,7‐diacetamido‐3,5,7,9‐tetradeoxy‐lglycero‐β‐lmanno‐nonulosonic acid:→4)‐β‐NonpA‐(2→4)‐β‐d‐Galp‐(1→4)‐β‐d‐GlcpNAc‐(1→
Structural studies of the O‐antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross‐reacting with Shigella dysenteriae type 3
Authors: Linnerborg Malin , Weintraub Andrej , Widmalm Göran 
Published: March 2003

The structure of the O‐antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure: Matrix assisted laser desorption ionization mass spectrometry (MALDI‐MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O‐antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross‐reactivity of this O‐antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.
Solution structure of a recombinant mouse major urinary protein
Authors: Lücke Christian , Franzoni Lorella , Abbate Francesco , Löhr Frank , Ferrari Elena , Sorbi Robert T. , Rüterjans Heinz , Spisni Alberto 
Published: March 2003

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism.In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild‐type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently.Here, we describe the refined high‐resolution three‐dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE‐derived distance restraints. A comparison with the crystal structure of the wild‐type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology.The secondary structure of the protein consists of eight antiparallel β‐strands forming a single β‐sheet and an α‐helix in the C‐terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the β‐strands. The tertiary fold of the β‐sheet creates a β‐barrel, common to all members of the lipocalin superfamily. The shape of the β‐barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.
Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter
Authors: Niedziela‐Majka Anita , Kochman Marian , Ożyhar Andrzej 
Published: March 2003

The functional 20‐hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E‐response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA‐binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half‐sites contribute differently to the binding of the heterodimer components; the 5′ half‐site exhibits higher affinity for both DBDs than the 3′ half‐site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5′ half‐site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5′‐UspDBD‐EcRDBD‐3′) on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C‐terminal sequences to the DNA‐binding and heterocomplex formation. This is supported by finding that deletion of the C‐terminal of EcRDBD region corresponding to the putative A‐helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T‐box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5′ half‐sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal .This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates.
The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli
Authors: van den Berg Willy A. M. , Hagen Wilfred R. , van Dongen Walter M. A. M. 
Published: March 2003

Hybrid‐cluster proteins (‘prismane proteins’) have previously been isolated and characterized from strictly anaerobic sulfate‐reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe–4S] cubane cluster with spin‐admixed S = 3/2 ground‐state paramagnetism and a novel type of hybrid [4Fe–2S–2O] cluster, which can attain four redox states.Genomic sequencing reveals that genes encoding putative hybrid‐cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid‐cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid‐cluster protein, however, give evidence for the presence of a [2Fe–2S] cluster instead of a [4Fe–4S] cluster. The hcp gene encoding the hybrid‐cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe–2S] cluster as cofactors. It catalysed the reduction of the hybrid‐cluster protein with NADH as an electron donor. Midpoint potentials (25 °C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: −320 mV) are transferred along the [2Fe–2S] cluster of the NADH oxidoreductase (Em = –220 mV) and the [2Fe–2S] cluster of the hybrid‐cluster protein (Em = –35 mV) to the hybrid cluster (Em = –50, +85 and +365 mV for the three redox transitions).The physiological function of the hybrid‐cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate‐and/or nitrite respiration.
Structural studies of the O‐specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92
Authors: Czaja Jolanta , Jachymek Wojciech , Niedziela Tomasz , Lugowski Czeslaw , Aldova Eva , Kenne Lennart 
Published: March 2003

The structure of the O‐specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix‐assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure:in which d‐β‐d‐Hepp is dglycero‐β‐dmanno‐heptopyranose and 6d‐β‐d‐Hep is 6‐deoxy‐β‐dmanno‐heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O‐antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high‐resolution magic angle spinning technique, 1H‐NMR spectra were also obtained for the O‐polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.
N‐Carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea
Authors: Bartoschek Stefan , Vorholt Julia A. , Thauer Rudolf K. , Geierstanger Bernhard H. , Griesinger Christian 
Published: March 2003

N‐Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from ‘MFR’(MFR + MFRH+) and ‘CO2’ (CO2 + H2CO3 + HCO3−+ CO32–) was found to be 7 m−1·s−1 (v1 = k1*[‘MFR’][‘CO2’]) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s−1 (v2 = k2*[carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/[‘MFR’][‘CO2’] was 0.6 m−1 at pH 7.0 corresponding to a free energy change ΔG°′ of + 1.3 kJ·mol−1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3− in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3− which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N‐carboxymethanofuran to N‐formylmethanofuran.From the concentrations of ‘CO2’ and of ‘MFR’ determined by 1D‐NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1[MFR][CO2]). The second order rate constant k1 was found to be approximately 1000 m−1·s−1 at 300 K and pH values between 7.0 and 8.0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.
Molecular cloning and characterization of murine and human N‐acetylglucosamine kinase
Authors: Hinderlich Stephan , Berger Markus , Schwarzkopf Martina , Effertz Karin , Reutter Werner 
Published: March 2003

N‐Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N‐acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino‐acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N‐acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N‐acetylglucosamine kinase activity as well as N‐acetylmannosamine kinase activity.The complete cDNA sequence of human N‐acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N‐acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino‐acid level. Amino‐acid sequences of murine and human N‐acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present.Tissue distribution of murine N‐acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.
The effect of tryptophanyl substitution on folding and structure of myoglobin
Authors: Sirangelo Ivana , Tavassi Simona , Martelli Pier Luigi , Casadio Rita , Irace Gaetano 
Published: March 2003

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A‐5) and 14 (A‐12) in the N‐terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site‐directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W→F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed.The acid‐induced denaturation curves of wild‐type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1‐anilino‐8‐naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W→Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W→F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.
Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture
Authors: Chigorno Vanna , Palestini Paola , Sciannamblo Mariateresa , Dolo Vincenza , Pavan Antonio , Tettamanti Guido , Sonnino Sandro 
Published: March 2003

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X‐100 and fractionated by sucrose‐gradient centrifugation to prepare a detergent‐insoluble membrane fraction known to be enriched in sphingolipid and caveolin‐1, i.e. of caveolae.The detergent‐insoluble membrane fraction prepared after feeding [1‐3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18‐fold). By feeding cells photoactivable radioactive GM3, after 2 h‐chase, caveolin‐1, CAV1, and proteins of high molecular mass became cross‐linked to GM3, the cross‐linking complexes being highly concentrated in the detergent‐insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time‐dependent, transient process so that CAV1 cross‐linking to GM3 was hardly detectable after a 24‐h chase followed the pulse time. After a 24‐h chase, only the high molecular mass proteins cross‐linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized.Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold‐conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti‐GM3 and anti‐CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals.Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.
A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides
Authors: Velayos Antonio , Eslava Arturo P. , Iturriaga Enrique A. 
Published: March 2003

Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M. circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi. This gene was identified using complementation tests among different classes of carotenoid mutants of M. circinelloides. The carRP gene product contains two domains: the R domain is located at the N‐terminus and determines lycopene cyclase activity; the P domain is located at the C‐terminus and displays phytoene synthase activity. The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function. The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp. Northern analyses show a co‐ordinated regulation of the expression of both genes by blue light. Several motifs found in this promoter region suggest a bi‐directional mode of transcription control.
Human brain GABA transaminase
Authors: Jeon Seong Gyu , Bahn Jae Hoon , Jang Joong Sik , Park Jinseu , Kwon Oh‐Shin , Cho Sung‐Woo , Choi Soo Young 
Published: March 2003

Human brain γ‐aminobutyrate transaminase is differentially expressed in a tissue‐specific manner. mRNA master dot‐blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the γ‐aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain γ‐aminobutyrate transaminase cDNA was expressed inEscherichia coliusing a pGEX expression vector system. Catalytically active γ‐aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well‐known antiepileptic drug vigabatrin. Values ofKiandkinactwere 1 mmand 0.35 min−1, respectively. Results from inactivation kinetics suggested that human γ‐aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.
Characterization of RCI‐1, a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators
Authors: Schaffrath Ulrich , Zabbai Frauke , Dudler Robert 
Published: March 2003

A full‐length lipoxygenase cDNA (RCI‐1) has been cloned from rice (Oryza sativa) whose corresponding transcripts accumulate in response to treatment of the plants with chemical inducers of acquired resistance such as benzo(1,2,3)thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH), 2,6‐dichloroisonicotinic acid (INA), and probenazol. In contrast, RCI‐1 transcript levels did not increase after inoculation with compatible and incompatible races of the rice blast fungus Magnaporthe grisea and the nonhost pathogen Pseudomonas syringae pv. syringae. RCI‐1 transcript levels also increased after exogenous application of jasmonic acid, but not upon wounding. Dose–response and time course experiments revealed a similar pattern of transcript accumulation and lipoxygenase activity in BTH‐treated rice leaves. Enzymatic analysis of recombinant RCI‐1 protein produced in Escherichia coli revealed that 13‐hydroperoxy‐octadecanoic acids were the predominant reaction products when either linoleic or linolenic acid used as a substrate. The RCI‐1 sequence features a putative chloroplast targeting sequence at its N‐terminus. Indeed, a protein consisting of the putative chloroplast transit peptide fused to green fluorescent protein was exclusively localized in chloroplasts, indicating that RCI‐1 is a chloroplastic enzyme.