bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial oxidative stress is modulated by oleic acid via an epidermal growth factor receptor-dependent activation of glutathione peroxidase
Authors: DUVAL Carine , AUGÉ Nathalie , FRISACH Marie-Françoise , CASTEILLA Louis , SALVAYRE Robert , NÈGRE-SALVAYRE Anne
Published: October 2002

Mitochondria generate reactive oxygen species (ROS) under various pathophysiological conditions. In isolated mitochondria, fatty acids (FA) exhibit an uncoupling effect of the respiratory activity and modulate ROS generation. The effect of FA on intact cultured cells remains to be elucidated. The present study reports that FA (buffered by BSA) decrease the level of cellular ROS generated by the mitochondrial respiratory chain in cultured cells incubated with antimycin A. Both saturated and unsaturated FA are effective. This fatty acid-induced antioxidant effect does not result from a decrease in ROS production, but is subsequent to cellular glutathione peroxidase (GPx) activation and enhanced ROS degradation. This fatty acid-induced GPx activation is mediated through epidermal growth factor receptor (EGFR) signalling, since this response is (i) abrogated by the EGFR inhibitor AG1478 or by a defect in EGFR (in EGFR-deficient B82L fibroblasts), (ii) restored in B82LK+ cells expressing EGFR and (iii) mimicked by epidermal growth factor. These findings indicate that FA contribute to enhance cellular antioxidant defences against mitochondrial oxidative stress through EGFR-dependent GPx activation.
Endogenous mono-ADP-ribosylation of the free Gβγ prevents stimulation of phosphoinositide 3-kinase-γ and phospholipase C-β2 and is activated by G-protein-coupled receptors
Authors: LUPI Rosita , DANI Nadia , DIETRICH Alexander , MARCHEGIANI Adriano , TURACCHIO Sabrina , BERRIE Christopher P. , MOSS Joel , GIERSCHIK Peter , CORDA Daniela , GIROLAMO Maria Di
Published: October 2002

We have recently demonstrated that the β subunit of the heterotrimeric G-proteins is endogenously mono-ADP-ribosylated in intact cells. The modified βγ heterodimer loses its ability to inhibit calmodulin-stimulated type 1 adenylate cyclase and, remarkably, is de-ADP-ribosylated by a cytosolic hydrolase that completes an ADP-/de-ADP-ribosylation cycle of potential physiological relevance. In the present study, we show that this ADP-ribosylation might indeed be a general mechanism for termination of βγ signalling, since the ADP-ribosylated βγ subunit is also unable to activate both phosphoinositide 3-kinase-γ and phospholipase C-β2. Moreover, we show that β subunit ADP-ribosylation is induced by G-protein-coupled receptor activation, since hormone stimulation of Chinese-hamster ovary plasma membranes leads to increases in β subunit labelling. This occurs when βγ is in its active heterodimeric conformation, since full inhibition of this modification can be achieved by binding of GDP-αi3 to the βγ heterodimer. Taken together, these findings delineate a pathway that arises from the activation of a G-protein-coupled receptor and leads to the inhibition of βγ activity through its reversible mono-ADP-ribosylation.
Urokinase upregulates matrix metalloproteinase-9 expression in THP-1 monocytes via gene transcription and protein synthesis
Authors: MENSHIKOV Mikhail , ELIZAROVA Eugenia , PLAKIDA Karina , TIMOFEEVA Angelika , KHASPEKOV Georgy , BEABEALASHVILLI Robert , BOBIK Alex , TKACHUK Vsevolod
Published: October 2002

Urokinase-type plasminogen activator (uPA) is suggested to exert its proliferatory, migratory and invasive action through binding with its membrane receptor, promoting pericellular proteolysis and mediating cell signal transduction. One of the possible actions of urokinase can be related to the regulation of activity and/or the expression of proteolytic enzymes participating in extracellular matrix degradation. In the present study, the role of uPA in regulating matrix metalloproteinase (MMP) expression and release by the monocyte cell line THP-1 was investigated. Recombinant uPA induced the release of MMP9/gelatinase B, as detected by zymography and Western blotting, and this release was abolished by actinomycin D and cycloheximide (inhibitors of DNA transcription and protein synthesis) and partially suppressed by monensin (an inhibitor of secretion). Proteolytically inactive urokinase with substitution of His204 for Gln was able to reproduce about 70% of the effect induced by the wild-type recombinant uPA. The reverse transcription-PCR and Northern blot data indicated that the action of r-uPA on THP-1 cells resulted in formation of MMP9 mRNA, which depended on time, within 6—48h, of the cell incubation with r-uPA. These results suggest that urokinase upregulates MMP9 expression in monocytes via MMP9 gene transcription and protein biosynthesis.
Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases
Authors: VECCHIO Pompea DEL , GRAZIANO Giuseppe , GRANATA Vincenzo , BARONE Guido , MANDRICH Luigi , ROSSI Mosè , MANCO Giuseppe
Published: October 2002

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge—charge interactions on the protein surface is a key factor for the stability of the two esterases.
Murine Nr4a1 and Herpud1 are up-regulated by Wnt-1, but the homologous human genes are independent from β-catenin activation
Authors: CHTARBOVA Slava , NIMMRICH Inko , ERDMANN Silke , HERTER Peter , RENNER Matthias , KITAJEWSKI Jan , MÜLLER Oliver
Published: October 2002

The Wnt signal transduction pathway regulates morphogenesis and mitogenesis of cells in multicellular organisms. A major downstream consequence of Wnt-1 signalling is the activation of β-catenin/T-cell factor (TCF)-mediated transcription. We compared Wnt-1-transformed murine mammary epithelial cells with control cells by subtractive hybridization. We found the two genes Nr4a1 and Herpud1 to be overexpressed in Wnt-1-transformed cells. Remarkably, the transcription levels of the two homologous human genes NR4A1 and HERPUD1 are neither activated in cells with activated β-catenin/TCF-mediated transcription nor can be induced by β-catenin transfection. These results indicate different regulation mechanisms of the two genes in murine and human cells.
Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo
Authors: HENRIKSSON Maria L. , SUNDIN Charlotta , JANSSON Anna L. , FORSBERG Åke , PALMER Ruth H. , HALLBERG Bengt
Published: October 2002

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.
Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin
Authors: VUJCIC Slavoljub , DIEGELMAN Paula , BACCHI Cyrus J. , KRAMER Debora L. , PORTER Carl W.
Published: October 2002

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N1-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N1-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N1-acetylspermine and that it failed to act on N1-acetylspermidine, spermidine or the preferred PAO substrate, N1,N12-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N1-(n-octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.
Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure
Authors: WACHTEL Marco , FREI Karl , EHLER Elisabeth , BAUER Christian , GASSMANN Max , GLOOR Sergio M.
Published: October 2002

During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O2 in serum- and glucose-free medium) for 5h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30min, strongly inhibited after 5h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.
New insights into long-chain fatty acid uptake by heart muscle: a crucial role for fatty acid translocase/CD36
Authors: BRINKMANN Joep F.F. , ABUMRAD Nada A. , IBRAHIMI Azeddine , vanderVUSSE Ger J. , GLATZ Jan F.C.
Published: October 2002

Long-chain fatty acids are an important source of energy for several cell types, in particular for the heart muscle cell. Three different proteins, fatty acid translocase (FAT)/CD36, fatty acid transport protein and plasma membrane fatty acid binding protein, have been identified as possible membrane fatty acid transporters. Much information has been accumulated recently about the fatty acid transporting function of FAT/CD36. Several experimental models to study the influence of altered FAT/CD36 expression on fatty acid homoeostasis have been identified or developed, and underscore the importance of FAT/CD36 for adequate fatty acid transport. These models include the FAT/CD36 null mouse, the spontaneously hypertensive rat and FAT/CD36-deficient humans. The fatty acid transporting role of FAT/CD36 is further demonstrated in mice overexpressing muscle-specific FAT/CD36, and in transgenic mice generated using a genetic-rescue approach. In addition, a wealth of information has been gathered about the mechanisms that regulate FAT/CD36 gene expression and the presence of functional FAT/CD36 on the plasma membrane. Available data also indicate that FAT/CD36 may have an important role in the aetiology of cardiac disease, especially cardiac hypertrophy and diabetic cardiomyopathy. This review discusses our current knowledge of the three candidate fatty acid transporters, the metabolic consequences of alterations in FAT/CD36 levels in different models, and the mechanisms that have been identified for FAT/CD36 regulation.
Activation of nuclear factor-κB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide
Authors: WANG Suwei , KOTAMRAJU Srigiridhar , KONOREV Eugene , KALIVENDI Shasi , JOSEPH Joy , KALYANARAMAN Balaraman
Published: October 2002

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor κB (NF-κB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-κB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-κB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H2O2, significantly decreased DOX-induced NF-κB activation and apoptosis. Inhibition of DOX-induced NF-κB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-κB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IκB mutant vector, another NF-κB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-κB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-κB activation is anti-apoptotic. Removal of intracellular H2O2 protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-κB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.
A re-evaluation of the role of histidine-426 within Pseudomonas aeruginosa exotoxin A
Authors: ROBERTS Tania M. , MERRILL A. Rod
Published: October 2002

CRM66 (cross-reactive 66kDa protein) is an inactive mutant form of Pseudomonas aeruginosa exotoxin A that has been isolated from a mutant strain of P. aeruginosa derived from nitrosoguanidine-based mutagenesis. The mutation within this enzyme toxin was previously identified as H426Y and it was shown to possess significantly reduced enzymic activity. Furthermore, it was previously suggested that His-426 may directly participate in the catalytic mechanism of the exotoxin A enzyme and that it may also play an important role in the binding of the protein substrate of exotoxin A, a critical protein factor in eukaryotic protein translation known as elongation factor-2. In order to more thoroughly characterize the role of His-426 in the enzyme mechanism of exotoxin A, amino acid substitutions were made within helix 1 of the enzyme domain in the vicinity of the His-426 residue. Analysis of the site-directed mutagenesis results involving kinetic and protein structural integrity measurements revealed that His-426 H-bonds to Tyr-502 and that replacement of His-426 with polar substitutions leads to structural alterations of the enzyme's folded conformation. Furthermore, it was shown that His-426 is not important for the binding of either of the two substrates of exotoxin A, NAD+ or elongation factor-2. In summary, these data show that His-426 is not an active-site residue and that it is not important for substrate binding or orientation, but that it plays an important structural role in helping to maintain the folded conformation of the enzyme toxin. Therefore, the role of His-426 would seem to be to tether helix 1 to the main body of the enzyme, and mutations resulting in the disruption of this region of the enzyme result in a significantly impaired enzyme.
Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines
Authors: ROTHEM Lilah , IFERGAN Ilan , KAUFMAN Yotam , PRIEST David G. , JANSEN Gerrit , ASSARAF Yehuda G.
Published: October 2002

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87—96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92—97% of parental [3H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.
Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line
Authors: TAKAHASHI Shigeru , MATSUURA Naomi , KUROKAWA Takako , TAKAHASHI Yuji , MIURA Takashi
Published: October 2002

We reported previously that the 5′-flanking region (nucleotides −1976 to −1655) of the human haem oxygenase-1 (hHO-1) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231—235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50bp fragment (-1793 to −1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to −1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.
The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits
Authors: ASTOUL Corinne HOULÈS , PEUMANS Willy J. , DAMME Els J.M. van , BARRE Annick , BOURNE Yves , ROUGÉ Pierre
Published: October 2002

Mannose-specific lectins with high sequence similarity to jacalin and the Maclura pomifera agglutinin have been isolated from species belonging to the families Moraceae, Convolvulaceae, Brassicaceae, Asteraceae, Poaceae and Musaceae. Although these novel mannose-specific lectins are undoubtedly related to the galactose-specific Moraceae lectins there are several important differences. Apart from the obvious differences in specificity, the mannose- and galactose-specific jacalin-related lectins differ in what concerns their biosynthesis and processing, intracellular location and degree of oligomerization of the protomers. Taking into consideration that the mannose-specific lectins are widely distributed in higher plants, whereas their galactose-specific counterparts are confined to a subgroup of the Moraceae sp. one can reasonably assume that the galactose-specific Moraceae lectins are a small-side group of the main family. The major change that took place in the structure of the binding site of the diverging Moraceae lectins concerns a proteolytic cleavage close to the N-terminus of the protomer. To corroborate the impact of this change, the specificity of jacalin was re-investigated using surface plasmon resonance analysis. This approach revealed that in addition to galactose and N-acetylgalactosamine, the carbohydrate-binding specificity of jacalin extends to mannose, glucose, N-acetylmuramic acid and N-acetylneuraminic acid. Owing to this broad carbohydrate-binding specificity, jacalin is capable of recognizing complex glycans from plant pathogens or predators.
A protein-domain microarray identifies novel protein–protein interactions
Authors: ESPEJO Alexsandra , CÔTÉ Jocelyn , BEDNAREK Andrzej , RICHARD Stephane , BEDFORD Mark T.
Published: October 2002

Protein domains mediate protein—protein interactions through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed glutathione S-transferase (GST) fusion proteins, with a focus on protein interaction domains, on to nitrocellulose-coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW (a domain with two conserved tryptophans), SH3 (Src homology 3), SH2, 14.3.3, FHA (forkhead-associated), PDZ (a domain originally identified in PSD-95, DLG and ZO-1 proteins), PH (pleckstrin homology) and FF (a domain with two conserved phenylalanines) domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can ‘fish’ proteins out of a total cell lysate; these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain-binding profile of the signalling molecule Sam68 (Src-associated during mitosis 68), and have identified a new binding profile for the core small nuclear ribonucleoprotein SmB′. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signal transduction pathways.
Mechanism by which metal cofactors control substrate specificity in pyrophosphatase
Authors: ZYRYANOV Anton B. , SHESTAKOV Alexander S. , LAHTI Reijo , BAYKOV Alexander A.
Published: October 2002

Family I soluble pyrophosphatases (PPases) exhibit appreciable ATPase activity in the presence of a number of transition metal ions, but not the physiological cofactor Mg2+. The results of the present study reveal a strong correlation between the catalytic efficiency of three family I PPases (from Saccharomyces cerevisiae, Escherichia coli and rat liver) and one family II PPase (from Streptococcus mutans) in ATP and tripolyphosphate (P3) hydrolysis in the presence of Mg2+, Mn2+, Zn2+ and Co2+ on the one hand, and the phosphate-binding affinity of the enzyme subsite P2 that interacts with the electrophilic terminal phosphate group of ATP on the other. A similar correlation was observed in S. cerevisiae PPase variants with modified P1 and P2 subsites. The effect of the above metal ion cofactors on ATP binding to S. cerevisiae PPase paralleled their effect on phosphate binding, resulting in a low affinity of Mg-PPase to ATP. We conclude that PPase mainly binds ATP and P3 through the terminal phosphate group that is attacked by water. Moreover, this interaction is critical in creating a reactive geometry at the P2 site with these bulky substrates, which do not otherwise fit the active site perfectly. We propose further that ATP is not hydrolysed by Mg-PPase, since its interaction with the terminal phosphate is not adequately strong for proper positioning of the nucleophile—electrophile pair.
A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus
Authors: TSAI Rong-Tzong , LEU Wei-Ming , CHEN Ling-Yun , HU Nien-Tai
Published: October 2002

The cytoplasmic membrane proteins XpsL, XpsM and XpsN are components required for type II secretion in Xanthomonas campestris pv. campestris. We performed metal-chelating chromatography to partially purify the His6-tagged XpsM (XpsMh)-containing complex. Immunoblot analysis revealed that both XpsL and XpsN co-eluted with XpsMh. The co-fractionated XpsL and XpsN proteins co-immune precipitated with each other, suggesting the existence of an XpsL—XpsM—XpsN complex. Ternary complex formation does not require other Xps protein components of the type II secretion apparatus. Further purification upon size-exclusion chromatography revealed that XpsN is prone to dissociate from the complex. Reassociation of XpsN with the XpsL—XpsMh complex immobilized on a nickel column is more effective than with XpsMh alone. Membrane-mixing experiments suggested that the XpsL—XpsMh complex and XpsN probably dissociate and reassociate in the membrane vesicles. Comparison of the half-lives of the XpsL—XpsMh—XpsN and XpsL—XpsMh complexes revealed that XpsL dissociates from the latter at a faster rate than from the former. Dissociation and reassociation between XpsL and XpsM were also demonstrated with membrane-mixing experiments. A dynamic model is proposed for the XpsL—XpsM—XpsN complex.
Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis
Authors: FUNA Nobutaka , OHNISHI Yasuo , EBIZUKA Yutaka , HORINOUCHI Sueharu
Published: October 2002

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys138, His270 and Asn303 of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr224 is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr224 by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala305 mutants and identification of their products showed that the substitution of Ala305 by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr224 (important for starter substrate selection) and Ala305 (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.
Membrane perturbations induced by the apoptotic Bax protein
Authors: EPAND Raquel F. , MARTINOU Jean-Claude , MONTESSUIT Sylvie , EPAND Richard M.
Published: October 2002

The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.
Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5
Authors: BROOKS Nicole D. , GRUNDY Jean E. , LAVIGNE Nadine , DERRY Mélanie C. , RESTALL Christina M. , MacKENZIE ROGER C. , WAISMAN David M. , PRYZDIAL Edward L.G.
Published: October 2002

Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca2+. Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus—aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2—A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca2+ in the millimolar range, had an apparent dissociation constant [Kd(app)] of 1nM at 2mM Ca2+ and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2—A5 complex formation was corroborated in an experiment, where [125I]A2 associated in a Ca2+-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2—A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus—aPL fusion.
Matrilin-2 interacts with itself and with other extracellular matrix proteins
Authors: PIECHA Dorothea , WIBERG Charlotte , MÖRGELIN Matthias , REINHARDT Dieter P. , DEÁK Ferenc , MAURER Patrik , PAULSSON Mats
Published: October 2002

Matrilin-2 is a component of extracellular filamentous networks. To study the interactions by which it can be integrated into such assemblies, full-length and truncated forms of matrilin-2 were recombinantly expressed in HEK-293 cells and purified from conditioned medium. The recombinant proteins, when used in interaction assays, showed affinity to matrilin-2 itself, but also to other collagenous and non-collagenous extracellular matrix proteins. The interaction between matrilin-2 and collagen I was studied in greater detail and could be shown to occur at distinct sites on the collagen I molecule and to have a KD of about 3×10-8M. Interactions with some non-collagenous protein ligands were even stronger, with matrilin-2 binding to fibrillin-2, fibronectin and laminin-1—nidogen-1 complexes, with KD values in the range of 10-8—10-11M. Co-localization of matrilin-2 with these ligands in the dermal-epidermal basement membrane, in the microfibrils extending from the basement membrane into the dermis, and in the dermal extracellular matrix, indicates a physiological relevance of the interactions in the assembly of supramolecular extracellular matrix structures.
Small ubiquitin-related modifier-1 (SUMO-1) modification of the glucocorticoid receptor
Authors: TIAN Sha , POUKKA Hetti , PALVIMO Jorma J. , JÄNNE Olli A.
Published: October 2002

Small ubiquitin-related modifier-1 (SUMO-1) is covalently attached to many cellular targets to regulate protein—protein and protein—DNA interactions, as well as localization and stability of the target protein. The SUMO-1-conjugating E2 enzyme Ubc9 is known to interact with the glucocorticoid receptor (GR), a ligand-dependent transcription factor. In the present study, we show that GR is post-translationally modified by SUMO-1 (sumoylated) in a ligand-enhanced fashion. We identify experimentally three consensus SUMO attachment sites, two in the N-terminal transactivation region and one in the ligand-binding domain of GR. The two N-terminal sites are the major acceptor sites for SUMO-1 attachment. Mutation of these sites enhances transcriptional activity of GR on minimal promoters, but has no clear effect on the more complex mouse mammary tumour virus promoter. Thus SUMO-1 modification of GR influences receptor function in a promoter context-dependent fashion.
Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the β-carbon of asparagine-803
Authors: McNEILL Luke A. , HEWITSON Kirsty S. , CLARIDGE Timothy D. , SEIBEL Jürgen F. , HORSFALL Louise E. , SCHOFIELD Christopher J.
Published: October 2002

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 α-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1α/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the β-carbon of Asn-803 and imply production of the threo-isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro-isomer.
About town: The city and the female reader, 1860–1900
Authors: Caesar Ann Hallamore
Published: October 2002

The period after Italian Unification saw a marked increase in the volume of publications, magazines and books intended specifically for a female readership which was made up of girls and married women. It also saw the rise of the professional woman writer and journalist. Drawing on two of the most popular genres, the novel (in particular the domestic novel) and conduct literature, this article examines their representations of the city and urban life. It notes that while the physical transformation of major towns and cities was bringing in its wake far-reaching changes to the experience of urban life, the literature for women treats the city as an almost entirely abstract entity with few distinctive characteristics. Instead, the focus of these writings is on the drawing up of rulebooks designed to enable women to negotiate urban life without bringing opprobrium to bear on themselves or their families
Traders, taxpayers, citizens: The lower middle classes from Liberalism to Fascism
Authors: Morris Jonathan
Published: October 2002

This article argues that it was through their common relationship to the commercial environment formed by the city that the independent lower middle classes were able to construct a collective identity. The article examines these identities first in the context of a comparison between Rome and Milan, and then within the provincial towns of Lombardy. It suggests that the concern of the petite bourgeoisie towards their city and its administration lay at the heart of an ‘apolitical’ conception of politics that privileged their own interests as traders, taxpayers and citizens over those of other residents. The consequences of this are explored in a final section devoted to the immediate post-war era.
Report on ACIS Conference, ‘The Importance of Italy’, Humanities Research Centre, Australian National University, September 2001
Authors: White Jonathan
Published: October 2002

Italian programmes can now be taken in fteen of Australia's thirty-nine universities-a contraction from their offer in twenty-six universities in 1990. In order to promote collaboration among Italianists and Italian scholars in both Australia and New Zealand, the Australasian Centre for Italian Studies (ACIS) was established in 2000, under a management representing seven universities. ACIS’ work includes the organization of conferences (the next to be held at the University of Western Australia in July 2003), sponsorship of collaborative research projects and the award of annual scholarships for Honours and postgraduate students to work in Italy.
‘Transition in Italian Politics’, Italian Politics Specialist Group panels held at the Political Studies Association's 52nd Annual Conference, 5–7 April 2002, University of Aberdeen
Authors: Albertazzi Daniele
Published: October 2002

Despite its hardly enticing title (‘Making Politics Count’) and the equally uninspiring choice of topic for its plenary lecture (‘Political Science After September 11’), the 52nd Annual Conference of the Political Studies Association (PSA), hosted by the University of Aberdeen, was an apparent success: it stimulated interdisciplinar y discussion among an international audience while also providing an arena for the presentation of comparative perspectives on issues, such as European politics and devolution, which are at the forefront of academic interest.
Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells
Authors: Lenz Johannes C. , Reusch H. Peter , Albrecht Nadine , Schultz Günter , Schaefer Michael
Published: October 2002

The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKCα accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKCε was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKCε, but not of coexpressed PKCα, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKCε than for PKCα. Subsequent photolysis of caged Ca2+ immediately recruited PKCα to the membrane, and DAG-bound PKCε was displaced. At low expression levels of PKCε, PKCα concentration dependently prevented the PKCε translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation.
The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans
Authors: Jospin Maëlle , Jacquemond Vincent , Mariol Marie-Christine , Ségalat Laurent , Allard Bruno
Published: October 2002

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.
Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3
Authors: Heremans Yves , Van De Casteele Mark , in't Veld Peter , Gradwohl Gerard , Serup Palle , Madsen Ole , Pipeleers Daniel , Heimberg Harry
Published: October 2002

Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/β2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for β-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and β-cell–specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/β2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells.
Down-regulation of Delta by proteolytic processing
Authors: Mishra-Gorur Ketu , Rand Matthew D. , Perez-Villamil Beatriz , Artavanis-Tsakonas Spyros
Published: October 2002

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.
Chromosomes are predominantly located randomly with respect to each other in interphase human cells
Authors: Cornforth Michael N. , Greulich-Bode Karin M. , Loucas Bradford D. , Arsuaga Javier , Vázquez Mariel , Sachs Rainer K. , Brückner Martina , Molls Michael , Hahnfeldt Philip , Hlatky Lynn , Brenner David J.
Published: October 2002

To test quantitatively whether there are systematic chromosome–chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome–chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.
Crystal structure of the TSP-1 type 1 repeats
Authors: Tan Kemin , Duquette Mark , Liu Jin-huan , Dong Yicheng , Zhang Rongguang , Joachimiak Andrzej , Lawler Jack , Wang Jia-huai
Published: October 2002

Thrombospondin-1 (TSP-1) contains three type 1 repeats (TSRs), which mediate cell attachment, glycosaminoglycan binding, inhibition of angiogenesis, activation of TGFβ, and inhibition of matrix metalloproteinases. The crystal structure of the TSRs reported in this article reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end. The front face of the TSR contains a right-handed spiral, positively charged groove that might be the “recognition” face, mediating interactions with various ligands. This is the first high-resolution crystal structure of a TSR domain that provides a prototypic architecture for structural and functional exploration of the diverse members of the TSR superfamily.