bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of immunoreactive octapeptides of human‐cytomegalovirus gp58
Authors: BONCI Allesandra , BRACCI Luisa , CAUDAI Cinzia , LOZZI Luisa , MOSCHETTINI Donatella , NICCOLAI Neri , SCARSELLI Maria , VALENSIN Pier Egisto , NERI Paolo
Published: March 2005

We have mapped continuous epitopes, for positions 591–673 of the human cytomegalovirus 58‐kDa glycoprotein using overlapping synthetic peptides and human sera. This region contains a fragment previously described as including the dominant site for induction of human‐cytomegalovirus antibodies. Since the selected sequence is highly conserved among herpes viruses, we have considered the possible presence of antigenic cross‐reactivity, particularly with the Epstein‐Barr virus. Several peptides in the studied region were antigenic and two main continuous epitopes have been identified. Serological cross‐reactions observed with Epstein‐Barr virus are discussed, focussing on the possible implications of structural features and sequence similarity between human‐cytomegalovirus and Epstein‐Barr‐virus glycoproteins.
Rat brain cytosol contains a factor which reconstitutes guanine‐nucleotide‐binding‐protein‐regulated phospholipase‐D activation in HL60 cells previously permeabilized with streptolysin O
Authors: GENY Blandine , FENSOME Amanda , COCKCROFT Shamshad
Published: March 2005

We report that guanosine 5′‐[γ‐thio]triphosphate (GTP[S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with steptolysin O. The ability of GTP[S] to stimulate PLD is impaired if the cells are previously permeabilized such that the majority of the cytosol has leaked out. Rat brain and HL60 cytosols were both found to restore GTP[S]‐stimulated PLD activity in a reconstitution assay consisting of previously permeabilized HL60 cells. Rat brain cytosol was fractionated on heparin agarose and assayed for reconstitution of GTP[S]‐stimulated PLD activity. The active fractions were pooled, concentrated and chromatographed on gel filtration to assess its molecular mass. The molecular mass of the reconstituting factor was found to be 16 kDa. Reconstitution by the cytosolic factor was dependent on GTP[S]. Ca2+ (pCa 5), MgATP and MgCl2 enhanced GTP[S]‐dependent reconstitution of PLD activity in the previously permeabilized HL60 cells. These results demonstrate the presence in rat brain cytosol of a factor which is an activator of GTP[S]‐stimulated PLD.
cDNA cloning of the sodium‐influx‐stimulating peptide in the mollusc, Lymnaea stagnalis
Authors: SMIT August B. , THIJSEN Steven F. T. , GERAERTS Wijnand P. M.
Published: March 2005

We isolated and characterized a cDNA clone, encoding the prohormone of the sodium‐influxstimulating (SIS) peptide of the freshwater snail Lymnaea stagnalis. The prohormone is cleaved to generate a signal peptide of 23 amino acids and a SIS peptide of 77 amino acids. The SIS peptide as encoded by the cDNA represents a novel and complex neuropetide, which controls the activity of sodium pumps in the integument, pericardium, ureter and nephridial gland. In situ hybridization showed that the SIS‐peptide gene is expressed by the neuroendocrine, so‐called Yellow Cells of the central nervous system.
1H‐NMR study of reduced heme proteins myoglobin and cytochrome P450
Authors: BANCI Lucia , BERTINI Ivano , MARCONI Simonetta , PIERATTELLI Roberta
Published: March 2005

The 1H‐NMR spectra of deoxymyoglobin and reduced cytochrome P450 are analyzed by NOE spectroscopy. Progress has been made in the assignment of the hyperfine‐shifted signals of deoxymyoglobin. The nuclear longitudinal‐relaxation‐time values indicate short electron‐relaxation times whereas Curie relaxation contributes significantly to the signals linewidths. For reduced cytochrome P450 the linewidths are larger due to the Curie‐relaxation contribution in a large protein. Therefore, the spectral information is poor. The electron‐relaxation rates are discussed in terms of possible electronic structure.
Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily
Authors: PARKER David B. , COE Imogen R. , DIXON Gordon H. , SHERWOOD Nancy M.
Published: March 2005

A cDNA that codes for two peptides in the glucagon superfamily has been isolated from sockeye salmon brain. The first peptide is related to growth hormone‐releasing hormone (GHRH), which has high sequence similarity with PACAP‐related peptide. The second peptide is structurally related to vasoactive intestinal peptide, which is also related to a newly identified peptide in mannals, pituitary adenylate‐cyclase‐activating polypeptide (PACAP). The salmon precursor contains 173 amino acids and has dibasic and monobasic enzyme‐processing sites for cleavage of a 45‐amino‐acid GHRH‐like peptide with a free C‐terminus and a 38‐amino‐acid PACAP with an amidated C‐terminus. The salmon GHRH‐like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP‐related peptide. The 38‐amino‐acid salmon PACAP is highly conserved (89–92% identity) with only three or four amino acid substitutions compared with the human, ovine and rat 38‐amino‐acid PACAP.

Not previously reported for mammalian species, a short precursor coding for only one peptide exists in salmon in addition to the long precursor coding for two peptides. In the short precursor, the coding region for GHRH is deleted leaving the PACAP‐coding region in a correct reading frame. This provides one possible control mechanism for an increased expression of one peptide (PACAP) without the concomitant increase in the other peptide (GHRH) as occurs in a double‐peptide precursor. The importance of the 3′non‐translated region of the salmon GHRH/PACAP precursor in the regulation of translation is suggested by its 70% nucleotide sequence identity to the 3′ non‐translated region of the mammalian PACAP precursors. The structural organization of the salmon GHRH/PACAP precursor provides a possible evolutionary scheme for precursors that contain tandem peptides in the glucagon superfamily.
Stereospecificity of the interaction of porcine pancreatic phospholipase A2 with micellar and monomeric inhibitors
Authors: VINCENT Michel , DEVEER Anne‐Mieke , DE HAAS Gerard H. , VERHEIJ Hubertus M. , GALLAY Jacques
Published: March 2005

The binding effect of enantiomeric substrate analogs under micellar form on the local conformation and dynamics of the N‐terminal region of porcine pancreas phospholipase A2 was examined by time‐resolved fluorescence measurements of its single tryptophan residue (Trp3). The complexity of the fluorescence intensity decay of the unliganded protein (four excited‐state lifetime populations) suggests a conformational heterogeneity of the N‐terminal region of the protein. A considerable simplification of the excited‐state lifetime profile was specifically observed in the complex with one of the stereoisomers [(R)‐2‐tetradecanoylamino)‐hexanol‐phosphocholine] at low inhibitor/protein/molar ratio of ∼ 9. This indicates the existence of a definite conformation of the N‐terminal region of the protein in the complex. No effect was detected for the S‐enantiomer. In parallel, the rotational mobility of the Trp residue in the complex with the R‐enantiomer was reduced. At a higher inhibitor/protein molar ratio of ∼ 130, the stereospecificity of the interaction was lost and complexes were formed with both stereoisomers. These complexes were, however, not similar to the specific one either in terms of the local Trp3 environment or of the volume of the rotating unit. The local effects of low amounts of monomeric inhibitors added to a preformed protein/micelle complex of a phospholipase A2 double mutant in which a Trp residue was genetically inserted near the active site at position 31 while the natural Trp3 was replaced by Phe [Kuipers, O., Vincent, M., Brochon, J. C., Verheij, H. M., de Haas, G. H. & Gallay, J. (1991) Biochemistry 30, 8771–8785], were also monitored by time‐resolved fluorescence of this single Trp residue. A stereospecific dependence of the local perturbations was again observed. These results support the idea that the active conformation of the protein is reached in solution only after formation of a ternary complex: protein‐interface‐inhibitor.
Tissue‐dependent variation in the expression of elongation factor‐1α isoforms: Isolation and characterisation of a cDNA encoding a novel variant of human elongation‐factor 1α
Authors: KNUDSEN Sanne M. , FRYDENBERG Jane , CLARK Brian F. C. , LEFFERS Henrik
Published: March 2005

A novel isoform of human elongation factor‐1α (EF‐1α2) has been characterised. It shows a high similarity to other EF‐1α proteins, especially to a rat EF‐1α variant and it has all the characteristics of a functional EF‐1α protein. The pattern of expression of both EF‐1α2 and EF‐1α was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF‐1α2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF‐1α was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.
In vitroformation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B
Authors: KUNKEL Tim , TOMIZAWA Ken‐Ichi , KERN Rainer , FURUYA Masaki , CHUA Nam‐Hai , SCHÄFER Eberhard
Published: March 2005

The light‐stable tobacco phytochrome apoprotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct. The spectral properties of the reconstituted PHYB‐phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies. The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far‐red‐light‐absorbing (Pfr) form and 356 nm and 658 nm for the red‐light‐absorbing (Pr) form. The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB‐phycocyanobilin adduct with the spectral properties of the Pr form. Kinetic analyses of thein vitroreconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first‐order rate constant of 2.8 × 10−2s−1. The similarity with the reported rate constant for the reconstitution of light‐labile phytochrome (PHYA) from oat [Li, L. & Lagarias, J. C. (1992) Phytochrome assembly,J. Biol. Chem. 267, 19 204–19 210] suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB.
A region of the C‐terminal part of the 11‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain
Authors: HEMRIKA Wieger , BERDEN Jan A. , GRIVELL Leslie A.
Published: March 2005

QCR8, the gene encoding the 11‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function. Resequencing shows that the previously published sequence [Maarse A. C. & Grivell L. A. (1987) Eur. J. Biochem 155, 419–425] lacks a C at position 185 of the coding sequence. As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa. Despite the altered C‐terminus, similarity to the corresponding beef heart subunit is not significantly altered.

One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo‐cytochrome b was lowered to 70% of that of the wildtype. Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively.

Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11‐kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired. Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206–217 of the coding sequence, resulting in the replacement of residues 69–73 (WWKNG) by a cysteine.

These results are discussed in terms of the 11‐kDa subunit contributing to the conformation of the Qout binding domain.
Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N‐terminal, head‐binding domain
Authors: DANNER Martina , FUCHS Albert , MILLER Stefan , SECKLER Robert
Published: March 2005

Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N‐terminally shortened protein fragment comprising amino‐acid residues 108–666 [Chen, B.‐L. & King, J. (1991) Biochemistry 30, 6260–6269]. In the present work, the 60‐kDa C‐terminal fragment was purified to homogeneity from the tryptic digest by gel‐fitration chromatography. As is the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS‐resistant, enzymatically active trimer. However, its unfolding in the presence of guanidinium chloride was accelerated at least 10‐fold compared to the complete, native tailspike protein. Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid‐urea‐unfolded fragment polypeptides with neutral buffer. Upon recombinant expression of the 60‐kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS‐resistant trimers. The refolding and assembly pathway of the N‐terminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels. As found for the complete tailspike protein, yields of refolding and assembly of the 60‐kDa fragments into SDS‐resistant trimers decreased with increasing temperature. The refolding yield of fragments derived from a temperature‐sensitive mutant (Gly244 → Arg) tailspike protein was affected in similar fashion as shown for the whole protein. We conclude that the N‐terminal domain (residues 1–107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.
Electrostatic evidence for the activation of the glutathione thiol by Tyr7 in π‐class glutathione transferases
Authors: KARSHIKOFF Andrej , REINEMER Peter , HUBER Robert , LADENSTEIN Rudolf
Published: March 2005

A number of spectrophotometric studies [Graminski, G. F., Kubo, Y. & Armstrong, R. N. (1989) Biochemistry 28, 3562–3568; Liu, S., Zhang, P., Ji, X., Johnson, W. W., Gilliland, G. L. & Armstrong, R. N. (1992) J. Biol. Chem. 267, 4296–4299] have recently shown that the glutathione (GSH) thiol is deprotonated when it is in complex with glutathione S‐transferase. Different models have been proposed for the activation of the glutathione Sy, all pointing out the key role of activesite residue Tyr7. It remains unclear, however, how Tyr7 is actually involved in this process. In this paper we present an analysis of the electrostatic potential in the region of the active site of a π‐class GSH transferase. This analysis provides evidence that the titration behaviour of the absorption band of the E · GSH complex with a pK between 6 and 7 [Liu, S., Zhang, P., Ji, X., Johnson, W. W., Gilliland, G. L. & Armstrong, R. N. (1992) J. Biol. Chem. 267, 4296–4299] should rather be explained by the protonation/deprotonation equilibrium of Tyr7 than by the protonation/deprotonation equilibrium of the GSH thiol group itself. On the basis of this conclusion, a mechanism for activation of GSH is proposed: the Tyr7 OH group is deprotonated by the influence of the protein charge constellation and the peptide dipoles. Thus it acts as a general base, promotes proton abstraction from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity.
Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe‐2S] centers
Authors: ROMÃO Maria J. , BARATA Belarmino A. S. , ARCHER Margarida , LOBECK Karin , MOURA Isabel , CARRONDO Maria A. , LeGALL Jean , LOTTSPEICH Friedrich , HUBER Robert , MOURA José J. G.
Published: March 2005

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe‐2S] centers. The enzyme was characterized by SDS/PAGE, gel‐filtration and analytical ultracentrifugation experiments. It was crystallized at 4°C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3‐nm (3.0‐Å) resolution and belong to space group P6122 or its enantiomorph, with cell dimensions a=b= 14.45 nm and c= 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 × 10−3 nm3/Da (2.5 Å3/Da), consistent with the experimental crystal density, p= 1.14 g/cm3. One dimer (approximately 2 × 100 kDa) is located on a crystallographic twofold axis.
Feedback regulation of mammalian ornithine decarboxylase
Authors: LÖVKVIST Eva , STJERNBORG Louise , PERSSON Lo
Published: March 2005

Ornithine decarboxylase catalyzes the first step in the biosynthesis of polyamines in mammalian cells. The enzyme is subject to various control mechanisms to maintain adequate intracellular levels of polyamines. Polyamines exert a strong feedback control on ornithine decarboxylase. In a recent study [van Daalen Wetters, T., Macrae, M., Brabant, M., Sittler, A. & Coffino, P. (1989) Mol. Cell. Biol. 9, 5484–5490], it was concluded that feedback control of ornithine decarboxylase is mainly, if not exclusively, a posttranslational phenomenon. The existence of a fast‐acting polyamine‐stimulated component of ornithine decarboxylase degradation that acts on newly synthesized monomeric forms of the enzyme was postulated. In the present study we have used a transient expression system to test this hypothesis. The expression of ornithine decarboxylase in mock‐transfected COS cells varied depending on the cellular supply of polyamines as has been found in other mammalian cells. Thus, supplementing the cells with exogenous spermidine resulted in a marked decrease in ornithine decarboxylase activity, whereas depletion of intracellular polyamines, using an ornithine decarboxylase inhibitor, gave a large increase in the cellular content of the enzyme. COS cells expressing an ornithine decarboxylase mRNA devoid of its 5′ non‐translated region did not exhibit any feedback control of the enzyme, neither in the presence of exogenous spermidine nor when the intracellular polyamine levels were depleted to the same extent as in the mock‐transfected COS cells. The results strongly suggest that the feedback control of ornithine decarboxylase is not merely a posttranslational phenomenon.
Identification and analysis of a matrix‐attachment region 5′ of the rat glutamate‐dehydrogenase‐encoding gene
Authors: DAS Atze T. , LUDÉRUS M. E. Eva , LAMERS Wouter H.
Published: March 2005

Eukaryotic chromatin is thought to be organized into independently regulated loop domains by interaction of matrix‐attachment regions (MAR) of the DNA to the nuclear matrix. To define the borders of the chromatin loop containing the glutamate dehydrogenase (GDH) gene, we screened the GDH gene and flanking regions for the presence of MAR sequences. We here report identification, mapping and sequencing of an (A+T)‐rich MAR located 2010–1397 bp upstream of the transcription initiation site of GDH, that mediates strong binding to the nuclear matrix. Smaller regions can also confer binding capacity, although at a lower affinity. This (A+T)‐rich MAR contained 11 bp and 12 bp (A+T)‐rich direct repeats, but not any of the sequences previously described to be associated with MAR activity. We here show that the presence of (A+T)‐rich domains of DNA is not sufficient to confer binding capacity, since (A+T)‐rich sequences located downstream of the identified MAR did not bind to the nuclear matrix. Moreover, a consensus topoisomerase‐II‐binding site located downstream of the MAR was found to be insufficient to mediate substantial binding. The number of binding sites in the nuclear matrix for MAR‐containing fragments was shown to be approximately 15 000/nucleus. Since organization of the entire rat genome in loops with an average loop size of 100 kbp would require 60 000 binding sites, this suggests that only part of the genome is organized in loops. Alternatively, we might have underestimated the number of binding sites. The GDH MAR, and MAR‐containing fragments derived from other species, were found to bind to the same binding sites in the nuclear matrix, although the affinity varied.
Inactivation of interleukin‐6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl‐transferase activities
Authors: LAOUAR Amale , VILLIERS Christian , SANCÉAU Josiane , MAISON Christel , COLOMB Maurice , WIETZERBIN Juana , BAUVOIS Brigitte
Published: March 2005

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N‐terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21‐kDa 125I‐interleukin‐6 (125I‐IL‐6) into 8‐kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cellproliferation bioassay. Degradation of 125I‐IL‐6 by plasma membranes was completely prevented by the serine‐protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by α1‐protease inhibitor and antibody against cathepsin G. A similar incubation of 125I‐IL‐6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8‐kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL‐6 using cell‐associated serine‐protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8‐kDa 125I‐IL‐6 fragments with purified U937 plasma membranes, led to a complete loss of IL‐6 activity related to the transformation of the 8‐kDa forms into a higher‐molecular‐mass complex (16 kDa). This complex was stable in SDS and 2‐mercaptoethanol at 100°C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non‐ester bond between the 8‐kDa 125I‐IL‐6‐derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I‐IL‐6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8‐kDa 125I‐IL‐6 peptide. The kinetics of complex formation (time dependence and plasmamembrane‐concentration dependence), as well as its inhibition by a specific inhibitor of N‐amino‐peptidase activity, bestatin, suggest the participation of peptidyl‐transferase activity in complex formation. Finally, a plasma‐membrane fraction, corresponding to a molecular mass ≥ 30 kDa, was able to convert the 8‐kDa 125I‐IL‐6 forms into the 125I‐labeled 16‐kDa complex, suggesting that a ≥ 30‐kDa peptidyl‐transferase enzyme catalyzes the reaction and provides the 125I‐labeled 16‐kDa peptide by dimerization of 8‐kDa 125I‐IL‐6‐derived intermediates. Further identification of the plasma‐membrane‐associated peptidyl transferase as a regulator of IL‐6 proteolysis may be of physiological relevance for the control of II‐6 biological activity.
Stereochemistry of the reaction catalysed by 2‐aminoethylphosphonate aminotransferase
Authors: LACOSTE Anne‐Marie , DUMORA Catherine , BALAS Laurence , HAMMERSCHMIDT Friedrich , VERCAUTEREN Joseph
Published: March 2005

(R)‐ and (S)‐2‐amino[2‐D1]ethylphosphonic acids ([2‐D1]AEP) were synthesised to investigate the stereochemistry of the reaction catalysed by 2‐aminoethylphosphonate aminotransferase from Pseudomonas aeruginosa. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2‐phosphonoacetaldehyde and alanine. The enzymic reaction proceeding through the abstraction of a proton from the Schiff‐base complex formed between the enzyme‐bound pyridoxal 5′‐phosphate, and the substrate, was carried out in an aqueous buffer at pH 8.5; it was followed by high‐field 1H‐NMR measurements (500 MHz, H2O) on an AMX 500 Bruker spectrometer. The spectra, recorded with chiral (R)‐ or (S)‐[2‐D1]AEP, both showed the methylenic signal (3.0 ppm), whereas (S)‐[2‐D1]AEP gave the additional aldehydic signal (CHO, 9.6 ppm). These data clearly show that AEP‐aminotransferase catalyses the abstraction of the pro‐S hydrogen atom at the prochiral C2 carbon of AEP. Furthermore, careful timing of NMR measurements over a 2‐hour period allows us to show the occurrence of an isotopic effect.
Properties of some nuclear nucleases of rat thymocytes and their changes in radiation‐induced apoptosis
Authors: NIKONOVA Larisa V. , BELETSKY Igor P. , UMANSKY Samuil R.
Published: March 2005

Three nuclease activites have been found and characterized in rat thymocyte nuclear extracts. A Mn2+‐dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCl. The enzyme is activated by Mn2+ but not by Mg2+, Ca2+, or both. Its molecular mass is 36–40 kDa when measured by gel filtration and 37 kDa by SDS/PAGE. An acidic nuclease is independent of divalent ions, produces DNA strand breaks with 5′‐OH ends, its molecular mass is about 37 kDa. Two fractions of Ca2+/Mn2+‐dependent nuclease, differing in binding to CM‐Sepharose but identical in other respects, are active in the presence of Mn2+ but can be additionally activated by Ca2+. They are inactive in the presence of Mg2+ or Ca2+ but cleave DNA in Ca2+/Mg2+‐containing medium. The molecular mass of the enzyme is 22 kDa as determined by both gel filtration and electrophoresis. The dependence of nuclease activities on pH, ions, and sulfhydryl reagents is described. Cycloheximide injection to both control and irradiated animals strongly inhibits the activities of Ca2+/Mn2+‐dependent nuclease from thymocyte nuclei separated by chromatography on CM‐Sepharose and does not change the activities of Mn2+‐dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in Ca2+/Mg2+‐containing and Ca2+/Mn2+‐containing media whereas there is no change in the activity of acidic nuclease. Ca2+/Mn2+‐dependent nuclease is extracted from thymocyte nuclei of irradiated rats with 0.35 M NaCl but from control nuclei only with 0.5 M NaCl. Possible reasons of labilization of Ca2+/Mn2+‐dependent nuclease binding to the nuclear structures in dying thymocytes are discussed.
Ciprofibrate, a carcinogenic peroxisome proliferator, increases the phosphorylation of epidermal‐growth‐factor receptor in isolated rat hepatocytes
Authors: ORELLANA Ariel , HOLUIGUE Loreto , HIDALGO Perla C. , FAÚNDEZ Víctor , GONZÁLEZ Alfonso , BRONFMAN Miguel
Published: March 2005

Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome‐proliferation effects in rat liver, was found to increase the phosphorylation of epidermal‐growth‐factor receptor in 32P‐labeled isolated rat hepatocytes. This effect was suppressed by protein‐kinase‐C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein‐kinase‐C phosphorylation of purified epidermal‐growth‐factor receptor was activated by ciprofibroyl‐CoA, the acyl‐CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal‐growth‐factor‐receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl‐CoA in liver cells would activate protein‐kinase‐C and produce changes in epidermal‐growth‐factor‐receptor function.
Inhibition of viral growth by an α‐oligonucleotide directed to the splice junction of Herpes simplex virus type‐1 immediate‐early pre‐mRNA species 22 and 47
Authors: JACOB Anne , DUVAL‐VALENTIN Guy , INGRAND Didier , THUONG Nguyen T. , HÉLÈNE Claude
Published: March 2005

A 13‐residue α‐anomeric oligonucleotide [α‐5′‐d(GGGCGTCCTCCTT)3′], 5′‐substituted with a psoralen derivative, Pso–α‐13 psoralen linked to the 5′ end of an α‐anomeric n‐residue oligonucleotide, was targeted to the acceptor splice junction of Herpes simplex virus type‐1 immediate‐early pre‐mRNA species 22 and 47. Inhibition of viral growth was observed upon irradiation of Vero cells infected with Herpes simplex virus type‐1 and treatment with Pso–α‐13. The virus titer was decreased by 80% at an oligonucleotide concentration of 0.5 μM and at a multiplicity of infection of 0.1 plaque‐forming units/cell. The 13‐residue oligonucleotide did not induce any cytotoxic effect after irradiation and the inhibition of viral growth was clearly sequence specific. A non‐specific 5′ Pso–α‐15 did not inhibit Herpes simplex virus type‐1 growth. The 5′ Pso–α‐13 targeted to the acceptor splice junction of Herpes simplex virus type 1, contained five mismatches with respect to the corresponding sequence of Herpes simplex virus type 2, and did not exhibit any inhibitory effects on Herpes simplex virus type‐2 growth. These results show that α‐oligonucleotides can exhibit a sequence‐specific antiviral effect and suggest that they may inhibit splicing reactions and be useful in targeting specific nucleic acid sequences within the cell nucleus.
Activation of the cloned platelet thrombin receptor decreases the pertussis‐toxin‐dependent ADP‐ribosylation of the membrane and soluble inhibitory guanine‐nucleotide‐binding‐α proteins
Authors: WILHELM Bernhard , SIESS Wolfgang
Published: March 2005

Thrombin binds at least to two sites of the platelet surface; to the recently cloned thrombin receptor [Vu, T. K., Hung, D. T., Wheaton, V. I. & Coughlin, S. R. (1991) Cell 64, 1057–1068] and to glycoprotein Ib. In the present study, the decrease of pertussis‐toxin‐dependent ADP‐ribosylation of membrane and soluble inhibitory guanine‐nucleotide‐binding α (Giα) proteins was measured after platelet stimulation with a thrombin‐receptor‐activating peptide (TRAP), and compared to stimulation with thrombin. Stimulation of intact platelets with TRAP decreased the pertussis‐toxin‐dependent ADP‐ribosylation of the major membrane 41‐kDa Giα protein and the minor soluble 40 kDa Giα protein recently described in platelets [Gennity, J. M. & Siess, W. (1991) Biochem. J. 279, 643–650]. The kinetics and extent of the decrease of pertussis‐toxin‐dependent ADP‐ribosylation after stimulation of TRAP were similar to the effect of thrombin. The decrease of pertussis‐toxin‐dependent ADP‐ribosylation of the soluble Giα protein was more pronounced and observed at lower agonist concentrations than the decrease of the membrane Giα protein. Desensitization of the thrombin receptor by incubating platelets with a low concentration of TRAP reduced the subsequent decrease of pertussis‐toxin‐dependent ADP‐ribosylation of Giα proteins, evoked by TRAP or thrombin. Platelet stimulation with γ‐thrombin that does not bind to glycoprotein Ib also showed a decrease in the pertussis‐toxin‐dependent ADP‐ribosylation of the soluble and membrane Giα proteins. Treatment of platelets with the stable prostacyclin analog, iloprost, reduced the decrease of pertussis‐toxin‐dependent ADP‐ribosylation of Giα proteins induced by TRAP or thrombin. Among other platelet stimuli tested (endoperoxide/thromboxane analog U44619, collagen, ADP, vasopressin), only U44619 decreased the pertussis‐toxin‐dependent ADP‐ribosylation of the soluble and membrane Giα proteins to a degree comparable to TRAP.

It is concluded that the thrombin‐induced activation of both the membrane and soluble Giα proteins in platelets occurs via stimulation of the recently cloned thrombin receptor and is independent of the binding of thrombin to glycoprotein Ib. Furthermore, the coupling thrombin receptor/Gi protein is reduced by intracellular cAMP.
Zinc coordination in mammalian sorbitol dehydrogenase
Authors: KARLSSON Christina , HÖÖG Jan‐Olov
Published: March 2005

Rat sorbitol dehydrogenase was expressed in Escherichia coli and purified to homogeneity, resulting in a protein with a specific activity of 4.7 U/mg, close to that of the enzyme isolated from mammalian liver.

A Glu residue has been postulated to replace the Cys of alcohol dehydrogenase as a ligand to the active‐site zinc atom of sorbitol dehydrogenase. This Glu (position 155 in the rat enzyme) was mutated both to Cys, in order to mimic the alcohol dehydrogenase relationships, and to Ala, as a control. A third mutation, Cys164 to Ala, was also performed since Cys has also been considered as a possible zinc ligand. With Ala at position 155, an inactive enzyme was obtained, showing that correct active‐site relationships have been destroyed. With Cys at position 155, the enzyme is still partly active, but rapidly looses activity unless stabilized by the addition of ZnSO4. The catalytic efficiency in the oxidation of sorbitol is 120‐fold less than that of the native form, and reduction of fructose is lost completely. In contrast, the activity of the Cys164Ala mutant is comparable with that of the native enzyme and, in fact, even increased in the oxidation of sorbitol. Combined, the results strongly suggest that Glu155 is a ligand to the active‐site zinc atom. Zinc analysis of the different variants of sorbitol dehydrogenase establishes that all contain one atom of zinc/subunit, also when the catalytic function is lost. Apparently, zinc remains coordinated even after replacement with an amino acid residue (Ala) unable to ligand metal atoms.
The major‐histocompatibility‐complex‐encoded β‐type proteasome subunits LMP2 and LMP7
Authors: FRENTZEL Stefan , KUHN‐HARTMANN Inge , GERNOLD Martina , GÖTT Peter , SEELIG Angela , KLOETZEL Peter‐M.
Published: March 2005

The proteasome (high‐molecular‐mass multicatalytic proteinase complex) is composed of a large number of non‐identical protein subunits of the α and β types. The mouse β‐type subunits LMP2 and LMP7 (LMP, low‐molecular‐mass protein) are encoded within the mouse major histocompatibility complex (MHC II) region, and are thought to connect the proteasome to the MHC class‐I antigen‐processing pathway. In the present communication, we have analysed the two proteasome subunits with regard to their identity within the proteasome complex, their protein levels, their amounts of mRNA in different mouse tissues and cell lines, and have investigated the intracellular localization of LMP2 and LMP7 subunits in thymus and liver by immunocytology. Our experiments indicate that LMP2 and LMP7 subunits are synthesized as precursor proteins of 24 kDa and 30 kDa, respectively, and that only the processed 21‐kDa and 23‐kDa subunits are part of the 20S proteasome complex. The proportion of LMP2‐subunit‐containing and LMP7‐subunit‐containing proteasome complexes, as well as LMP2 and LMP7 mRNA levels, vary strongly and are shown to be dependent on the tissues or cell lines analysed. Furthermore, high LMP2 and LMP7 mRNA levels do not always correlate with high protein levels, suggesting a specific translational mechanism which controls proteasome subunit synthesis. Generally, mRNA levels appear to be particularly high in those tissues which are known to be involved in MHC class‐I antigen presentation. Immunocytological analysis shows a strong nuclear localization of the subunits in cells of the thymus, while in the liver they appear to be evenly distributed between the two cellular compartments. Our data support the idea that both LMP2 and LMP7 proteins are non‐essential proteasome subunits which are probably involved in the regulation of proteasome activities. The function of the two subunits, however, may not be restricted to the proposed role of proteasomes in antigen presentation.
The solution structure of the histidine‐containing protein (HPr) from Staphylococcus aureus as determined by two‐dimensional 1H‐NMR spectroscopy
Authors: KALBITZER Hans Robert , HENGSTENBERG Wolfgang
Published: March 2005

The three‐dimensional solution structure of the heat‐stable phosphocarrier protein HPr from Staphylococcus aureus was determined from two‐dimensional NMR data by restrained molecular dynamics. It consists of a large twisted antiparallel β‐pleated sheet with four strands A, B, C, and D of amino acids 2–7, 34–37, 40–42 and 60–65. Three right‐handed helices A, B, C (amino acids 18–27, 47–53 and 71–85) are positioned on top of this sheet. The aromatic ring of His15 is located in a cleft formed by amino acids 12–17 and 55–58, only the nitrogen (Nδ1) atom which can be phosphorylated by enzyme I is exposed to the water. The side chains of Thr12 and Arg17 are located close to the histidine ring. The regulatory serine residue (Ser46) is located in a hydrophobic patch, its hydroxyl group is water‐accessible but forms hydrogen bonds with the amide groups of the backbone. The general features of the three‐dimensional structure are similar to those found in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis and Streptococcus faecalis.
Comparative study of high‐mannose‐type oligosaccharides in membrane glycoproteins of rat hepatocytes and different rat hepatoma cell lines
Authors: NUCK Rolf , PAUL Christine , WIELAND Barbara , HEIDRICH Claudia , GEILEN Christoph C. , REUTTER Werner
Published: March 2005

A comparative study was undertaken to characterize the oligosaccharides released by endo‐β‐N‐acetylglucosaminidase H (endo H) from the membrane glycoproteins of rat hepatocytes and three different Morris hepatoma cell lines (NA‐MH 7777, HTC and MH1C1). It is shown that the membrane glycoproteins of hepatocytes and hepatoma cells contain markedly different quantities and forms of high‐mannose‐type carbohydrate chains.

After radiolabelling of the cells with d‐[2‐3H]mannose, in the absence and presence of 1 mM 1,5‐dideoxy‐1,5‐imino‐d‐mannitol (1‐deoxymannojirimycin), high‐mannose‐type oligosaccharides were released from delipidated membrane glycoproteins by enzymic digestion with endo H. The carbohydrate chains were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride, then further analysed by HPLC using an APS‐2 Hypersil column.

In the absence of 1‐deoxymannojirimycin, up to 10% of the radiolabelled oligosaccharides were released by endo H‐treatment of the membrane glycoprotein fraction from rat hepatocytes. In contrast, the quantity of radiolabelled high‐mannose‐type carbohydrate chains released by endo H‐treatment from tumour‐cell membrane glycoproteins of hepatoma cell lines NA‐MH 7777 (31,5%), MH1C1‐MH 7795 (37,2%) and HTC‐MH 7288c (48%) was increased up to fivefold. The formation of higher‐mannosylated structures after oligosaccharide analysis was observed in all hepatoma cell lines, with Man8GlcNAcOH as the major component, whereas in hepatocytes Man5GlcNAcOH was the predominant high‐mannose‐type structure.

In contrast, in the presence of the Golgi α‐d‐mannosidase I inhibitor, 1‐deoxymannojirimycin, no significant differences were observed between the distribution of high‐mannose‐type oligosaccharides in the membrane glycoproteins of hepatocytes and hepatoma cells. However, in the presence of this inhibitor, the proportion of radiolabelled glycans sensitive to deglycosylation by endo H was greatly increased (>85%) in all the cell lines investigated, the predominant structures being Man8–9−GlcNAcOH. This study shows that an increased content of high‐mannose‐type sugar chains is a general characteristic of membrane‐bound glycoproteins for malignant transformed hepatocytes.
Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate
Authors: SEVCIK Jozef , ZEGERS Ingrid , WYNS Lode , DAUTER Zbigniew , WILSON Keith S.
Published: March 2005

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exoguanosine 2′, 3′‐cyclophosphorothioate has been refined against 0.2‐nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2′‐GMP complex. The refinement was based on all data over 1.0–0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2′, 3′‐cyclophosphorothioate, which is a thio analogue of the intermediate of the two‐step reaction. However, exo guanosine 2′, 3′‐cyclophosphorothioate is bound in a non‐functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.
Cytochrome c6 from Monoraphidium braunii
Authors: CAMPOS António P. , AGUIAR António P. , HERVÁS Manuel , REGALLA Manuela , NAVARRO José A. , ORTEGA José M. , XAVIER António V. , DE LA ROSA Miguel A. , TEIXEIRA Miguel
Published: March 2005

A soluble monoheme c‐type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class‐I c‐type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H‐NMR, ultraviolet/visible, EPR and Mössbauer spectroscopies. The pH equilibria are complex, several pKa values and pH‐dependent forms being observed. The amino acid sequence, the reduction‐potential value and the visible and NMR spectroscopies data in the pH range 4–9 indicate that the heme iron has a methionine‐histidine axial coordination. However, the EPR and Mössbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz= 3.27, gy= 2.05 and gx= 1.05; form II, gz= 2.95, gy= 2.29 and gx= 1.43. While form I has crystal‐field parameters typical of a methionine‐histidine coordination, those associated with form II would suggest a histidine‐histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine‐histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c‐type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233–236], corroborating our proposal. Since both forms, with very distinct crystal‐field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme‐iron ligand field cannot be directly correlated with the heme‐reduction potential.

The pH‐dependence studies have also shown that the form I and form II are interconvertible, with pKa∼ 5. To establish a possible physiological significance for this process in particular for the interaction of the cytochrome with the membrane‐bound electron‐transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.
Two adjacent E box elements and a M‐CAT box are involved in the muscle‐specific regulation of the rat acetylcholine receptor β subunit gene
Authors: BERBERICH Christof , DÜRR Ingolf , KOENEN Michael , WITZEMANN Veit
Published: March 2005

We have isolated and analysed the 5′ flanking region of the rat acetylcholine receptor (AChR) β subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA‐box‐less promoter region containing an initiator motif. An 85‐bp fragment has been shown to promote high muscle‐specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle‐specific promoter element carrying a M‐CAT box that is flanked at the 5′ end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M‐CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle‐specific and developmental expression of the rat AChR β subunit gene requires both regulatory elements, the M‐CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR β subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.
Proteins of the Golgi apparatus
Authors: BENDIAK Brad , WARD Larry D. , SIMPSON Richard J.
Published: March 2005

The Golgi marker enzyme, UDP‐galactose: N‐acetylglucosamine β1‐4galactosyltransferase (β1‐4GalT) was purified 44300‐fold in its intact, membrane‐bound form from rat liver membranes. The protein was isolated from detergent extracts as a high‐Mr form, having a Stokes radius approximating a globular protein of Mr 440000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an Mr near 51000, and does not have intermolecular disulfide crosslinks. N‐terminal sequencing of the enzyme demonstrated that it contains an N‐terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5′ end of the mRNA [Russo, R. N, Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324–3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N‐terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low‐Mr forms which were fully catalytically active and which, upon sequencing, were missing a 66‐amino‐acid stretch from the N‐terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with Mr near 50000. The N‐terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non‐protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.
Purification and characterization of an NADH–rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas
Authors: CHEN Liang , LIU Ming‐Yih , LEGALL Jean , FARELEIRA Paula , SANTOS Helena , XAVIER António V.
Published: March 2005

An NADH–rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109–115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid‐point redox potentials of –295 mV and –325mV, this FMN‐ and FAD‐containing protein can induce the specific reduction of D. gigas rubredoxin. In contrast, rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half‐inhibited by 4.2μM p‐chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.