bluuebunny/crossref_metadata_2025_split huggingface dataset:
The structure of the nonreducing terminal groups in the O‐specific polysaccharides from two strains of Bordetella bronchiseptica
Authors: Vinogradov Evgeny , Peppler Mark S. , Perry Malcolm B. 
Published: March 2003

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of α‐(1–4)‐linked 2,3‐diacetamido‐2,3‐dideoxy‐l‐galacturonic acid repeating units. Polysaccharides from both strains have 2,3,4‐triamino‐2,3,4‐trideoxy‐α‐galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N‐formyl‐l‐alanyl or l‐alanyl substituents at N‐2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2‐methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H‐13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.
CB1 receptor–G protein association
Authors: Mukhopadhyay Somnath , Howlett Allyn C. 
Published: March 2003

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C‐terminal domain (CB1401–417). Guanosine 5′‐O‐(3‐thio)triphosphate binding to G proteins can be stimulated by both peptide CB1401–417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol.53, 504–510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry38, 3447–3455]. In Chaps‐solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB1 receptor–Gαi association and attenuated the CB1401–417‐induced inhibition of adenylate cyclase. CB1401–417 significantly reduced the CB1 receptor association with Gαi3, but not with Gαi1 or Gαi2. In contrast, third intracellular loop peptides significantly reduced the CB1 receptor association with Gαi1 and Gαi2, but not Gαi3. These interactions are specific for the CB1 receptor because a peptide corresponding to the juxtamembrane C‐terminal domain of the CB2 receptor failed to compete for the association of the CB1 receptor with any of the Giα subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB1 receptor direct the interaction with specific G protein subtypes.
Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNATyr are conserved but expressed differently
Authors: Fechter Pierre , Rudinger‐Thirion Joëlle , Tukalo Mikhail , Giegé Richard 
Published: March 2003

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl‐tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base‐pair C1–G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild‐type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNATyr transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1–G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.
Interaction of Escherichia coli hemolysin with biological membranes
Authors: Schindel Christine , Zitzer Alexander , Schulte Berit , Gerhards Arnd , Stanley Peter , Hughes Colin , Koronakis Vassilis , Bhakdi Sucharit , Palmer Michael 
Published: March 2003

Escherichia coli hemolysin (HlyA) is a membrane‐permeabilizing protein belonging to the family of RTX‐toxins. Lytic activity depends on binding of Ca2+ to the C‐terminus of the molecule. The N‐terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane‐inserting domain. In this study, 13 HlyA cysteine‐replacement mutants were constructed and labeled with the polarity‐sensitive fluorescent probe 6‐bromoacetyl‐2‐dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane‐bound toxin. Binding effected a major blue shift in the emission of six residues within the N‐terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2+, suggesting that Ca2+ is not required for the toxin to enter membranes. However, binding of Ca2+ to HlyA in solution effected conformational changes in both the C‐terminal and N‐terminal domain that paralleled activation. Our data indicate that binding of Ca2+ to the toxin in solution effects a conformational change that is relayed to the N‐terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.
Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus
Authors: Ivens Andreas , Mayans Olga , Szadkowski Halina , Wilmanns Matthias , Kirschner Kasper 
Published: March 2003

Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady‐state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 Å resolution and preliminary X‐ray characterization was carried out. The crystals are suitable for structure determination.
Identification and characterization of the genes for N‐acetylglucosamine kinase and N‐acetylglucosamine‐phosphate deacetylase in the pathogenic fungus Candida albicans
Authors: Yamada‐Okabe Toshiko , Sakamori Yoshimi , Mio Toshiyuki , Yamada‐Okabe Hisafumi 
Published: March 2003

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc‐dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S‐transferase (GST), CaNag5p produced GlcNAc‐P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST–CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.
Samui, a novel cold‐inducible gene, encoding a protein with a BAG domain similar to silencer of death domains (SODD/BAG‐4), isolated from Bombyx diapause eggs
Authors: Moribe Yoriko , Niimi Teruyuki , Yamashita Okitsugu , Yaginuma Toshinobu 
Published: March 2003

Cellular responses to cold‐acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 °C, chilling at 5 °C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 °C, we isolated a novel cold‐inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 °C for 5–6 days, lasted for another 30 days and then weakened. Exposure to 25 °C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 °C, Samui was also up‐regulated, although the expression was weaker. Samui contained nuclear localization‐signals, a ssDNA‐binding motif and a BAG domain similar to that of SODD/BAG‐4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the ‘5 °C signal’ for SDH expression in diapause eggs, while also protecting against cold‐injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up‐regulated by cold.
2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans
Authors: Brock Matthias , Darley Dan , Textor Susanne , Buckel Wolfgang 
Published: March 2003

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2‐methylisocitrate to succinate and pyruvate is catalysed by 2‐methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo‐2‐methylisocitrate; the erythro‐diastereomer was not active. 2‐Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (≈ 48 kDa). 2‐Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2‐methylisocitrate lyases have a native homotetrameric structure as identified by size‐exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2‐methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3‐bromopyruvate whereby the activity was lost and one amino‐acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.
The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system
Authors: Olsthoorn‐Tieleman Lian N. , Plooster Lizanne J. , Kraal Barend 
Published: March 2003

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)‐Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF‐Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome‐bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF‐Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6‐tagged EF‐Tu1 from a S. coelicolor cell‐free extract. Translational activity of this system is totally dependent on the addition of purified EF‐Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid‐borne tuf gene. Using this EF‐Tu‐dependent translation system, we have established that S. coelicolor EF‐Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF‐Tu sequence at positions involved in either aminoacyl‐tRNA binding or interaction with the guanine‐nucleotide exchange factor EF‐Ts.
Grass group I pollen allergens (β‐expansins) lack proteinase activity and do not cause wall loosening via proteolysis
Authors: Li Lian‐Chao , Cosgrove Daniel J. 
Published: March 2003

Group I grass pollen allergens make up a subgroup of the β‐expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain‐like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of β‐expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load‐bearing components of the plant cell wall. Third, we tested the sensitivity of β‐expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N‐ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of β‐expansins do not loosen cell walls via a proteolytic mechanism.
Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants
Authors: Dirnberger Dietmar , Steinkellner Herta , Abdennebi Latifa , Remy Jean‐Jacques , van de Wiel Dick 
Published: March 2003

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N‐glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single‐chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc‐bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc‐bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex‐type N‐glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native‐like structure of sc‐bFSH and a sufficient extent of N‐glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc‐bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.
Mapping divalent metal ion binding sites in a group II intron by Mn2+‐ and Zn2+‐induced site‐specific RNA cleavage
Authors: Hertweck Maren , Mueller Manfred W. 
Published: March 2003

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+‐ and Zn2+‐induced site‐specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI–DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single‐stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+‐/Zn2+‐induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild‐type precursor and multiple splicing‐deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first‐step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5′ splice site, but does not require exon‐binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the ‘first‐step active site’.
Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene
Authors: Erent Muriel , Gonin Philippe , Cherfils Jacqueline , Tissier Pierre , Raschellà Giuseppe , Giartosio Anna , Agou Fabrice , Sarger Claude , Lacombe Marie‐Lise , Konrad Manfred , Lascu Ioan 
Published: March 2003

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia‐blast crisis primary cells. The over‐expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N‐terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CΔ. NDP kinase CΔ had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CΔ, based on the crystal structure of NDP kinase B, indicated that NDP kinase CΔ had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CΔ readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.
A Method for Simulating Signal Evolution Using Real Animals
Authors: Forkman Björn , Enquist Magnus 
Published: March 2003

AbstractWe studied response biases to visual stimulation using a new experimental technique. The subjects (hens, Gallus gallus domesticus) were confronted with several rewarding and non‐rewarding patterns on a computer screen. In contrast with standard discrimination tasks the rewarding patterns were not identical and varied in a dimension differentiating them from the non‐rewarding patterns. The rewarding patterns changed in response to hens' biases in selection of patterns. The aim of the study was to examine the possibility of receivers being a driving force in signal evolution. In one of the experiments a clear‐cut result was obtained. During the course of the experiment the rewarding patterns became gradually more different from the non‐rewarding one, a result expected from theoretical studies of the effect of response bias in signal evolution. A second similar experiment was less conclusive, with ceiling and floor effects influencing the results.
Distraction Displays in Meadow Pipit (Anthus pratensis) Females in Central and Northern Europe
Authors: Pavel Václav , Bureš Stanislav , Weidinger Karel , Kovařík Petr 
Published: March 2003

AbstractDistraction displays to a human were studied in the Meadow Pipit (Anthus pratensis) females in Central (Jeseníky Mountains–Czech Republic; 50°N 17°E) and Northern European mountains (Tydal area–Central Norway; 63°N 12°E) between 1995 and 1998. Three risk‐dependent variables were scored and we found that flushing and settling distances of pipit females decreased with increasing intensity of display both in the Central and the Northern Europe locations. Parental risk taking did not markedly differ between studied populations, although, in particular, larger brood size and reduced opportunity to re‐nest should contribute to a higher intensity of nest defence in the Northern than in the Central European mountains. Moreover, the level of risk taken was not significantly dependent on the air temperature, age of brood and brood size in both study areas, when compared separately. These results do not support the findings of most previous studies on nest defence in birds. Possible causes of our contradictory results are discussed with respect to the reproductive value and the feedback hypotheses.