bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential pre‐mRNA utilisation of an upstream cryptic 5′ splice site created by a single base deletion mutation in exon 37 of the FBN‐1 gene
Authors: Gibson Mark A. , Ellis Sarah L. , Ades Lesley C. , Haan Eric , Cleary Edward G.
Published: March 2003

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin‐1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5′ splice site in exon 37 which was utilised in preference to the normal 5′ splice site during pre‐mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48‐bp deletion from the 3′ end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16‐amino‐acid sequence from a central eight‐cysteine repeat motif of the fibrillin‐1 molecule. Interestingly, the cryptic 5′ splice site in exon 37 and the normal 5′ splice site had equally strong consensuses for splice‐site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre‐mRNA splicing. Analysis by reverse‐transcription PCR indicated that, in the patients skin fibroblasts, the steady‐state level of the mis‐spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse‐chase biosynthetic labelling indicated that close to normal amounts of fibrillin‐1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin‐1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin‐1 molecules appeared to be exerting a powerful dominant‐negative effect on the incorporation of normal fibrillin‐1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the ’classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.
Expression of membrane‐bound transferrin‐like protein p97 on the cell surface of chondrocytes
Authors: Kawamoto Takeshi , Pan Haiou , Yan Weiqun , Ishida Hideyuki , Usui Emiko , Oda Ryo , Nakamasu Kazuko , Noshiro Mitsuhide , Kawashima‐Ohya Yoshie , Fujii Masashi , Shintani Hideaki , Okada Yasunori , Kato Yukio
Published: March 2003

A concanavalin‐A‐binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N‐terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour‐associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse‐transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.
Functional characterization of the human thiopurine S‐methyltransferase (TPMT) gene promoter
Authors: Fessing Michael Y. , Krynetski Eugene Y. , Zambetti Gerard P. , Evans William E.
Published: March 2003

Thiopurine S‐methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S‐methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently isolated the human TPMT promoter, which does not contain TATA box or CCAAT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis‐regulatory elements. Here, we report the functional characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Sp1) as an important trans‐activator essential for constitutive activity in cell culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed positive cis‐regulatory elements located in the regions −85 to −75, −68 to −58, −58 to −51 and +34 to +60 relative to the transcription start site. DNaseI footprinting analysis and cotransfection in Drosophila Schneider SL2 cells documented that Sp1 binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive transcription of the TPMT gene involves a limited upstream GC‐rich DNA sequence, containing multiple GC boxes, and that transcription factor Sp1 [or related protein(s)] is an important trans‐activator of this TATA‐less promoter.
Phorbol ester causes ligand‐independent activation of the androgen receptor
Authors: Darne Christian , Veyssiere Georges , Jean Claude
Published: March 2003

We have examined the human androgen receptor (hAR) for its ability to activate AR‐dependent transcription of a transgene in a ligand‐independent manner. The transcriptional activity was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in T47D cells cotransfected with a plasmid expressing the hAR and a natural AR‐regulated promoter (the MVDP androgen‐dependent enhancer) ligated to the reporter CAT gene. In this study, the effects of the protein kinase C (PKC) activator 12‐O‐tetradecanoyphorbol‐13 acetate (TPA) on AR activity were tested. We demonstrated that in the absence of androgen, TPA enhanced AR‐mediated transactivation by 10−12‐fold. This effect was specific of the PKC pathway since stimulation to the PKA pathway did not activate the unliganded AR. This ligand‐independent pathway can function through another androgen‐regulated promoter as shown by the use of the mouse mammary tumor virus MMTV‐CAT reporter. The human glucocorticoid receptor (hGR) and the rabbit progesterone receptor (rPR) could not be activated by TPA, indicating that the effects are not universal for steroid receptors. A reporter plasmid containing the MVDP androgen response element (ARE) in front of the thymidine kinase promoter ligated to the CAT gene was activated by DHT but not by TPA, indicating that the context of the natural promoter is critical for ligand‐independent activation of the AR. Exogenous c‐jun enhanced transcriptional activation by the AR in a ligand‐dependent manner, but had no effect in the absence of DHT. Base pair substitutions in both AR‐binding (5′‐TGTTCT‐3′ to 5′‐TTTTTT‐3′) and NF1‐binding (5′‐GTGGCTG‐3′ to 5′‐GTTTTTG‐3′) sites resulted in a loss of TPA responsiveness. Our results suggest that ligand‐independent activation of the AR by TPA results from interaction of unliganded AR with other proteins in the transcription machinery.
Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria
Authors: Bjørgo Elisa , Knappskog Per M. , Martinez Aurora , Stevens Raymond C. , Flatmark Torgeir
Published: March 2003

The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose‐binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20 % (R252Q, tetramer), 44 % (L255V, tetramer) and 4.4 % (R252G, dimer) of the activity for the respective wild‐type (wt) forms. When expressed by a coupled in vitro transcription‐translation system, all the mutant enzymes were recovered as a mixture of non‐phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11 % of wt‐hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.
Menaquinone‐dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential
Authors: Schirawski Jan , Unden Gottfried
Published: March 2003

Succinate dehydrogenases from bacteria and archaea using menaquinone (MK) as an electron acceptor (succinate/menaquinone oxidoreductases) contain, or are predicted to contain, two heme‐B groups in the membrane‐anchoring protein(s), located close to opposite sides of the membrane. All succinate/ubiquinone oxidoreductases, however, contain only one heme‐B molecule. In Bacillus subtilis and other bacteria that use MK as the respiratory quinone, the succinate oxidase activity (succinate←O2), and the succinate/menaquinone oxidoreductase activity were specifically inhibited by uncoupler (CCCP, carbonyl cyanide m‐chlorophenylhydrazone) or by agents dissipating the membrane potential (valinomycin). Other parts of the respiratory chains were not affected by the agents. Succinate oxidase or succinate/ubiquinone oxidoreductase from bacteria using ubiquinone as an acceptor were not inhibited. We propose that the endergonic electron transport from succinate (E+′ = +30 mV) to MK (E+′ ≅ −80 mV) in succinate/menaquinone oxidoreductase includes a reversed electron transport across the cytoplasmic membrane from the inner (negative) to the outer (positive) side via the two heme‐B groups. The reversed electron transport is driven by the proton or electrical potential, which provides the driving force for MK reduction.
Bacterial pro‐transglutaminase from Streptoverticillium mobaraense
Authors: Pasternack Ralf , Dorsch Simone , Otterbach Jens T. , Robenek Isabella R. , Wolf Sabine , Fuchsbauer Hans‐Lothar
Published: March 2003

The zymogen of bacterial transglutaminase was found during cultivation of Streptoverticillium mobaraense (DSMZ strain) using rabbit antibodies raised against the active enzyme. Ion‐exchange chromatography at pH 5.0 yielded a highly purified pro‐enzyme. Structure information was obtained by means of Edman degradation and analysis of PCR amplified nucleotide fragments. The data revealed an excess of negatively charged amino acids in the pro‐region resulting in a decreased isoelectric point of the zymogen. Additionally, the new sequence gave rise to some modifications to the previously published hypothetical structure of prepro‐transglutaminase derived from genomic DNA [Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M. & Takeuchi, K. (1994) Biosci. Biotechnol. Biochem. 58, 82−87]. Inactive transglutaminase, which carries an activation peptide of 45 amino acids, has a calculated molecular mass of 42 445 Da. Its pro‐region provides for both suppression of activity and increased thermostability. Furthermore, it could be shown that the micro‐organism produces a protease which cleaves pro‐transglutaminase at the C‐side of Pro45. Rapid transformation of the mature enzyme also occurs by addition of other proteases. During conversion, 43 and 41 amino acid peptides are released by bovine trypsin and dispase from Bacillus polymyxa, respectively. The detection of endogenous substrates in the murein layer makes discussion of the physiological role of bacterial transglutaminases necessary.
Structural analysis of the chicken hydroxyindole‐O‐methyltransferase gene
Authors: Grechez‐Cassiau Aline , Bernard Marianne , Ladjali Kafia , Rodriguez Ignacio R. , Voisin Pierre
Published: March 2003

Hydroxyindole‐O‐methyltransferase (HIOMT) catalyzes the final step of melatonin synthesis, a neurohormone involved in photoperiodism and produced specifically in the pineal gland and in the retina. In the chicken, HIOMT gene transcription appears to be controlled by a circadian oscillator located in the pineal gland. We have characterized the chicken HIOMT gene over 17 kb, including 2.9 kb of 5′‐flanking sequence. The major transcript (1.6 kb) is composed of eight exons distributed over 7.5 kb of genomic DNA. A ninth alternative exon was identified 6 kb downstream of exon 8. It was found in minor transcripts in the pineal gland and in the retina. Sequence similarity between exons 8 and 9 suggests their origin by exon duplication. Due to early stop codons, inclusion of exon 9 truncates the open reading frame by up to 33 %. A restriction fragment length polymorphism was detected for a BglII site in intron 8. Fluorescence hybridization localized the HIOMT gene on chicken chromosome 1q22. The 5′‐flanking region contains GATTAA and TAATCC sequences that may be related to tissue‐specific expression. An ATTTAAAT sequence at position −29 would play the role of a TATA box, as evidenced by electrophoretic mobility shift assay. Information obtained in this study open the way to further studies aimed at analyzing the circadian rhythm of transcription at promoter level.
The high degree of internal flexibility observed for an oligomannose oligosaccharide does not alter the overall topology of the molecule
Authors: Woods Robert J. , Pathiaseril Ahammadunny , Wormald Mark R. , Edge Christopher J. , Dwek Raymond A.
Published: March 2003

The conformational properties of oligosaccharides are important in determining their biological properties, such as recognition by proteins. The structural and dynamic properties of many oligosaccharides are poorly understood both because of a lack of experimental data (usually obtained from solution NMR parameters) and because of gross approximations frequently invoked in theoretical models. To characterise the oligomannose oligosaccharide Man9GlcNAc2 we have acquired a more extensive NMR data set and performed the first unrestrained molecular dynamics (MD) simulation in water of this large oligosaccharide (employing the GLYCAM—93 parameter set with the AMBER force field). Good agreement is seen between the computed dynamics data and the results of both an isolated spin pair (ISPA) analysis of short mixing time NOE data and NOE build‐up curves for mixing times from 100 to 2000 ms. The number of experimental conformational constraints obtained in this study are in principle sufficient to fully define a rigid structure. The fact that this could not be done indicates a high degree of internal flexibility and/or the presence of multiple conformations about the glycosidic linkages. Independently, the same conclusions are reached from an analysis of the MD results. In addition, the theoretical results allow the overall topology of the molecule and its intra‐molecular and solvent‐mediated hydrogen bonding pattern to be defined. Extensive re‐organisation of solvent and inter‐residue hydrogen bonds is shown to be required for significant conformational changes to occur, resulting in relatively long life‐times for distinct glycosidic linkage conformations, despite the high local flexibility of the glycosidic linkages. This factor is also seen in the overall topology of the molecule, where the considerable internal flexibility is not translated into gross changes in structure. The control exerted by the solvent over both the flexibility and overall topology of an oligosaccharide has important implications for recognition processes and for the conformational properties of glycans attached to glycoproteins.
Rejoining of DNA double‐strand breaks in vitro by single‐strand annealing
Authors: Göttlich Bernd , Reichenberger Susanne , Feldmann Elke , Pfeiffer Petra
Published: March 2003

Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double‐strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error‐prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End‐joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single‐strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase ε, FEN‐1 endonuclease, and exonuclease activities of 5′‐3′ and 3′‐5′ directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA‐dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error‐prone NHEJ pathway that creates deletions by microhomology‐driven SSA.
Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer
Authors: Kress Stefan , Reichert Jeannette , Schwarz Michael
Published: March 2003

The environmental contaminant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD or dioxin) induces gene transcription, a process that requires binding of the activated aryl hydrocarbon receptor (AhR) to dioxin‐responsive elements (DREs) within the enhancer region of responsive genes. Most of what is known about the molecular mechanism of AhR‐dependent gene activation results from studies on the murine prototype TCDD‐responsive gene cytochrome P4501A1 (CYP1A1). Much less is known, however, about the regulation of human TCDD‐responsive genes. We have therefore conducted a detailed analysis of the enhancer region of the human CYP1A1 gene. From the ten DRE core motifs investigated within a stretch of 1400 bp in two human tumor cell lines using a ligation‐mediated PCR technique, five motifs displayed a TCDD‐inducible in vivo footprint. Four of these sites were functional enhancer sequences as demonstrated by a transient expression assay. Based on these data, a distinct functional consensus sequence for DRE motifs within the human CYP1A1 gene is suggested. After introduction of the four functional sites into various mouse hepatoma cell lines, only three exhibited a functional response, suggesting some species differences in CYP1A1 gene regulation. In addition to the footprints at DRE sites, we also detected protein‐DNA interactions at three G‐rich domains located within the enhancer region of the human CYP1A1 gene. Our data show that, besides some similarities in the regulation of the human and mouse CYP1A1 genes, there also exist some distinct differences, including number, location, and functional consensus sequences of DRE motifs, as well as quantity and location of footprinted G‐rich domains.
Glutamate dehydrogenase, the marker protein of Plasmodium falciparum
Authors: Wagner Jan T. , Lüdemann Heike , Färber Petra M. , Lottspeich Friedrich , Krauth‐Siegel R. Luise
Published: March 2003

The gene of an NADP+‐specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern‐blot analysis indicates a single‐copy gene. The gene encodes a protein with 470 residues which has 50 % of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by
D‐glutamate and glutarate, but not by chloroquine. Like other coenzyme‐specific glutamate dehydrogenases, but in contrast to the dual‐specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western‐blot analysis of stage‐specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.
Isolation, amino acid sequence determination and binding properties of two fatty‐acid‐binding proteins from axolotl (Ambistoma mexicanum) liver
Authors: Di Pietro Santiago M. , Veerkamp Jacques H. , Santomé José A.
Published: March 2003

Up until now, the primary structure of fatty‐acid‐binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L‐FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb‐FABPs). The finding of both L‐FABP and Lb‐FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of ≈ 694 million years ago. The ligand‐binding properties of axolotl liver FABPs were studied by means of parinaric‐acid‐binding and parinaric‐acid‐displacement assays. L‐FABP binds two fatty acids per molecule but Lb‐FABP displays a fatty‐acid‐conformation‐dependent binding stoichiometry; L‐FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb‐FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue‐expression pattern is different, L‐FABP is present in liver and intestinal mucosa while the expression of Lb‐FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.
Primary structure of a cadmium‐induced metallothionein from the insect Orchesella cincta (Collembola)
Authors: Hensbergen Paul J. , Donker , Marianne H. , Van Velzen , Martin J. M. , Roelofs , Dick , Van Der Schors , Roel C. , Hunziker , Peter E. , Van Straalen Nico M.
Published: March 2003

The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd‐binding, cysteine‐rich peptides were isolated from whole‐body homogenates, using gel filtration and reversed‐phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989‐Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139‐Da protein was unsuccessful, probably due to N‐terminal blockage. Using different PCR techniques (3′ and 5′ RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C‐terminal part of this protein. The 4139‐Da protein is probably encoded by the N‐terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post‐translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.
Selective formation of Gsα–MHC I complexes after desensitization of human platelets with iloprost
Authors: Ferreira Patrik , Meyer Ilse , Mollner Stefan , Frank Rainer , Pfeuffer Thomas
Published: March 2003

Prolonged treatment of human platelets with the adenylate cyclase‐stimulating prostacyclin analog iloprost leads to reduction in cAMP formation. Previous studies have demonstrated that this may be ascribed to modification of both receptor and Gsα function rather than of the catalytic component of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur. J. Biochem.210, 539–544]. Iloprost‐induced desensitization was accompanied by the formation of a Gsα‐containing 90‐kDa product in membranes treated with the bifunctional cross‐linker 1,6‐bismaleimidohexane. The cAMP‐inducing prostanoid PGD2, which does not promote desensitization, did not cause formation of the 90‐kDa species either. The long‐term effect of the common G‐protein activator [AlF4]− on human platelet adenylate cyclase was shown in many respects to be comparable with that of iloprost. However, [AlF4]− treatment also failed to induce the 90‐kDa species, showing that different mechanisms of desensitization were operating. Treatment of the cross‐linked 90‐kDa complex with PNGase F demonstrated the glycoprotein nature of the Gsα‐associated component. The 90‐kDa cross‐linked product was purified by consecutive immunoaffinity chromatography and preparative PAGE to apparent homogeneity. Analysis of the purified protein by MS suggested that, besides Gsα, the heavy chain of MHC I (HLA‐A2) was part of the complex. This was confirmed by coprecipitation of Gsα by the monoclonal anti‐(MHC I) antibody W6/32.
Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol‐3‐phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan
Authors: Denise Hubert , Giroud Christiane , Barrett Michael Peter , Baltz Théo
Published: March 2003

A 36‐kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol‐3‐phosphate dehydrogenase.  Trypanosomal glycerol‐3‐phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose‐dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol‐3‐phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.
Requirement for cAMP‐response element (CRE) binding protein/CRE modulator transcription factors in thyrotropin‐induced proliferation of dog thyroid cells in primary culture
Authors: Uyttersprot Nathalie , Costagliola Sabine , Dumont Jacques E. , Miot Françoise
Published: March 2003

In several cell types, mostly of epithelial origin, activation of the cAMP pathway triggers DNA synthesis and cell division. Regulation of gene expression by cAMP involves phosphorylation by pyruvate kinase A and activation of cAMP‐response element binding protein (CREB)/CRE modulator (CREM) transcription factors which bind DNA to CRE sites. On the other hand, several CREM isoforms are transcriptional repressors, such as the inducible cAMP early repressor (ICER) transcription factors, which are synthesized from an intronic promoter of the CREM gene. This study investigated the potential role of CREB/CREM transcription factors in the cAMP mitogenic pathway, using an experimental model of epithelial cells in primary culture, i.e. dog thyroid cells stimulated by thyroid‐stimulating hormone (TSH). In response to TSH, CREB/CREM transcription factors were phosphorylated on the serine residue of the pyruvate kinase A consensus site. In addition, the synthesis of ICER mRNAs was strongly induced by TSH. This transient upregulation of ICER expression correlated with increased protein levels. It was restricted to the cAMP pathway, as neither epidermal growth factor nor phorbol myristate acetate, which are potent mitogens for dog thyroid cells, induced ICER expression. On the other hand, increased expression of ICER mRNAs was not detected in dog thyroids chronically stimulated by TSH in vivo. The requirement for CREB/CREM transcription factors in the mitogenic effect of TSH was assessed by transfecting expression vectors encoding CREM repressors into dog thyrocytes in order to interfere with CRE‐mediated gene transcription. The ectopic expression of ICER Iγ or CREM α isoforms inhibited DNA replication in dog thyrocytes stimulated by TSH. This inhibitory effect was dependent on the ability of CREM repressors to form dimers but did not involve their DNA‐binding capacity. Together these results show that CREB/CREM transcription factors are tightly regulated, at the transcriptional and post‐translational levels, by TSH in dog thyroid cells, and provide clear evidence that their activity is required for the cAMP‐dependent proliferation of cells in primary culture. Moreover, the transient induction of ICER transcription factors during mitogenic stimulation by TSH raises questions about the role of these potent repressors of CRE‐dependent transcription as timers of cellular proliferation.
High level accumulation of single‐chain variable fragments in the cytosol of transgenic Petunia hybrida
Authors: De Jaeger Geert , Buys Emmanuel , Eeckhout Dominique , De Wilde Chris , Jacobs Anni , Kapila Jyoti , Angenon Geert , Van Montagu Marc , Gerats , Tom , Depicker Ann
Published: March 2003

The accumulation of five murine single‐chain variable fragments, binding to dihydroflavonol 4‐reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv‐encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen‐binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.
Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family
Authors: Ito Yuji , Yoshikawa Ayako , Hotani Takuji , Fukuda Seiko , Sugimura Kazuhisa , Imoto Taiji
Published: March 2003

Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N‐terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme‐like enzymatic features.
Domain structure, GTP‐hydrolyzing activity and 7S RNA binding of Acidianus ambivalens Ffh‐homologous protein suggest an SRP‐like complex in archaea
Authors: Moll Ralf , Schmidtke Silke , Schäfer Günter
Published: March 2003

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP‐like ribonucleoprotein complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies.

 A. ambivalens Ffh encodes a 50.4‐kDa protein that is structured by three distinct regions: the N‐terminal hydrophilic N‐region (N), the GTP/GDP‐binding domain (G) and a C‐terminal located C‐domain (C). The A. ambivalens Ffh sequence shares 44–46% sequence similarity with Ffh of methanogenic archaea, 34–36% similarity with eukaryal SRP54 and 30–34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A. ambivalens. Recombinant Ffh has a melting point of tm = 89 °C. Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts. Highest rates of GTP hydrolysis have been achieved at 81 °C in presence of 0.1–1 mm Mg2+. GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents. The Km for GTP is 13.7 µm at 70 °C and GTP hydrolysis is strongly inhibited by GDP (Ki = 8 µm). A. ambivalens Ffh, which includes an RNA‐binding motif in the C‐terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus.

 Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic crenarchaea which strongly supposes recognition events by an Ffh containing SRP‐like particle in these organisms.
Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse
Authors: Rus’d Ahmed Abu , Ikejiri Yasuko , Ono Hiroyuki , Yonekawa Toshihiro , Shiraiwa Masakazu , Kawada Akira , Takahara Hidenari
Published: March 2003

Peptidylarginine deiminases (PADs), a group of post‐translational enzymes, catalyze the conversion of protein‐bound arginine residues to citrulline residues in a calcium ion‐dependent manner and are widely distributed in various organs of vertebrates. Although the existence of four isoforms of PAD (types I, II, III, and IV) is reported in rodents, the relative functions of the isoforms with respect to their colocation in the tissues have yet to be explored. In this study, we cloned the full‐length cDNA encoding mouse PAD type I by screening a uterine cDNA library and using the RACE method. This cDNA consists of an open reading frame of 1989 bases encoding 662 amino acids (73 823 Da), a 5′‐untranslated region of 127 bases and a 3′‐untranslated region of 1639 bases. Comparative reverse transcription‐PCR and Northern‐blot analyses detected PAD type I mRNA only in the epidermis and uterus. Administration of estrogen to adult ovariectomized mice increased the content of PAD type I mRNA in the uterus, providing evidence that its expression is under the control of the sex steroid hormone. We also cloned the full‐length cDNAs of mouse PAD type III and type IV by the reverse transcription‐PCR and RACE methods. The primary structure of PAD type III contains 664 amino acids (75 098 Da) deduced from the coding region of 1995 bases, and the primary structure of PAD type IV consists of 666 amino acids (74 475 Da) deduced from the coding region of 2001 bases. Comparison of the deduced amino acid sequences of all four isoforms of PAD showed about 50% identity with each other, the 3′ regions being highly homologous compared with the 5′ regions.
Enzymatic properties of mouse 25‐hydroxyvitamin D3 1α‐hydroxylase expressed in Escherichia coli
Authors: Sakaki Toshiyuki , Sawada Natsumi , Takeyama Ken‐ichi , Kato Shigeaki , Inouye Kuniyo
Published: March 2003

Renal 25‐hydroxyvitamin D3 1α‐hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially‐expressed enzyme catalyzes the 1α‐hydroxylation of 25‐hydroxyvitamin D3 with a Michaelis constant, Km, value of 2.7µm. Unexpectedly, the enzyme also hydroxylates the 1α‐position of 24,25‐dihydroxyvitamin D3 with a Km of 1.3µm, and a fourfold higher Vmax/Km compared with the 25‐hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25‐dihydroxyvitamin D3 is a better substrate than 25‐hydroxyvitamin D3 for 1α‐hydroxylase. In addition, the enzyme showed 1α‐hydroxylase activity toward 24‐oxo‐25‐hydroxyvitamin D3. However, it showed only slight activity towards 23,25‐dihydroxyvitamin D3 and 24‐oxo‐23,25‐dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27‐tetranor‐23‐hydroxyvitamin D3. These results suggest that the 25‐hydroxyl group of vitamin D3 is essential for the 1α‐hydroxylase activity and the 24‐hydroxyl group enhances the activity, but the 23‐hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1α‐hydroxylated products, suggesting the presence of a redox partner of 1α‐hydroxylase in E. coli cells.
A Kazal‐type trypsin inhibitor from the protochordate Ciona intestinalis
Authors: Ødum Lars , Bundgaard Jens R. , Johnsen Anders H.
Published: March 2003

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion‐exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N‐terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4‐Cys37, Cys8‐Cys30 and Cys16‐Cys51. The structure of the inhibitor is homologous to Kazal‐type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 n
m whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C‐terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C‐terminal sequence is unusual for Kazal‐type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal‐type inhibitors.
Solution structure of the α‐subunit of human chorionic gonadotropin
Authors: Erbel Paul J. A. , Karimi‐Nejad Yasmin , De Beer Tonny , Boelens Rolf , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: March 2003

The three‐dimensional solution structure of the α‐subunit in the α,β heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo‐β‐N‐acetylglucosaminidase‐B (dg‐αhCG), was determined using 2D homonuclear and 2D heteronuclear 1H, 13C NMR spectroscopy at natural abundance in conjunction with the program package xplor. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in αhCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein‐carbohydrate and 3 intraresidual NOEs. The solution structure of dg‐αhCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg‐αhCG exhibits: (a) an increased structural disorder (residues 33–57); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the β‐subunit. Consequently, in free dg‐αhCG, compared to the intact dimer, the two hairpin loops 20–23 and 70–74 are arranged differently with respect to each other. The β‐GlcNAc(78) is tightly associated with the hydrophobic protein‐core in between the β‐hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N‐acetyl protons of β‐GlcNAc(78) to the protein‐core. The hydrophobic protein‐core between the β‐hairpins is thereby shielded from the solvent.
Optimal sequences for non‐phosphate‐directed phoshorylation by protein kinase CK1 (casein kinase‐1) – a re‐evaluation
Authors: Pulgar Victor , Marin Oriano , Meggio Flavio , Allende Catherine C. , Allende Jorge E. , Pinna Lorenzo A.
Published: March 2003

A variety of synthetic peptides derived from either the inhibitor‐2 (I‐2) phosphoacceptor sites or the optimal sequences selected in an oriented peptide library have been compared for their susceptibility to phosphorylation by protein kinase CK1 (also termed casein kinase‐1). The I‐2‐derived peptides are by far preferred over the library peptides by both rat liver CK1 (and by the α/β, γ and δ/ε isoforms immunoprecipitated from it) and recombinant Xenopus laevis CK1α. The superiority of the I‐2‐derived peptides over the library ones is reflected by Vmax values one to two orders of magnitude higher while the Km values are comparable. Individual substitutions of any of the aspartic acids with alanine in the I‐2‐derived peptide RRKHAAIGDDDDAYSITA is detrimental, producing both a fall in Vmax and an increase in Km which are more pronounced at position n –3, but also quite significant at positions n –4, n –5 and, to a lesser extent, n –6. The unfavourable effect of these substitutions is more evident with rat liver CK1 than with recombinant Xenopus laevis CK1α. The chimeric peptide IGDDDDAY‐S‐IIIFFA, resulting from the combination of the N‐terminal acidic sequence of the I‐2 (Ser86) site and the C‐terminal hydrophobic cluster selected in the library peptides (MAEFDTG‐S‐IIIFFAKKK and MAYYDAA‐S‐IIIFFAKKK) is phosphorylated as efficiently as the I‐2‐derived peptide in terms of both Km and Vmax. These combined data strongly support the conclusion that, at variance with the optimal sequences selected in the library, optimal non‐phosphate‐directed phosphorylation of peptide substrates by CK1 critically relies on the presence of a cluster of acidic residues (preferably aspartic acid) upstream from position n –2, while the highly hydrophobic region downstream from serine selected in the library appears to be dispensable. The reason for these discrepancies remains unclear. The possibility that the library data are biased by the invariant elements forming its scaffold (MA‐x‐x‐x‐x‐x‐SI‐x‐x‐x‐x‐AKKK) would be consistent with the observation that the library‐selected peptides, despite their low Km values, fail to compete against the phosphorylation of protein and peptide substrates by CK1, suggesting that they bind to elements partially distinct from those responsible for substrate recognition.
Investigation of bax‐induced release of cytochrome c from yeast mitochondria
Authors: Priault Muriel , Chaudhuri Bhabatosh , Clow Angela , Camougrand Nadine , Manon Stéphen
Published: March 2003

Recent studies that attempt to explore the action of pro‐ and anti‐apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so‐called permeability transition pore. We show in this paper: (a) that bax‐induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer‐membrane voltage‐dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax‐induced release of cytochrome c or in the prevention of this release by bcl‐xL. (c) Bax devoid of its C‐terminal putative hydrophobic α‐helix is as efficient as full‐length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane‐targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria.