bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and mechanistic aspects of transcriptional induction of cytochrome P450 1A1 by benzimidazole derivatives in rat hepatoma H4IIE cells
Authors: Backlund Maria , Weidolf Lars , Ingelman‐Sundberg Magnus
Published: March 2003

The effect of several structurally different benzimidazole compounds on CYP1A1 expression at the transcriptional, mRNA and protein levels was investigated in the rat hepatoma H4IIE cell line. Omeprazole, thiabendazole, carbendazim, 2‐mercaptobenzimidazole and 2‐mercapto‐5‐methoxybenzimidazole caused a dose‐dependent increase in CYP1A1 protein levels that reached maximum effect at 250 µm, as measured by Western blot. In addition, hydroxyomeprazole, 2‐aminobenzimidazole and 2‐mercapto‐5‐nitro‐benzimidazole caused a notable increase in CYP1A1 protein expression, whereas 5‐O‐desmethylomeprazole, 2‐hydroxybenzimidazole, 2‐benzimidazole propionic acid and 5‐benzimidazole carboxylic acid were ineffective. Thus, benzimidazole substituted with a thiol or an amino group in the 2‐position were active inducers. Northern blot analysis confirmed an extensive increase of CYP1A1 mRNA induced by omeprazole and 2‐mercapto‐5‐methoxybenzimidazole which was 32% and 49% of maximal induction by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) respectively, whereas thiabendazole and carbendazim showed ≈15% increase as compared to TCDD. Transient transfection of H4IIE cells, with a XRE‐pGL3 reporter gene construct revealed a 2.3–4.3‐fold induction by carbendazim, thiabendazole, and 2‐mercapto‐5‐methoxybenzimidazole as compared to a 3.3‐ and 23‐fold induction by omeprazole and TCDD, respectively. Thus, these data indicate that the benzimidazoles utilize the aryl hydrocarbon receptor–arnt–XRE‐mediated signal‐transduction pathway for induction of the CYP1A1 gene.
Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule‐deficient strain of Haemophilus influenzae Rd
Authors: Risberg Anna , Masoud Hussein , Martin Adèle , Richards James C. , Moxon E. Richard , Schweda Elke K. H.
Published: March 2003

Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influenzae, strain Rd, a capsule‐deficient type d strain, has been achieved by using high‐field NMR techniques and electrospray ionization‐mass spectrometry (ESI‐MS) on delipidated LPS and core oligosaccharide samples. It was found that this organism expresses heterogeneous populations of LPS of which the oligosaccharide (OS) epitopes are subject to phase variation. ESI‐MS of O‐deacylated LPS revealed a series of related structures differing in the number of hexose residues linked to a conserved inner‐core element, l‐α‐d‐Hepp‐(1→2)‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)‐]‐l‐α‐d‐Hepp‐(1→5)‐α‐Kdo, and the degree of phosphorylation. The structures of the major LPS glycoforms containing three (two Glc and one Gal), four (two Glc and two Gal) and five (two Glc, two Gal and one GalNAc) hexoses were substituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and were determined in detail. In the major glycoform, Hex3, a lactose unit, β‐d‐Galp‐(1→4)‐β‐d‐Glcp, is attached at the O‐2 position of the terminal heptose of the inner‐core element. The Hex4 glycoform contains the PK epitope, α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp while in the Hex5 glycoform, this OS is elongated by the addition of a terminal β‐d‐GalpNAc residue, giving the P antigen, β‐d‐GalpNAc‐(1→3)‐α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐d‐Glcp. The fully extended LPS glycoform (Hex5) has the following structure.

The structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. influenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd− derived mutant strain (RM.118–28) [Risberg, A., Schweda, E. K. H. & Jansson, P‐E. (1997) Eur. J. Biochem.243, 701–707].
Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C
Authors: Reiffert Silke , Maytum Robin , Geeves Michael , Lohmann Karin , Greis Thomas , Blüggel Martin , Meyer Helmut E. , Heilmeyer Ludwig M. G. , Jaquet Kornelia
Published: March 2003

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP‐dependent protein kinase A at two adjacently located serine residues within the heart‐specific N‐terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild‐type cardiac troponin I (cTnI‐WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD‐spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild‐type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI‐WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50‐values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.
Linear free‐energy model description of the conformational stability of uracil‐DNA glycosylase inhibitor
Authors: Reddy G. Bhanuprakash , Purnapatre Kedar , Lawrence Rajendran , Roy Sudipta , Varshney Umesh , Surolia Avadhesha
Published: March 2003

The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal‐induced and guanidine hydrochloride (GdnCl)‐induced denaturation. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity ΔCp that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration. The unfolding of Ugi is a simple two‐state, reversible process. The protein undergoes both low‐temperature and high‐temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant. The data are consistent with the linear free‐energy model and with a temperature independent ΔCp over the large temperature range of unfolding. The small ΔCp (6.52 kJ·mol−1·K−1) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein. Despite a relatively low value of ΔG(H2O), 7.40 kJ·mol−1 at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.
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%).
Identification of Δ12‐fatty acid desaturase from arachidonic acid‐producing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae
Authors: Sakuradani Eiji , Kobayashi Michihiko , Ashikari Toshihiko , Shimizu Sakayu
Published: March 2003

Based on the sequence information for the ω3‐desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Δ9, Δ12–18 : 2) to α‐linolenic acid (Δ9, Δ12, Δ15–18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S‐4, which is used industrially to produce arachidonic acid. Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal ω6‐desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal ω3‐desaturase (from soybean). The evolutionary implications of these enzymes will be discussed. The cloned cDNA was confirmed to encode a Δ12‐desaturase, which was involved in the desaturation of oleic acid (Δ9–18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S. cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%). Genomic Southern blot analysis of the transformants with the MortierellaΔ12‐desaturase gene as a probe confirmed integration of this gene into the genome of A. oryzae. The M. alpina 1S‐4 Δ12‐desaturase is the first example of a cloned nonplant Δ12‐desaturase.
Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24
Authors: Sakaki Toshiyuki , Sawada Natsumi , Nonaka Yasuki , Ohyama Yoshihiko , Inouye Kuniyo
Published: March 2003

Previously we expressed rat 25‐hydroxyvitamin D3 24‐hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three‐step monooxygenation towards 25‐hydroxyvitamin D3 and 1α,25‐dihydroxyvitamin D3[Akiyoshi‐Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335–343]. In this study, we demonstrate further oxidation by CYP24 including four‐ and six‐step monooxygenation towards 25‐hydroxyvitamin D3 and 1α,25‐dihydroxyvitamin D3, respectively. When the substrate 25‐hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24,25‐dihydroxyvitamin D3, 24‐oxo‐25‐hydroxyvitamin D3, 24‐oxo‐23,25‐dihydroxyvitamin D3 and 24,25,26,27‐tetranor‐23‐hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four‐step monooxygenation toward 25‐hydroxyvitamin D3. Furthermore, in‐vivo and in‐vitro metabolic studies on 1α,25‐dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six‐step monooxygenation to convert 1α,25‐dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1α,25‐dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25‐hydroxyvitamin D3 and 1α,25‐dihydroxyvitamin D3.
Antisense‐mediated depletion of potato leaf ω3 fatty acid desaturase lowers linolenic acid content and reduces gene activation in response to wounding
Authors: Martín Marta , León José , Dammann Christian , Albar Juan‐Pablo , Griffiths Gareth , Sánchez‐Serrano Jose J.
Published: March 2003

Fatty acid ω3 desaturases act on membrane lipids to catalyse the formation of trienoic fatty acids, the most abundant in plant tissues being α‐linolenic acid. This fatty acid is a precursor of jasmonic acid, a plant growth regulator involved in the control of wound‐induced gene activation in plants and in the induction of tuberization in potato. We isolated a potato ω3 desaturase cDNA, possibly encoding a plastidial isoform, and used it to investigate its expression pattern throughout plant development and in response to wounding. Plastidial ω3 desaturase gene transcripts accumulate rapidly upon wounding, preceding the jasmonate‐dependent induction of the wound‐responsive proteinase inhibitor II gene. We generated transgenic potato plants constitutively expressing an antisense RNA to this plastidial ω3 desaturase. Selected transgenic lines in which the cognate ω3 desaturase mRNA is largely depleted show a marked reduction, of up to 60%, in trienoic acids in leaves and tubers. In these lines, a corresponding reduction in jasmonate content and proteinase inhibitor II expression is observed upon wounding. Our results indicate that a reduction in ω3 desaturase mRNA levels compromises the wound‐induced activation of proteinase inhibitor II, suggesting that wound‐induced synthesis of linolenic acid is required for jasmonic acid production. The antisense‐mediated depletion of fatty acid ω3 desaturases is a viable alternative for reducing trienoic fatty acid content in plant species in which a mutant screening approach is not applicable.
Sex‐specific transcripts of the Dstpk61 serine/threonine kinase gene in Drosophila melanogaster
Authors: MacDougall Colin N. , Clyde Dorothy , Wood Timothy , Todman Martin , Harbison Diane , Bownes Mary
Published: March 2003

We describe the characterization of several transcripts of the Drosophila serine/threonine protein kinase 61 (Dstpk61) gene. Dstpk61 produces at least four transcripts, including a 3.0‐kb testis‐specific transcript, a 4.5‐kb female‐specific carcass transcript, a 3.5‐kb ovary‐specific transcript, and a 4.7‐kb non‐sex‐specific transcript. Two cDNAs, a 4.5‐kb cDNA (cDNAB) and a 3.0‐kb cDNA (cDNAA), likely to correspond to either the non‐specific or the female‐specific carcass and the testis‐specific transcript, respectively, were fully sequenced and found to encode a novel OPA‐repeat‐containing serine/threonine‐specific protein kinase. cDNAA and cDNAB both contain the entire ORF that encodes this predicted protein, but differ in the untranslated regions. The cDNAs contain translational control elements which are found in transcripts under male germline‐specific translational control, and doublesex‐like 13‐nucleotide repeat elements, which are required for transformer/transformer‐2‐mediated splicing of the female doublesex transcript. The complex tissue and sex‐specific transcripts, differing in the untranslated regions which are likely to be crucial in translational control, suggest that this kinase may have both general and sex‐specific functions. The protein is homologous to human 3‐phosphoinositide dependent protein kinase, which is involved in transduction of insulin signalling.
In vivo post‐translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130
Authors: Li Yong , Chen Jianfeng , Jiang Weihong , Mao Xiang , Zhao Guoping , Wang Enduo
Published: March 2003

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7‐amino cephalosporanic acid (GL‐7ACA) to produce 7‐amino cephalosporanic acid (7‐ACA). The acylase from Pseudomonas sp. 130 (CA‐130) is highly active on GL‐7ACA and glutaryl 7‐aminodesacetoxycephalosporanic acid (GL‐7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by α‐ and β‐subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the β‐subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N‐terminal nucleophile hydrolases) described recently.
Analysis of ectatomin action on cell membranes
Authors: Pluzhnikov Kirill , Nosyreva Elena , Shevchenko Ludmila , Kokoz Yuri , Schmalz Dirk , Hucho Ferdinand , Grishin Evgeniy
Published: March 2003

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four α‐helix bundle. At concentrations of 0.05–0.1 µm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 × 10−7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L‐type calcium current was studied. Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole‐cell perforated patch‐clamp technique. It was shown that ectatomin at concentrations of 0.01–10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of β‐adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol‐ and forskolin‐sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 μm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.
Complete nucleotide sequence, origin of isoform and functional characterization of the mouse hepsin gene
Authors: Kawamura Shunsuke , Kurachi Sumiko , Deyashiki Yoshihiro , Kurachi Kotoku
Published: March 2003

Hepsin, a type‐II membrane‐associated serine protease, has been implicated in cell growth and developement as well as possible initiation of blood coagulation. Here, we report on the complete nucleotide sequence, functional characterization of key structural features and the promoter of the mouse hepsin gene. The gene has a size of ≈17 kb, and is composed of 12, 13, or 14 exons depending on alternative intron splicings – one in the 5′‐UTR and the other two in the second intron. The latter two, which occur in approximately half of the hepsin transcripts, generate a hepsin mRNA species with an extra exon, which is responsible for producing a hepsin isoform with a unique 20‐residue sequence inserted in the cytoplasmic portion of hepsin. Most hepsin transcripts have the 5′‐UTR intron spliced, and its splicing can occur independently of the other alternative splicings. The transcriptional initiation site was determined to be 636 bp upstream of the first ATG site in a cytidine‐rich region. The 5′‐flanking region of hepsin up to nucleotide 274 showed a substantial promoter activity in HepG2 cells, with its expression activity sevenfold higher in the presence of the 5′‐UTR intron sequence in comparison to that without the intron sequence. The basal promoter region contains potential binding sites for several transcription factors including SP1, AP2, C/EBP, LF‐A1, and E box, which may be responsible for ubiquitous, but liver‐ and kidney‐preferred tissue expression of the hepsin gene.
Expression and characterization of bispecific single‐chain Fv fragments produced in transgenic plants
Authors: Fischer Rainer , Schumann Detlef , Zimmermann Sabine , Drossard Jürgen , Sack Markus , Schillberg Stefan
Published: March 2003

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single‐chain antibodies, scFv24 and scFv29, connected by theTrichoderma reesicellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429‐cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429‐apoplast). A third construct contained the C‐terminal KDEL sequence for retention in the ER (biscFv2429‐KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast‐targeted biscFv2429‐apoplast and ER‐retained biscFv2429‐KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant‐expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single‐chain antibodies for use in medical and biological applications.
The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare
Authors: Martin Gilbert , Sorokine Odile , Moniatte Marc , Bulet Philippe , Hetru Charles , Van Dorsselaer Alain
Published: March 2003

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC‐purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino‐acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N‐glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C‐peptide (50 amino acids). These results were confirmed by matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex‐determination. In invertebrates, there is no other example where sex‐differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti‐Müllerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.
The single cysteine residue of the Sud protein is required for its function as a polysulfide‐sulfur transferase in Wolinella succinogenes
Authors: Klimmek Oliver , Stein Torsten , Pisa René , Simon Jörg , Kröger Achim
Published: March 2003

The periplasmic Sud protein which is induced in Wolinella succinogenes growing by polysulfide respiration, has been previously proposed to serve as a polysulfide binding protein and to transfer polysulfide‐sulfur to the active site of polysulfide reductase [Klimmek, O, Kreis, V., Klein, C., Simon, J., Wittershagen, A. & Kröger, A. (1998) Eur. J. Biochem.253, 263–269.]. The results presented in this communication suggest that polysulfide‐sulfur is covalently bound to the single cysteine residue (Cys109) of the Sud monomer, and that Cys109 is required for tight binding of polysulfide‐sulfur and for sulfur transfer. A modified Sud protein [(C109S)Sud‐His6] in which the cysteine residue was replaced by serine, did not catalyze sulfur transfer from polysulfide to cyanide and did not stimulate electron transport to polysulfide, in contrast to Sud‐His6. The polysulfide‐sulfur bound to (C109S)Sud‐His6 was fully removed upon dialysis against sulfide. After this treatment, Sud‐His6 retained one sulfur atom per monomer; thiocyanate was formed upon addition of cyanide to the preparation. After incubation of Sud‐His6 with polysulfide, a proportion of the Sud‐His6 monomers carried one or two sulfur atoms, as shown by matrix‐assisted laser desorption ionization mass spectrometry. The sulfur atoms were absent from monomers derived from Sud‐His6 treated with cyanide and from (C109S)Sud‐His6 incubated with polysulfide.
Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum
Authors: van den Hooven Henno W. , Appelman Arno W. J. , Zey Thomas , de Wit Pierre J. G. M. , Vervoort Jacques
Published: March 2003

The race‐specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28‐residue β‐sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60–70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three‐disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15‐fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28‐residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.
Refinement and evaluation of a model of Mg2+ buffering in human red cells
Authors: Raftos Julia E. , Lew Virgilio L. , Flatman Peter W.
Published: March 2003

The total Mg2+ content of human red cells ([Mg]T,i) is partitioned between free and bound forms. The main cytoplasmic Mg2+ buffers are ATP and 2,3 bisphosphoglycerate. Haemoglobin binds free ATP and bisphosphoglycerate, preferentially in the deoxygenated state. Thus, the free ionized Mg2+ concentration ([Mg2+]i) oscillates with the oxy‐deoxy condition of the cells. The binding reactions are also modulated by the pH changes that accompany the oxygenation–deoxygenation transitions. The complex interactions between Mg2+, its ligands and Hb can be encoded in a set of equilibrium equations representing all the known binding reactions of the system. To develop a comprehensive understanding of the Mg2+ homeostasis of intact red cells it is necessary to correct and refine the equations and parameters of the model by systematic comparisons between model predictions and measured cytoplasmic Mg2+ buffering curves under a variety of experimental conditions. Earlier models largely underestimated total Mg2+ binding in intact cells. We carried out experiments in which [Mg]T,i and [Mg2+]i were controlled over a wide range ([Mg]T,i between 0.1 and 23 mm) by the use of the ionophore A23187, under diverse metabolic conditions, and the results were used to interpret the adjustments required for good model fits. By the inclusion of low‐affinity Mg2+ binding to ATP and bisphosphoglycerate, and also binding of Mg2+ to haemoglobin (four ions per tetramer) with an apparent dissociation constant of 45 mm we were able to realistically model, for the first time, all the experimentally observed changes in [Mg2+]i in human red cells under diverse metabolic conditions.
Biosynthesis of bitter acids in hops
Authors: Goese Markus , Kammhuber Klaus , Bacher Adelbert , Zenk Meinhart H. , Eisenreich Wolfgang
Published: March 2003

The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope‐incorporation experiments using 13C‐labelled glucose or 2H2O. 13C enrichments, 2H enrichments and 13C13C coupling patterns identify isovaleryl‐CoA, malonyl‐CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.
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→
31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida
Authors: Rager Marie Noëlle , Binet Marie R. B. , Bouvet Odile M. M.
Published: March 2003

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell‐free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden–Meyerhof and pentose phosphate pathways. The P. multocida fructose 6‐phosphate phosphotransferase activity (the key enzyme of the Embden–Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by α glycerophosphate. This inhibition is consistent with the very low fructose 6‐phosphate phosphotransferase activity found in cell‐free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6‐phosphate and fructose 6‐phosphate. We propose a new metabolic pathway in P. multocida where fructose 6‐phosphate is reduced to mannitol 1‐phosphate by fructose 6‐phosphate reductase. Mannitol 1‐phosphate produced is then converted to mannitol by mannitol 1‐phosphatase.
Identification of a linear epitope of interferon‐α2b recognized by neutralizing monoclonal antibodies
Authors: Blank Viviana C. , Sterin‐Prync Aída , Retegui Lilia , Vidal Alejandro , Criscuolo Marcelo , Roguin Leonor P.
Published: March 2003

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon‐α2b (IFN‐α2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN‐α2b binding to immobilized mAbs was completely inhibited by IFN‐α2b and IFN‐α2a but neither IFNβ nor IFNγ showed any effect. Gel‐filtration HPLC of the immune complexes formed by incubating [125I]IFN‐α2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN‐α2b anti‐viral and anti‐proliferative activities as well as [125I]IFN‐α2b binding to WISH cell‐membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN‐α2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107–132 or 107–146. As this zone includes the sequence 123–140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.
Cell adhesion to tenascin‐X
Authors: Elefteriou Florent , Exposito Jean‐Yves , Garrone Robert , Lethias Claire
Published: March 2003

Adhesive properties of tenascin‐X (TN‐X) were investigated using TN‐X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type‐III domain, and the fibrinogen‐like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN‐X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type‐III domains 9 and 10 (FNX 9‐10) but not to the FNX 10 domain alone. This adhesion to FNX 9‐10 was shown to be mediated by αvβ3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against αvβ3 integrin had no effects on cell adhesion to purified TN‐X, we suggest that the RGD sequence is masked in intact TN‐X. Cell attachment to the recombinant TN‐X fibrinogen domain (FbgX) and to purified TN‐X was greater for MG63 than for ECV304 cells. A β1‐containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN‐X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.
Ectoplasmic insertion of a glycosylphosphatidylinositol‐anchored protein in glycosphingolipid‐ and cholesterol‐containing phosphatidylcholine vesicles
Authors: Nosjean Olivier , Roux Bernard
Published: March 2003

Glycosylphosphatidylinositol (glycosyl‐PtdIns)‐anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl‐PtdIns‐protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose‐specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium‐dependent for its catalytic activity and was inhibited after incubation of ALP‐containing liposomes in a magnesium‐free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.
Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature
Authors: Kojoh Kanehisa , Matsuzawa Hiroshi , Wakagi Takayoshi
Published: March 2003

Ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 has a 36‐residue extra domain at its N‐terminus and a 67‐residue core domain carrying two iron–sulfur clusters. A zinc ion is held at the interface of the two domains through tetrahedral coordination of three histidine residues (−6, −19 and −34) and one aspartic acid residue (−76) [Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka, N. & Oshima, T. (1997) Biochemistry36, 1505–1513]. To elucidate the roles of the novel zinc ion and the extra N‐terminal domain, a series of truncated mutants was constructed: G1, V12, S17, G23, L31 and V38, which lack residues 0, 11, 16, 22, 30 and 37 starting from the N‐terminus, respectively. A mutant with two histidine residues each replaced by an alanine residue, H16A/H19A, was also constructed. All the mutant ferredoxins had two iron–sulfur clusters, while zinc was retained only in G1 and V12. The thermal stability of the proteins was investigated by monitoring A408; the melting temperature (Tm) was ≈109 °C for the natural ferredoxin, ≈109 °C for G1, 97.6 °C for V12, 89.0 °C for S17, 89.2 °C for G23, 89.3 °C for L31, 82.1 °C for V38, and 89.4 °C for H16A/H19A. Km and Vmax values of 2‐oxoglutarate:ferredoxin oxidoreductase for natural ferredoxin, G1, S17 and L31 were similar, suggesting that electron‐accepting activities were not affected by the deletion. The combination of CD and fluorescent spectroscopic analyses with truncated mutant S17 indicated that not only the clusters but also the secondary and tertiary structures were simultaneously degraded at a Tm around 89 °C. These results unequivocally demonstrate that the zinc ion and certain parts, but not all, of the extra sequence stretch in the N‐terminal domain are responsible not for function but for thermal stabilization of the molecule.
Alternative promoters direct tissue‐specific expression of the mouse protein phosphatase 2Cβ gene
Authors: Ohnishi Motoko , Chida Naoki , Kobayashi Takayasu , Wang Hong , Ikeda Shoko , Hanada Masahito , Yanagawa Yuchio , Katsura Koji , Hiraga Akira , Tamura Shinri
Published: March 2003

Type 2C protein phosphatases (PP2Cs), a class of ubiquitous and evolutionally conserved serine/threonine protein phosphatases, are encoded in at least four distinct genes and implicated in the regulation of various cellular functions. Of these four PP2C genes, the expression of the PP2Cβ gene has been reported to be tissue‐specific and development‐dependent. To understand more precisely the regulatory mechanism of this expression, we have isolated and characterized overlapping mouse genomic λ clones. A comparison of genomic sequences with PP2Cβ cDNA sequences provided information on the structure and localization of intron/exon boundaries and indicated that PP2Cβ isoforms with different 5′ termini were generated by alternative splicing of its pre‐mRNA. The 5′‐flanking region of exon 1 had features characteristic of a housekeeping gene: it was GC‐rich, lacked TATA boxes and CAAT boxes in the standard positions, and contained potential binding sites for the transcription factor SP1. In the 5′‐flanking region of exon 2, several consensus sequences were found, such as a TATA‐like sequence and negative regulatory element box‐1, ‐2 and ‐3. Subsequent analysis by transient transfection assay with a reporter gene showed that these regions act as distinct promoters. Analysis of PP2Cβ transcripts by reverse transcriptase‐PCR showed that exon‐1 transcripts were expressed ubiquitously in all of the tissues examined, whereas exon‐2 transcripts were predominantly expressed in the testis, intestine and liver. These results suggest that the alternative usage of two promoters within the PP2Cβ gene regulates tissue‐specific expression of PP2Cβ mRNA.
NMR structure of active and inactive forms of the sterol‐dependent antifungal antibiotic bacillomycin L
Authors: Volpon Laurent , Besson Françoise , Lancelin Jean‐Marc
Published: March 2003

The antifungal antibiotic lipopeptide bacillomycin L[cyclo‐(l‐Asp1‐d‐Tyr2‐d‐Asn3‐l‐Ser4‐l‐Gln5‐d‐Ser6‐l‐Thr7‐β‐amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol‐phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (l‐Asp1, d‐Tyr2) di‐O‐methylated form. The structures were calculated under NMR‐derived restraints using molecular‐dynamic simulated‐annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral φ and ψ angles defining a type‐II′β‐turn including amino acids 5–8, in certain cases stabilized by a 8HN‐5CO hydrogen bond, whereas a minority of structures adopt an inverse γ‐turn including amino acids 6–8, stabilized in all cases by an 8HN‐6CO hydrogen bond. The di‐O‐methylation of l‐Asp1 and d‐Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved d‐Tyr2 position.
Cyclin A‐dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis
Authors: Findeisen Marco , El‐Denary Medhat , Kapitza Thomas , Graf Roney , Strausfeld Ulrich
Published: March 2003

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin‐dependent kinase activities. High cyclin A‐dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin‐bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.
Co‐operating ATP sites in the multiple drug resistance transporter Mdr1
Authors: Buxbaum Engelbert
Published: March 2003

The ATPase activity of the multiple drug resistance transporter Mdr1 (P‐glycoprotein, gp170) depended on the concentration of ATP with both positive and negative co‐operativity both in the absence and in the presence of verapamil. Four co‐operating binding sites for ATP were required to adequately model the experimental findings. The activation energy for the ATPase activity increased from ≈ 385 kJ·mol−1 at 10 µm ATP to 512 kJ·mol−1 at 1600 µm, while changes in verapamil concentration had little effect. This indicates that the reaction mechanism of ATP hydrolysis depends on ATP concentration and is further evidence for co‐operation of ATP binding sites. Free ATP in higher concentration was inhibitory; however, this inhibition could be reduced by complexing the ATP with Mg2+. Free Mg2+ had little effect on Mdr1 apart from complexing ATP.
Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact
Authors: Achleitner G. , Gaigg B. , Krasser A. , Kainersdorfer E. , Kohlwein S. D. , Perktold A. , Zellnig G. , Daum G.
Published: March 2003

Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer‐aided three‐dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria‐associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re‐associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem.270, 29836–29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy‐independent in the reconstituted yeast system.