bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of mutations of residue 340 in the large subunit polypeptide of Rubisco from Anacystis nidulans
Authors: Madgwick Pippa J. , Parmar Saroj , Parry Martin A. J.
Published: March 2003

Residues 338−342 at the C‐terminal end of loop 6 in the large subunit β/α barrel structure of Rubisco influence specificity towards CO2 and O2. In Anacystis nidulans Rubisco, replacement of alanine 340 by tyrosine or histidine increased the specificity factor by 12−13 %, accompanied by a 25−33 % fall in Vc, the rate of carboxylation, while replacement by asparagine increased the specificity factor by 9 % and Vc by 19 %. Other mutations did not significantly alter specificity. Alanine 340 does not interact directly with the bisphosphate substrate, thus replacing it with other residues must have indirect effects on the specificity factor and rate of carboxylation.
Cloning, purification and characterisation of the lipase from Staphylococcus epidermidis
Authors: Simons Jan‐Willem F. A. , van Kampen Muriel D. , Riel Sabine , Götz Friedrich , Egmond Maarten R. , Verheij Hubertus M.
Published: March 2003

On the chromosome of Staphylococcus epidermidis RP62A the lipase gene (gehSE1) is immediately flanked by the icaAA′BC operon, which is involved in biofilm formation. Since lipase production might play a role in staphylococcal skin colonisation as well, we studied the biochemical properties of the staphylococcal lipases more closely. The DNA sequence and the deduced protein sequence revealed that gehSE1 is very similar to the lipase sequence of S. epidermidis strain 9. Like other staphylococcal lipases, gehSE1 is organised as a preproenzyme. The part of gehSE1 coding for the mature lipase was cloned and overexpressed as a fusion protein with an N‐terminal histidine tag in Escherichia coli. The lipase was purified to homogeneity using a combination of precipitation techniques, metal‐affinity chromatography and gel filtration. Biochemical characterisation showed that this lipase is closely related to the lipase from Staphylococcus aureus NCTC8530. Both enzymes have a pH optimum around 6, are very stable at low pH, and need calcium as a cofactor for catalytic activity. The preferred substrates are small triacylglycerols, with a maximum activity toward tributyrylglycerol. Comparison of the substrate selectivity with those of other microbial lipases showed that phospholipids are generally poor substrates for lipases. An exception is the lipase from Staphylococcus hyicus, which prefers phospholipids as a substrate, distinguishing this staphylococcal lipase from other microbial lipases. These results are discussed in view of the structure/function relationships of staphylococcal lipases, and the possible involvement of these enzymes in biological processes such as skin colonisation and pathogenesis.
Cloning of two cDNAs encoding three small serine protease inhibiting peptides from the desert locust Schistocerca gregaria and analysis of tissue‐dependent and stage‐dependent expression
Authors: Vanden Broeck Jozef , Chiou Shean‐Jaw , Schoofs Liliane , Hamdaoui Ahmed , Vandenbussche Frank , Simonet Gert , Wataleb Said , De Loof Arnold
Published: March 2003

This study describes the cloning of two cDNAs encoding three serine‐protease‐inhibiting peptides, SGPI I, II and III, which were recently identified from ovarian extracts of the desert locust, Schistocerca gregaria. The first cDNA codes for the precursor polypeptides of SGPI I and SGPI II; the second encodes only a single inhibitor, SGPI III.

Northern‐blot analysis revealed an approximate length of 0.8 kb for SGPI‐I/II mRNA and 0.6 kb for SGPI‐III mRNA. The transcripts are present in several locust tissues, but they could not be detected in the midgut. The gene for SGPI‐I/II is abundantly transcribed during all larval and adult stages, whereas SGPI‐III mRNA is mainly present in adults. Northern‐blot hybridization also revealed important changes in the SGPI‐mRNA content during the molting cycle and during the adult reproductive cycle. Moreover, a differential hormonal control was observed in adult females which had been treated with precocene, juvenile hormone or ecdysone.
Different derivations of knowledge‐based potentials and analysis of their robustness and context‐dependent predictive power
Authors: Rooman Marianne , Gilis Dimitri
Published: March 2003

The possibility of defining effective potentials from known protein structures, which are sufficiently accurate to be used for protein‐structure‐prediction purposes, is investigated. Three types of distance potentials and three types of backbone torsion potentials are defined, based on propensities of amino acid pairs to be separated by a given spatial distance or to be associated to a backbone torsion angle domain. Their differences reside in the way the physical correlations between the amino acids and the conformational states are extracted from the bulk interactions due to the presence of many residues in a protein. For the distance potentials, a physical meaning can be associated to the different definitions, given that some of the potentials favor hydrophobic interactions and others favor interactions between oppositely charged residues. The performance of the different torsion and distance potentials in structure prediction procedures, in particular native‐fold recognition and evaluation of protein stability changes upon point mutations, is analyzed. It appears to differ according to the specific proteins and protein environments. In particular, one of the distance potentials performs better than the others for membrane proteins and in protein regions involving charged residues, but less well in other protein regions. Furthermore, the dependence of the potentials on the characteristics of the proteins from which they are derived is analyzed. It is shown that the dependence of the potentials on the length, amino acid composition and secondary‐structure content of the proteins from the dataset is either very limited or rather strong, according to the type of potential. The results obtained suggest that the main problem limiting the performance of database‐derived potentials is their lack of universality : each potential describes with satisfactory accuracy only the interactions present in certain protein environments.
Clamping down on clamps and clamp loaders
Authors: Mossi Romina , Hübscher Ulrich
Published: March 2003

DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non‐protein cofactors. Among them three main accessory proteins, replication factor C (RF‐C), proliferating‐cell nuclear antigen (PCNA) and replication protein A (RP‐A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF‐C is a complex consisting of five polypeptides with distinct functions. RF‐C can bind to a template‐primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF‐C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S‐phase checkpoint regulation, apoptosis, differentiation and telomere‐length regulation.
Sialic‐acid‐binding lectin from the slug Limax flavus
Authors: Kurachi Sumiko , Song Zhiwei , Takagaki Masaru , Yang Qing , Winter Harry C. , Kurachi Kotoku , Goldstein Irwin J.
Published: March 2003

A cDNA library of Limax flavus was constructed and screened for sialic‐acid‐specific lectins. Complementary DNA clones were categorized into seven groups corresponding to closely related but different sequences. Group 1 clones contained an ORF encoding 199 amino acids including a sequence identical to the partial amino acid sequence obtained from the lectin protein. Within its 1074‐bp 3′ untranslated region, ten closely related 60‐bp sequence repeats were found. Group 2 clones contained an ORF encoding a polypeptide chain of the same number of amino acid residues, with 89.1  % overall identity to that of the group 1 and eight 60‐bp repeat sequences in the 3′ untranslated region. The remaining groups of clones contained ORF with highly similar full or partial sequences, with or without 60 bp repeats in the 3′ untranslated region. The large number of closely related but different cDNA clones obtained indicated that the slug sialic‐acid‐specific lectin gene is a member of a multigene family. The lectin amino acid sequence showed significant similarity with the fibrinogen domain of human tenascin‐C, with a human C‐type serum lectin, and with pig ficolin. Immunostaining analysis of slug tissue for the lectin indicated that it is present primarily on the epidermal surface and in mucous glands. Recombinant slug lectin protein lacking the 20‐amino‐acid N‐terminal signal sequence produced in a bacterial expression system from a group‐1 clone accumulated as aggregates in inclusion bodies, suggesting that large‐scale production of the active agglutinin may be possible.
Iron‐regulatory protein‐1 (IRP‐1) is highly conserved in two invertebrate species
Authors: Muckenthaler Martina , Gunkel Niki , Frishman Dmitrij , Cyrklaff Anna , Tomancak Pavel , Hentze Matthias W.
Published: March 2003

Iron‐regulatory protein‐1 (IRP‐1) plays a dual role as a regulatory RNA‐binding protein and as a cytoplasmic aconitase. When bound to iron‐responsive elements (IRE), IRP‐1 post‐transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate‐primer PCR strategy and the screening of data bases, we now identify the homologues of IRP‐1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP‐1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP‐1 homologues (Drosophila IRP‐1A and Drosophila IRP‐1B), displaying 86  % identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP‐2. In contrast to the mammalian system where the two IRP (IRP‐1 and IRP‐2) are differentially expressed, Drosophila IRP‐1A and Drosophila IRP‐1B are not preferentially expressed in specific organs. The localization of Drosophila IRP‐1A to position 94C1‐8 and of Drosophila IRP‐1B to position 86B3‐6 on the right arm of chromosome 3 and the availability of an IRP‐1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.
Cloning, sequencing, temporal expression and tissue‐specificity of two serine proteases from the midgut of the blood‐feeding fly Stomoxys calcitrans
Authors: Lehane Stella M. , Assinder Susan J. , Lehane Michael J.
Published: March 2003

Using highly degenerate, serine‐protease‐specific PCR primers on a midgut‐specific cDNA library it was estimated that a minimum of 24 independent serine proteases were expressed in the midgut of Stomoxys calcitrans. The relative abundance of these 24 independent serine proteases has been estimated by restriction analysis of PCR products, showing that 69  % fall into six almost equally abundant groups. Two highly abundant serine protease cDNAs (Ssp1 and Ssp2) were isolated and sequenced. They encode preproenzymes of 272 amino acids (Mr 28 521) and 255 amino acids (Mr 27 097) with putative signal peptides of 17 amino acids and 16 amino acids, putative activation peptides of 15 amino acids and 10 amino acids and mature enzymes of 239 amino acids (Mr 25 322; pI 4.89) and 228 amino acids (Mr 24 182; pI 7.59), respectively. Both deduced amino acid sequences contain the Asp/His/Ser catalytic triad and the highly conserved sequences surrounding it. Ssp2 also has the aspartate and two glycine residues in the specificity pocket, marking this as a typical trypsin. The positioning of the residues in the specificity pocket of Ssp1 is unusual; aspartate and glycine residues are present, which is typical of trypsin, but both are separated from surrounding conserved residues by additional amino acids; the second glycine found in the specificity pocket of trypsin is replaced by a serine, which is typical of chymotrypsin. Although a serine protease, the precise substrate specificity of Ssp1 remains to be determined. Northern analysis shows that both serine proteases are expressed constitutively with only a 20  % change in the levels of expression of Ssp1 and Ssp2 through the digestive cycle, and that expression occurs predominantly in the opaque region of the midgut, the region responsible for secretion of digestive enzymes.
A family of flavoproteins in the domains Archaea and Bacteria
Authors: Wasserfallen Alain , Ragettli Sandra , Jouanneau Yves , Leisinger Thomas
Published: March 2003

A family of flavoproteins, called A‐type flavoproteins, is described. It consists of 14 protein sequences of 385−597 amino acids in length, 7 from methanogens (domain : Archaea), 5 from phototrophic prokaryotes, one from Escherichia coli, and a partial sequence from the sulfate reducer Desulfovibrio gigas (domain : Bacteria). No similar sequence could be found in the domain Eucarya. All sequences show significant similarity over a 385−400 amino acid portion overlapping a recognizable flavodoxin signature starting at positions 245−285 of the common core sequence. Cofactor analysis and, to some extent, analysis of the primary structure of six A‐type flavoproteins, three of which are structurally characterized here, support the existence of four sub‐families : (a) simple flavoproteins binding only FMN; (b) diflavin flavoproteins binding FMN and FAD; (c) a flavorubredoxin binding FMN and iron; (d) a hemoflavoprotein. The possible involvement of A‐type flavoproteins in the metabolism of oxygen, as suggested for D. gigas hemoflavoprotein [Gomes, C. M., Silva, G., Oliveira, S., LeGall, J., Liu, M.‐Y., Xavier, A. V., Rodrigues‐Pousada, C. & Teixeira, M. (1997) J. Biol. Chem.272, 22 502−22 508], is discussed.
Interferon‐α activates signal transducers and activators of transcription 5 and 6 in Daudi cells
Authors: Fasler‐Kan Elizaveta , Pansky Andreas , Wiederkehr Michèle , Battegay Manuel , Heim Markus H.
Published: March 2003

In most cells studied so far, interferon‐α (IFN‐α) activates signal transducer and activator of transcription (Stat1), Stat2 and Stat3, whereas interferon‐γ (IFN‐γ) induces Stat1 only. In general, each of the several dozens of cytokines, growth factors and hormones that signal through the Janus kinases‐signal transducers and activators of transcription (Jak‐STAT) pathway activates a distinct subset of STATs, and this selectivity is thought to be essential for the specificity of the cellular responses toward these ligands. Here, we have studied the pattern of STAT activation in the human lymphoblastoid cell line Daudi in response to IFN‐α. In addition to Stat1, Stat2 and Stat3 activation, IFN‐α was found to directly induce activation of Stat5 and Stat6. Cell‐type‐specific activation of additional STATs could be responsible for cell‐type‐specific responses to IFN‐α.
Interaction of caldesmon with actin subdomain‐2
Authors: Bartegi Aghleb , Roustan Claude , Bertrand Raoul , Kassab Ridha , Fattoum Abdellatif
Published: March 2003

The polymerization‐resistant maleimidobenzoyl−G‐actin (MBS−G‐actin), which behaves as a functional analogue of native G‐actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028−6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085−9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross‐linking reactions. MBS−G‐actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 +C, with a high affinity (Kd≈0.04 μM) and with a 1 : 1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS−G‐actin cleaved at the subdomain‐2 loop with subtilisin decreased fivefold (Kd≈0.20 μM). Conversely, caldesmon strongly protected subdomain‐2 of MBS−G‐actin from tryptic digestion at the susceptible peptide bond at positions 68−69. Furthermore, caldesmon induced the dissociation of native G‐actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS−G‐actin−caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS−G‐actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain‐2, to caldesmon, producing in high yield a 205‐kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS−F‐actin. MBS−G‐actin could be also cross‐linked to caldesmon by 1‐ethyl‐3[3‐(dimethylamino)propyl]carbodiimide, producing a three‐band pattern identical to that of F‐actin and caldesmon and previously shown to reflect the covalent union between the NH2‐terminal segment of actin and the COOH‐terminal actin‐binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain‐2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain‐1 as well as the top portion of its subdomain‐2.
Mouse fibroblast‐activation protein
Authors: Niedermeyer Joachim , Enenkel Barbara , Park John E. , Lenter Martin , Rettig Wolfgang J. , Damm Klaus , Schnapp Andreas
Published: March 2003

The human fibroblast‐activation protein (FAP), a member of the serine protease family, was discovered as an inducible type‐II cell‐surface glycoprotein selectively expressed by reactive stromal fibroblasts of epithelial cancers and healing wounds. Antibodies directed against human FAP have a clinical use for antibody‐based tumor imaging. As part of an effort to generate animal models of FAP expression in epithelial tumorigenesis and wound healing, we previously cloned the cDNA encoding the mouse FAP homolog. In this study, we used PCR/restriction‐fragment length polymorphism, identified in interspecific back‐crosses between Mus musculus and Mus spretus, to map the Fap gene locus to a region of mouse chromosome 2, known to be syntenic to the previously identified FAP gene locus on human chromosome 2q23. The Fap gene spans approximately 60 kb and contains 26 exons ranging in size from 46 bp to 195 bp. This genomic organization is very similar to that of the human FAP locus. Similar to the gene encoding dipeptidyl peptidase IV (DPP IV), the nucleotides encoding the serine protease consensus motif, WGWSYGG, are split between two exons, a feature distinct from classical serine proteases. Consistent with the similarity to DPP IV, a chimeric FAP fusion protein expressed in a baculovirus system has dipeptidyl peptidase activity.
Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis‐inhibitory factor
Authors: Morinaga Tomonori , Nakagawa Nobuaki , Yasuda Hisataka , Tsuda Eisuke , Higashio Kanji
Published: March 2003

The human osteoprotegerin (OPG)‐osteoclastogenesis‐inhibitory factor (OCIF) gene has been cloned and characterized. The OPG‐OCIF gene is a single‐copy gene consisting of five exons, and spans 29 kb of the human genome. All the exon/intron boundaries comply with the GT/AG rule. The translation‐termination codon is present in exon 5 and a typical poly(A)‐addition signal resides 173‐nucleotides downstream of the translation‐termination codon. A major transcription‐initiation site is present 67‐nucleotides upstream of the initiation ATG codon. Two minor sites are present further upstream. The 4.2‐kb and 6.5‐kb transcripts detected in IMR‐90 cells were found to contain the 3′‐half of intron 2 and the entire intron 2, respectively. In the OPG‐OCIF gene, a single intron divides the stretch that encode four Cys‐rich motifs, implying diversity from the other members of the tumor necrosis factor receptor (TNFR) family. Two death domain homologous regions (DDHs) present in tandem in OPG‐OCIF are encoded separately by exons 4 and 5. The conservation of amino‐acid sequences suggests that exon 4 is produced by a duplication of a portion of exon 5.
The lack of a stress response in Hydra oligactis is due to reduced hsp70 mRNA stability
Authors: Brennecke Thomas , Gellner Klaus , Bosch Thomas C. G.
Published: March 2003

Synthesis and degradation of hsp70 mRNA was examined and compared in Hydra species living in different habitats and showing different heat‐shock response. Hydra oligactis is restricted to habitats of low temperature and relatively stable pH. We have shown previously that this species is unable to acquire thermotolerance [Bosch, T., Krylow, S., Bode, H. & Steele, R. (1988) Proc. Natl Acad. Sci. USA 85, 7927−7931] and synthesizes significantly less heat‐shock protein and hsp70 mRNA [Gellner, K., Praetzel, G. & Bosch, T. C. G. (1992) Eur. J. Biochem. 210, 683−691] in response to stress than related species, such as Hydra vulgaris or Hydra magnipapillata, which are adapted to habitats of wide temperature range and variable water quality. To examine the mechanisms responsible for the differential heat‐shock responses in these species, a construct containing H. magnipapillata hsp70 regulatory sequences fused to firefly luciferase was introduced into H. oligactis and H. magnipapillata polyps, and expression of luciferase examined. The results showed that luciferase can be expressed equally well in a heat‐inducible manner in both species, suggesting that H. oligactis heat‐shock factor can interact with H. magnipapillata heat‐shock elements. Northern blots of α‐amanitin‐treated polyps demonstrated that the half‐life of hsp70 mRNA in heat‐shocked H. oligactis is drastically shorter than in H. magnipapillata. Thus, differences in hsp70 mRNA stability appear to be responsible for the habitat‐correlated differences in the stress response in Hydra species.
Pro‐oxidant role of superoxide dismutase in ultraviolet‐A‐induced lipid peroxidation in cultured normal human skin fibroblasts
Authors: Morlière Patrice , Santus René
Published: March 2003

Exposure of cultured normal human skin fibroblasts to ultraviolet A triggers lipid peroxidation. In sharp contrast with the tert‐butylhydroperoxide‐induced lipid peroxidation, the ultraviolet‐A‐induced lipid peroxidation is inhibited by treating cells with diethyldithiocarbamate. Diethyldithiocarbamate decreases superoxide dismutase activity and, to a lesser extent, the total glutathione level. Catalase and glutathione peroxidase, however, are unaffected. The decrease in the superoxide dismutase activity parallels an inhibition of H2O2 formation in both irradiated and unirradiated cells. The protection against lipid peroxidation may thus be associated with superoxide dismutase inhibition. Membrane damage revealed by neutral red uptake is not prevented by diethyldithiocarbamate.
Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step
Authors: Labit‐Le Bouteiller Christine , Jamme Marie Françoise , David Martine , Silve Sandra , Lanau Colette , Dhers Christiane , Picard Claudine , Rahier Alain , Taton Maryse , Loison Gérard , Caput Daniel , Ferrara Pascual , Lupker Jan
Published: March 2003

SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil‐binding protein, have been characterised molecularly. Only the σ1 receptor has been shown to bind (Z)N‐cyclohexyl‐N‐ethyl‐3‐(3‐chloro4‐cyclohexylphenyl)propen‐2‐ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T‐cell proliferation was consistent with a sigma1 receptor‐mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil‐binding protein is a new SR31747A‐binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a Δ8‐cholesterol isomer at the expense of cholesterol in SR31747A‐treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the Δ8‐Δ7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil‐binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti‐proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol‐isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.
Characterization of an acetyl‐11‐keto‐β‐boswellic acid and arachidonate‐binding regulatory site of 5‐lipoxygenase using photoaffinity labeling
Authors: Sailer Eckart‐Roderich , Schweizer Stephan , Boden Sven E. , Ammon Hermann P. T. , Safayhi Hasan
Published: March 2003

AKBA (acetyl‐11‐keto‐β‐boswellic acid), a natural pentacyclic triterpene, is an orally active leukotriene‐synthesis inhibitor, which acts by a 5‐lipoxygenase−directed, non‐redox, non‐competitive mechanism. It is the only leukotriene‐synthesis inhibitor so far identified that inhibits 5‐lipoxygenase activity as an allosteric regulator and not by a reducing or competitive mechanism. To characterize AKBA's effector site we prepared azido125I‐KBA (4‐azido‐5‐125iodo‐salicyloyl‐β‐alanyl‐11‐keto‐β‐boswellic acid) as a photoaffinity analogue, which inhibited 5‐lipoxygenase activity as efficiently as the lead compound and specifically labeled human 5‐lipoxygenase protein. The labeling of 5‐lipoxygenase by azido‐125I‐KBA strictly depended on the presence of calcium ([Ca2+]free > 500 nM) and was abolished by heat denaturation or by priorincubation with a series of pentacyclic triterpenes (e.g., amyrin, β‐boswellic acid, AKBA and 18α‐glycyrrhetinic acid). In contrast, 18‐β‐glycyrrhetinic acid and competitive 5‐lipoxygenase inhibitors (e.g., ZM‐230,487 and L‐739,010) did not affect labeling. Arachidonic acid, in enzyme‐activity‐inhibiting concentrations, reduced photoincorporation (IC50 about 10 μM), whereas a variety of other long‐chain fatty acids and their derivatives (e.g., arachidinic acid, arachidonic acid methyl ester, lipoxins A4 and B4) had no effect. The inhibitory arachidonate action on labeling was not affected by blocking the substrate‐binding site by micromolar amounts of the competitive inhibitor L‐739,010. Therefore, we suggest that AKBA binds in presence of calcium to a site which is distinct from the substrate binding site of 5‐lipoxygenase. The AKBA‐binding site is likely to be identical with a regulatory, second arachidonate binding site of the enzyme.
Sulfated di‐, tri‐ and tetraantennary N‐glycans in human Tamm‐Horsfall glycoprotein
Authors: van Rooijen Johannes J. M. , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: March 2003

The primary structures of 32 sulfated di‐, tri‐ and tetraantennary N‐glycans of human Tamm‐Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase‐F and fractionated via FPLC on Resource Q, HPLC on LiChrosorb‐NH2, and high‐pH anion‐exchange chromatography on CarboPac PA‐1. Characterizations were performed using 500‐MHz and 600‐MHz 1H‐NMR spectroscopy, in combination with sialidase treatments. The type of characterized N‐glycans ranged from monosulfated to trisulfated N‐glycans, whereby the sulfate groups were present as 3‐O‐sulfated Gal (Gal3S) and 4‐O‐sulfated GalNAc (GalNAc4S). A compilation of the established structures is shown below. /d/im3/ejbS620/S11620Formel01.tif.lay,0,0,0,‐1>
Bioenergetic consequences of accumulating the common 4977‐bp mitochondrial DNA deletion
Authors: Porteous William K. , James Andrew M. , Sheard Philip W. , Porteous Carolyn M. , Packer Michael A. , Hyslop Serena J. , Melton Julian V. , Pang Cheng‐Yoong , Wei Yau‐Huei , Murphy Michael P.
Published: March 2003

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (Δ‐mtDNA) lacking a specific 4977‐bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns‐Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of Δ‐mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of Δ‐mtDNA on cell function. The 4977‐bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of Δ‐mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (@gR0) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0−86 %Δ‐mtDNA and all had volumes, protein contents, plasma‐membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating Δ‐mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50−55 %Δ‐mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of Δ‐mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of Δ‐mtDNA in the target tissues of patients with mtDNA diseases.
Corticotropin‐releasing factor receptor type 1 from Tupaia belangeri
Authors: Palchaudhuri Monika R. , Wille Sandra , Mevenkamp Gregor , Spiess Joachim , Fuchs Eberhard , Dautzenberg Frank M.
Published: March 2003

A cDNA clone encoding corticotropin‐releasing factor (CRF) type 1 (CRF‐R1) has been isolated from the tree shrew Tupaia belangeri with a PCR‐based approach. The full‐length cDNA encoded a 415‐amino‐acid protein with highest sequence identity (≈98 %) to human CRF‐R1 and slightly less identity to rat or mouse CRF‐R1 (≈97 %). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the N‐terminus, residue 232 in transmembrane domain 4 and residue 410 in the C‐terminus) differed between tree shrew CRF‐R1 (tCRF‐R1) and human CRF‐R1 (hCRF‐R1). tCRF‐R1 mRNA was detected by semiquantitative RT‐PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF‐R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF‐R1 showed that the CRF agonists ovine CRF (KD = 1.28 nM), human/rat CRF (KD = 1.09 nM), urocortin (KD = 0.37 nM) and sauvagine (KD = 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (KD = 12.4 nM). In agreement with the binding data half maximum effective EC50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF‐R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF‐R1.
The solution structure of parsley [2Fe‐2S]ferredoxin
Authors: Im Sang‐choul , Liu Gaohua , Luchinat Claudio , Sykes A. Geoffrey , Bertini Ivano
Published: March 2003

The [2Fe‐2S]ferredoxin I (Fd I) from parsley leaves (Mr = 10 500; 96 amino acids) in the Fe(III)Fe(III) oxidized form has been studied by 1H‐NMR spectroscopy. Sequence‐specific 1H‐NMR assignments were obtained through two‐dimensional classical double‐quantum‐filtered‐COSY, NOESY and TOCSY spectra. NOEs between protons as close as 5.6 Å from the paramagnetic Fe(III) atoms were observed at 800 MHz. A total of 3066 NOEs (of which 2533 are meaningful) and 18 distance constraints taken from X‐ray crystallography of the Fe2S2 active site were used to obtain the solution structure. From inversion recovery NOESY experiments, 33 longitudinal relaxation rate (ρpara) constraints were used for the structural refinement. The final structure was obtained by a process of restrained energy minimization. Root‐mean‐square (rmsd) deviation values obtained for the family of 18 structures (with reference to the average structure) are 0.52 ± 0.10 Å and 0.91 ± 0.12 Å for backbone and all heavy atoms respectively. The structure consists of seven‐strands of β‐sheets and four short α‐helices. The quality of the present solution structure is among the best of those reported for [2Fe‐2S]ferredoxins. The secondary structure and overall folding are compared with those of Anabaena variabilis Fd and the higher plant Equistum arvense (horse tail) protein determined through X‐ray crystallography. The groups believed to be responsible for electron transfer have been analysed.
A low‐spin iron with CN and CO as intrinsic ligands forms the core of the active site in [Fe]‐hydrogenases
Authors: Pierik Antonio J. , Hulstein Marco , Hagen Wilfred R. , Albracht Simon P. J.
Published: March 2003

In this report the first high‐quality infrared spectra of [Fe]‐hydrogenase are presented. Analyses of these spectra obtained under a variety of redox conditions strongly indicate that [Fe]‐hydrogenases contain a low‐spin Fe ion in the active site with one CN− group and one CO molecule as intrinsic, non‐protein ligands. When in the ferric state, the presence of such an ion can explain the enigmatic EPR properties (the rhombic 2.10 signal) of the active, oxidised enzyme. To account for other, well‐characterised properties of the active site, we propose that the active site of [Fe]‐hydrogenases consists of this low‐spin Fe ion bound to an unusual [4Fe‐4S] cluster via bridges with sulphur atoms.
The roles of the N‐linked glycans and extension regions of soybean β‐conglycinin in folding, assembly and structural features
Authors: Maruyama Nobuyuki , Katsube Tomoyuki , Wada Yusuke , Oh Moon Hun , Barba De La Rosa Ana Paulina , Okuda Eiko , Nakagawa Shuko , Utsumi Shigeru
Published: March 2003

β‐Conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, α, α′ and β. All subunits are N‐glycosylated and α and α′ contain extension regions in addition to the core regions common to all subunits. Non‐glycosylated individual subunits and deletion mutants (αc and α.es.cf1.′.cf5..rb.eic.rb) lacking the extension regions of α and α′ were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density‐gradient centrifugation and gel‐filtration profiles, indicating that the N‐linked glycans and extension regions are not essential for the folding and the assembly into trimers of β‐conglycinin. Density‐gradient centrifugation, gel‐filtration and differential scanning calorimetry profiles of the recombinant proteins and the native β‐conglycinin indicated that the N‐linked glycans and extension regions contribute to the dimension of β‐conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the α and α′ subunits were soluble at lower ionic strength (μ < 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N‐linked glycans.
Competition and cooperation amongst yeast elongation factors
Authors: Kovalchuke Oleh , Kambampati Ravi , Pladies Erika , Chakraburtty* Kalpana
Published: March 2003

Elongation factor 3 (EF‐3) is an essential requirement for translation in fungi. We previously reported activation of EF‐3‐ATPase by yeast ribosomes. EF‐3 interacts with both ribosomal subunits and shows high affinity for 60S subparticles. Translational inhibitors α‐sarcin, ricin and auto‐immune antibodies to GTPase‐activation center inhibit binding of EF‐2 but not of EF‐3 to yeast ribosomes. EF‐2 competes with EF‐3 for the ribosomal binding sites and inhibits EF‐3‐ATPase activity. Neomycin relieves the inhibitory effect of EF‐2 on EF‐3 function. The apparent competition between EF‐2 and EF‐3 may represent binding of these two proteins to specific conformational states of the ribosome.

EF‐3 stimulates ternary complex binding to yeast ribosomes. Neither the binding of EF‐3 to ribosomes, nor the ribosome‐dependent EF‐3‐ATPase activity are influenced by EF‐1α. Three lines of experimental evidence suggest a direct interaction between EF‐1α and EF‐3. A polyclonal antibody to EF‐3 immunoprecipitates EF‐1α along with EF‐3. EF‐1α co‐migrates with GST‐EF‐3 on glutathione‐Sepharose columns. ELISA tests demonstrate an interference of EF‐3/anti‐EF‐3 interaction by EF‐1α but not by EF‐2. These results strongly suggest that the stimulatory effect of EF‐3 on the ternary complex binding to yeast ribosomes involves a direct interaction between EF‐1α and EF‐3.
Characterization and molecular cloning of the lectin from  Helianthus tuberosus
Authors: Van Damme Els J. M. , Barre Annick , Mazard Anne‐Marie , Verhaert Peter , Horman Alistair , Debray Henri , Rouge Pierre , Peumans Willy J.
Published: March 2003

A lectin called Helianthus tuberosus agglutinin or Heltuba has been isolated from tubers of the Jerusalem artichoke, a typical representative of the Asteraceae family. Heltuba is a tetrameric protein composed of four identical subunits of 15.5 kDa and exhibits a preferential specificity towards oligomannosides. Cloning of the corresponding cDNAs revealed that the mature lectin polypeptide comprises the entire open reading frame of the cDNA suggesting that the primary translation product is not processed and that the lectin is a cytosolic protein. Searches in the databases revealed sequence similarity with lectins from the taxonomically unrelated Convolvulaceae and Moraceae species. Therefore, the discovery of Heltuba is of great importance in view of the occurrence and molecular evolution of the jacalin‐related lectins.
Biosynthesis of indole‐3‐acetic acid in Azospirillum brasilense
Authors: Zakharova Elena A. , Shcherbakov Alexander A. , Brudnik Vitaly V. , Skripko Nataliya G. , Bulkhin Nail Sh. , Ignatov Vladimir V.
Published: March 2003

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole‐3‐acetic acid (IAA). The standard free energy changes (ΔG°′sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole‐3‐acetamide (IAM), indole‐3‐pyruvic acid (IPyA), and indole‐3‐acetonitrile (IAN). The ΔG°′sum for Trp synthesis from chorismic acid was – 402 (– 434) kJ·mol–1 (values in parentheses were calculated by PM3). The ΔG°′sum for IAA synthesis from Trp were − 565 (– 548) kJ·mol–1 for the IAN pathway, – 481 (– 506) kJ·mol–1 for the IAM pathway, and – 289 (– 306) kJ·mol–1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp‐independent pathway.
Mouse Rt6.1 is a thiol‐dependent arginine‐specific ADP‐ribosyltransferase
Authors: Hara Nobumasa , Badruzzaman , Muhammad , Sugae , Takashi , Shimoyama , Makoto , Tsuchiya Mikako
Published: March 2003

Mouse T‐cell antigens Rt6.1 and Rt6.2 are glycosylphosphatidylinositol‐anchored arginine‐specific adenosine diphosphate (ADP)‐ribosyltransferases. In the present study, we obtained evidence that an arginine‐specific ADP‐ribosyltransferase activity liberated from BALB/c mouse splenocytes by phosphatidylinositol‐specific phospholipase C increased fivefold in the presence of dithiothreitol and that the activity was immunoprecipitated by polyclonal antibodies generated against recombinant rat RT6.1. When mouse Rt6.1 was expressed as a recombinant protein, the transferase activity of Rt6.1 was stimulated by dithiothreitol, and inhibited by N‐ethylmaleimide, while activities of recombinant mouse Rt6.2 and the Glu‐207 mutant of rat RT6.1 [Hara, N., Tsuchiya, M., and Shimoyama, M. (1996) J. Biol. Chem.271, 29552–29555] were unaffected by either agent. In addition to four cysteine residues conserved among mouse Rt6 and rat RT6 antigens, Rt6.1 has two extra cysteine residues at positions 80 and 201. To investigate a contribution of these extra cysteines in mouse Rt6.1 to thiol dependency of Rt6.1 transferase activity, Cys‐80 and Cys‐201 of Rt6.1 were replaced with serine and phenylalanine, respectively, the corresponding residues of mouse Rt6.2 and rat RT6.1. Transferase activity of the Phe‐201 mutant of Rt6.1 lost thiol dependency while that of the Ser‐80 mutant remained thiol‐dependent. Thus, we conclude that mouse Rt6.1 is a thiol‐dependent arginine‐specific ADP‐ribosyltransferase, and that Cys‐201 confers thiol dependency on Rt6.1 transferase. Our study indicates that arginine‐specific ADP‐ribosyltransferase activity detected on BALB/c mouse splenocytes is attributed to Rt6.1 and that Rt6.1 differs from Rt6.2 in enzymatic property of the transferase and perhaps in immunoregulatory functions.
Structural changes of horseradish peroxidase in presence of low concentrations of urea
Authors: Haque Md. Emdadul , Debnath Dilip , Basak Soumen , Chakrabarti Abhijit
Published: March 2003

The presence of very low concentrations of the widely used denaturant urea induces structural changes in the monomeric heme‐containing enzyme, horseradish peroxidase (HRP). Structural alterations in the protein were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern–Volmer quenching constants measured in presence of urea, even in concentrations below 100 mm, were higher than those measured in absence of the denaturant. The fluorescence emission maximum of 1,8‐ANS, used as a probe for monitoring conformational changes in the enzyme, was blue‐shifted from 530 nm in aqueous buffer to 518 nm when incorporated in native HRP. This blue shift increased further by 3 nm in presence of HRP preincubated with 100 mm urea, whereupon it steadily decreased with increasing urea concentration to become zero at 8 m urea. The mean fluorescence lifetime of 1,8‐ANS incorporated in HRP was much higher than that of ANS in aqueous buffer, and showed continuous variation with the concentration of urea in which the enzyme was incubated. Systematic changes in the microenvironment of the heme moiety in HRP were also reflected in the visible CD spectra of the enzyme incubated with low concentrations of urea. These results are consistent with those of our earlier studies performed with the denaturant guanidinium chloride and indicate structural relaxation of HRP, with retention of enzymatic activity and native‐like secondary structure, in presence of millimolar concentrations of urea.
Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations
Authors: James Andrew M. , Sheard Philip W. , Wei Yau‐Huei , Murphy Michael P.
Published: March 2003

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke‐like episodes) and MERRF (myoclonic epilepsy and ragged‐red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudyet al. (1995)PNAS,92, 729–733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60–88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+‐ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+‐ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2–20‐fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.
Sequestration of dopamine D2 receptors depends on coexpression of  G‐protein‐coupled receptor kinases 2 or 5
Authors: Ito Kazumitsu , Haga Tatsuya , Lameh Jelveh , Sadée Wolfgang
Published: March 2003

We examined the agonist‐dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS‐7 and HEK 293 cells with or without G‐protein‐coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride‐binding activity from the cell surface and by transfer of [3H] spiperone‐binding activity from the membrane fraction to the light vesicle fraction in sucrose‐density gradients. In COS‐7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10–4 m dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN‐GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist‐dependent and GRK2‐dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 m sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist‐dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK‐mediated pathway.