bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s
Authors: Baek Hwa Jin , Kwon So Yeon , Kim Sunkyu , Kim Sukwon S. , Oh Utaek , Hwang Seongbin , Chang Hyeun Wook , Lee Byeong Jae
Published: March 2003

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog‐skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse‐phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand–receptor binding occurs specifically. Their N‐terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2‐R1 and PLA2‐R2, respectively. In addition, their ligand‐binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.
Activation and regulation of Hsp32 and Hsp70
Authors: Stuhlmeier Karl M.
Published: March 2003

Endothelial cells (EC) play a key role in the propagation of inflammatory responses. Better understanding of inflammatory processes in EC might provide new ways of controlling inflammation. We report here that the known antioxidant pyrrolidinedithiocarbamate (PDTC) leads to time and dose dependent activation of heat‐shock protein 70 (Hsp70) as well as Hsp32 in EC. We further demonstrate that PDTC activates heat‐shock factor 1 (HSF1), one of several transcription factor involved in the upregulation of heat‐shock proteins. And more importantly, we demonstrate that Hsp32 as well as Hsp70 can be upregulated independently of the transcription factor nuclear factor kappaB (NF‐κB). The presented data provide further insight into the mechanism of Hsp32 and Hsp70 regulation, as well as further distinguishing these genes from other so called ‘protective genes’ whose upregulation depends on the activation of NF‐κB. These findings indicate that Hsp32 and Hsp70 might be ideal candidates among protective genes. Hsp32 and Hsp70 provide many desirable protective effects but, being independent of NF‐κB, would leave open the option to interfere with the upregulation of proinflammatory genes by modulating the activation of NF‐κB.
Cloning, overexpression and characterization of micro‐myoglobin, a minimal heme‐binding fragment
Authors: Grandori Rita , Schwarzinger Stephan , Müller Norbert
Published: March 2003

We report the cloning and expression of micro‐myoglobin, a 78‐amino‐acid fragment containing residues 29–105 of sperm whale myoglobin, and spanning the region from mid‐helix B to mid‐helix G of the globin fold. In contrast to full‐length myoglobin and to mini‐myoglobin (residues 32–129), the micro‐myoglobin apoprotein is almost unfolded. However, circular dichroism and absorption spectroscopy data indicate that this fragment is capable of folding into a functional heme‐binding unit forming a complex with the prosthetic group with characteristics similar to native myoglobin. Therefore, this case represents a new example of cofactor‐assisted folding. The experimental data suggest independence between myoglobin subdomains.
Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide
Authors: Bogumil Ralf , Namgaladze Dmitry , Schaarschmidt Daniel , Schmachtel Tilman , Hellstern Simon , Mutzel Rupert , Ullrich Volker
Published: March 2003

Calcineurin (CaN) is a Ca2+‐and calmodulin (CaM)‐dependent serine/threonine phosphatase containing a dinuclear Fe–Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A‐subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3–8 µm and the inactivation was reversed by 2,3‐dimercapto‐1‐propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration‐dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4‐dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A‐subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+–Zn2+ dinuclear centre. Upon H2O2‐mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.
Study of the subunit interactions in myosin phosphatase by surface plasmon resonance
Authors: Tóth Attila , Kiss Enikö , Herberg Friedrich W. , Gergely Pál , Hartshorne David J. , Erdödi Ferenc
Published: March 2003

The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N‐terminal half (residues 1–511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin‐biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11–296 > MYPT11–38 > MYPT123–38. No binding was detected with MYPT11–34, suggesting a critical role for residues 35–38, i.e. the PP1c binding motif. Binding of residues 1–22 was inferred from: a higher affinity binding to PP1c for MYPT11–38 compared to MYPT123–38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11–38, but not by MYPT123–38. Residues 40–296 (ankyrin repeats) in MYPT11–296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nm), whereas MYPT11–38, MYPT123–38 or MYPT11–34 were without effect. MYPT140–511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c–MYPT11–38 and PP1c–MYPT123–38. The inhibitory effect of MYPT140–511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11–38. The binding of MYPT1304–511 to complexes of PP1c and MYPT11–38, or MYPT11–296, was detected by SPR. These results suggest that within the N‐terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c‐binding motif and the other interactions are facilitated in an ordered and cooperative manner.
Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis
Authors: Nayak Surendra K. , Shveta  , Batra Janendra K.
Published: March 2003

Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single‐chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N‐ and C‐terminus in its activity, we have systematically deleted amino‐acid residues from both the termini. Three N‐terminal deletions removing 8, 15 and 30 amino acids, and three C‐terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N‐terminal or four C‐terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N‐ and C‐termini are required for the optimum functional activity of restrictocin.
Functional expression of a locust visual pigment in transgenic Drosophila melanogaster
Authors: Engels Armin , Reichert Heinrich , Gehring Walter J. , Gärtner Wolfgang
Published: March 2003

The cDNA encoding a visual pigment of the locust Schistocerca gregaria has been inserted into the germline of the ninaE mutant of Drosophila melanogaster by P‐element‐mediated transformation. Functional expression has been documented by recording light‐regulated electroretinograms in transgenic flies. The spectral properties of the expressed visual pigment were determined with detergent‐solubilized material, prepared from the eyecups of the transgenic D. melanogaster. The recombinant locust pigment, as well as the genuine pigment of the fruitfly (Rh1) that served as a control for transformation/expression, showed photoreversibility between the pigment and metapigment forms. The absorptions of the difference spectra identify the locust visual pigment as a short wavelength‐absorbing, blue‐light‐sensitive photoreceptor. The absorption maxima are similar to those recorded on living locust animals. These results show that, although locust visual pigments contain 11‐cis retinal as chromophore, the expressed protein is able to adopt 3‐hydroxyretinal that is provided by the transgenic fruitflies. The electrophysiological recordings reveal that the locust visual pigment is able to induce phototransduction in the fruitfly. The reported results have two important consequences: On the one hand, the binding site of the locust opsin is apparently able to interact with the 3‐hydroxyretinal from Drosophila in a way that the biological signal generated by the photoisomerization of the chromophore can be used by the protein to adopt a physiologically active conformation. On the other hand, despite the relatively large phylogenetic distance between both insect species, the extent of conservation between the protein domains thought to be involved in G‐protein activation is striking.
Intracellular site of γ‐secretase cleavage for Aβ42 generation in Neuro 2a cells harbouring a presenilin 1 mutation
Authors: Sudoh Shinji , Hua Gang , Kawamura Yuuki , Maruyama Kei , Komano Hiroto , Yanagisawa Katsuhiko
Published: March 2003

Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid β‐protein (Aβ)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Aβ42–γ‐secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Aβ42–γ‐secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP–C100 encoding the C‐terminal β‐amyloid precursor protein (APP) fragment truncated at the N terminus of Aβ (C100); C100 requires only γ‐secretase cleavage to yield Aβ. Mutated PS1 (M146L)‐induced Neuro 2a cells showed enhanced Aβ1–42 generation from transiently expressed C100 as well as from full‐length APP, whereas the generation of Aβ1–40 was not increased. The intracellular generation of Aβ1–42 from transiently expressed C100 in both mutated PS1‐induced and wild‐type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Aβ1–42 and Aβ1–40 from a C100 mutant containing a di‐lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of γ‐secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Aβ1–42/40 from C100 was not influenced by monensin treatment, and the level of Aβ1–42/40 generated from C100 carrying a sorting signal for the trans‐Golgi network was higher than that generated from wild‐type C100. These results using PS1‐mutation‐harbouring and wild‐type Neuro 2a cells suggest that Aβ42/40–γ‐secretase cleavages occur in the Golgi compartment and the trans‐Golgi network, and that the PS1 mutation does not alter the intracelluar site of Aβ42–γ‐secretase cleavage in the normal APP proteolytic processing pathway.
Turkey cytochrome c oxidase contains subunit VIa of the liver type associated with low efficiency of energy transduction
Authors: Hüttemann Maik , Arnold Susanne , Lee Icksoo , Mühlenbein Nicole , Linder Dietmar , Lottspeich Friedrich , Kadenbach Bernhard
Published: March 2003

Cytochrome c oxidase was isolated from turkey liver, heart and breast skeletal muscle and separated by SDS/PAGE. The N‐terminal amino‐acid sequence of subunit VIa from all tissues and internal sequences from the skeletal muscle enzyme show homology to the mammalian liver‐type subunit VIaL, which was verified by isolation and sequencing of the cDNA of turkey subunit VIa. No cDNA corresponding to subunit VIaH (mammalian heart‐type) could be found by RACE‐PCR with mRNA from all turkey tissues. Measurement of proton translocation with the reconstituted enzymes from turkey liver and heart revealed H+/e− ratios below 0.5 that were independent of the intraliposomal ATP/ADP ratio, as previously found with the bovine liver enzyme. Under identical conditions, the bovine heart enzyme revealed H+/e− ratios of 0.85 at low and 0.48 at high intraliposomal ATP/ADP ratios. The results suggest that in birds the lower H+/e−ratio of cytochrome c oxidase participates in elevated resting metabolic rate and thermogenesis.
Characterization and crystallization of an active N‐terminally truncated form of the Escherichia coli glycogen branching enzyme
Authors: Hilden Ida , Leggio Leila L. , Larsen Sine , Poulsen Peter
Published: March 2003

The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N‐terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino‐acid residues with unknown function. In order to characterize the function of this region, the 728 amino‐acid residue, full‐length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino‐acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion‐exchange chromatography, and a second ammonium sulphate precipitation. Purified full‐length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg·mL−1. In contrast, the truncated form could be concentrated to 6 mg·mL−1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full‐length GBE and is therefore a suitable model for the study of the enzymes’ catalytic mechanism.
Deduced amino‐acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus
Authors: Hatada Yuji , Saito Kazuhiro , Koike Kenzo , Yoshimatsu Tadashi , Ozawa Tadahiro , Kobayashi Tohru , Ito Susumu
Published: March 2003

The nucleotide sequence of the gene for a highly alkaline, low‐molecular‐mass pectate lyase (Pel‐15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino‐acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel‐15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site‐directed mutagenesis of the Pel‐15 gene, we replaced Lys20 in the N‐terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C‐terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel‐15 in the N‐terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel‐15 in the C‐terminal regions. The substituted PelB segment could also express β‐elimination activity in the chimeric molecule, confirming that Pel‐15 and PelB share a similar active‐site topology.
Structure of the O polysaccharide and serological classification of Pseudomonas syringae pv. ribicola NCPPB 1010
Authors: Ovod Vladimir V. , Zdorovenko Evelina L. , Shashkov Aleksander S. , Kocharova Nina A. , Knirel Yuriy A.
Published: March 2003

The O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) of a phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 was studied by sugar and methylation analyses, Smith degradation, and 1H‐ and 13C‐NMR spectroscopy, including 2D COSY, TOCSY, NOESY and H‐detected 1H,13C HMQC experiments. The OPS structure was elucidated, and shown to be composed of branched pentasaccharide repeating units (O repeats) of two types, major (1) and minor (2), differing in the position of substitution of one of the rhamnose residues. Both O repeats form structurally homogeneous blocks within the same polysaccharide molecule.

Although P. syringae pv. ribicola NCPPB 1010 demonstrates genetic relatedness and similarity in the OPS chemical structure to some other P. syringae pathovars, it did not cross‐react with any OPS‐specific mAbs produced against heterologous P. syringae strains. Therefore, we propose to classify P. syringae pv. ribicola NCPPB 1010 in a new serogroup, O8.
Inhibition of inositol uptake in astrocytes by antisense oligonucleotides delivered by pH‐sensitive liposomes
Authors: Lubrich Beate , van Calker Dietrich , Peschka‐Süss Regine
Published: March 2003

An oligonucleotide of 20 bases, complementary to a region of the sodium/myo‐inositol cotransporter (SMIT) mRNA, was used to investigate the uptake efficiency and activity of transferred antisense oligonucleotides with regard to substrate uptake. We compared the efficiency of oligonucleotide delivery after application of either free or liposome‐encapsulated material. Delivery of liposome‐encapsulated material (marker or oligonucleotides) into astrocytoma cells and primary astrocyte cultures was more effective with pH‐sensitive liposomes [dioleoylphosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS)] than with non‐pH‐sensitive liposomes (soy lecithin) or free material in solution. Antisense activity was evaluated by determination of myo‐inositol uptake and detection of SMIT transcripts by RT‐PCR. Encapsulation of oligonucleotides in pH‐sensitive liposomes increased the inhibition of inositol uptake at least 50‐fold compared with application of free oligonucleotides in solution.
Investigation of the haem–nicotinate interaction in leghaemoglobin
Authors: Patel Neesha , Jones Deborah K. , Raven Emma Lloyd
Published: March 2003

A strategic assessment of the contributions of two active‐site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen‐bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, µ = 0.10 m, 25.0 ± 0.1 °C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 ± 0.3 µm, 19 ± 1 µm and 11 ± 1 µm, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 ± 1 mm, 50 ± 2 mm and 12 ± 1 mm, and for binding of pyridine were 260 ± 50 µm, 4.5 ± 0.5 µm and 66 ± 8 µm, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH‐dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 ± 0.1 and 6.7 ± 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb–nicotinate derivatives were 29 ± 2 mV (pH 5.40, 25.0 °C, µ = 0.10 m) and − 65 ± 2 mV (pH 5.42, 25.0 °C, µ = 0.10 m), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.
Functional and structural differences between the prion protein from two alleles prnpa and prnpb of mouse
Authors: Brown David R. , Iordanova Ilina K. , Wong Boon‐Seng , Vénien‐Bryan Catherine , Hafiz Farida , Glasssmith Leslie L. , Sy Man‐Sun , Gambetti Pierluigi , Jones Ian M. , Clive Christine , Haswell Stephen J.
Published: March 2003

The prion protein is a glycoprotein expressed by neurones and other cells. In its holo‐form it binds copper and exhibits superoxide dismutase activity. Studies in mice have led to the description of two distinct alleles. Differences in these alleles are linked to long and short incubation times following infection with scrapie. We studied recombinant mouse protein corresponding to the products of either allele and two intermediates carrying single amino‐acid residue substitutions. The different forms of the prion protein exhibited differences in superoxide dismutase (SOD) activity and conformation. Intermediates with single substitutions were less stable than either allelic product. The findings provide insight into the differences between the two alleles and might have consequences for understanding differences in susceptibility to prion disease.
Purification, molecular cloning and ethylene‐inducible expression of a soluble‐type epoxide hydrolase from soybean (Glycine max[L.] Merr.)
Authors: Arahira Masaomi , Nong Van Hai , Udaka Kyoko , Fukazawa Chikafusa
Published: March 2003

A soybean protein was purified from mature dry seeds. Amino‐acid sequencing of the nine internal peptides derived from this N‐terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23‐mer oligonucleotides with sequences corresponding to an internal region of eight amino‐acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332‐bp cDNA obtained was found to have an open‐reading frame encoding the seed epoxide hydrolase‐like precursor consisting of 341 amino‐acid residues, suggesting that 25 amino‐acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase‐like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide‐diol converting activity using styrene oxide as substrate. The Vmax and Km values for styrene oxide are 1.36 µmol·min−1·mg−1 and 1500 µm, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12‐kb genomic clone was selected and the sequence of a 1933‐bp fragment from this clone was found to cover the entire coding region together with 5′‐ and 3′‐flanking regions of the soybean epoxide hydrolase gene. The coding region of the gene, interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up‐regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up‐regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble‐type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self‐defense systems of plant.
Enzyme‐mediated sulfide production for the reconstitution of [2Fe−2S] clusters into apo‐biotin synthase of Escherichia coli
Authors: Tse Sum Bui Bernadette , Escalettes Franck , Chottard Geneviève , Florentin Dominique , Marquet Andrée
Published: March 2003

We previously showed that biotin synthase in which the (Fe–S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226–230]. We therefore proposed that the source of sulfur was very likely the (Fe–S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated.

In this report, we show that the NifS protein from Azotobacter vinelandii and C‐DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe−2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme.

When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S‐reconstituted enzyme.

NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe–Se) cluster.

However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.
Three pheromone‐binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi
Authors: Maida Rosario , Krieger Jürgen , Gebauer Therese , Lange Ute , Ziegelberger Gunde
Published: March 2003

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone‐binding proteins (PBPs) could be identified in pheromone‐sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI‐TOF MS of sensillum lymph droplets from pheromone‐sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant‐binding proteins, immunoreactivity was found only with an anti‐(Apol PBP) serum. Free‐flow IEF, ion‐exchange chromatography and Western blot analyses revealed at least three anti‐(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N‐Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215–2218] and a new PBP having only 57% identity with this amino‐acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N‐terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin‐labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277–284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)‐6,11‐hexadecadienyl acetate (AC1) and the (E,Z)‐6,11‐hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H‐labelled acetate, whereas no binding of the 3H‐labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.
A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein
Authors: Deswal Renu , Pandey Girdhar K , Chandok Meena Rani , Yadav Nagendra , Bhattacharya Alok , Sopory Sudhir K.
Published: March 2003

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nm) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM–Sepharose. There was neither inhibition of the kinase activity in the presence of W‐7 (a CaM antagonist), KN‐62 (a specific calcium/CaM kinase inhibitor) and anti‐CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM‐kinase specific substrate, syntide‐2, proved to be a poor substrate for the BjCCaBPk compared with histone III‐S. The phosphorylation of histone III‐S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.
Conformational variants of human α2‐macroglobulin are reflected in a C‐terminal ‘switch region’
Authors: Gunnarsson Martin , Stigbrand Torgny , Jensen Poul Erik H.
Published: March 2003

Human α2‐macroglobulin displays extensive conformational changes when induced to transform into new quaternary structures, which are eliminated from the systemic circulation by receptor‐mediated endocytosis. One major region involved in these conformational changes is located in a segment of 30 amino acids from Glu1314 to Ser1343 (‐Glu‐Glu‐Phe‐Pro‐Phe‐Ala‐Leu‐Gly‐Val‐Gln‐Thr‐Leu‐Pro‐Gln‐Thr‐Cys‐Asp‐Glu‐Pro‐Lys‐Ala‐His‐Thr‐Ser‐Phe‐Gln‐Ile‐Ser‐Leu‐Ser‐), which we term the ‘switch region’ of α2‐macroglobulin, as deduced by immunochemical techniques. Monoclonal antibodies were generated using either native, methylamine‐treated or the 18‐kDa C‐terminal receptor‐binding fragment as the immunogen. From an extensive number of obtained hybridomas, 11 mAbs were selected because of their capacity to bind to the C‐terminal fragment. Irrespective of the original configuration of the antigen used for immunization, seven of the antibodies were shown to be reactive with a set of overlapping epitopes, closely positioned within the ‘switch region’, as confirmed by the use of synthetic peptides covering the entire C‐terminal fragment. The specificities of the seven individual antibodies, as determined by ELISA and BIAcore technologies, revealed a pronounced conformational pleomorphism in the ‘switch region’. The results indicate that the ‘switch region’ may be involved in the exposure of the receptor recognition site and can be used as an indicator region for different conformational states of α2‐macroglobulin.
Expression patterns of the regulatory proteins G protein‐coupled receptor kinase 2 and β‐arrestin 1 during rat postnatal brain development
Authors: Penela Petronila , Álvarez‐Dolado Manuel , Muñoz Alberto , Mayor Federico
Published: March 2003

G protein‐coupled receptor kinase 2 (GRK2) and β‐arrestin 1 are key regulatory proteins that modulate the desensitization and resensitization of a wide variety of G protein‐coupled receptors (GPCRs) involved in brain functions. In this report, we describe the postnatal developmental profile of the mRNA and protein levels of GRK2 and β‐arrestin 1 in rat brain. The expression levels of GRK2 and β‐arrestin 1 display a marked increase at the second and third week after birth, respectively, consistent with an involvement of these proteins in brain maturation processes. However, the expression attained at birth and during the first postnatal week with respect to adult values (45–70% for GRK2, ≈ 30% for β‐arrestin 1) is relatively high compared to that reported for several GPCRs, indicating the existence of changes in the ratio of receptors to their regulatory proteins during brain development. On the other hand, we report that experimental hypothyroidism results in changes in the patterns of expression of GRK2 and β‐arrestin 1 in cerebral cortex, leading to a 25–30% reduction in GRK2 levels at several stages of development. Such changes could help to explain the alterations in GPCR signaling that occur during this pathophysiological condition.
Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Δ strains
Authors: DiRusso Concetta C. , Connell Elise J. , Færgeman Nils J. , Knudsen Jens , Hansen Jan K. , Black Paul N.
Published: March 2003

Saccharomyces cerevisiae is an ideal model eukaryote for studying fatty‐acid transport. Yeast are auxotrophic for unsaturated fatty acids when grown under hypoxic conditions or when the fatty‐acid synthase inhibitor cerulenin is included in the growth media. The FAT1 gene encodes a protein, Fat1p, which is required for maximal levels of fatty‐acid import and has an acyl CoA synthetase activity specific for very‐long‐chain fatty acids suggesting this protein plays a pivotal role in fatty‐acid trafficking. In the present work, we present evidence that Fat1p and the murine fatty‐acid transport protein (FATP) are functional homologues. FAT1 is essential for growth under hypoxic conditions and when cerulenin was included in the culture media in the presence or absence of unsaturated fatty acids. FAT1 disruptants (fat1Δ) fail to accumulate the fluorescent long‐chain fatty acid fatty‐acid analogue 4,4‐difluoro‐5‐methyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐dodecanoic acid (C1‐BODIPY‐C12), have a greatly diminished capacity to transport exogenous long‐chain fatty acids, and have very long‐chain acyl CoA synthetase activities that were 40% wild‐type. The depression in very long‐chain acyl CoA synthetase activities were not apparent in cells grown in the presence of oleate. Additionally, β‐oxidation of exogenous long‐chain fatty acids is depressed to 30% wild‐type levels. The reduction of β‐oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Δ strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Δ strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Δ cells in the presence of cerulenin and under hypoxic conditions. Furthermore, fatty‐acid transport was restored and was found to be chain length specific: octanoate, a medium‐chain fatty acid was transported in a Fat1p‐ and FATP‐independent manner while the long‐chain fatty acids myristate, palmitate, and oleate required either Fat1p or FATP for maximal levels of transport. Lignoceryl CoA synthetase activities were restored to wild‐type levels in fat1Δ strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild‐type levels of β‐oxidation of exogenous long‐chain fatty acids. These data show that Fat1p and FATP are functionally equivalent when expressed in yeast and play a central role in fatty‐acid trafficking.
Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain‐of‐function allele of gene YAP1
Authors: Jungwirth Helmut , Wendler Franz , Platzer Barbara , Bergler Helmut , Högenauer Gregor
Published: March 2003

We have found that YAP1‐mediated diazaborine resistance in the yeast Saccharomyces cerevisiae requires two efflux pumps, i.e. the major‐facilitator‐superfamily transporter Flr1p, which is located in the cytoplasmic membrane and the ATP‐binding‐cassette transporter Ycf1p which is present in the vacuolar membrane. Both these transporters are known to be under the control of the transcriptional transactivator Yap1p which explains our earlier finding that overexpression of YAP1 mediates diazaborine resistance. Overexpression of YAP1 in a Δflr1Δycf1 double disruptant strain does not mediate any diazaborine resistance, showing that these pumps are the only ones involved in detoxification of this drug. We also found a new mechanism of diazaborine resistance which is caused by an allelic form of YAP1, designated YAP1‐11. This allele of YAP1 carries a mutation that leads to a C620F exchange in the C‐terminal cysteine‐rich‐domain region and is the first mutant of YAP1 that was isolated by a conventional genetic screen for drug resistance. The protein encoded by the gain‐of‐function allele may transactivate by a different mechanism from the wild‐type protein when overexpressed because it does not enhance YCF1 mRNA and still mediates diazaborine resistance in a Δflr1Δycf1 background.
Simulation of electrostatic effects in Fab–antigen complex formation
Authors: Fogolari Federico , Ugolini Raffaella , Molinari Henriette , Viglino Paolo , Esposito Gennaro
Published: March 2003

A model based on the Poisson–Boltzmann equation has been used to model electrostatics in Anti‐p24 (HIV‐1) Fab–antigen association. The ionization state at different pH values has been simulated and the results have been used to estimate the stability at different pH values and to generate electrostatic potential maps at physiological ionic strength. The analysis of the electrostatic potential at the solvent‐accessible surface shows that residues involved in binding are mostly found in the highest, but also in lowest potential regions. Brownian dynamics simulations have been used to estimate the enhancement of the association rate due to electrostatics which appears limited (≈ 2 at 150 mm ionic strength and ≈ 3 at 15 mm ionic strength). A much more pronounced effect is observed upon increase of the charge of the diffusing particle. These results compare well with results obtained previously in similar studies on different systems and may serve to estimate the expected order of magnitude of electrostatic effects on association rates in antibody–antigen systems.
Evaluating limited specificity of drug pumps
Authors: Jongsma A. P. M. , Riethorst A. , Lankelma J. , Dekker H. , Westerhoff H. V.
Published: March 2003

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P‐glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.
The structure of a complex glycosylphosphatidyl inositol‐anchored glucoxylan from the kinetoplastid protozoan Leptomonas samueli
Authors: Jones Christopher , Wait Robin , Previato José O. , Mendonça‐Previato Lucia
Published: March 2003

The structure of a glycosylphosphatidyl inositol‐anchored glucoxylan (GPI‐glucoxylan) synthesized by the monogenetic trypanosomatid Leptomonas samueli has been determined. The glucoxylan is anchored to the membrane by phytoceramide and an oligosaccharide core, the structure of which is identical to glycoinositolphospholipids (GIPLs) expressed by this protozoan. The glucoxylan chain is linear, containing →4Glcα1→, →4Xylβ1→ and →3Xylβ1→ residues. A well defined sequence heterogeneity was analysed in terms of a series of overlapping trisaccharide substructures. A proportion of the chains are capped with a GlcAα1→3Glcα1→ sequence. While an average GlcA‐capped chain contained 10 Glc and 16 Xyl residues, uncapped chains have a higher molecular mass with an average of 30 Glc and 50 Xyl per chain. We propose a mode of biosynthesis based on the observed structural heterogeneity.
The LCCL module
Authors: Trexler Mária , Bányai László , Patthy László
Published: March 2003

Here we show that Lgl1 protein, cub‐1‐related proteins, coch‐5b2‐related proteins, coagulation factor C of horse‐shoe crab and a predicted protein of Plasmodium falciparum share a homologous domain. Since this domain‐type was first identified in Limulus factor C, Coch‐5b2 and Lgl1 we propose the name LCCL for this domain‐family. The LCCL module of coch‐5b2 is of special biological interest because it has been shown recently that mutations affecting this module cause the deafness disorder DFNA9 in humans. With a view to defining the structure and function of the LCCL domain of human coch‐5b2 protein, we have expressed it in Escherichia coli and subjected it to preliminary structural characterization. Structure prediction and circular dichroism studies on the recombinant protein indicate that the domain possesses both α helices and β strands. It is shown that the mutations which cause hearing loss in humans affect residues that are critical for the integrity of the LCCL module of the coch‐5b2 protein.
Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane
Authors: De Geyter Carine , Wattiez Ruddy , Sansonetti Philippe , Falmagne Paul , Ruysschaert Jean‐Marie , Parsot Claude , Cabiaux Véronique
Published: March 2003

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid‐binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.
Adenovirus type 7 penton
Authors: Bal Harshawardhan P. , Chroboczek Jadwiga , Schoehn Guy , Ruigrok Rob W. H. , Dewhurst Stephen
Published: March 2003

Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino‐acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C‐terminal fusion with glutathione S‐transferase (GST–Ad7PB) and was purified by single‐step affinity chromatography. Both GST–Ad7PB and cleaved (GST‐free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST–Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C‐terminus of this linker peptide. GST–Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD‐containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin‐mediated uptake of the GST–Ad7PB–FK20–DNA complexes. We propose that Ad7 pentons can be developed into integrin‐specific gene delivery agents.