bluuebunny/crossref_metadata_2025 huggingface dataset:
Branched Synthetic Constructs that Mimic the Physico‐Chemical Properties of Apolipoprotein AI in Reconstituted High‐Density Lipoproteins
Authors: Demoor Ludovic , Boutillon Christophe , Fievet Catherine , Vanloo Berlinda , Baert Johan , Rosseneu Maryvonne , Fruchart Jean‐Charles , Tartar André
Published: July 2004

Amphipathic helical repeats are considered as the structural units of numerous apolipoproteins and have been described as being responsible for the interaction of apolipoproteins with phospholipids in high‐density lipoproteins (HDL). Furthermore, apolipoproteins, and especially apolipoprotein AI (apoAI), are involved in various biological functions of these circulating particles in plasma. Studies with synthetic peptides corresponding to domains of the apoAI sequence have however shown that short 39‐residue fragments do not interact strongly enough with phospholipids to generate particles that correctly mimic the physico‐chemical properties of HDL reconstituted with native apoAI [Vanloo, B., Demoor, L., Boutillon, C., Lins, L., Baert, J., Fruchart, J. C., Tartar, A. & Rosseneu, M. (1995) Association of synthetic peptide fragments of human apolipoprotein A‐I with phospholipids, J. Lipid Res. 36, 1686–1696.]. Here we show that synthetic branched multimeric peptides, often used as carriers for the design of synthetic vaccines (multiple‐antigen peptides), can be used to mimic the physicochemical properties of apoAI in HDL. This type of molecule is obtained by using a small core matrix of Lys residues bearing radially branched synthetic peptides as dendritic arms. We compared the lipid‐binding capacities and the structural properties of a linear peptide corresponding to residues 145–183 of apoAI [apoAI‐(145–183)‐peptide] with those of two multimeric peptides consisting respectively of three [trimeric apoAI‐(145–183)] and four copies [tetrameric apoAI‐(145–183)] of the selected sequence, branched on a covalent core matrix. This paper provides evidence for the increased abilities of the multimeric peptides to associate with phospholipids compared with the short linear peptides. Moreover, the trimeric apoAI‐(145–183) peptide was most efficient in mimicking the physico‐chemical and structural properties of native apoAI in reconstituted HDL. As tools adequate to unravel the structure/function relationship of separate apolipoprotein domains are still missing, these multimeric peptides might constitute an alternative approach to linear peptides which are poor mimetics and to protein mutants which are difficult to produce and only provide information about the total sequence.
Evidence for Propeptide‐Assisted Folding of the Calcium‐Dependent Protease of the Cyanobacterium Anabaena
Authors: Baier Kerstin , Nicklisch Sabine , Lockau Wolfgang
Published: July 2004

The Ca2+‐dependent protease of the cyanobacterium Anabaena variabilis is a cytoplasmic enzyme with a substrate specificity like trypsin. Its previously published DNA sequence [Maldener, I., Lockau, W., Cai, Y. & Wolk, C. P. (1991)Mol. Gen. Genet. 225, 113–120] contained a sequencing error. Here we report the corrected sequence which shows, that the Ca2+‐protease belongs to the family of subtilases (subtilisin‐like serine proteases). Consistent with its cytoplasmic localization, a pre‐sequence is not found.

The enzyme is produced as a precursor with a large amino‐terminal propeptide. Expression of the pro‐region and mature region (protease domain) in Escherichia coli cells in trans demonstrates that formation of the active enzyme requires the propeptide. The results demonstrate that propeptide‐assisted protein folding also occurs with cytoplasmic enzymes, in support of the hypothesis that this mechanism is a widespread phenomenon.
Phosphorylation and Dephosphorylation in the Proline‐Rich C‐Terminal Domain of Microtubule‐Associated Protein 2
Authors: Sánchez Carlos , Tompa Peter , Szücs Kornélia , Friedrich Peter , Avila Jesús
Published: July 2004

The C‐terminal domain of microtubule‐associated protein 2 (MAP2) contains a proline‐rich region and the tubulin‐binding domain. We have generated antibodies to follow the phosphorylation state of the proline‐rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline‐rich region of MAP2 and is phosphorylated in vitro by at least three different proline‐directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen‐synthase kinase 3) α/β The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C‐terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high‐molecular‐mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein‐phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.
Ole e 3, An Olive‐Tree Allergen, Belongs to a Widespread Family of Pollen Proteins
Authors: Batanero Eva , Villalba Mayte , Ledesma Amalia , Puente Xosé S. , Rodríguez Rosalía
Published: July 2004

An allergen has been isolated from a saline extract of olive tree (Olea europaea) pollen. The protein consists of a single polypeptide chain of 9.2‐kDa, as determined by mass spectrometry. It contains neither tryptophan nor tyrosine residues, and displays an acidic isoelectric point. The secondary structure of the protein, estimated from the analysis of the circular‐dichroism spectrum in the peptide‐bond region, is composed of 52% a‐helix, 10%β‐strand, 29%βturn and 9% non‐regular conformation. The N‐terminal end of the protein is blocked. Amino‐acid‐sequence data have been obtained from peptides produced by CNBr treatment of the native allergen. A partial sequence of 36 amino acids has thus been elucidated. The protein exhibits sequence similarity with pollen allergens from Brassica species and contains a Ca2+‐binding motif. The isolated protein displays IgE‐binding activity against sera of patients allergic to olive‐tree pollen. It has been named Ole e 3, according to the recommendations of the IUIS Nomenclature Committee. IgG ELISA inhibition assays with polyclonal antibodies specific for Ole e 3 reveal the presence of proteins similar to Ole e 3 in the pollen from non‐related plant species, which may explain allergic cross‐reactivity processes.
The Primary Structure and Characterization of Carbohydrate Chains of the Extracellular Glycoprotein Proteinase Inhibitor from Latex of Carica Papaya
Authors: Odani Shoji , Yokokawa Yayoi , Takeda Hiroshi , Abe Sachiko , Odani Sumiko
Published: July 2004

A secretory proteinase inhibitor was isolated from the latex of green fruits of papaya (Carica papaya). The protein exhibited stoichiometric inhibition of bovine trypsin and α‐chymotrypsin by the same site or overlapping binding sites. The complete covalent structure consisting of 184 amino acids and two disulfide bonds was determined by protein analysis. During the structural analysis, a procedure was established to separate very hydrophilic peptides by reverse‐phase HPLC. The result revealed that the latex protein belongs to an extensively diverse plant protein family that includes inhibitors of serine, cysteine and aspartic proteases, a taste‐modifying protein, wound responsive proteins, storage proteins, amylase inhibitors and even an oxidoreductase. In this superfamily, the latex proteinase inhibitor is most similar to the curious protein, miraculin, which makes sour food taste sweet.

Two carbohydrate chains, each probably composed of (mannose)5, (xylose)1, (fucose)0‐2, and (N ‐acetylglucosamine)2 residues, were attached to asparagine 84 and 90. Mass‐spectrometric and compositional analysis suggested that they may represent a new class of plant xylose‐containing carbohydrate chains with five mannose residues.
Pfmrk, A MO15‐Related Protein Kinase from Plasmodium falciparum
Authors: Li Ji‐Liang , Robson Kathryn J. H. , Chen Ji‐Li , Targett Geoffrey A. T. , Baker David A.
Published: July 2004

Cyclin‐dependent kinases (Cdks) play a central role in the regulation of the eukaryotic cell cycle. A novel gene encoding a Cdk‐like protein, Pfmrk, has been isolated from the human malaria parasite Plasmodium falciparum. The gene has no introns and comprises an open reading frame encoding a protein of 324 amino acids with a predicted molecular mass of 38 kDa. Database searches revealed a striking similarity to the Cdk subfamily with the highest similarity to human MO15 (Cdk7). The overall sequence of Pfmrk shares 62% similarity and 46% identity with human MO15, in comparison to the 49–58% similarity and 34–43% identity with other human Cdks. Pfmrk contains two unique inserts: one consisting of 5 amino acids just before the cyclin‐binding motif and the other composed of 13 amino acids within the T‐loop equivalent region. Southern blots of genomic DNA digests and chromosomal separations showed that Pfmrk is a single‐copy gene conserved between several parasite strains and is located on chromosome 10. A 2500‐nucleotide transcript of this gene is expressed predominantly in the sexual blood stages (gametocytes), suggesting that Pfmrk may be involved in sexual stage development.
An Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases
Authors: Mazauric Marie‐Hélène , Reinbolt Joseph , Lorber Bernard , Ebel Christine , Keith Gérard , Giegé Richard , Kern Daniel
Published: July 2004

Glycyl‐tRNA synthetase (Gly‐tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five‐step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D. T., Mazauric, M.‐H., Kern, D. & Moras, D. (1995)EMBO J. 14, 4156–4167]. Molecular‐mass determinations of the native and denatured protein indicate an oligomeric structure of the α2 type consistent with that found for eukaryotic Gly‐tRNA synthetases (yeast and Bombyx mori), but different from that of Gly‐tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are α2β2 tetramers. N‐terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both α and β chains of the prokaryotic enzymes (E. coli, Haemophilus influenzue and Coxiella burnetii) albeit the enzyme is deprived of the N‐terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly‐tRNA synthetase combines strong structural homologies of eukaryotic Gly‐tRNA synthetases with a feature of prokaryotic synthetases.

Heat‐stability measurements show that this synthetase keeps its ATP‐PPi exchange and aminoacylation activities up to 70°C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNAGly does not induce protection.

Cross‐aminoacylations reveal that the thermophilic Gly‐tRNA synthetase charges heterologous E. coli tRNAGly(GCC) and tRNAGly(CCC) and yeast tRNAGly(GCC) as efficiently as T. thermophilus tRNAGly. All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly‐tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity.

The results are discussed in the context of the three‐dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.
Anoxia‐Induced Gene Expression in Turtle Heart
Authors: Cai Q. , Storey Kenneth B.
Published: July 2004

A cDNA library constructed from heart of anoxia‐exposed adult turtles (Trachemys scripta elegans) was differentially screened with 32P‐labeled single‐stranded cDNA probes from heart of control versus anoxic animals to clone genes induced by anoxia stress. Four cDNA clones, pBTaR20, pBTaR34, pBTaR63 and pBTaR914 were obtained and confirmed to be upregulated in response to anoxic submergence (20 h in N2‐bubbled water at 7°C). Two clones, pBTaR20 and pBTaR63, were characterized by sequence analysis and in vivo expression. The clone pBTaR20 had a 1597‐bp cDNA sequence and pBTaR63 contained a 1837‐bp sequence. The pBTaR20 sequence contained a single open reading frame that was very close to full length and could potentially encode a polypeptide with 508 amino acids. The deduced polypeptide sequence showed approximately 83% of the residues identical with the sequence of cytochrome c oxidase subunit 1 (COI) that is encoded by a mtDNA gene Coxl. The clone pBTaR63 contained a single potentially full‐length open reading frame that could encode a polypeptide of 591 residues. This was similar to another mitochondrial protein, NADH‐ubiquinone oxidoreductase subunit 5 (NDS), which is encoded by mtDNA gene Nad5 Analysis of the time course of expression of Coxl and Nad5 by northern hybridization analysis showed that mRNA transcripts for both accumulated rapidly (within 1 h) in response to anoxia exposure. Both showed similar increases in their transcript content after 1 h of anoxia but with longer anoxia exposures (5 or 20 h) Nad5 mRNA levels remained high whereas Coxl mRNA content declined somewhat. Northern‐blot hybridization also revealed differential expression of these two genes in five other organs of T: s. elegans during anoxia exposure (brain, kidney, liver, red and white skeletal muscle), with a particularly large increase in mRNA transcript levels of both genes in anoxic red muscle. Organ‐specific analysis of these genes in a freeze‐tolerant turtle species (Chrysemys picta marginata) also showed that differential expression of these genes occurred in response to the ischemia induced by plasma freezing.
Arabidopsis Formaldehyde Dehydrogenase
Authors: Martínez M. Carmen , Achkor Hakima , Persson Bengt , Fernández M. Rosario , Shafqat Jawed , Farrés Jaume , Jörnvall Hans , Parés Xavier
Published: July 2004

A glutathione‐dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) has been characterized from Arabidopsis thulium. This plant enzyme exhibits kinetic and molecular properties in common with the class III forms from mammals, with a Km, for S‐hydroxymethylglutathione of 1.4 μM, an anodic electrophoretic mobility (PI: 5.3–5.6) and a cross‐reaction with anti‐(rat class III alcohol dehydrogenase) antibodies. The enzyme structure, deduced from the cDNA sequence, fits into the complex system of alcohol dehydrogenases and shows that all life forms share the class III protein type. The corresponding mRNA is 1.4 kb and present in all plant organs; a single copy of the gene is found in the genome. The class III structural variability is different from that of the ethanol‐active enzyme types in both vertebrates (class I) and plants (class P), although class P conserves more of the class III properties than class I does. Also the enzymatic properties differ between the two ethanol‐active classes. Active‐site variability and exchanges at essential residues (Leu/Gly57, Asp/Arg115) may explain the distinct kinetics. These patterns are consistent with two different metabolic roles for the ethanol‐active enzymes, a more constant function, reduction of acetaldehyde during hypoxia, for class P, and a more variable function, the detoxication of alcohols and participation in metabolic conversions, for class I. A sequence motif, Pro‐Xaa‐Ile/Val‐Xaa‐Gly‐His‐Glu‐Xaa‐Xaa‐Gly, common to all medium‐chain alcohol dehydrogenases is defined.
Origin of the Two Carbonyl Oxygens of Bacteriochlorophyll a
Authors: Porra Robert J. , Schäfer Wolfram , Gad'on Nasr , Katheder Ingrid , Drews Gerhart , Scheer Hugo
Published: July 2004

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10–20‐fold. This adaptation was performed in the presence of either H218O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3‐acetyl and 131‐oxo oxygen atoms: both were derived from water.

The derivation of the 131‐oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13‐propionic acid methylester side chain of Mg2+‐protoporphyrin IX mono‐methylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 131‐oxo group is derived from molecular oxygen via an oxygenase.

In contrast to adapting R. sphaeroides cells, the 131‐oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3‐acetyl group was not labelled by 18O2. Thus, while the 131‐oxo group has different origins in R. sphaeroides and R. denitrificans, the 3‐acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.
Bacterial Triterpenoids of the Hopane Series from the Prochlorophyte Prochlorothrix Hollandica and their Intracellular Localization
Authors: Simonin Pascale , Jürgens Uwe J. , Rohmer Michel
Published: July 2004

35‐O‐β–Galacturonopyranosyl‐, 35‐O‐β– 3,5‐anhydrogalacturonopyranosyl‐ and 35‐O‐α‐altruronopyranosylbacteriohopanetetrol accompanied by their 2β‐methyl homologues have been isolated from Prochlorothrix hollandica. We report here C35 triterpenoids of the hopane series in a prochlorophyte, a group of prokaryotic oxigenic phototrophs of the cyanobacterial lineage. Like many cyanobacteria, P. hollandica contains a mixture of non‐methylated as well as 2D‐methylhopanoids. After side‐chain cleavage by periodic acid oxidation followed by sodium borohydride reduction, these hopanoids could be localized in cell walls and thylakoids, in accordance with their role as membrane stabilizers.
Arachidonic Acid Up‐Regulates and Prostaglandin E2 Down‐Regulates the Expression of Pancreatic‐Type Phospholipase A2 and Prostaglandin‐Endoperoxide Synthase 2 in Uterine Stromal Cells
Authors: Prigent‐Tessier Anne , Pageaux Jean‐François , Fayard Jean‐Michel , Lagarde Michel , Laugier Christian , Cohen Hélène
Published: July 2004

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two‐series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic‐type phospholipase A2(PLA2 I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line U111 which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1 which is expressed at a high level in U111 cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up‐regulated by increasing arachidonate concentrations (10–10 μM). The maximal response was obtained at 24 h, reaching a 2.3‐fold and 2.6‐fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate‐induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose‐dependent manner, the maximal response being reached at 1 μM. We conclude that arachidonic acid up‐regulates the expression of PLA2I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down‐regulating enzyme expression.
The Overall Partitioning of Anthracyclines into Phosphatidyl‐Containing Model Membranes Depends Neither on the Drug Charge Nor the Presence of Anionic Phospholipids
Authors: Gallois Laurence , Fiallo Marina , Laigle Alain , Priebe Waldemar , Garnier‐Suillerot Arlette
Published: July 2004

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P‐glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P‐glycoprotein‐mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point.

The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions.

Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles.

The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3′‐hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13‐hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol.

We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.
The Presence of Rotenone‐Sensitive NADH Dehydrogenase in the Long Slender Bloodstream and the Procyclic Forms of Trypanosoma Brucei Brucei
Authors: Beattie Diana S. , Howton Marilyn M.
Published: July 2004

The mitochondrial electron‐transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80–90% by antimycin A or cyanide, 15–19% by salicylhydroxamic acid, and 30–35% by rotenone. Cytochrom‐c–reductase activity, with proline or glycerol 3‐phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome‐c–reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone‐sensitive NADH dehydrogenase (complex 1) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3‐phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone‐sensitive NADH‐dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.
Striking Differences Between the Kinetics of Regulation of Respiration by ADP in Slow‐Twitch and Fast‐Twitch Muscles In Vivo
Authors: Kuznetsov Andrei V. , Tiivel Toomas , Sikk Peeter , Kaambre Tuuli , Kay Laurence , Daneshrad Zoya , Rossi André , Kadaja Lumme , Peet Nadya , Seppet Enn , Saks Valdur A.
Published: July 2004

The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow‐twitch skeletal muscle (soleus) and fast‐twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin‐skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. The results of these experiments confirm the observation of Veksler et al. [Veksler, V. I., Kuznetsov, A. V., Anflous, K., Mateo, P., van Deursen, J., Wieringa, B. & Ventura‐Clapier, R. (1995)J. Biol. Chem. 270, 19921–19929], who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent Km for respiration regulation was 300–400 μM and decreased to 50–80 μM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10–20 μM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 μg/ml) for 15 min decreased the apparent Km for ADP in cardiac and soleus muscle fibres to 40–98 μM without significant alteration of Vmax or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent Km, for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 μM enzyme. These results are taken to show that in cardiac and slow‐twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial‐ligand conduction. This protein factor is not expressed in fast‐twitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow‐twitch muscles.
Selenium‐Dependent Peroxidases Suppress 5‐Lipoxygenase Activity in B‐Lymphocytes and Immature Myeloid Cells
Authors: Werz Oliver , Steinhilber Dieter
Published: July 2004

Differentiation of HL‐60 cells by dimethylsulfoxide induces 5‐lipoxygenase protein expression, but only low cellular 5‐lipoxygenase activity. Similarly, B‐lymphocytes express 5‐lipoxygenase protein and show activity in cell homogenates but not in intact cells. Here, we demonstrate that suppression of cellular 5‐lipoxygenase activity in these cell lines is serum dependent and that the serum effect can be mimicked by selenium. Selenium‐dependent inhibition of 5‐lipoxygenase activity was also observed in the corresponding cell homogenates or 100000×g supernatants when dithiothreitol or glutathione (GSH) was added. The properties of the endogenous selenium‐dependent inhibitor, i.e. molecular mass, utilization of GSH and dithiothreitol as substrates, sensitivity to iodacetate, inhibition of 5‐lipoxygenase activity in the presence of the GPx‐1 inhibitor mercaptosuccinate, suggest that a selenoenzyme with properties of the phospholipid hydroperoxide glutathione peroxidase (GPx‐4) is responsible for the 5‐lipoxygenase inhibition in BL41‐E95‐A and immature HL‐60 cells.

Differentiation of HL‐60 cells in the presence of 1,25‐dihydroxyvitamin D3 and transforming growth factor‐β(TGFβ) upregulated cellular 5‐lipoxygenase activity regardless of whether the cells were grown with or without serum or selenium. Also, 5‐lipoxygenase activity in homogenates or 100000×g supernatants of 1,25‐dihydroxyvitamin D3/TGFβ differentiated HL‐60 cells and of human granulocytes was not inhibited by dithiothreitol or GSH. Thus, after 1,25‐dihydroxyvitamin D3/TGFβ differentiation, HL‐60 cells resemble normal granulocytes with respect to the high 5‐lipoxygenase activity in intact cells and to the dithiothreitol effects in broken cell preparations. Combination experiments with 100000×g supernatants of BL41‐E95‐A cells and neutrophils revealed that the high 5‐lipoxygenase activity of granulocytes is due to stability of the 5‐lipoxygenase catalytic activity against selenium‐dependent peroxidases, but not to low peroxidase activity. Our data suggest that the capability of mature myeloid cells to release large amounts of leukotrienes after stimulation is due to a peroxidase‐insensitive 5‐lipoxygenase catalytic activity.
Isoforms of Myosin in Growing Muscles of Ky (kyphoscoliotic) Mice
Authors: Maréchal Georges , Beckers‐Bleukx Godelieve , Berquin Anne , Coulton Gary
Published: July 2004

Kyphoscoliotic (ky) mice are spontaneous mutants of the BDL strain whose postural muscles atrophy during post‐natal growth, resulting in extensive kyphoscoliosis in adult animals. At 21 days of age, the seven muscles examined were already well differentiated into fast, slow and mixed type on the basis of the proportions of their native myosin isoforms or their subunits. During post‐natal growth, from 21 to 120 days of age, the normal pattern of myosin maturation was essentially respected by the ky mutation: fast muscles became faster, slow muscles became slower and mixed muscles specialized in both directions. However, the post‐natal increases of myosin heavy chain 2B and fast myosin light chain LC3f were depressed in ky muscles, whilst there was novel expression of slow myosin light chains, LC1s and LC2s, in muscles which normally did not express them. Intermediate native myosin IM was absent in adult ky soleus, but it increased in adult ky tibialis anterior. We conclude that the ky mutation depresses the normal post‐natal transition towards faster muscles and results in adult muscles whose myosin isoforms are generally shifted in a fast‐to‐slow direction.
Contribution of Individual Tryptophan Residues to the Structure and Activity of θ‐Toxin (Perfringolysin O), A Cholesterol‐Binding Cytolysin
Authors: Sekino‐Suzuki Naoko , Nakamura Megumi , Mitsui Ken‐ichiro , Ohno‐Iwashita Yoshiko
Published: July 2004

θ‐Toxin (perfringolysin O), secreted by Clostridium perfringens, shares with other known thiol‐activated toxins a conserved undecapeptide, ECTGLAWEWWR, located in the C‐terminal region of the protein and containing the unique cysteine of the molecule. Single and double amino acid substitutions were created in the θ‐toxin molecule to investigate the role of individual tryptophan residues in the lytic activity of θ‐toxin. Wild‐type and mutant θ‐toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified. The relative hemolytic activities of four mutant toxins, each with a Trp to Phe substitution outside the common Cys‐containing region, were more than 60% that of wild‐type θ‐toxin. In contrast, mutant toxins with Phe replacements within the Cys‐containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte‐membrane‐binding activities. The largest reduction in binding affinity, more than 100‐fold, was observed for Trp438 mutant toxins. However, the mutants retain binding specificity for cholesterol and the ability to form arc‐shaped and ring‐shaped structures on membranes. These results indicate that the low hemolytic activities of these mutant toxins can be ascribed, at least in part, to reduced binding activities. With respect to protease susceptibility and far‐ultraviolet circular‐dichroism spectra, only the W436→F mutant toxin, showed any considerable difference from wild‐type toxin in secondary or higher‐order structures, indicating that Trp436 is essential for maintenance of toxin structure.
Strained Template Under the Thumbs
Authors: Hermann Thomas , Heumann Hermann
Published: July 2004

In retroviruses, such as human immunodeficiency virus type 1 (HIV‐1), the reverse transcriptase (RT) copies single‐stranded viral RNA into complementary DNA, which is then used as a template for synthesis of the second DNA strand. The resulting double‐stranded DNA is integrated into the host genome. How RT translocates on the different templates is the subject of this study. We have developed a theoretical model for RT translocation during processive DNA synthesis. The model is based on the assumption that there are two template‐binding sites, namely the helix clamps, located in the thumb subdomains of RT subunits p66 and p51. Flexibility of the p66 thumb provides undisrupted template‐binding during polymerase translocation. Coordinated association and dissociation of the template at the thumbs, triggered by nucleotide incorporation, is assumed, which ensures template contact with at least one subdomain throughout translocation. We suggest that coordination between the sites is effected by stress in the template region located between the thumbs. Translocation of HIV‐1 RT proceeds continuously but with different processivities on RNA and DNA templates. These findings are explained in detail by the proposed model.
Analysis of the Solution Structure of the Homeodomain of Rat Thyroid Transcription Factor 1 by 1H‐NMR Spectroscopy and Restrained Molecular Mechanics
Authors: Esposito Gennaro , Fogolari Federico , Damante Giuseppe , Formisano Silvestro , Tell Gianluca , Leonardi Antonio , Di Lauro Roberto , Viglino Paolo
Published: July 2004

The solution structure of the rat thyroid transcription factor 1 (TTF‐1) homeodomain has been elucidated by 1H‐NMR and restrained modeling. The TTF‐1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix II and helix III. The typical assembly of the hydrophobic core is maintained and N‐capping motifs are identified in helix I and helix II. The N‐terminal stretch of helix II exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N‐terminal decapeptide and the C‐terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C‐terminal extension of helix III, which is either disordered or helically folded. In the TTF‐1 homeodomain, the C‐terminal extension of helix III (residues 51–59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOEs and hydrogen‐deuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix III, which is introduced by a tightening or a kink of residues 51–53.
Inhibition by Gangliosides of Bacillus cereus Phospholipase C Activity Against Monolayers, Micelles and Bilayer Vesicles
Authors: Daniele José J. , Maggio Bruno , Bianco Ismael D. , Goñi Félix M. , Alonso Alicia , Fidelio Gerardo D.
Published: July 2004

The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.
Hepatic 5‐Aminolevulinic Acid Synthase mRNA Stability is Modulated by Inhibitors of Heme Biosynthesis and by Metalloporphyrins
Authors: Cable Edward E. , Gildemeister Otto S. , Pepe Joyce A. , Donohue Susan E. , Lambrecht Richard W. , Bonkovsky Herbert L.
Published: July 2004

Hepatic 5‐aminolevulinic acid synthase, the first and normally rate‐controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5‐aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5‐aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5‐aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6‐dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5‐aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 μM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5‐aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 μM) was due to inhibition of heme oxygenase, rather than a direct, heme‐like, effect of zinc mesoporphyrin on 5‐aminolevulinic acid synthase mRNA; (d) Among the several non‐heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5‐aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA.
Studies on Protein Processing for Membrane‐Bound Spinach Leaf Mitochondrial Processing Peptidase Integrated into the Cytochrome bc1, Complex and the Soluble Rat Liver Matrix Mitochondrial Processing Peptidase
Authors: Sjöling Sara , Waltner Mary , Kalousek Frantisek , Glaser Elzbieta , Weiner Henry
Published: July 2004

The plant mitochondrial processing peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane‐bound enzyme that constitutes an integral part of the bc1 complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc1/MPP with rat liver MPP using synthetic signal peptides and different mitochondrial precursor proteins. Inhibition studies of processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker‐deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plumbaginifolia F1β also showed a similar inhibitory pattern with rat MPP as with spinach MPP processing. In‐vitro synthesised precursors of plant N. plumbaginifolia F1β and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of processing was higher with the homologous precursor. Linker‐deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in processing by either MPP. These results show that similar higher‐order structural elements upstream from the cleavage site are important for processing by both the membrane‐bound plant and the soluble mammalian MPP.
Dietary Modulation of the mRNA Stability of Trypsin Isozymes and the Two Forms of Secretory Trypsin Inhibitor in the Rat Pancreas
Authors: Carreira Suzanne , Fueri Christian , Chaix Jean‐Claude , Puigserver Antoine
Published: July 2004

The stability of the mRNAs encoding pancreatic trypsin isozymes, namely the cationic form and the two anionic forms I and II, as well as that of the secretory trypsin inhibitors I and II, were studied in rats fed on either a high‐protein diet, or a protein‐free diet compared with a standard diet for a 10‐day period. Either immediately or 3 h and 6 h after injecting the transcription inhibitor, actinomycin D, the mRNA levels were quantified by performing dot‐blot hybridization with specific oligonucleotide probes. Under high‐protein dietary conditions, the stability of the mRNAs coding for anionic trypsin II and cationic trypsin showed no change, whereas that of anionic trypsin I and the two forms of secretory trypsin inhibitor were affected. The mRNA half‐life of anionic trypsin I and trypsin inhibitor II increased, in sharp contrast with that of trypsin inhibitor I, which decreased. When rats were fed on a protein‐free diet, the stabilities of both anionic trypsin forms and trypsin inhibitor I increased, whereas that of trypsin inhibitor II decreased and that of cationic trypsin remained unchanged. The present results show the existence of differences in the mechanisms whereby gene expression of trypsin isozymes and secretory trypsin inhibitors is regulated, although they are synthesized in parallel in the pancreatic acinar cell and stored in zymogen granules before being secreted into the intestinal lumen.
Amino Acid Sequence of the Maize Ferredoxin: Thioredoxin Reductase Variable Subunit
Authors: Iwadate Hiromoto , Tsugita Akira , Chow Lu‐Ping , Kizuki Kazuyuki , Stritt‐Etter Anne‐Lise , Li Junsheng , Schürmann Peter
Published: July 2004

The ferredoxin: thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe‐4S] cluster and a redox‐active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N‐terminus, one in the middle of the subunit and one near the C‐terminus. We have obtained some evidence indicating that the N‐terminal consensus domain is possibly involved in the interaction with the catalytic subunit.
Evaluation of the Secondary Structure of Vaccinia‐Virus Thymidine Kinase by Circular‐Dichroism Spectroscopy of Overlapping Synthetic Peptides
Authors: Behrends Henrik W. , Beck‐Sickinger Annette G. , Folkers Gerd
Published: July 2004

A new approach is reported that includes multiple‐peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia‐virus thymidine kinase (TK). We divided the sequence of the vaccinia‐virus TK into 82 peptides of IS residues that overlapped by 13 residues and covered the complete sequence of vaccinia‐virus TK. All peptides were synthesized by solid‐phase multiple‐peptide synthesis by means of the Fmoc/tert ‐butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary‐structure evaluation led to determination of a vaccinia‐virus‐TK secondary‐structure pattern.

Consecutive peptides with α‐helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of α‐helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear β‐sheet conformation, or CD spectra of mixed secondary‐structure content were observed for β‐sheets. Therefore, the exact localization of β‐sheet‐containing residues was deduced by comparison with isofunctional sequence‐dissimilar proteins. We identified seven α‐helices and six β‐sheet‐containing regions, which we used for a secondary‐structure model of the vacciniavirus TK protein.
Characterization and Ion Channel Activities of Novel Antibacterial Proteins from the Skin Mucosa of Carp (Cyprinus carpio)
Authors: Lemaître Christelle , Orange Nicole , Saglio Philippe , Saint Nathalie , Gagnon Jean , Molle Gérard
Published: July 2004

A detergent‐solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non‐ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31‐kDa and 27‐kDa proteins. In contrast to the 27‐kDa protein, which was glycosylated, the 31‐kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27‐kDa protein and 500 pS for the 31‐kDa protein, and selectivity measurements gave PCl/PK ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018–0.18 μM) against different strains of gram‐negative and gram‐positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31‐kDa protein was protected on its N‐terminal extremity in contrast to the 27‐kDa protein, which had a 19‐amino‐acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.