bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Saccharomyces cerevisiae ARG7 Gene Encoding Ornithine Acetyltransferase, An Enzyme also Endowed with Acetylglutamate Synthase Activity
Authors: Crabeel Marjolaine , Abadjieva Agnes , Hilven Pierre , Desimpelaere Joke , Soetens Oriane
Published: September 2004

We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2‐encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine‐leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine.

Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y.‐S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291–296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non‐cleavable‐type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence.

Yeast Arg7p shares between 32–43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl‐CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing the overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p‐linked acetylglutamate synthase activity was measurable in an assay. The yeast enzyme is thus clearly, albeit modestly, bifunctional.

As with several bacterial ornithine acetyltransferases, the activity of Arg7p was practically insensitive to arginine but strongly inhibited by ornithine, which behaved as a competitive inhibitor.
Modulation of Platelet‐Activating‐Factor Production by Incorporation of Naturally Occurring 1‐O‐Alkylglycerols in Phospholipids of Human Leukemic Monocyte‐Like THP‐1 Cells
Authors: Hichami Aziz , Duroudier Valérie , Leblais Véronique , Vernhet Laurent , Le Goffic François , Ninio Ewa , Legrand Alain
Published: September 2004

1‐O‐Alkylglycerols (alkyl‐Gro), naturally occurring compounds abundant in shark liver oil, protect patients from radiotherapy side‐effects. However, the protection mechanism is not well understood. It might be mediated by alkyl‐Gro incorporation into pools of platelet‐activating factor (PAF) precursor and subsequent modification of PAF biosynthesis. Using a 3H‐labelled or unlabelled natural alkyl‐Gro mixture, in which prominent alkyl chains were C18:1(9) (54–65%), C16:1(7) (5–15.5%), and C16:0 (5–10%), we investigated the incorporation of alkyl‐Gro into phospholipids of human leukemic monocyte‐like THP‐1 cells. Incubation of cells for 24 h with [3H]alkyl‐Gro (10 μM) resulted in their incorporation into 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine (1097 ± 25.1 pmol/2×106 cells) and into 1‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine (640.4 + 12.5 pmol/2×106 cells) with a total yield of 6.5%. Such incorporation induced production of 1‐O‐[3H]alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine ([3H]PAF), which was increased after stimulation by the calcium ionophore A23187. HPLC analysis of the [3H]PAF molecular species indicated that the three major [3H]alkyl‐Gro were used for [3H]PAF synthesis in ratios similar to that of the mixture. Total production of biologically active PAF, as measured by the platelet‐aggregation bioassay, was also increased by alkyl‐Gro incorporation in resting (+20%) and in A23187‐stimulated (+59%) THP‐1 cells. HPLC analysis of the [3H]PAF produced in the presence of [3H]acetate, confirmed that levels of PAF, but not of its 1‐acyl analog, were increased by alkyl‐Gro incorporation in resting and stimulated cells. However, the rise in [3H]acetyl‐PAF, which resulted mainly from C16:0 PAF, was reduced by about 50% in the presence of the PAF‐receptor antagonist SR 27417, providing evidence that stimulation of total PAF synthesis was caused by the increase in the precursor pool and autocrine amplification of PAF‐induced PAF production. Thus, the supplementation of THP‐1 cells in culture with naturally occurring alkyl‐Gro led to the incorporation of alkyl‐Gro into ether‐containing phospholipids, which were subsequently used for PAF synthesis. Furthermore, alkyl‐Gro incorporation resulted in a significant rise in PAF production by THP‐1 cells under resting and stimulated conditions. These results may be of importance for modulating PAF production in several pathophysiological conditions, such as peroxysome deficiencies, that are associated with a lack of ether lipid synthesis.
Structure, Catalysis and Supramolecular Assembly of Adenylate Kinase from Maize
Authors: Wild Klemens , Grafmüller Rita , Wagner Edgar , Schulz Georg E.
Published: September 2004

The crystal structure of adenylate kinase from maize ligated with an inhibitor has been determined by molecular replacement and refined to 3.5‐Å resolution. The enzyme keeps the ATP/ADP/AMP equilibrium in the cell. In the C4 plant maize, it has the special task to recycle the AMP produced in large amounts in primary CO2 assimilation. The established structure explains the side reaction with CMP. Moreover, it shows infinite rods that can be readily discerned in the crystal packing. In comparison with homologues, two structural differences that are crucial for this supramolecular assembly are evident. We propose that the rods represent a natural inactive storage form that assembles at night when maize stops CO2 assimilation and thus most of the AMP production in its C4 cycle. The enzyme is particularly abundant in mesophyll chloroplasts, where such an assembly would release appreciable amounts of water that can be used in other processes during the night.
Cytochrome ba3 from Natronobacterium pharaonis
Authors: Mattar Stephan , Engelhard Martin
Published: September 2004

Cytochrome ba3, a terminal oxidase was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis. NH2‐terminal sequence information of two subunits with apparent molecular masses of 40 and 36 kDa was used to generate a DNA probe by polymerase chain reaction. Cloning and sequencing of two overlapping genomic fragments revealed four genes forming a transcriptional unit. The policystronic messenger RNA of this cbaDBAC gene locus was identified by RNA analysis. The genes cbaC and cbaD code for small hydrophobic peptides with 81 and 54 amino acids. The genes cbaB and cbaA code for cytochrome oxidase subunit II (calculated molecular mass = 18.6 kDa) and I (calculated molecular mass = 63.8 kDa) respectively. Five potential CuA ligands for subunit II and six His residues for subunit I located in conserved positions indicate cytochrome ba3 to be a c‐type oxidase. Sequence comparison and phylogenetic analysis place the natronobacterial enzyme together with the archaeal quinol oxidase Sox‐ABCD from Sulfolobus acidocaldarius and the eubacterial ba3‐type oxidase from Thermus thermophilus into a distinct evolutionary group. All three members are missing residues which are functionally important for vectorial proton translocation. The four‐subunit enzyme complex was also identified on the protein level using chromatographic buffers containing ethylene glycol for purification.
Transcriptional Regulation of the Rat Poly(ADP‐ribose) Polymerase Gene by Sp1
Authors: Bergeron Marie‐Josée , Leclerc Steeve , Laniel Marc‐André , Poirier Guy G. , Guérin Sylvain L.
Published: September 2004

Expression of the gene encoding poly(ADP‐ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US‐1 and F1–F4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site‐directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions ‐101 and ‐60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T‐lymphoblastoid and Ltk− fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site‐directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis‐acting elements by transcription factors other than Sp1.
Human Amyloid Precursor‐Like Protein 1
Authors: Paliga Krzysztof , Peraus Gisela , Kreger Sylvia , Dürrwang Ulrike , Hesse Lars , Multhaup Gerd , Masters Colin L. , Beyreuther Konrad , Weidemann Andreas
Published: September 2004

Amyloid precursor‐like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell‐free system displays an apparent molecular mass of ≈80 kDa in SDS‐containing gels and becomes N‐glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS‐7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N‐ and O‐linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy‐terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1‐specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.
Functional Properties of the Quinol Oxidase from Acidianus ambivalens and the Possible Catalytic Role of its Electron Donor
Authors: Giuffrè Alessandro , Gomes Cláudio M. , Antonini Giovanni , D'itri Emilio , Teixeira Miguel , Brunori Maurizio
Published: September 2004

The aa3 quinol oxidase has been purified from the thermoacidophilic archaea Acidianus ambivalens as a three‐redox‐centers enzyme. The functional properties of this oxidase both as purified and in its most integral form (i.e. in native membranes and in intact cells) were investigated by stopped‐flow spectrophotometry. The results suggest that the enzyme interacts in vivo with a redox‐active molecule, which favours the electron entry via heme a and provides the fourth electron demanded for catalysis.

We observe that the purified enzyme has two hemes with apparent redox potentials 215 ± 20 mV and 415 ± 20 mV at pH 5.4, showing redox‐Bohr effect, and a heme a3‐CuB center with an affinity for carbon monoxide (Ka= 5.7 × 104 M−1 at 35°C) much lower than that reported for the mammalian enzyme (Ka= 4 × 106 M−1 at 20deg;C). The reduction by dithionite is fast and monophasic when the quinol oxidase is in the native membranes, whereas it is slow and biphasic in the purified enzyme (with heme a3 being reduced faster than heme a). The oxygen reaction of the reduced purified enzyme is fast (few milliseconds), but yields an intermediate (likely ferryl) clearly different from the fully oxidized enzyme. In contrast, the same reaction performed in intact cells leads to the fully oxidized enzyme.

We postulate that caldariella quinol, the physiological electron donor, is in vivo tightly bound to the enzyme, providing the fourth redox active center lacking in the purified enzyme.
Characterization of a Nuclear Localization Signal of Canine Parvovirus Capsid Proteins
Authors: Vihinen‐Ranta Maija , Kakkola Laura , Kalela Anne , Vilja Pekka , Vuento Matti
Published: September 2004

We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N‐terminal residues 4–13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor‐mediated process). Transport into the nucleus was an energy‐dependent and temperature‐dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.
Dof DNA‐Binding Domains of Plant Transcription Factors Contribute to Multiple Protein‐Protein Interactions
Authors: Yanagisawa Shuichi
Published: September 2004

Dof proteins are a family of plant transcription factors that have a strongly conserved DNA‐binding domain, designated the Dof domain. This domain has the potential to form a single zinc finger. This report describes the self‐association of a maize Dof protein, Dof1 (previously designated MNB1a). Affinity chromatography revealed that Dof1 also interacted with another maize Dof protein, Dof2, as well as with high‐mobility‐group (HMG) protein 1. Results of mapping of the region required for the protein‐protein interactions of Dof1 suggested that these interactions may be mediated by the Dof domain. When gel mobility shift assays were performed with purified recombinant Dof proteins, homomeric and heteromeric complexes of Dof proteins on DNA were detected. It seems possible that formation of complexes of different Dof proteins through direct protein‐protein interactions might be involved in the regulation of transcription. Evidence is also presented that HMG1 has an effect on the binding of Dof1 to DNA. Therefore, it appears that the Dof domain is a multifunctional domain that is involved not merely in binding to DNA but also in multiple protein‐protein interactions.
Identification of Stat 5B as a Substrate of the Insulin Receptor
Authors: Sawka‐Verhelle Dominique , Filloux Chantal , Tartare‐Deckert Sophie , Mothe Isabelle , Van Obberghen Emmanuel
Published: September 2004

We have screened a human placenta library using the yeast two‐hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin‐receptor Kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960‐motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen‐activated protein Kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells.

Taken together, our finding suggest that Stat 5B corresponds to a substrate for the insulin‐receptor Kinase, and this widens the repertoire of insulin‐signaling pathways.
Examining F‐Actin Interaction with Intact Talin and Talin Head and Tail Fragment using Static and Dynamic Light Scattering
Authors: Goldmann Wolfgalng H. , Guttenberg Zeno , Kaufmann Stefan , Hess Daniel , Ezzell Robert M. , Isenberg Gerhard
Published: September 2004

We examined the binding kinetics of intact talin and talin head and tail fragment with F‐actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F‐actin cross‐linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530–537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross‐linking/bundling events with intact talin and talin and talin tail fragment. No binding of the talin head fragment with F‐actin was detected by either method.
Interaction of F‐Actin with Synthetic Peptides Spanning the Loop Region of Human Cardiac β‐Myosin Heavy Chain Containing Arg403
Authors: Bartegi Aghleb , Roustan Claude , Chavanieu Alain , Kassab Ridha , Fattoum Abdellatif
Published: September 2004

The atomic model of the F‐actin–myosin subfragment 1 complex (acto‐S‐1) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50‐kDa subdomain of myosin subfragment 1 (S‐1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the β‐heavy chain sequence, a residue whose mutation into Gln is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F‐actin with this particular S‐1 loop region, we have synthesized four 17‐residue peptides corresponding to the sequence Gly398–Gly414 of the human β‐cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross‐linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F‐actin, at a nearly physiological ionic strength, with almost identical affinities (Kd≈40 μM). The binding strength of the F‐actin‐GG17 peptide complex was increased fivefold (Kd= 8 μM) in the presence of subsaturating concentrations of added skeletal S‐1 relative to actin, without apparent competition between the peptide and S‐1. Each of the three actin‐binding peptides inhibited the steady‐state actin‐activated MgATPase of skeletal S–1 by specifically decreasing about twofold the Vmax of the reaction without changing the actin affinity for the S‐1–ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F‐actin that uncouples its binding to S‐1 from ATP hydrolysis, probably by interfering with the proper attachment of the S‐1 loop segment to the interdomain connection of actin.
Organization of the Proximal Promoter of the Hatching‐Enzyme Gene, the Earliest Zygotic Gene Expressed in the Sea Urchin Embryo
Authors: Ghiglione Christian , Emily‐Fenouil Francoise , Lhomond Guy , Gache Christian
Published: September 2004

The hatching enzyme (HE) gene is the earliest zygotic gene expressed in the sea urchin embryo. To investigate the regulation of the HE gene activity, 5· flanking DNA and the 5· untranslated leader were inserted upstream of reporter genes whose expression was monitored in vivo during development after transfer into eggs. By deletion analysis we showed that no more than 3 kb of flanking sequence are required for correct expression of transgenes. The proximal region of 0.5 kb does not precisely control spatial restriction but drives expression at a nearly maximal level. The proximal promoter was searched extensively for sites of protein‐DNA interactions by DNAse protection and gel‐shift methods. The 12 sites identified form 3 groups: core promoter; central region; and distal region. The central region bears three sites that contain a direct or inverted CCAAT box. Mutation and deletion analysis showed that, in addition to the core‐promoter elements, the two most‐distal CCAAT‐containing sites are indispensable for promoter activity. These sites bind the same set of proteins, which are abundant in the nuclei of cleavage embryos.
Molybdate‐Uptake Genes and Molybdopterin‐Biosynthesis Genes on a Bacterial Plasmid
Authors: Menéndez Cástor , Otto Annegret , Igloi Gabor , Nick Peter , Brandsch Rainer , Schubach Berthold , Böttcher Bettina , Brandsch Roderich
Published: September 2004

A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high‐affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin‐dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo‐dependent and the MoCo‐biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44 500 Da, associated into high‐molecular‐mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP · pmol protein−1, min−1). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C‐terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.
Structure of an Acidic Exopolysaccharide of Burkholderia pseudomallei
Authors: Nimtz Manfred , Wray Victor , Domke Tobias , Brenneke Birgit , Häussler Susanne , Steinmetz Ivo
Published: September 2004

A recently described water‐soluble exopolysaccharide of Burkholderia pseudomallei recognized by the IgG 1 monoclonal antibody 3015 [Steinmetz, I., Rohde, M. & Brenneke, B. (1995) Infect. Immun. 63, 3959–3965] was isolated by repetitive ethanol‐precipitation steps and by anion‐exchange chromatography. The structure of the polysaccharide was determined by a combination of chemical‐derivatization and mass‐spectrometric techniques (compositional and methylation analysis, GC/MS, and electrospray‐ionization‐MS/MS of reduced and permethylated hydrolytic fragments), and two‐dimensional 1H‐NMR methods (COSY, TOCSY and NOESY) and confirmed by isolation and structural characterization of the depolymerized repeating unit of the polysaccharide. The combined structural data established a linear tetrasaccharide repeating unit consisting of three galactose residues, one bearing a 2‐linked O‐acetyl group, and a 3‐deoxy‐D‐manno‐2‐octulosonic acid residue.
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Structural Studies of the Putative O‐Specific Polysaccharide of Acinetobacter baumannii O24 Containing 5,7‐Diamino‐3,5,7,9‐Tetradeoxy‐L‐Glycerol‐D‐Galacto‐Nonulosonic Acid
Authors: Haseley Simon R. , Wilkinson Stephen G.
Published: September 2004

A polysaccharide containing D‐GlcN, 2‐amino‐2,6‐dideoxy‐L‐galactose (L‐FucN), and 7‐acetamido‐5‐acylamino‐3,5,7,9‐tetradeoxy‐L‐glycerol‐D‐galacto‐nonulosonic acid (LegAX), in which the acyl group (X) is either S‐3‐hydroxybutyryl (50%) or acetyl (50%), was isolated by mild acid hydrolysis treatment, followed by gel‐permeation chromatography, of the water‐soluble lipopolysaccharide from Acinetobacter baumannii serogroup O24. The polysaccharide, characterised by means of monosaccharide analyses, partial acid hydrolysis, methylation analysis and NMR studies, was shown to have a linear tetrasaccharide repeating unit, as depicted below. Serological tests indicated that the polymer corresponded to the O24 antigen.
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Surface Charge Control of Electropermeabilization and Glycophorin Electroinsertion with 1,2‐Diacyl‐sn‐Grycero‐3‐Phosphocholine (lecithin) Liposomes
Authors: Raffy Sophie , Teissié Justin
Published: September 2004

Back insertion of a solubilized membrane protein, glycophorin A, has been obtained in lipid multilamellar vesicles by applying calibrated electric field pulses on a lipid/protein mixture. Experimental evidence for insertion is given by means of immunofluorescence. Insertion was obtained only under field conditions that induced the leakage of a soluble hydrophilic molecule, calcein, which was trapped between the lipid layers. Studies were performed on mixed liposomes where charged species were present. The critical permeabilizing field is the same whatever the composition, but with overcritical fields the associated calcein transmembraneous flow is higher with positively charged lipids. Field conditions that where prone to trigger glycophorin insertion were similar to those that induced electropermeabilization. No electroinsertion has been obtained with stearylamine (SteNH2)/1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (Pam2GroPCho) liposomes under the same conditions. Calcein efflux as well as glycophorin insertion are controlled by the electric surface charge of the host liposome. These observations confirm our previous conclusions that spontaneous membrane protein insertion is obtained when the host membrane is brought to its electropermeabilized state, but show that a strong control due to the surface charges is nevertheless present.
Thermomechanical design in the automotive industry
Authors: THOMAS J. J. , VERGER L. , BIGNONNET A. , CHARKALUK E.
Published: September 2004

In this paper thermomechanical fatigue assessment in the automotive industry is discussed. The design strategy is based upon a consistent approach of the thermomechanical loading, the mechanical constitutive law of the material, the damage parameters and the fatigue strength criteria. The good understanding of these different steps allows one to perform predictive calculations of automotive parts subjected to thermomechanical loading. The main hypotheses and modelling choices are presented and results are illustrated by a series of computations on real 3D structures. Cracked area and lifetime prediction are described in the case of aluminium alloy cylinder heads subjected to transient thermal loadings.
A simple model for the prediction of the fatigue delamination growth of impacted composite panels
Authors: KATERELOS D. G. , PAIPETIS A. , KOSTOPOULOS V.
Published: September 2004

The fatigue behaviour of composite panels that have been subjected to low‐velocity impact was studied. Impacted specimens were tested under compression–compression fatigue. A delamination propagation model based on the derivation of the strain energy release rate was used. The stress distribution around the initially induced delamination was derived analytically. The shape of the delamination was experimentally monitored by c‐scan imaging and is assumed to be an ellipse. The orientation and aspect ratio of the ellipse were used to calculate the corresponding strain energy‐release rates, which were subsequently used to predict the direction of delamination propagation.
A local approach to cleavage fracture in ferritic steels following warm pre‐stressing
Authors: HADIDI‐MOUD S. , MIRZAEE‐SISAN A. , TRUMAN C. E. , SMITH D. J.
Published: September 2004

Quantification of the enhancement in cleavage fracture toughness of ferritic steels following warm pre‐stressing has received great interest in light of its significance in the integrity assessment of such structures as pressure vessels. A Beremin type probability distribution model, i.e., a local stress‐based approach to cleavage fracture, has been developed and used for estimating cleavage fracture following prior loading (or warm pre‐stressing, WPS) in two ferritic steels with different geometry configurations. Firstly, the Weibull parameters required to match the experimental scatter in lower shelf toughness of the candidate steels are identified. These parameters are then used in two‐ and three‐dimensional finite element simulations of prior loading on the upper shelf followed by unloading and cooling to lower shelf temperatures (WPS) to determine the probability of failure. Using both isotropic hardening and kinematic hardening material models, the effect of hardening response on the predictions obtained from the suggested approach has been examined. The predictions are consistent with experimental scatter in toughness following WPS and provide a means of determining the importance of the crack tip residual stresses. We demonstrate that for our steels the crack tip residual stress is the pivotal feature in improving the fracture toughness following WPS. Predictions are compared with the available experimental data. The paper finally discusses the results in the context of the non‐uniqueness of the Weibull parameters and investigates the sensitivity of predictions to the Weibull exponent, m, and the relevance of m to the stress triaxiality factor as suggested in the literature.
A new model for describing a stable cyclic stress–strain relationship under non‐proportional loading based on activation state of slip systems
Authors: ITOH T. , KAMEOKA M. , OBATAYA Y.
Published: September 2004

This paper proposes a new simple model for cyclic incremental plasticity based on activation states of slip systems describing stable cyclic stress–strain relationships under non‐proportional loading. In the model, the magnitude and the direction of incremental plastic strain are estimated by (1+αfNP) and Q, respectively. Here, α is the constant related to the dependence of material on additional hardening and fNP the intensity factor expressing the severity of non‐proportional loading. Q is the second‐order tensor describing the activation states of slip systems in polycrystalline metals and is given by the calculation using a virtual specimen. The model was examined by application to the prediction of the stable cyclic stress–strain relationship in extensive non‐proportional low cycle fatigue tests for type 304 stainless steel and 6061 aluminium alloy. The simulated results showed that the model gave a satisfactory prediction of the stable cyclic stress–strain relationship under complex non‐proportional multiaxial loadings for the two materials.
The use of multiaxial fatigue models to predict fretting fatigue life of components subjected to different contact stress fields
Authors: ARAÚJO J. A. , NOWELL D. , VIVACQUA R. C.
Published: September 2004

This work describes the application of multiaxial fatigue criteria based on critical plane and mesoscopic (Dang Van, 1973, Sciences et Techniques de lÁrmement,47, 647—722) approaches to predict the fatigue initiation life of fretted components. To validate the analysis, several tests under closely controlled laboratory conditions are carried out in a Ti‐6Al‐4V alloy. These classical Hertzian tests reveal a size effect where fretting fatigue lives vary with contact size. Experimentally available data for fretting fatigue of an Al‐4Cu alloy are also used to assess the models. Neither the critical plane models nor the mesoscopic criterion considered can account for the effects of different contact stress fields on the initiation life, if the calculation is based only on highly stressed points on the surface. It is shown, however, that satisfactory results can be achieved if high values of the fatigue parameters are sustained over a critical volume.
Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida
Authors: Duque Estrella , García Vanina , De La Torre Jesús , Godoy Patricia , Bernal Patricia , Ramos Juan‐Luis
Published: September 2004

The cyoABCDE gene cluster of Pseudomonas putida DOT‐T1E encodes a terminal cytochrome oxidase. A 500‐bp ‘cyoB’ DNA fragment was cloned in pCHESIΩKm and used to generate a cyoB knock‐out mutant in vivo. The mutant strain was not limited in the generation of proton‐motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild‐type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild‐type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent‐induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.
An Empirical Analysis of Productivity in Philippine Agriculture, 1974–2000
Authors: Teruel Romeo G. , Kuroda Yoshimi
Published: September 2004

Using recent agricultural data sets, we estimate a non‐neutral and non‐homothetic translog variable cost function to empirically investigate Philippine agricultural productivity. Overall, the annual average productivity growth rate is 0.51 percent. The highest productivity level is observed during the post‐Green Revolution period (1974–1980). Thereafter, productivity growth shows a discernible decline up until the late 1990s. This indicates that productivity level during the Green Revolution era has not been sustained or paralleled, despite substantial policy changes put in place since 1986 to invigorate the agricultural sector. The government should therefore continue to promote technological progress in Philippine agriculture; however, this may require redirection of its present policy by placing more emphasis on productivity‐enhancing investments, such as research and development, irrigation and other infrastructure.
Increasing Student Learning through Volitional Control
Authors: Mccann Erin J. , Turner Jeannine E.
Published: September 2004

Students’ engagement and persistence on academic learning tasks often require their use of volitional strategies to protect against distractions and facilitate task completion. In this article we discusses factors that both positively and negatively influence students’ ability to regulate their learning activities and the supporting role that volitional strategies play in helping students to develop positive work habits for tackling academic assignments. Using the Academic Volitional Strategy Inventory (AVSI), we present teachers with approaches for helping students who need to support their learning efforts. Included are ways to use the AVSI (1) as a diagnostic tool, (2) to incorporate instruction in and modeling of volitional strategies in regular curriculum assignments, and (3) to promote student reflection and peer group interaction in facilitating self-regulatory action.
Promoting Effective Task Interpretation as an Important Work Habit: A Key to Successful Teaching and Learning
Authors: Butler Deborah L. , Cartier Sylvie C.
Published: September 2004

In this article we argue that to be successful in an academic arena, students must adopt a consistent approach to completing academic work (i.e., a work habit) that includes very carefully interpreting the demands of tasks that are presented to them in schools. To clarify why task interpretation is so critical to student success, and is thus an important instructional objective for teachers, we begin by presenting two vignettes illustrating links between task interpretation and students’ successful engagement in academic work. Then, we take a step back to describe what we mean by academic work and engagement and to explain how and why students’ knowledge about, conceptions of, and interpretations of tasks are so foundational to performance. We also describe how students’ task interpretation breaks down and why such breakdowns often occur. Finally, we close by advancing practical suggestions for teachers regarding how to structure activities, instruction, and evaluation to promote students’ adoption of task interpretation as an important work habit in the pursuit of effective learning.

Sally is teaching a ninth grade English class. Her goal is for students to learn how to write various kinds of paragraphs. After showing some examples and talking with the class about the structure of a narrative paragraph, Sally asks the students to write one themselves. She writes the assignment on the chalkboard as she explains it to the class but notices that quite a number of students start talking to each other while her back is turned. As students work on the assignment, Sally circulates around the room. She reexplains the task to some students and reminds others to focus on their work. She notices that many students seem lost and that only a few students are doing a good job of following her very detailed instructions.

Amy is an eighth grade student taking science. One day, Amy arrives home with her science textbook and instructions to answer the questions at the end of Chapter 6. Amy sits down, turns her book to the end of the chapter, finds the first question and looks for key words. She then searches for those key words in the chapter to find information relevant to the question. She copies the information word for word onto a piece of paper, then moves onto the remaining questions. When she is challenged by her teacher the next day, who asks if she has answered the questions in her own words, Amy replies that she does not need to understand the information. She shows her teacher how she has received 100% on each of her previous homework assignments, which she completed in the same way.
Family Help and Homework Management in Urban and Rural Secondary Schools
Authors: Xu Jianzhong
Published: September 2004

This article calls attention to developmental and home conditions that affect the development of good work habits through homework. The first section examines recent studies that have alluded to the possibility of developing good work habits through family involvement with secondary school homework. The second section describes two survey studies, in urban and rural secondary schools, that explicitly link homework management to family help and grade level. The data suggest that secondary students could still benefit from clear expectations from adult assistance regarding how to foster the development of homework management strategies, regardless of helper's educational background. The data also suggest, however, that such help is overshadowed by increasing internal distractions students encounter as they move from middle school into high school. The final section discusses implications for future research and practice regarding how to foster adolescents’ work habits through homework, particularly for high school students.
Self-Regulated Learning and Internet Searching
Authors: Rogers Donna , Swan Karen
Published: September 2004

As the Internet becomes a resource in the classroom, students must learn to glean maximally from that information using skills that enhance their understandings. Students engage in activities involved with Internet searching to the degree that they are skilled and motivated. This article reports on a research study that demonstrates that the model of self-regulated learning developed by Corno and Mandinach relative to traditional classroom activities can be applied to Internet searching behaviors. The study also found that not all Internet searchers are equal; thus, the article suggests that students need to be helped to become better at it, and the article also suggests ways this might be accomplished, which are linked to the findings and research on self-regulated learning in general. In particular, it gives curricular and instructional recommendations for accomplishing and supporting student learning in both Internet searches and regulation of their own searching behaviors.
Teachers as Self-Regulated Learners
Authors: Randi Judi
Published: September 2004

This article describes how preservice teachers are afforded opportunities for developing self-regulation as they learn to transition to “the other side of the desk” in their teacher education program. The article focuses on self-regulated learning as it applies to teachers’ work in the classroom and distinguishes volitional work styles and student study habits from learning-oriented volitional strategies teachers engage to learn from teaching. To illustrate teachers’ strategy use and to reify volitional constructs as they are embodied in practice, this article presents practical examples from one teacher education program. Finally, the article considers ways that teacher preparation programs might structure learning environments, affording teachers opportunities to learn self-regulation and then apply those strategies to learn from their own teaching.
Putting Volition to Work in Education
Authors: Winne Philip H.
Published: September 2004

Playing the role of constructive critic of articles in this special issue, I adopt the term self-reliance to refer to work habits, work styles, volition, and self-regulated learning. Starting with the belief that developing self-reliance is an important goal of education, I examine key issues that influence the likelihood of reaching this goal. I assume first that learners are agents. This human quality ubiquitously affects theorizing, researching, and teaching. I examine the nature of this influence in relation to three issues: How learners experiment to develop self-reliance, conditions that influence how learners express and extend self-reliance, and considerations about how to advance theory and practice in the service of enhancing self-reliance.
Tests for Cycling in a Signalling Pathway
Authors: Müller T. G. , Faller D. , Timmer J. , Swameye I. , Sandra O. , Klingmüller U.
Published: September 2004

Cellular signalling pathways, mediating receptor activity to nuclear gene activation, are generally regarded as feed forward cascades. We analyse measured data of a partially observed signalling pathway and address the question of possible feed-back cycling of involved biochemical components between the nucleus and cytoplasm. First we address the question of cycling in general, starting from basic assumptions about the system. We reformulate the problem as a statistical test leading to likelihood ratio tests under non-standard conditions. We find that the modelling approach without cycling is rejected. Afterwards, to differentiate two different transport mechanisms within the nucleus, we derive the appropriate dynamical models which lead to two systems of ordinary differential equations. To compare both models we apply a statistical testing procedure that is based on bootstrap distributions. We find that one of both transport mechanisms leads to a dynamical model which is rejected whereas the other model is satisfactory.
Robust Designs for Linear Mixed Effects Models
Authors: Berger Martijn P. F. , Tan Frans E. S.
Published: September 2004

In health sciences, medicine and social sciences linear mixed effects models are often used to analyse time-structured data. The search for optimal designs for these models is often hampered by two problems. The first problem is that these designs are only locally optimal. The second problem is that an optimal design for one model may not be optimal for other models. In this paper the maximin principle is adopted to handle both problems, simultaneously. The maximin criterion is formulated by means of a relative efficiency measure, which gives an indication of how much efficiency is lost when the uncertainty about the models over a prior domain of parameters is taken into account. The procedure is illustrated by means of three growth studies. Results are presented for a vocabulary growth study from education, a bone gain study from medical research and an epidemiological decline in height study. It is shown that, for the mixed effects polynomial models that are applied to these studies, the maximin designs remain highly efficient for different sets of models and combinations of parameter values.
Hedge Funds and the Technology Bubble
Authors: K. BRUNNERMEIER MARKUS , NAGEL STEFAN
Published: September 2004

This paper documents that hedge funds did not exert a correcting force on stock prices during the technology bubble. Instead, they were heavily invested in technology stocks. This does not seem to be the result of unawareness of the bubble: Hedge funds captured the upturn, but, by reducing their positions in stocks that were about to decline, avoided much of the downturn. Our findings question the efficient markets notion that rational speculators always stabilize prices. They are consistent with models in which rational investors may prefer to ride bubbles because of predictable investor sentiment and limits to arbitrage.
Cash and Corporate Control
Authors: FALEYE OLUBUNMI
Published: September 2004

The takeover market is often suggested as appropriate for containing the agency problems of excessive corporate cash holdings. However, recent studies report contradictory evidence. I focus on the takeover‐deterrence effects of corporate liquidity and suggest the proxy contest as an effective alternative control mechanism. I find that proxy fight targets hold 23% more cash than comparable firms, and that the probability of a contest is significantly increasing in excess cash holdings. Proxy fight announcement return also is positively related to excess cash. Following a contest, executive turnover and special cash distributions to shareholders increase, while cash holdings significantly decline.
Dividend Changes and the Persistence of Past Earnings Changes
Authors: KOCH ADAM S. , SUN AMY X.
Published: September 2004

We examine whether the market interprets changes in dividends as a signal about the persistence of past earnings changes. Prior to observing this signal, investors may believe that past earnings changes are not necessarily indicative of future earnings levels. We empirically investigate whether a change in dividends alters investors' assessments about the valuation implications of past earnings. Results confirm the hypothesis that changes in dividends cause investors to revise their expectations about the persistence of past earnings changes. This effect varies predictably with the magnitude of the dividend change and the sign of the past earnings change.