bluuebunny/crossref_metadata_2025 huggingface dataset:
Selenium‐containing xanthine dehydrogenase from Eubacterium barkeri
Authors: Schräder Thomas , Rienhöfer Annette , Andreesen Jan R.
Published: March 2003

A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39‐fold to apparent homogeneity using a three‐step purification scheme. It exhibited an NADP‐dependent specific activity of 164 µmol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH‐dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum‐complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid‐labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide‐inactivated enzyme and with chloramphenicol‐treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.
Sites on the cytoplasmic region of phospholamban involved in interaction with the calcium‐activated ATPase of the sarcoplasmic reticulum
Authors: Levine Barry A. , Patchell Valerie B. , Sharma Parveen , Gao Yuan , Bigelow Diana J. , Yao Qing , Goh Swee , Colyer John , Drago Guido A. , Perry Samuel V.
Published: March 2003

Proton NMR studies have shown that when a peptide corresponding to the N‐terminal region of phospholamban, PLB(1–20), interacts with the Ca2+ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C‐terminal residues of PLB(1–20). In the phosphorylated peptide weakened interaction occurs with residues at the N‐terminus of PLB(1–20). PLB(1–20) is shown to interact with a peptide corresponding to residues 378–405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C‐terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1–20 of PLB. They also suggest that phospholamban residues 1–20 straddle separate segments of the cytoplasmic domain of SERCA with the N‐terminus of PLB associated with a region other than that corresponding to SERCA2a(378–405).
Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2
Authors: Duggan Brendan M. , Dyson H. Jane , Wright Peter E.
Published: March 2003

Nematode anticoagulant proteins (NAPs) from the hematophagous nematode Ancylostoma caninum inhibit blood coagulation with picomolar inhibition constants, and have been targeted as novel pharmaceutical agents. NAP5 and NAP6 inhibit factor Xa by binding to its active site, whereas NAPc2 binds to factor Xa at a different, as yet unidentified, site and the resultant binary complex inhibits the tissue factor–factor VIIa complex. We have undertaken NMR studies of NAPc2, including the calculation of a solution structure, and found that the protein is folded, with five disulfide bonds, but is extremely flexible, especially in the acidic loop. The Hα secondary shifts and 3JHNHα coupling constants indicate the presence of some β structure and a short helix, but the intervening loops are highly conformationally heterogeneous. Heteronuclear NOE measurements showed the presence of large amplitude motions on a subnanosecond timescale at the N‐terminus and C‐terminus and in the substrate‐binding loop, indicating that the conformational heterogeneity observed in the NMR structures is due to flexibility of the polypeptide chain in these regions. Flexibility may well be an important factor in the physiological function of NAPc2, because it must interact with other proteins in the inhibition of blood coagulation. We suggest that this inhibitor is likely to become structured on binding to factor Xa, because the inhibition of the tissue factor–factor VIIa complex requires both NAPc2 and factor Xa.
Structural studies on lipopolysaccharides of serologically non‐typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants
Authors: Knirel Yuriy A. , Kocharova Nina A. , Hynes Sean O. , Widmalm Göran , Andersen Leif P. , Jansson Per‐Erik , Moran Anthony P.
Published: March 2003

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewisx (Lex) and Lewisy (Ley) antigenic determinants in the O‐specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non‐typable with anti‐Lex and anti‐Ley antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H‐NMR spectroscopy and electrospray ionization‐mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O‐specific polysaccharide chains. The O‐specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly‐N‐acetyl‐β‐lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Lex chain terminating with Lex or Ley units:

where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0–2, 6–7 in strain AF1 and m = 0–2, 6–7 or ≈ 40 in strain 007, the medium‐size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti‐Lex and anti‐Ley may reflect the presence of a polymeric Lex chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a dglycerodmanno‐heptose residue in the outer core varied in the two strains, and an extended dd‐heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core lglycerodmanno‐heptose region of both strains carried a 2‐aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.
Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp
Authors: Destoumieux Delphine , Bulet Philippe , Strub Jean‐Marc , van Dorsselaer Alain , Bachère Evelyne
Published: March 2003

Penaeidins are 5.5‐ to 6.6‐kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline‐rich N‐terminus and of a C‐terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen‐2 and Pen‐3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen‐2 and ‐3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large‐scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen‐2 and ‐3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram‐positive bacteria, with a strain‐specific inhibition mechanism. Despite a better efficiency of Pen‐3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen‐2 and ‐3a. Finally, no synergistic effect could be observed between the two molecules.
Localization and light‐dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in Neurospora crassa
Authors: Schwerdtfeger Carsten , Linden Hartmut
Published: March 2003

In Neurospora crassa only two white collar (wc) mutants, wc‐1 and wc‐2, have been described that seem to be insensitive to light. The pleiotropic phenotypes of these mutants suggest that they represent two central components of blue light signal transduction. The WC proteins have several characteristics of transcription factors consistent with an involvement in transcriptional control of light‐regulated genes. Here, we present a biochemical analysis of WC1 and WC2 polypeptides in N. crassa. Using specific antisera against WC1 and WC2, respectively, the subcellular localization of the WC polypeptides was investigated. The WC1 protein was localized exclusively in the nucleus, whereas WC2 was detected in both the nuclear and cytoplasmic fractions. The nuclear localization of WC1 and WC2 was shown to be independent of light and dimerization between the two proteins. In addition, WC1 and WC2 are phosphorylated in response to light. The phosphorylation of WC1 and WC2 was dependent on functional WC1 and WC2 proteins, respectively, which clearly indicated a correlation between the light‐dependent phosphorylation and the function of WC1 and WC2 in blue light signaling. However, the light‐specific phosphorylation of the WC proteins revealed different kinetics. The phosphorylation of WC1 was transient whereas the WC2 phosphorylation was shown to be stable under constant light conditions. The analysis of the light‐dependent phosphorylation of WC1 and WC2 in wc‐2 and wc‐1 mutants revealed an epistatic relationship for WC1 and WC2 with WC2 acting downstream of WC1 in the signal transduction pathway of blue light.
Structure of a 2‐aminoethyl phosphate‐containing O‐specific polysaccharide of Proteus penneri 63 from a new serogroup O68
Authors: Shashkov Aleksander S. , Kondakova Anna N. , Senchenkova Sof’ya N. , Zych Krystyna , Toukach Filip V. , Knirel Yuriy A. , Sidorczyk Zygmunt
Published: March 2003

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O‐specific polysaccharide that was isolated by gel‐permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two‐dimensional 1H,1H COSY, TOCSY rotating‐frame NOE spectroscopy, H‐detected 1H,13C and 1H,31P heteronuclear multiple‐quantum coherence (HMQC), and 1H,13C HMQC‐TOCSY experiments, demonstrated the following structure of the polysaccharide:

where FucNAc is 2‐acetamido‐2,6‐dideoxygalactose and PEtn is 2‐aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of d‐GlcNAc6PEtn and an α‐L‐FucNAc‐(1→3)‐d‐GlcNAc disaccharide, with other Proteus O‐specific polysaccharides. A marked cross‐reactivity of P. penneri 63 O‐antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O‐specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O‐antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.
cDNA cloning and analysis of tissue‐specific expression of mouse peroxisomal straight‐chain acyl‐CoA oxidase
Authors: Nöhammer Christa , El‐Shabrawi Yosuf , Schauer Silvia , Hiden Michaela , Berger Johannes , Forss‐Petter Sonja , Winter Elke , Eferl Robert , Zechner Rudolf , Hoefler Gerald
Published: March 2003

Straight‐chain acyl‐CoA oxidase is the first and rate limiting enzyme in the peroxisomal β‐oxidation pathway catalysing the desaturation of acyl‐CoAs to 2‐trans‐enoyl‐CoAs, thereby producing H2O2. To study peroxisomal β‐oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl‐CoA oxidase. It consists of 3778 bp, including a 1983‐bp ORF encoding a polypeptide of 661 amino‐acid residues. Like the rat and human homologue the C‐terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino‐acid homology with rat (96%) and human (87%) acyl‐CoA oxidase in addition to minor homology (≈ 40%) with other related proteins, such as rabbit trihydroxy‐cholestanoyl‐CoA oxidase, human branched chain acyl‐CoA oxidase and rat trihydroxycoprostanoyl‐CoA oxidase. Acyl‐CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl‐CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal β‐oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator‐activated receptor alpha and acyl‐CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator‐activated receptor alpha mRNA in proportion to acyl‐CoA oxidase mRNA was found. Our data show that acyl‐CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.
The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium
Authors: Hessel Elke , Herrmann Andreas , Müller Peter , Schnetkamp Paul P. M. , Hofmann Klaus‐Peter
Published: March 2003

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll‐floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A‐discs, B‐discs C‐discs, respectively). In all cases, spin‐labelled phospholipid analogues (SL‐PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL‐PL addition, as deduced from the disappearance of spin‐label from the suspension medium and from the specific ESR spectrum of membrane‐associated spin‐label. Using BSA extraction, the amount of SL‐PL in the outer leaflet of the bilayer was determined. It decreased with a mean half‐time of < 5 min at 25 °C, indicating rapid redistribution of all spin‐labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N‐ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy‐consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.
Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae
Authors: Böcking Till , Barrow Kevin D. , Netting Andrew G. , Chilcott Terry C. , Coster Hans G. L. , Höfer Milan
Published: March 2003

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5α,6α‐epoxy‐(22E)‐ergosta‐8,22‐dien‐3β,7α‐diol (8‐DED). 9(11)‐dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8‐DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen‐mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8‐DED. Ergosterol 7‐hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.
Cloning of MMP‐26
Authors: Benoit de Coignac Amélie , Elson Greg , Delneste Yves , Magistrelli Giovanni , Jeannin Pascale , Aubry Jean‐Pierre , Berthier Odile , Schmitt Daniel , Bonnefoy Jean‐Yves , Gauchat Jean‐François
Published: March 2003

A cDNA encoding a novel human matrix metalloproteinase (MMP), named MMP‐26, was cloned from fetal cDNA. The deduced 261‐amino‐acid sequence is homologous to macrophage metalloelastase (51.8% identity). It includes only the minimal characteristic features of the MMP family: a signal peptide, a prodomain and a catalytic domain. As with MMP‐7, this new MMP does not comprise the hemopexin domain, which is believed to be involved in substrate recognition. A study of MMP‐26 mRNA steady states levels reveals, among the tissue examined, a specific expression in placenta. MMP‐26 mRNA could also be detected in several human cell lines such as HEK 293 kidney cells and HFB1 lymphoma cells. Recombinant MMP‐26 was produced in mammalian cells and used to demonstrate a proteolytic activity of the enzyme on gelatin and β‐casein.
Thermal unfolding and refolding of β‐lactoglobulin
Authors: Bhattacharjee Chaitali , Das K. P.
Published: March 2003

The conformational features of β‐lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85–90 °C, β‐lactoglobulin does not completely lose its folded structure. The unfolding and refolding of β‐lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native β‐lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1‐anilino 8‐naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded β‐lactoglobulin is observed compared to that of the native form. Our results indicate that β‐lactoglobulin, refolded after heating to above 70 °C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native β‐lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open.
A small‐angle X‐ray solution scattering study of bovine α‐crystallin
Authors: Vanhoudt Jos , Abgar Saı¨d , Aerts Tony , Clauwaert Julius
Published: March 2003

The native high molecular mass form of α‐crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 °C in dilute solutions were investigated by synchrotron radiation small‐angle X‐ray scattering. The α‐crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm.

In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z‐average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of α‐crystallin yield bean‐like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data.

We emphasized the apparent contradiction between hydrodynamic and molecular properties of α‐crystallin. An explanation was put forward based on the presence of solvent‐exposed flexible C‐terminal extensions. We present two bead models (‘hollow globule with tentacles’ and ‘bean with tentacles’) based on NMR and cryo‐electron microscopy studies and discuss how well they correspond with our data from X‐ray scattering, light scattering and analytical ultracentrifugation.
Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures
Authors: Moraes Trevor F. , Plaxton William C.
Published: March 2003

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104‐kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi‐sufficient (+Pi) and Pi‐starved (–Pi) cells to electrophoretic homogeneity and final specific activities of 37–40 µmol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and –Pi PEPCs are both homotetramers composed of an identical 104‐kDa subunit. Respective pH–activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to l‐malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and –Pi cells exists mainly in its dephosphorylated (l‐malate sensitive) form. Thus, up‐regulation of PEPC activity in –Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6‐phosphate and inhibition by l‐malate, l‐aspartate, l‐glutamate, dl‐isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. l‐Aspartate and l‐glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.
Cross‐talk between olfactory second messenger pathways
Authors: Vogl Alexander , Noé Johannes , Breer Heinz , Boekhoff Ingrid
Published: March 2003

The second messengers 3′‐5′‐cyclic‐monophosphate (cAMP) and inositol 1,4,5‐trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U‐73122 led to an augmentation of odor‐induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor‐induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor‐induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN‐(2‐phenylcyclopentyl)azacylotridec‐1‐en‐2‐amine (MDL‐12,330 A) results in potentiation of odor‐induced InsP3 formation. Concerning the molecular mechanism involved in cross‐interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor‐induced InsP3 signaling by cAMP may be mediated by a PKA‐controlled reaction.
Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry
Authors: Teusink Bas , Passarge Jutta , Reijenga Corinne A. , Esgalhado Eugenia , van der Weijden Coen C. , Schepper Mike , Walsh Michael C. , Bakker Barbara M. , van Dam Karel , Westerhoff Hans V. , Snoep Jacky L.
Published: March 2003

This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured.

In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.
Hadrurin, a new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus
Authors: Torres‐Larios Alfredo , Gurrola Georgina B. , Zamudio Fernando Z. , Possani Lourival D.
Published: March 2003

A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G‐50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino‐acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three‐dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two α‐helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C‐terminal amidated, and all d‐enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N‐terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide–lipid interactions.
Glutathione metabolism in brain
Authors: Dringen Ralf , Gutterer Jan M , Hirrlinger Johannes
Published: March 2003

The cells of the adult human brain consume ≈ 20% of the oxygen utilized by the body although the brain comprises only 2% of the body weight. Reactive oxygen species, which are produced continuously during oxidative metabolism, are generated at high rates within the brain. Therefore, the defense against the toxic effects of reactive oxygen species is an essential task within the brain. An important component of the cellular detoxification of reactive oxygen species is the antioxidant glutathione. The main focus of this short review is recent results on glutathione metabolism of brain astrocytes and neurons in culture. These two types of cell prefer different extracellular precursors for glutathione. Glutathione is involved in the disposal of exogenous peroxides by astrocytes and neurons. In coculture astrocytes protect neurons against the toxicity of reactive oxygen species. One mechanism of this interaction is the supply by astrocytes of glutathione precursors to neurons.
Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8
Authors: Abbott Catherine A. , Yu Denise M. T. , Woollatt Erica , Sutherland Grant R. , McCaughan Geoffrey W. , Gorrell Mark D.
Published: March 2003

Dipeptidyl peptidase (DPP) IV has roles in T‐cell costimulation, chemokine biology, type‐II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern‐blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full‐length DPP8 cDNA codes for an 882‐amino‐acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N‐linked or O‐linked glycosylation. Western blots and confocal microscopy of transfected COS‐7 cells showed DPP8 to be a 100‐kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala‐Pro, Arg‐Pro and Gly‐Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T‐cell activation and immune function.
Structural and functional analysis of the two haemoglobins of the Antarctic seabird Catharacta maccormicki
Authors: Tamburrini Maurizio , Riccio Antonio , Romano Maurizio , Giardina Bruno , di Prisco Guido
Published: March 2003

The amino‐acid sequence and the oxygen‐binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1α, G6α and HC3α, is located between the two α chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two β chains by means of a site–site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.
Physiological functions of thioredoxin and thioredoxin reductase
Authors: Arnér Elias S. J. , Holmgren Arne
Published: March 2003

Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione‐disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin–thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF‐κB or the Ref‐1‐dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co‐cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70‐kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112–130 kDa), selenium‐dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C‐terminal elongation with a cysteine–selenocysteine sequence forming a redox‐active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.
Synthesis of the translational apparatus is regulated at the translational level
Authors: Meyuhas Oded
Published: March 2003

The synthesis of many mammalian proteins associated with the translational apparatus is selectively regulated by mitogenic and nutritional stimuli, at the translational level. The apparent advantages of the regulation of gene expression at the translational level are the speed and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking unnecessary energy wastage. Likewise, when amino acids are replenished or mitogenic stimulation is applied, then cells can rapidly respond in resuming the costly biosynthesis of the translational apparatus. A structural hallmark, common to mRNAs encoding many components of the translational machinery, is the presence of a 5’ terminal oligopyrimidine tract (5’TOP), referred to as TOP mRNAs. This structural motif comprises the core of the translational cis‐regulatory element of these mRNAs. The present review focuses on the mechanism underlying the translational control of TOP mRNAs upon growth and nutritional stimuli. A special emphasis is put on the pivotal role played by ribosomal protein S6 kinase (S6K) in this mode of regulation, and the upstream regulatory pathways, which might be engaged in transducing external signals into activation of S6K. Finally, the possible involvement of pyrimidine‐binding proteins in the translational control of TOP mRNAs is discussed.
Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells
Authors: Leschelle Xavier , Delpal Serge , Goubern Marc , Blottière Hervé M , Blachier François
Published: March 2003

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT‐29 Glc−/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT‐29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk‐cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl‐CoA synthesis is not required for the growth‐restraining effect of this SCFA. Furthermore, HT‐29 cells modestly incorporated the 14C‐labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when d‐glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT‐29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C‐labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT‐29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl‐CoA synthesis. In contrast, the metabolism downstream of acetyl‐CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.
Oxidative degradation of bilirubin produces vasoactive compounds
Authors: Kranc Kamil R. , Pyne Gail J. , Tao Limei , Claridge Tim D. W. , Harris David A. , Cadoux‐Hudson Thomas A. D. , Turnbull Jonathan J. , Schofield Christopher J. , Clark Joseph F.
Published: March 2003

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO‐1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivatives, 4‐methyl‐5‐oxo‐3‐vinyl‐(1,5‐dihydropyrrol‐2‐ylidene)acetamide and 3‐methyl‐5‐oxo‐4‐vinyl‐(1,5‐dihydropyrrol‐2‐ylidene)acetamide and the third was 4‐methyl‐3‐vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.
Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis
Authors: Chauhan Hitesh J. , Domingo Gonzalo J. , Jung Hyo‐Il , Perham Richard N.
Published: March 2003

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (α2β2) of E1α and E1β polypeptide chains. The domain structure of the E1α and E1β chains, and the protein–protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1α in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1α, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (α2β2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit‐binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild‐type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.
Sulfoxidation mechanism of vanadium bromoperoxidase from Ascophyllum nodosum
Authors: ten Brink Hilda B. , Schoemaker Hans E. , Wever Ron
Published: March 2003

We have previously shown that vanadium bromoperoxidase from Ascophyllum nodosum mediates production of the (R)‐enantiomer of methyl phenyl sulfoxide with 91% enantiomeric excess. Investigation of the intrinsic selectivity of vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidation of methyl phenyl sulfide in a purely enantioselective manner. The Km of the enzyme for methyl phenyl sulfide was determined to be ≈ 3.5 mm in the presence of 25% methanol or tert‐butanol. The selectivity of the sulfoxidation of methyl phenyl sulfide is optimal in the temperature range 25–30 °C and can be further optimized by increasing the enzyme concentration, yielding selectivities with up to 96% enantiomeric excess. Furthermore, we established for the first time that vanadium bromoperoxidase is functional at temperatures up to 70 °C. A detailed investigation of the sulfoxidation activity of this enzyme using 18O‐labeled hydrogen peroxide shows that vanadium bromoperoxidase mediates the direct transfer of the peroxide oxygen to the sulfide. A schematic model of the vanadium haloperoxidase sulfoxidation mechanism is presented.
Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor
Authors: Andersson Dick , Carlsson Uno , Freskgård Per‐Ola
Published: March 2003

The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180–305 nm). The sum of the individual spectra of each Trp residue in the near‐UV region was qualitatively identical to the wild‐type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far‐UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp‐free CD spectrum obtained after subtraction of all individual Trp spectra from the wild‐type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native‐like tertiary structure by kinetic near‐UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side‐chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.
Expression and induction of a large set of drug‐metabolizing enzymes by the highly differentiated human hepatoma cell line BC2
Authors: Gómez‐Lechón María J. , Donato Teresa , Jover Ramiro , Rodriguez Cristina , Ponsoda Xavier , Glaise Denize , Castell José V. , Guguen‐Guillouzo Christiane
Published: March 2003

The BC2 cell line derived from the human hepatocarcinoma, HGB, undergoes a spontaneous sharp differentiation process in culture as it becomes confluent, remains stably differentiated for several weeks, and may return to proliferation thereafter under appropriate density conditions. The relevance of the line as an hepatic model has been evaluated. Cells synthesize a large number of plasma proteins, and rates of glycogen and urea synthesis increase with time of confluency and become sensitive to insulin, reflecting the process of differentiation. Differentiated BC2 cells express the most relevant cytochrome P‐450 (CYP) isozyme activities (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4) and conjugating enzymes (glutathione S‐transferase and UDP‐glucuronyltransferase) and also respond to model inducers. Methylcholanthrene induced an increase in CYP1A1/2 enzyme activity (eightfold), phenobarbital induced CYP2B6 activity (1.7‐fold), and dexamethasone induced CYP3A4 activity (fivefold). In parallel, expression of the most relevant liver‐enriched transcription factors, HNF‐4, HNF‐1, C/EBP‐α and C/EBP‐β mRNAs, was significantly increased in differentiated cultures. This increase was largest in HNF‐1 and HNF‐4, which supports the idea that a redifferentiation process towards the hepatic phenotype takes place. BC2 is an hepatic cell line that is able to express most hepatic functions, especially the drug‐biotransformation function, far more efficiently than any previously described human hepatoma cell line.