bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of differences in mesangial cell phenotype between diabetic and normal rats: Role of culture passages
Authors: Ouardani Mohammed , Travo Pierre , Bastie Marie‐Jose´ , Mornet Dominique , Neff Ste´phanie , Leung‐Tack Jeanne
Published: July 2002

Summry— In mesangial cells (MC) isolated from streptozotocin (STZ)‐induced diabetic rat kidneys, sensitivity to bradykinin (BK) for the induction of cell division and collagen synthesis, was found to be lower than in normal MC. Nevertheless, decreased activities could be reverted in vitro by insulin, at non‐proliferative concentration (Girolami et al (1995), Can J Physiol Pharmacol 73, 848–853). The aim of the present study was to determine whether differences in the properties of diabetic MC could be ascribed to the diabetic state per se, and/or to experimental conditions, ie culture replating. Through successive cell replating, normal and diabetic types of MC were compared in terms of proliferation, contraction, free calcium concentration in response to KCl depolarization, and in relation to the expression of two cytoskeleton proteins specific to muscle cells, myosin and dystrophin. Studies of proliferation, contraction and free calcium concentration consistently showed that passage 5 was a limit beyond which differences between the two MC types were very small and sometimes non‐significant. We found that the mean maximum contraction (MMC) and especially the proportion of contractile cells (PCC) among diabetic cells was lower than in normal MC. In addition, loss in proliferation activity and in [Ca2+]i concentrations were also found to occur during these five early passages. Dystrophin, a new marker of contractile phenotype recently described in smooth muscle cell (Leis et al (1994) Cell Biol Toxicol 10, 305), was first localized in MC and was compared with myosin also expressed in MC. However, during the course of cell replating and/or with the diabetic state, no visible quantitative changes were detected in the expression of the two contractile proteins. We conclude that cultured mesangial cells undergo phenotype modulations, as observed in other cells, in particular smooth muscle cells and consequently, comparative studies between normal and diabetic MC should not be carried out after the 5th passage.
Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: Effects of maturation and a GABA agonist
Authors: Elster Lisbeth , Hansen Gert H. , Belhage Bo , Fritschy Jean M. , Möhler Hanns , Schousboe Arne
Published: July 2002

Quantitative analysis of the density of 1and β2/3 GABAA receptor subunits was performed at the electron microscope level after indirect pre‐embedding immunogold labeling with subunit‐specific antibodies of rat cerebellar granule cell cultures grown for 4 or 8 days and in the presence or absence of the GABAA receptor agonist 4,5,6,7‐tetrahydroisoxazolo[5,4c]pyridin‐3‐ol (THIP). THIP (150 μM) induced a 2‐fold increase in the number of α1and β2/3 subunits in both cell bodies and processes in 4‐day‐old cultures. Extending the culture period to 8 days led to a polarization of the receptor expression, since the increase in the number of subunits selectively was observed in the processes. Moreover, a general subcellular differentiation of the receptor population was observed in all culture conditions, since the ratio between the two subunits (β2/3;α1) was four times higher in cell bodies compared to processes. A detailed analysis of the less mature (4‐day‐old) cultures revealed the existence of two populations of neurons exhibiting differences in the average number of receptors. During maturation neurons with few receptors developed into cells with a higher density of receptors resulting in a single population of the latter neurons, a process enhanced by exposure to THIP. This may indicate that receptor development is a discontinuous process with individual neurons following different temporal patterns. In double‐labeling experiments, a spatially close association of the α1and β2/3 subunits could be seen, but the subunits were more frequently found separated from each other. In spite of the fact that exposure of the neurons to THIP increased the total number of receptor subunits, its presence apparently prevented formation of receptors with this subunit composition. Interestingly, receptor subunit clusters, consisting of α1 alone, were more frequently observed than composite (1and β2/3) clusters. This substantiates the view that receptors not having α1and β2/3 subunits in the same complex may exist.
Vav, a GDP/GTP nucleotide exchange factor, interacts with GDIs, proteins that inhibit GDP/GTP dissociation
Authors: Groysman Maya , Russek Chen Shifrin Niva , Katzav Shulamit
Published: July 2002

Vav functions as a specific GDP/GTP nucleotide exchange factor which is regulated by tyrosine phosphorylation in the hematopoietic system. Loss of the amino‐terminus sequences of Vav was sufficient to control its transforming potential and its function in T cells. We report here the identification of the hematopoietic GDP dissociation inhibitor protein, Ly‐GDI, as a protein that interacts with the amino‐terminus of Vav. Further analysis confirmed that Vav and Ly‐GDI interact both in in vitro and in in vivo assays. This association is maximal only when the amino region of Vav is intact and requires an intact carboxy‐terminus of Ly‐GDI. The interaction between Vav and Ly‐GDI is not dependent on the tyrosine phosphorylation status of Vav. In addition, Rho‐GDI, the highly homologous protein to Ly‐GDI, associates with Vav as well. The contribution of the interaction between Vav and GDIs, proteins that are involved in the GDP/GTP exchange processes, to the biological function of Vav is further discussed.
Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase
Authors: Balzarini Jan , Degrève Bart , Esteban-Gamboa Antonio , Esnouf Robert , De Clercq Erik , Engelborghs Yves , Camarasa Maria-José , Pérez-Pérez Maria-Jesús
Published: July 2002

A kinetic analysis was performed for the novel 1‐(8‐phosphonooctyl)‐6‐amino‐5‐bromouracil and 1‐(8‐phosphonooctyl)‐7‐deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6‐amino‐5‐bromouracil and 7‐deazaxanthine behaved as non‐competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1‐(8‐phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)‐ and phosphate‐binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.
Binding of coatomer by the PEX11 C‐terminus is not required for function
Authors: Maier Alexander G. , Schulreich Sebastian , Bremser Martina , Clayton Christine
Published: July 2002

Microbodies are single membrane‐bound organelles found in eukaryotes from trypanosomes to man. Although they have diverse roles in metabolism, the mechanisms and molecules involved in membrane biogenesis and matrix protein import are conserved. Similarly, the basic mechanisms and structures involved in vesicular transport are similar throughout eukaryotic evolution. The PEX11 proteins are required for the division of microbodies in trypanosomes, yeast and mammals, and a role of coatomer in this process has been suggested. We show here that the binding of trypanosome, yeast and bovine coatomers to selected peptides is identical. Coatomer binds to the C‐termini of trypanosome PEX11 and rat Pex11α, but not yeast Pex11p or human Pex11β. Mutations of the C‐terminus of trypanosome PEX11 that eliminated coatomer binding did not affect function in yeast or trypanosomes. Thus binding of coatomer to the C‐terminus of PEX11 is not required for PEX11 function.
Genomic Exploration of the Hemiascomycetous Yeasts: 2. Data generation and processing
Authors: Artiguenave François , Wincker Patrick , Brottier Phillippe , Duprat Simone , Jovelin Fabien , Scarpelli Claude , Verdier Jean , Vico Virginie , Weissenbach Jean , Saurin William
Published: July 2002

The generation of sequencing data for the hemiascomycetous yeast random sequence tag project was performed using the procedures established at GENOSCOPE. These procedures include a series of protocols for the sequencing reactions, using infra‐red labelled primers, performed on both ends of the plasmid inserts in the same reaction tube, and their analysis on automated DNA sequencers. They also include a package of computer programs aimed at detecting potential assignation errors, selecting good quality sequences and estimating their useful length.
Genomic Exploration of the Hemiascomycetous Yeasts: 9. Saccharomyces kluyveri
Authors: Neuvéglise Cécile , Bon Elisabeth , Lépingle Andrée , Wincker Patrick , Artiguenave François , Gaillardin C. , Casarégola Serge
Published: July 2002

The genome of Saccharomyces kluyveri was explored through 2528 random sequence tags with an average length of 981 bp. The complete nuclear ribosomal DNA unit was found to be 8656 bp in length. Sequences homologous to retroelements of the gypsy and copia types were identified as well as numerous solo long terminal repeats. We identified at least 1406 genes homologous to Saccharomyces cerevisiae open reading frames, with on average 58.1% and 72.4% amino acid identity and similarity, respectively. In addition, by comparison with completely sequenced genomes and the SwissProt database, we found 27 novel S. kluyveri genes. Most of these genes belong to pathways or have functions absent from S. cerevisiae, such as the catabolic pathway of purines or pyrimidines, melibiose fermentation, sorbitol utilization, or degradation of pollutants. The sequences are deposited in EMBL under the accession numbers AL404849–AL407376.
The substitution of the C‐terminus of bax by that of bcl‐xL does not affect its subcellular localization but abrogates its pro‐apoptotic properties
Authors: Oliver Lisa , Priault Muriel , Tremblais Karine , LeCabellec Marie-Thérèse , Meflah Khaled , Manon Stéphen , Vallette Francois M
Published: July 2002

The interaction of the anti‐apoptotic members of the Bcl‐2 family with mitochondria, through their hydrophobic C‐terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C‐terminal end of pro‐apoptotic bax by that of anti‐apoptotic bcl‐xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C‐terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.
In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast
Authors: Song Youtao , Azakami Hiroyuki , Hamasu Mika , Kato Akio
Published: July 2002

The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild‐type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N‐glycosylation signal sequence (Asn‐X‐Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non‐glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37°C whereas the non‐glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the β‐strand of amyloidogenic lysozymes from the intermolecular cross‐β‐sheet association, thus improving the solubility of amyloidogenic lysozymes.
Circadian and photic regulation of MAP kinase by Ras‐ and protein phosphatase‐dependent pathways in the chick pineal gland
Authors: Hayashi Yuichiro , Sanada Kamon , Fukada Yoshitaka
Published: July 2002

Chick pineal mitogen‐activated protein kinase (MAPK) exhibits circadian activation and light‐dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf‐1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras‐MAPK pathway in a circadian manner. In contrast, the light‐dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light‐dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.
Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids
Authors: Santabarbara S. , Neverov K.V. , Garlaschi F.M. , Zucchelli G. , Jennings R.C.
Published: July 2002

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment–protein complexes may be involved.
Anticoagulants and inhibitors of platelet aggregation derived from leeches
Authors: Salzet Michel
Published: July 2002

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.
Carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2α phosphorylation and translation inhibition in PC12 cells
Authors: Muñoz F. , Martı́n M.E. , Salinas M. , Fando J.L.
Published: July 2002

We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2‐fold increase in intracellular Ca2+ concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the α subunit of eIF2 (eIF2α) (25% vs. 7%, for FCCP‐treated and control cells, respectively) and a 1.7‐fold increase in the double‐stranded RNA‐dependent protein kinase activity. No changes in the PKR endoplasmic reticulum‐related kinase or eIF2α phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.
A putative proton binding site of plasma membrane H+‐ATPase identified through homology modelling
Authors: Bukrinsky Jens T. , Buch-Pedersen Morten J. , Larsen Sine , Palmgren Michael G.
Published: July 2002

We have used the 2.6 Å structure of the rabbit sarcoplasmic reticulum Ca2+‐ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647–655], to build models by homology modelling of two plasma membrane (PM) H+‐ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1. We propose that in both yeast and plant PM H+‐ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684AHA2/D730PMA1), and three backbone carbonyls in M4 (I282AHA2/I331PMA1, G283AHA2/I332PMA1 and I285AHA2/V334PMA1) comprise a binding site for H3O+, suggesting a previously unknown mechanism for transport of protons. Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C‐terminal auto‐inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region.
Mitochondrial uncoupling proteins and phylogenesis – UCP4 as the ancestral uncoupling protein
Authors: Hanák Petr , Ježek Petr
Published: July 2002

We searched for the previously defined uncoupling protein (UCP) signatures [Ježek, P. and Urbánková, E. (2000) IUBMB Life 49, 63–70] in genomes of Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and Arabidopsis thaliana. We identified four UCPs in Drosophila and one in Caenorhabditis or Dictyostelium as close relatives of human UCP4 (BMCP), but distant from UCP1, UCP2, UCP3, and two plant UCPs of Arabidopsis. But the third Arabidopsis UCP is the closest UCP4 relative. This suggests that UCP4 represents the ancestral UCP from which other mammalian and plant UCPs diverged. Speculations on UCP4 participation in apoptosis are thus supported by its early phylogenetic occurrence.
Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration
Authors: Nakayama Toru , Sato Takuya , Fukui Yuko , Yonekura-Sakakibara Keiko , Hayashi Hideyuki , Tanaka Yoshikazu , Kusumi Takaaki , Nishino Tokuzo
Published: July 2002

Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4‐monohydroxy or 3,4‐dihydroxy B‐ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3‐oxygenation. However, it exhibited virtually no PPO activity toward non‐chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO‐catalyzed reactions and conclude that the enzyme is a chalcone‐specific PPO specialized for aurone biosynthesis.
Vav is associated with signal transducing molecules gp130, Grb2 and Erk2, and is tyrosine phosphorylated in response to interleukin‐6
Authors: Lee Ihn-Sook , Liu Yin , Narazaki Masashi , Hibi Masahiko , Kishimoto Tadamitsu , Taga Tetsuya
Published: July 2002

Vav is a hematopoietic cell‐specific proto‐oncogene. We show that interleukin‐6 (IL‐6) induces transient tyrosine phosphorylation of Vav in a human myeloma cell line, U266. A membrane‐distal part of the cytoplasmic region of gp130 is critical for association between Vav and gp130, and the IL‐6‐induced tyrosine phosphorylation of Vav. Mitogen‐activated protein kinase (MAPK) (p42MAPK or extracellular signal‐regulated kinase 2 (Erk2)) is coprecipitated with Vav. MAPK activity in the anti‐Vav immunoprecipitates is upregulated by IL‐6 stimulation. Furthermore Vav is associated with Grb2 which is known as an adapter protein leading to Ras activation. The results imply that Vav may link gp130 activation to downstream MAPK activation in hematopoietic cells.
Cooperative and competitive interactions of regulatory elements are involved in the control of divergent transcription of human Col4A1 and Col4A2 genes
Authors: Pollner Reinhold , Schmidt Cornelia , Fischer Gudrun , Kühn Klaus , Pöschl Ernst
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Reconstitution in vitro of the components and conditions required for the oxidative cross‐linking of extracellular proteins in French bean (Phaseolus vulgaris L.)
Authors: Wojtaszek Przemyslaw , Trethowan Jonathan , Bolwell G.Paul
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A novel putative G‐protein‐coupled receptor expressed in lung, heart and lymphoid tissue
Authors: Kvingedal Ane M , Smeland Erlend B
Published: July 2002

cDNA encoding a novel putative G‐protein‐coupled receptor, named LyGPR (
mphocyte derived
‐
rotein‐coupled
eceptor) was cloned using a reverse transcription‐PCR approach. The LyGPR amino acid sequence is 375 residues long and shows similarity (about 30–35% identity) both to the angiotensin receptors and members of the chemokine receptor family. Northern blot analysis revealed a 3.1‐kb LyGPR transcript expressed predominantly in lung, heart and lymphoid tissues. LyGPR expression was also detected in the pre‐B acute lymphoblastoid leukemia cell lines Reh and Nalm‐6, in the Burkitt's lymphoma line Daudi, and in hematopoietic progenitor cells from bone marrow, as well as in B cells, T cells and monocytes from peripheral blood.
Biochemistry and genetics of inositol phosphate metabolism in Dictyostelium
Authors: van Haastert Peter J.M , van Dijken Peter
Published: July 2002

Biochemical and genetic data on the metabolism of inositol phosphates in the microorganism Dictyostelium are combined in a scheme composed of in five subroutes. The first subroute is the inositol cycle as found in other organisms: inositol is incorporated into phospholipids that are hydrolysed by PLC producing Ins(1,4,5)P3 which is dephosphorylated to inositol. The second subroute is the sequential phosphorylation of inositol to InsP6; the Ins(3,4,6)P3 intermediate does not release Ca2+. The third subroute is the sequential phosphorylation of Ins(1,4,5)P3 to InsP6 in a nucleus associated fraction, whereas the fourth subroute is the dephosphorylation of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 at the plasma membrane. This last route mediates Ins(1,4,5)P3 formation in cells with a disruption of the single PLC gene. Finally, we recognize the formation of InsP7 and InsP8 as the fifth subroute.
Transfer of CD26/dipeptidyl peptidase IV (E.C. 3.5.4.4) from prostasomes to sperm
Authors: Arienti Giuseppe , Polci Andrea , Carlini Enrico , Palmerini Carlo A
Published: July 2002

Prostasomes are vesicles present in human semen. They are secreted by the prostate and contain large amounts of cholesterol and sphingomyelin. Some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous papers we demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at neutral or slightly acidic pH. In this paper we demonstrate that CD26/dipeptidyl peptidase IV, an enzymatic activity absent in sperm, is transferred to sperm from prostasomes. This may be of particular interest since, by this procedure, sperm may acquire new membrane‐bound enzymes and modify the catalytic activity of their surface.
The origin and utility of histone deacetylases
Authors: Khochbin Saadi , Wolffe Alan P
Published: July 2002

A large region of two distinct yeast histone deacetylases, RPD3 and HDA1, is highly homologous to several prokaryotic enzymes that catalyze reactions involving various acetylated substrates. Proteins sharing this homology domain are found also in many higher eukaryotes and they all appear to be related to the RPD3 family of histone deacetylases. In each member of the family, the `prokaryotic homology' domain covers almost two thirds of the protein, with the remaining portion containing the most divergent sequences. These sequences are located at the C‐terminal region allowing for a clear definition of variants. Since the involvement of deacetylase members in different distinct regulatory complexes is now well established, the above observation suggests that the C‐terminal domain may confer specificity to different members of the family. The RPD3 histone deacetylases thus appear as members of a family with a large conserved domain involved in enzymatic activity targeted to a short C‐terminal domain, which probably confers functional specificity. The potential for deacetylases to be involved in multiple regulatory pathways provides an attractive counterpoint to the role of multiple histone acetyltransferases as coactivators.
Protection against apoptosis by monoamine oxidase A inhibitors
Authors: Malorni W , Giammarioli A.M , Matarrese P , Pietrangeli P , Agostinelli E , Ciaccio A , Grassilli E , Mondovi' B
Published: July 2002

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO‐A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.
Expression of Hex mRNA in early murine postimplantation embryo development
Authors: Keng Vincent W. , Fujimori Kazuhiro E. , Myint Zaw , Tamamaki Nobuaki , Nojyo Yoshiaki , Noguchi Tamio
Published: July 2002

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.
Primary structure of a potassium channel toxin from the sea anemone Actinia equina
Authors: Minagawa Sonomi , Ishida Masami , Nagashima Yuji , Shiomi Kazuo
Published: July 2002

A potassium channel toxin (AeK) was isolated from the sea anemone Actinia equina by gel filtration on Sephadex G‐50 and reverse‐phase HPLC on TSKgel ODS‐120T. AeK and α‐dendrotoxin inhibited the binding of 125I‐α‐dendrotoxin to rat synaptosomal membranes with IC50 of 22 and 0.34 nM, respectively, indicating that AeK is about sixty‐five times less toxic than α‐dendrotoxin. The complete amino acid sequence of AeK was elucidated; it is composed of 36 amino acid residues including six half‐Cys residues. The determined sequence showed that AeK is analogous to the three potassium channel toxins from sea anemones (BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS from Anemonia sulcata), with an especially high sequence homology (86%) with AsKS.
APO2 ligand: a novel lethal weapon against malignant glioma?
Authors: Rieger Johannes , Naumann Ulrike , Glaser Tamara , Ashkenazi Avi , Weller Michael
Published: July 2002

APO2L (TRAIL) is a novel CD95L (Fas/APO‐1‐L) homologous cytotoxic cytokine that interacts with various receptors which transmit (DR4, DR5) or inhibit (DcR1, DcR2) an apoptotic signal. Here, we report that human glioma cell lines preferentially express mRNAs for agonistic death receptors DR4 (8/12) and DR5 (11/12) rather than the death‐inhibitory decoy receptors DcR1 (4/12) and DcR2 (2/12). Ten of 12 cell lines are susceptible to APO2L‐induced apoptosis. The resistant cell lines, U138MG and U373MG, are cross‐resistant to CD95L‐induced apoptosis. Similar to CD95L‐induced apoptosis, APO2L‐induced apoptosis is inhibited by ectopic expression of the caspase inhibitor, crm‐A, or of bcl‐2, or by coexposure to the corticosteroid, dexamethasone, or the lipoxygenase inhibitor, nordihydroguaretic acid. There is no correlation between p53 genetic status of the cell lines and their susceptibility to APO2L‐induced apoptosis, but the latter is moderately enhanced by ectopic expression of wild‐type p53. APO2L targeting may be a promising approach for selectively targeting apoptosis to human malignant glioma cells.
Pervanadate inhibits mitogen‐activated protein kinase kinase‐1 in a p38MAPK‐dependent manner
Authors: Daum Günter , Kalmes Andreas , Levkau Bodo , Wang Yunxia , Davies Mark G , Clowes Alexander W
Published: July 2002

In baboon smooth muscle cells (SMCs), pervanadate has a biphasic dose‐dependent effect on MEK‐1 activity. After a 30 min incubation period, low concentrations (1–10 μM) activate, while higher doses (30–100 μM) fail to stimulate MEK‐1. One possibility is that higher doses of pervanadate induce an additional signaling pathway that inhibits MEK‐1. Three lines of investigations provide support for the conclusion that this inhibitory effect is mediated by p38MAPK. First, pervanadate induces p38MAPK activity at concentrations that fail to activate MEK‐1. Second, pervanadate‐stimulated p38MAPK activity is maximal after a 10 min incubation, at a time, when MEK‐1 activity disappears. Third, addition of the specific p38MAPK inhibitor SB203580 preserves MEK‐1 activation by 100 μM pervanadate. The inhibitory effect of p38MAPK is probably not due to a phosphorylation of MEK‐1 although we can not rule out that other p38MAPK isoforms such as SAPK3 and SAPK4 may be involved, and may directly phosphorylate and inhibit MEK‐1.
Functional genomics: the search for novel neurotransmitters and neuropeptides
Authors: Civelli Olivier
Published: July 2002

Functional genomics can be defined as the search for the physiological role of a gene for which only its primary sequence is known. Most of the genes encoding proteins containing seven hydrophobic stretches code for G protein‐coupled receptors (GPCRs). Although many of these have been shown to interact with known natural ligands, several bind ligands which have not been thus far isolated. These are the so‐called orphan GPCRs. As an example of functional genomics, an `orphan receptor strategy' has been developed to identify the natural ligands of orphan GPCRs. The application of this strategy is bound to revolutionize our understanding of the diversity of the primary messengers which modulate synaptic transmission. This review discusses the basic concepts and some of the particular problems associated with the orphan receptor strategy. The strategy's potential is exemplified by its successes which culminated in the discovery of the neuropeptides `orphanin FQ/nociceptin' and `orexins/hypocretins'. The steps that led to the characterization of these neuropeptides are discussed as are some of the further studies that have addressed the roles of these neuropeptides. To conclude, some of the implications of the application of the orphan receptor strategy are discussed.
Potentiation of nitric oxide synthase expression by superoxide in interleukin 1β‐stimulated rat mesangial cells
Authors: Beck Karl-Friedrich , Eberhardt Wolfgang , Walpen Sebastian , Apel Martina , Pfeilschifter Josef
Published: July 2002

Exposure of mesangial cells to superoxide, generated by the hypoxanthine/xanthine oxidase system or by the redox cycler 2,3‐dimethoxy‐1,4‐naphthoquinone caused a concentration‐dependent amplification of interleukin (IL)‐1β‐stimulated nitrite production. The effect of superoxide was accompanied by an increase in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA levels. Incubation of mesangial cells with superoxide alone did not induce iNOS expression. To elucidate whether the increase of iNOS expression is due to transcriptional upregulation we fused a 4.5‐kb genomic iNOS fragment that contains the transcriptional start site of the rat iNOS gene to a luciferase reporter gene. In transient transfection studies, superoxide caused a 10‐fold augmentation of iNOS promoter activity in IL‐1β‐challenged mesangial cells. Our data identify superoxide as a co‐stimulatory factor amplifying cytokine‐induced iNOS gene expression and subsequent nitric oxide (NO) synthesis.
Simulated microgravity inhibits the genetic expression of interleukin‐2 and its receptor in mitogen‐activated T lymphocytes
Authors: Walther Isabelle , Pippia Proto , Meloni Maria Antonia , Turrini Franco , Mannu Franca , Cogoli Augusto
Published: July 2002

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin‐2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin‐2 and of its receptor in concanavalin A‐activated lymphocytes with the RT‐PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL‐2 and IL‐2Rα genes is significantly inhibited in simulated 0×g. Thus full activation is prevented.
Angiotensinogen cleavage by renin: importance of a structurally constrained N‐terminus
Authors: Streatfeild-James Rosa M.A , Williamson David , Pike Robert N , Tewksbury Duane , Carrell Robin W , Coughlin Paul B
Published: July 2002

Angiotensinogen, a plasma serpin, functions as a donor of the decapeptide angiotensin I, which is cleaved from the N‐terminus by renin. To assess the contribution of the serpin framework to peptide cleavage we produced a chimaeric molecule of α1‐antitrypsin carrying the angiotensinogen N‐terminus and determined the kinetic parameters for angiotensin I release. The K
m for plasma angiotensinogen was 18‐fold lower than for the chimaeric protein while the catalytic efficiency was four‐fold higher. We also show that Cys‐18 participates in a disulphide bond and propose that constraints on the N‐terminus profoundly affect the interaction with renin.
Synthetic as compared with natural vitamin E is preferentially excreted as α‐CEHC in human urine: studies using deuterated α‐tocopheryl acetates
Authors: Traber Maret G. , Elsner Angelika , Brigelius-Flohé Regina
Published: July 2002

α‐CEHC (2,5,7,8‐tetramethyl‐2(2′‐carboxyethyl)‐6‐hydroxychroman) is a urinary vitamin E metabolite with a truncated phytyl tail. To test whether natural and synthetic vitamin E are similarly converted to α‐CEHC, 6 humans consumed 150 mg each RRR‐α‐[5‐(C2H3)]‐ and all rac‐α‐[5,7(C2H3)2]‐tocopheryl acetates (d3
RRR‐α‐ and d6
all rac‐α‐tocopheryl acetates, respectively). Plasma was collected at 0, 6, 12 and 24 h; urine (24 h) at 0, 1, 2, 3, 4 and 8 days. Following dosing, plasma was enriched with d3
RRR‐α‐tocopherol, while urine was enriched with α‐CEHC derived from d6
all rac‐α‐tocopherol. Thus, synthetic compared with natural vitamin E is preferentially metabolized to α‐CEHC and excreted.
Taraxalisin – a serine proteinase from dandelion Taraxacum officinale Webb s.l
Authors: Rudenskaya G.N. , Bogacheva A.M. , Preusser A. , Kuznetsova A.V. , Dunaevsky Ya.E. , Golovkin B.N. , Stepanov V.M.
Published: July 2002

Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp‐Ala‐Ala‐Leu‐pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose‐Ala‐Ala‐Leu‐mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6–9 in the presence of 2 mM Ca2+, temperature optimum is at 40°C; K
m=0.37±0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B‐chain is comparable with that of two other subtilisin‐like serine proteinases, cucumisin and macluralisin. The taraxalisin N‐terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.
The helix‐loop‐helix transcription factors Id1 and Id3 have a functional role in control of cell division in human normal and neoplastic chondrocytes
Authors: Asp Julia , Thornemo Maria , Inerot Sven , Lindahl Anders
Published: July 2002

The expression and localization of the helix‐loop‐helix transcription factors Id1 and Id3, as well as localization of the E12‐protein, were studied in cells isolated from human articular cartilage and chondrosarcoma. Serum withdrawal down‐regulated Id1 and Id3 expression in chondrocytes but not the Id1 expression in chondrosarcoma cells. Antisense oligonucleotides directed against Id1 and Id3 decreased BrdU labeling in both cell types. E12 was localized to the nucleus in chondrocytes and non‐confluent tumor cells and in confluent tumor cells, E12 had a cytoplasmic localization. This study suggests a functional role for Id1 and Id3 in the control of proliferation and differentiation of cartilage.
Oxidative injury and survival during endotoxemia
Authors: Basu S , Eriksson M
Published: July 2002

This study investigates the plasma levels of 8‐iso‐PGF2α, a non‐enzymatic, and 15‐K‐DH‐PGF2α, a cyclooxygenase catalyzed oxidation product of arachidonic acid in an experimental porcine endotoxemic shock model. A significant (P<0.001) and rapid appearance and disappearance of PGF2α metabolite after endotoxin infusion was very similar in both non‐survival and survival groups indicating an acute progression and recession of inflammation. When oxidative injury was assessed by measuring free 8‐iso‐PGF2α the levels in plasma increased significantly up to 2 h and remained at this level until death among the non‐survivors. This was apparently different from the survivors where the 8‐iso‐PGF2α levels increased to its height at 1 h, then decreased to the basal levels after 5 h. Thus, free radical and cyclooxygenase catalyzed oxidation of arachidonic acid occurs during endotoxemia. Free radical dependent oxidative injury following endotoxin induced inflammation may be the major cause of organ failure and increased mortality.
Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney
Authors: Uwai Yuichi , Okuda Masahiro , Takami Kenji , Hashimoto Yukiya , Inui Ken-ichi
Published: July 2002

The functional characteristics of rat organic anion transporter OAT1 were investigated using Xenopus laevis oocytes. Uptake of p‐aminohippurate (PAH) by the oocytes expressing OAT1 was markedly inhibited by glutarate, α‐ketoglutarate and probenecid, moderately inhibited by folate and methotrexate, but not inhibited by taurocholate or tetraethylammonium. Methotrexate and folate were transported by OAT1, but probenecid, a typical inhibitor of organic anion transporter, was not transported. Inhibition of PAH uptake by aliphatic dicarboxylates with various alkyl chain lengths was maximal at 5 (glutarate) and 6 (adipate) carbon atoms. OAT1‐mediated PAH uptake was markedly inhibited by phorbol 12‐myristate 13‐acetate (PMA), phorbol 12,13‐dibutyrate and mezerein, but not by 4α‐phorbol 12,13‐didecanoate. The inhibitory effect of PMA was attenuated in the presence of staurosporine, suggesting that OAT1 is regulated by protein kinase C. These results suggest that the substrate recognition of OAT1 is comparable to that of renal basolateral organic anion transporter, and the transport activity is regulated by protein kinase C.
Functional identification of a fatty acid Δ5 desaturase gene from Caenorhabditis elegans
Authors: Michaelson Louise V. , Napier Johnathan A. , Lewis Mervyn , Griffiths Gareth , Lazarus Colin M. , Stobart A.Keith
Published: July 2002

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid Δ5 desaturase. Saccharomyces cerevisiae expressing the full‐length cDNA was able to convert di‐homo‐γ‐linolenic acid to arachidonic acid, thus confirming Δ5 desaturation. The 1341 bp Δ5 desaturase sequence contained an N‐terminal cytochrome b
5 domain and was located within a kilobase of the C. elegans Δ6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a Δ5 desaturase gene isolated from an animal.
Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens
Authors: Kawasaki Yoshimi , Fujii Junichi , Miyazawa Nobuko , Hoshi Ayumu , Okado Ayako , Tano Yasuo , Taniguchi Naoyuki
Published: July 2002

The glycation reaction by fructose, as well as that by glucose, in control and diabetic rat lens was analyzed by using antibodies which specifically recognize adducts of lysine with fructose and with glucose. Levels of fructose adducts in diabetic rat lens were 2.5 times that of the control, and correlated with sorbitol levels. This was mainly due to enhanced glycation of β‐ and γ‐crystallins by fructose under diabetic conditions. These data suggest that glycation by fructose may also play a role in cataract formation under conditions of diabetes and aging.
Identification of caspases that cleave presenilin‐1 and presenilin‐2
Authors: van de Craen Marc , de Jonghe Chris , van den Brande Ilse , Declercq Wim , van Gassen Geert , van Criekinge Wim , Vanderhoeven Inge , Fiers Walter , van Broeckhoven Christine , Hendriks Lydia , Vandenabeele Peter
Published: July 2002

Mutations in the presenilin (PS) genes PS1 and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis‐associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase‐8, ‐6 and ‐11, cleaved PS1 after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase‐3, ‐7 and ‐1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C‐terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase‐8, ‐3, ‐1, ‐6 and ‐7 proteolysed PS2 at the recognition site D326SYD329. Caspase‐8 and ‐3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase‐2 and PS2 also not by caspase‐11. None of five missense mutations affected the sensitivity of PS1 to caspase‐mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase‐dependent processing.
MDM2 interacts with MDMX through their RING finger domains
Authors: Tanimura Shyu , Ohtsuka Satoshi , Mitsui Kaoru , Shirouzu Kazuo , Yoshimura Akihiko , Ohtsubo Motoaki
Published: July 2002

The N‐terminus of MDM2 proto‐oncoprotein interacts with p53 and down modulates p53 activity by inhibiting transcriptional activity and promoting p53 degradation. MDMX is structurally related to MDM2 and also binds to p53. However, the function of MDMX has not been clarified yet. We found that MDM2 hetero‐oligomerized with MDMX through their C‐terminal RING finger domains. Yeast two‐hybrid analysis revealed that the hetero‐oligomerization between MDMX and MDM2 was more stable than the homo‐oligomerization of each protein. MDM2 has been shown to be degraded by the ubiquitin‐proteasome pathway, while MDMX was a stable protein. Interaction of MDMX with MDM2 through the C‐terminal RING finger domains resulted in inhibiting degradation of MDM2. These data indicate that MDMX functions as a regulator of MDM2.
H (0) blood group determinant is present on soluble human L‐selectin expressed in BHK‐cells
Authors: Gohlke Martin , Mach Ulrich , Nuck Rolf , Volz Barbara , Fieger Claudia , Tauber Rudolf , Reutter Werner
Published: July 2002

In the present study we show that the H (0) blood group determinant Fucα1‐2Galβ1‐4GlcNAcβ1‐R is present on N‐linked glycans of soluble human L‐selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fucα1‐2Galβ1‐4GlcNAcβ1 was detected for bi‐, 2,4 branched tri‐ and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.
Differential expression of β1,3galactosyltransferases in human colon cells derived from adenocarcinomas or normal mucosa1
Authors: Bardoni Anna , Valli Maurizia , Trinchera Marco
Published: July 2002

Two β1,3galactosyltransferases are detected in human colon cells: one corresponds to β3GalT1, the other (β3GalTx) is found to be different from any cloned β3GalT since in vitro it utilizes GlcNAc very efficiently under specific reaction conditions. Expression of β3GalT1 transcript is high in normal colon mucosa and control neuroectodermal cells, which do not express sialyl‐Lewis a antigen, and low in colon adenocarcinoma cells, as assessed by competitive RT‐PCR. β3GalTx activity is high in adenocarcinoma cells expressing sialyl‐Lewis a and undetectable in all other cells, suggesting differential involvement and opposite regulation of such enzymes during carcinogenesis.
14‐3‐3 proteins control proteolysis of nitrate reductase in spinach leaves
Authors: Weiner Hendrik , Kaiser Werner M
Published: July 2002

To test a possible role of 14‐3‐3 proteins in the degradation of nitrate reductase (NR) in leaves, we monitored 14‐3‐3s bound to NR in leaf extracts. The amount of 14‐3‐3s that coimmunoprecipitated with serine 543 phospo‐NR (p‐NR) increased upon a light/dark transition. This was accompanied by a similar increase in the protein turnover rate of NR in leaves. Purified NR was degraded in extracts from darkened but not from illuminated leaves. Removal of 14‐3‐3s from such extracts prevented NR degradation. We conclude that the availability of 14‐3‐3s for p‐NR regulates the stability of NR.
A gene family encoding glutathione peroxidase homologues in Hordeum vulgare (barley)
Authors: Churin Yuri , Schilling Silke , Börner Thomas
Published: July 2002

We have isolated and characterised three barley cDNAs encoding glutathione peroxidase (GPX) homologues, designated HVGPH1, HVGPH2 and HVGPH3. HVGPH1 may represent a cytosolic form of GPX. The structure of the HVGPH2 N‐terminal domain is typical for a plastid transit peptide. A potential peroxisomal targeting sequence occurs near the N‐terminus of HVGPH3. Transcript levels of HVGPH1 and HVGPH2 were increased in leaves undergoing stress. In contrast, HVGPH3 mRNA accumulation showed a negative response to stress. Our data indicate that the barley genome bears a small gene family encoding GPX homologues differing in their function and cellular localisation.