bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in troglitazone action on glucose metabolism in freshly isolated vs long‐term incubated rat skeletal muscle
Authors: Gras Florian , Brunmair Barbara , Roden Michael , Waldhäusl Werner , Fürnsinn Clemens
Published: April 2003

Exposure of isolated skeletal muscle to troglitazone has resulted in inconsistent findings ranging from inhibition to stimulation of fuel oxidation and the glycogenic pathway. To better understand such variation in outcome, the present study used isolated rat soleus muscle strips to examine the interdependent influences of prolonged maintenance in vitro and of troglitazone exposure.

If freshly isolated muscle strips were exposed to troglitazone (1 μmol l−1) for 24 h, glucose oxidation was markedly reduced (−26±1%, P<0.0001), whereas glycogen synthesis remained unaffected (+9±7%, n.s.).

In contrast, extended exposure to troglitazone for 72 h increased both glucose oxidation (+65±28%, P<0.05) and glycogen synthesis (+46±11%, P<0.005), and a similar stimulatory effect was also observed in muscles exposed to troglitazone only during the last 24 h of their 72 h preincubation period (glucose oxidation: +61±15%, P<0.001; glycogen synthesis: +43±15%, P<0.01).

Troglitazone thus stimulated glucose utilization in long‐term incubated muscle independent of the duration of exposure (24 or 72 h), whereas it inhibited glucose utilization in freshly isolated muscle.

The observed differences in troglitazone action on freshly isolated vs long‐term incubated muscle suggest that findings on muscle tissue subject to prolonged maintenance in vitro cannot be extrapolated to native muscle in vivo.

British Journal of Pharmacology (2003) 138, 1140–1146. doi:10.1038/sj.bjp.0705162
Stress‐responsive JNK mitogen‐activated protein kinase mediates aspirin‐induced suppression of B16 melanoma cellular proliferation
Authors: Ordan Orly , Rotem Ronit , Jaspers Ilona , Flescher Eliezer
Published: April 2003

Available anticancer drugs do not seem to modify the prognosis of metastatic melanoma. Salicylate and acetyl salicylic acid (aspirin) were found to suppress growth in a number of transformed cells, that is, prostate and colon. Therefore, we studied the direct effects of aspirin on metastatic B16 melanoma cells.

Aspirin at a plasma‐attainable and nontoxic level suppressed the proliferation of B16 cells.

Aspirin induced the activation of p38 and c‐Jun N‐terminal kinase (JNK) mitogen‐activated protein kinases.

Inhibition of JNK, but not p38, decreased the suppressive effect of aspirin upon the proliferation of B16 cells.

The aspirin‐induced reduction in B16 proliferation was cumulative over time.

Aspirin and the chemotherapeutic drug 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) induced B16 cell death synergistically.

In addition to the murine B16 cell line, the proliferation of SK‐28 human melanoma cells was also suppressed by aspirin.

In conclusion, aspirin suppresses the proliferation of metastatic B16 cells in a JNK‐dependent mechanism.

British Journal of Pharmacology (2003) 138, 1156–1162. doi:10.1038/sj.bjp.0705163
Inhibition of EDHF by two new combinations of K+‐channel inhibitors in rat isolated mesenteric arteries
Authors: Hinton Jane M , Langton Philip D
Published: April 2003

It is widely established that in rat mesenteric arteries, endothelium‐derived hyperpolarizing factor (EDHF)‐mediated relaxation evoked by acetylcholine is abolished by a combination of charybdotoxin plus apamin. 4‐Aminopyridine, an inhibitor of voltage‐gated (Kv) K+‐channels, in combination with apamin had moderate effects on the EDHF‐mediated relaxation. Maurotoxin (MTX), an inhibitor of Kv and intermediate‐conductance Ca2+‐activated K+‐channels (IK), had no effect on EDHF‐mediated relaxation. However, MTX in combination with apamin completely abolished EDHF‐mediated relaxation and endothelial cell hyperpolarization. The selective IK inhibitor 2‐(2‐chlorophenyl)‐2,2‐diphenyl acetonitrile (TRAM‐39) had no significant effect on EDHF‐mediated relaxation. EDHF‐mediated vasorelaxation and hyperpolarization was abolished by a combination of TRAM‐39 and apamin. These data demonstrate two new combinations of K+‐channel inhibitors for the investigation of EDHF. Furthermore, by using TRAM‐39, a potent selective inhibitor of IK channels, we provide the first direct evidence that abolition of EDHF requires the simultaneous presence of intermediate‐ and small‐conductance Ca2+‐activated K+‐channel inhibitors.

British Journal of Pharmacology (2003) 138, 1031–1035. doi:10.1038/sj.bjp.0705171
Inhibition of G protein‐activated inwardly rectifying K+ channels by fluoxetine (Prozac)
Authors: Kobayashi Toru , Washiyama Kazuo , Ikeda Kazutaka
Published: April 2003

The effects of fluoxetine, a commonly used antidepressant drug, on G protein‐activated inwardly rectifying K+ channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays.

In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration‐dependence, a weak voltage‐dependence and a slight time‐dependence with a predominant effect on the instantaneous current elicited by voltage pulses and followed by slight further inhibition. Furthermore, in oocytes expressing GIRK1/2 channels and the cloned Xenopus A1 adenosine receptor, GIRK current responses activated by the receptor were inhibited by fluoxetine. In contrast, ROMK1 and IRK1 channels in other Kir channel subfamilies were insensitive to fluoxetine.

The inhibitory effect on GIRK channels was not obtained by intracellularly applied fluoxetine, and not affected by extracellular pH, which changed the proportion of the uncharged to protonated fluoxetine, suggesting that fluoxetine inhibits GIRK channels from the extracellular side.

The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine.

We demonstrate that fluoxetine, at low micromolar concentrations, inhibits GIRK channels that play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate through activation of various G‐protein‐coupled receptors. The present results suggest that inhibition of GIRK channels by fluoxetine may contribute to some of its therapeutic effects and adverse side effects, particularly seizures in overdose, observed in clinical practice.

British Journal of Pharmacology (2003) 138, 1119–1128. doi:10.1038/sj.bjp.0705172
Long‐patch DNA repair synthesis during base excision repair in mammalian cells
Authors: Sattler Ulrike , Frit Philippe , Salles Bernard , Calsou Patrick
Published: April 2003

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub‐pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either ‘short‐patch’ BER (one nucleotide) or ‘long‐patch’ BER (LP‐BER; more than one nucleotide). To investigate the occurrence of LP‐BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single‐nucleotide mismatch, at varying distances on the 3′ side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch‐repair‐deficient cells. Repair patches longer than one nucleotide were observed for 55–80% or 80–100% of the plasmids with a mean length of 2–6 or 6–12 nucleotides for 8‐oxo‐7,8‐dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP‐BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP‐BER sub‐pathway.
Glycopeptide specificity of the secretory protein folding sensor UDP–glucose glycoprotein:glucosyltransferase
Authors: Taylor Sean C , Thibault Pierre , Tessier Daniel C , Bergeron John J M , Thomas David Y
Published: April 2003

Secretory and membrane N‐linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N‐linked Man9‐GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9‐GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino‐acid residues close to N‐linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.
Monitoring global messenger RNA changes in externally controlled microarray experiments
Authors: van de Peppel Jeroen , Kemmeren Patrick , van Bakel Harm , Radonjic Marijana , van Leenen Dik , Holstege Frank C P
Published: April 2003

Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.
Differential oxidative damage and expression of stress defence regulons in culturable and non‐culturable Escherichia coli cells
Authors: Desnues Benoît , Cuny Caroline , Grégori Gérald , Dukan Sam , Aguilaniu Hugo , Nyström Thomas
Published: April 2003

Potentially pathogenic bacteria, such as Escherichia coli and Vibrio cholerae, become non‐culturable during stasis. The analysis of such cells has been hampered by difficulties in studying bacterial population heterogeneity. Using in situ detection of protein oxidation in single E. coli cells, and using a density‐gradient centrifugation technique to separate culturable and non‐culturable cells, we show that the proteins in non‐culturable cells show increased and irreversible oxidative damage, which affects various bacterial compartments and proteins. The levels of expression of specific stress regulons are higher in non‐culturable cells, confirming increased defects relating to oxidative damage and the occurrence of aberrant, such as by amino‐acid misincorporation, proteins. Our data suggest that non‐culturable cells are produced due to stochastic deterioration, rather than an adaptive programme, and pinpoint oxidation management as the ‘Achilles heel’ of these cells.
ORFB is a subunit of F1 FO‐ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower
Authors: Sabar Mohammed , Gagliardi Dominique , Balk Janneke , Leaver Christopher J
Published: April 2003

ORFB is the product of a gene that is conserved in plant mitochondrial genomes, and which, on the basis of sequence motif and structural similarity, is predicted to be the homologue of yeast and mammalian ATP8, part of the FO component of the F1 FO‐ATP synthase. We have shown that, in sunflower, orfB transcripts are edited, increasing the similarity of the predicted protein to ATP8 proteins from non‐plant species. Blue‐native polyacrylamide gel electrophoresis and peptide sequencing confirm that ORFB localizes to the ATP synthase complex. The predicted amino‐terminal 19 amino acids of ORFB are identical to those in the chimeric mitochondrial ORF522 protein, which is associated with cytoplasmic male sterility (CMS) in sunflower. Assays comparing respiratory complexes from a male‐sterile line expressing ORF522 with those from a male‐fertile line show a specific decrease in ATP hydrolysis by the ATP synthase. These observations allow us to propose a mechanism underlying CMS that is associated with the expression of chimeric open reading frames containing part of the orfB gene.
Integrin‐linked kinase regulates chondrocyte shape and proliferation
Authors: Grashoff Carsten , Aszódi Attila , Sakai Takao , Hunziker Ernst B , Fässler Reinhard
Published: April 2003

The interaction of chondrocytes with the extracellular‐matrix environment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin‐linked kinase (Ilk). Ilk binds the cytoplasmic domain of β1‐, β2‐ and β3‐integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3‐β (GSK3‐β). Here, we show that mice with a chondrocyte‐specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory distress. The chondrodysplasia was characterized by abnormal chondrocyte shape and decreased chondrocyte proliferation. In addition, Ilk‐deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3‐β is unaffected in Ilk‐deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3‐β.
Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons
Authors: Pilz Andreas , Ramsauer Katrin , Heidari Hamid , Leitges Michael , Kovarik Pavel , Decker Thomas
Published: April 2003

Stat1 (signal transducer and activator of transcription 1) regulates transcription in response to the type I interferons IFN‐α and IFN‐β, either in its dimerized form or as a subunit of the interferon‐stimulated gene factor 3 (Isgf3) complex (consisting of Stat1, Stat2 and interferon‐regulating factor 9). Full‐length Stat1‐α and the splice variant Stat1‐β, which lacks the carboxyl terminus and the Ser727 phosphorylation site, are found in all cell types. IFN‐induced phosphorylation of Stat1‐α on Ser727 occurs in the absence of the candidate kinase, protein kinase C‐δ. When expressed in Stat1‐deficient cells, Stat1‐β and a Stat1‐S727A mutant both restored the formation of Stat1 dimers and of the Isgf3 complex on treatment with IFN‐β. By contrast, only Stat1‐α restored the ability of IFN‐β to induce high levels of transcription from target genes of Stat1 dimers and Isgf3 and to induce an antiviral state. Our data suggest an important contribution of the Stat1 C terminus and its phosphorylation at Ser727 to the transcriptional activities of the Stat1 dimer and the Isgf3 complex.
Impaired gametogenesis in mice that overexpress the RNA‐binding protein HuR
Authors: Levadoux‐Martin Marilyne , Gouble Agnès , Jégou Bernard , Vallet‐Erdtmann Virginie , Auriol Jacques , Mercier Pascale , Morello Dominique
Published: April 2003

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA‐binding protein HuR increases the half‐life of some messenger RNAs that contain adenylate/uridylate‐rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.
Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains
Authors: Yeung Man‐Lung , Tam Tammy S M , Tsang Anthony C C , Yao Kwok‐Ming
Published: April 2003

PDZD2 (PDZ‐domain‐containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi‐PDZ‐domain protein. We have shown that PDZD2, which shows extensive homology to pro‐interleukin‐16 (pro‐IL‐16), is localized mainly to the endoplasmic reticulum (ER). Pro‐IL‐16 is cleaved in a caspase‐3‐dependent mechanism to generate the secreted cytokine IL‐16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro‐IL‐16, suggests that similar post‐translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope‐tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi‐PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains.
A glucocorticoid‐induced leucine‐zipper protein, GILZ, inhibits adipogenesis of mesenchymal cells
Authors: Shi Xingming , Shi Weibin , Li Qingnan , Song Buer , Wan Mei , Bai Shuting , Cao Xu
Published: April 2003

Mesenchymal stem cells have the potential to differentiate into various cell lineages, including adipocytes and osteoblasts. The induction of adipocyte differentiation by glucocorticoids (GCs) not only causes the accumulation of fat cells in bone marrow, but also depletes the supply of osteoblasts for new bone formation, thus leading to osteoporosis. We have shown that a GC‐induced leucine‐zipper protein (GILZ) antagonizes adipocyte differentiation. GILZ binds to a tandem repeat of CCAAT/enhancer‐binding protein (C/EBP) binding sites in the promoter of the gene encoding peroxisome‐proliferator‐activated receptor‐γ2 (PPAR‐γ2), and inhibits its transcription as a sequence‐specific transcriptional repressor. We have also shown that ectopic expression of GILZ blocks GC‐induced adipocyte differentiation. Furthermore, adipogenic marker genes (for example, those encoding PPAR‐γ2, C/EBP‐α, lipoprotein lipase and adipsin) are also inhibited by GILZ. Our results reveal a novel GC antagonistic mechanism that has potential therapeutic applications for the inhibition of GC‐induced adipocyte differentiation.
Cell senescence and hypermitogenic arrest
Authors: Blagosklonny Mikhail V
Published: April 2003

A diverse range of conditions, from mitogenic stimuli to cytotoxic stress, can induce cell senescence. Here, I propose that simultaneous stimulation of mitogen‐activated pathways and downstream inhibition of cyclin‐dependent kinases leads, ultimately, to cell senescence. This model distinguishes between two types of growth arrest: first, exit to G0 phase, which is caused by the withdrawal of mitogens and can lead to apoptosis; and second, hypermitogenic arrest, which is stimulated by mitogens and can lead to senescence. The concept of hypermitogenic arrest defines cell senescence as a functionally active, stable and conditionally reversible state.
Self‐assembly of minimal COPII cages
Authors: Antonny Bruno , Gounon Pierre , Schekman Randy , Orci Lelio
Published: April 2003

The small G‐protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self‐assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40–60 nm. Self‐assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60–120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these stuctures, which represent a minimal COPII cage, mimic the molecular organization of the membrane‐associated COPII coat.
Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase
Authors: Shao Yuan , Elly Chris , Liu Yun‐Cai
Published: April 2003

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine‐nucleotide exchange activity of C3G were all increased in Cbl−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin‐mediated cell adhesion was also seen in Cbl−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis‐independent E3‐ubiquitin‐ligase activity of Cbl that modulates protein–protein interactions.
Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications
Authors: BERTOLDI Mariarita , CELLINI Barbara , PAIARDINI Alessandro , Di SALVO Martino , BORRIVOLTATTORNI Carla
Published: April 2003

To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with l- and d-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429nm and of a band absorbing at 498nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, kcat and Km, for l-alanine are 1.05±0.03s−1 and 10±1mM respectively, whereas those for d-alanine are 1.4±0.1s−1 and 10±1mM. During the reaction of cystalysin with l- or d-alanine, a time-dependent loss of β-elimination activity occurs concomitantly with the conversion of the pyridoxal 5′-phosphate (PLP) coenzyme into pyridoxamine 5′-phosphate (PMP). The catalytic efficiency of the half-transamination of l-alanine is found to be 5.3×10−5 mM−1·s−1, 5-fold higher when compared with that of d-alanine. The partition ratio between racemization and half-transamination reactions is 2.3×103 for l-alanine and 1.4×104 for d-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK values of 6.5–6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of β-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4′ of both (4′S)- and (4′R)-[4′-3H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of l- and d-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.
A peptide mimetic of human interferon (IFN)-beta
Authors: SATO Atsushi , SONE Saburo
Published: April 2003

Type I interferons (IFNs) are cytokines that are used clinically as antiviral and antitumour agents. The interaction of IFNs with their heterodimeric type I IFN receptor comprised of IFNAR1 and IFNAR2 is a first step to inducing biological actions. Here, we describe the successful mimicry of IFN-β by a peptide isolated by phage-display screening using a neutralizing anti-IFN-β monoclonal antibody. The 15-mer peptide, designated SYR6, was shown to compete with IFN-β for binding to type I IFN receptor in a concentration-dependent manner, and was shown to elicit antiviral activity on cultured cells. This antiviral activity was not eliminated in the presence of neutralizing monoclonal antibodies to IFN-α, -β and -γ, and a low concentration of soluble type I IFN receptor, suggesting that it was not due to IFN contamination or the induction of endogenous IFNs by SYR6. This peptide might be a potent agonist to provide a mechanism of activating heterodimeric cytokine receptors.
Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates
Authors: DI PAOLO Maria Luisa , STEVANATO Roberto , CORAZZA Alessandra , VIANELLO Fabio , LUNELLI Lorenzo , SCARPA Marina , RIGO Adelio
Published: April 2003

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4–10.2) and ionic strength (10–200mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in kcat values (approx. one order of magnitude) accompanied by marked changes in Km and kcat/Km values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L-H+, G-H+ and IH+, characterized by pKa values of 6.2±0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231–2239], 8.2±0.3 and 7.8±0.4 respectively. The electrostatic interaction between the protonated substrates and the enzyme containing the residues L-H+, G-H+ and IH+ in the deprotonated form, the on/off role of the IH+ residue and the role of hydrophobic interactions with substrates characterized by apolar chains are discussed.
Tyrosinases from crustaceans form hexamers
Authors: JAENICKE Elmar , DECKER Heinz
Published: April 2003

Tyrosinases, which are widely distributed among animals, plants and fungi, are involved in many biologically essential functions, including pigmentation, sclerotization, primary immune response and host defence. In the present study, we present a structural and physicochemical characterization of two new tyrosinases from the crustaceans Palinurus elephas (European spiny lobster) and Astacus leptodactylus (freshwater crayfish). In vivo, the purified crustacean tyrosinases occur as hexamers composed of one subunit type with a molecular mass of approx. 71kDa. The tyrosinase hexamers appear to be similar to the haemocyanins, based on electron microscopy. Thus a careful purification protocol was developed to discriminate clearly between tyrosinases and the closely related haemocyanins. The physicochemical properties of haemocyanins and tyrosinases are different with respect to electronegativity and hydrophobicity. The hexameric nature of arthropod tyrosinases suggests that these proteins were the ideal predecessors from which to develop the oxygen-carrier protein haemocyanin, with its allosteric and co-operative properties, later on.
Transcriptional regulation of the human Sp1 gene promoter by the specificity protein (Sp) family members nuclear factor Y (NF-Y) and E2F
Authors: NICOLÁS Marta , NOÉ Vèronique , CIUDAD Carlos J.
Published: April 2003

We analysed in detail the minimal promoter of transcription factor Sp1, which extends 217bp from the initiation of transcription. Within this sequence we identified putative binding sites for Sp1, nuclear factor Y (NF-Y), activator protein 2 ('AP-2'), CCAAT/enhancer-binding protein ('C/EBP') and E2F transcription factors. In one case, the boxes for Sp1 and NF-Y are overlapping. Gel-shift and supershift assays demonstrated specific binding of Sp1, Sp3 and NF-Y proteins. Transient transfections and luciferase assays revealed activation of the Sp1 minimal promoter upon overexpression of Sp1 itself, NF-Y and E2F. Whereas overexpression of NF-Y or E2F had an additive effect on Sp1 overexpression, the activation of Sp1 transcription due to Sp1 was counteracted by Sp3 overexpression. Mutagenesis analysis of the NFY/Sp1-overlapping box revealed that both factors compete for this box, and that when the NF-Y site of this overlapping box is specifically mutated there is an increase in Sp1 binding, thus increasing transcriptional activity. These results help to explain the complex regulation of the Sp1 gene, which depends on the relative amounts of Sp1, Sp3, E2F and NF-Y proteins in the cell.
Inhibition of oxygen evolution in Photosystem II by Cu(II) ions is associated with oxidation of cytochrome b559
Authors: BURDA Kvetoslava , KRUK Jerzy , SCHMID Georg H. , STRZALKA Kazimierz
Published: April 2003

We have found that elevated copper concentrations, apart from the inhibition of oxygen evolution, changed the initial states distribution of the oxygen-evolving complex. Already at low concentrations, copper ions oxidized the low-potential form of cytochrome b559 and also its high-potential form at higher concentrations at which fluorescence quenching was observed. We suggest that the primary target sites in Photosystem II for copper is tyrosinez, both cytochrome b559 forms and chlorophyllz, and that these sites are the source of the copper-induced fluorescence quenching and oxygen evolution inhibition in Photosystem II.
Regulation of transcription by the heterogeneous nuclear ribonucleoprotein E1B-AP5 is mediated by complex formation with the novel bromodomain-containing protein BRD7
Authors: KZHYSHKOWSKA Julia , RUSCH Andre , WOLF Hans , DOBNER Thomas
Published: April 2003

E1B-AP5 was initially identified as a target of the early adenovirus E1B-55kDa protein during the course of lytic infection. E1B-AP5 belongs to the heterogeneous nuclear ribonucleoprotein family and was demonstrated to be involved in mRNA processing and transport [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960–7971]. In the present paper, we demonstrate that E1B-AP5 differentially regulates basic and ligand-dependent transcription. We found that E1B-AP5 represses basic transcription driven by several virus and cellular promoters, and mapped the repression activity to the N-terminal part of the protein. In contrast with basic repression, E1B-AP5 activated the glucocorticoid-dependent promoter in the absence of dexamethasone, but did not contribute to the dexamethasone-induced activation. Mutant analysis indicated the presence of an additional cellular factor that modulates E1B-AP5 transcriptional activity. Using yeast two-hybrid screening, we identified a novel chromatin-associated bromodomain-containing protein, BRD7, as an E1B-AP5 interaction partner. We confirmed E1B-AP5–BRD7 complex formation in vivo and in vitro. We found that, although BRD7 binds to histones H2A, H2B, H3 and H4 through its bromodomain, this domain was not necessary for the interaction with E1B-AP5. Indeed, the triple complex formation of E1B-AP5, BRD7 and histones was demonstrated. Disruption of the E1B-AP5–BRD7 complex increased E1B-AP5 repression activity for basic transcription and converted it from being an activator of the hormone-dependent promoter into being a strong repressor. We conclude that complex formation between BRD7 and E1B-AP5 links chromatin events with mRNA processing at the level of transcriptional regulation.
Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate
Authors: ANNEREAU Jean Philippe , KO Young Hee , PEDERSEN Peter L.
Published: April 2003

Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major ‘hotspot’ for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7min−1] and an apparent affinity for ATP (Km, 8.7μM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co2+, followed by Mn2+ and Mg2+. In contrast, Ca2+ is ineffective and Cd2+ is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.
A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells
Authors: MOYANO José V. , MAQUEDA Alfredo , ALBAR Juan P. , GARCIA-PARDO Angeles
Published: April 2003

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with α5β1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with α5β1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7–FNIII10 (FNIII7–10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7–10 or adding soluble HBP/III5 to cells prespread on FNIII7–10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to α5β1 for the progression of the cytoskeletal response and that these include activation of RhoA.
Identification of galectin I and thioredoxin peroxidase II as two arsenic-binding proteins in Chinese hamster ovary cells
Authors: CHANG Kwang Ning , LEE T. Chang , TAM Ming F. , CHEN Y. Chin , LEE L. Wen , LEE Shin Ying , LIN Pei Jung , HUANG Rong Nan
Published: April 2003

In this study, we report the identification of two arsenic-binding proteins from Chinese hamster ovary (CHO) cells. The crude extract derived from CHO and SA7 (arsenic-resistant CHO cells) was applied to a phenylarsine oxide–agarose affinity column, and after extensive washing, the absorbed proteins were eluted with buffers containing 20mM 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). Three differentially expressed proteins, galectin 1 (Gal-1; in the 2-ME-eluted fraction from CHO cells), glutathione S-transferase P-form (GST-P) and thioredoxin peroxidase II (TPX-II), respectively in the 2-ME- and DTT-eluted fractions from SA7 cells, were identified by partial amino acid sequence analysis after separation by SDS/PAGE. The GST-P protein has been previously shown to facilitate the excretion of sodium arsenite [As(III)] from SA7 cells. TPX II was detected predominately in SA7 cells [routinely cultured in As(III)-containing medium], but not in CHO or SA7N (a revertant of SA7 cells cultured in regular medium) cells. In contrast, Gal-1 was specifically identified in CHO and SA7N cells, but not in SA7 cells. The preferential expression of Gal-1 in CHO cells and TPX-II in SA7 cells was further illustrated by quantitative PCR analysis. The binding of Gal-1 and TPX-II with As(III) was further verified by both co-immunoprecipitation and co-elution of Gal-1 and TPX-II with As(III). It is suggested that Gal-1 and TPX-II are two proteins that serve as high-affinity binding sites for As(III) and thus both may be involved in the biological action of As(III).
Osmotic regulation of insulin-induced mitogen-activated protein kinase phosphatase (MKP-1) expression in H4IIE rat hepatoma cells
Authors: LORNEJAD-SCHÄFER Mohammad Reza , SCHÄFER Christine , GRAF Dirk , HÄUSSINGER Dieter , SCHLIESS Freimut
Published: April 2003

A contribution of intracellular dehydration to insulin resistance has been established in human subjects and in different experimental systems. Here the effect of hyperosmolarity (405mosmol/l) on insulin-induced mitogen-activated protein (MAP) kinase phosphatase (MKP)-1 expression was studied in H4IIE rat hepatoma cells. Insulin induces robust MKP-1 expression which correlates with a vanadate-sensitive decay of extracellular-signal-regulated kinase (Erk-1/Erk-2) activity. Hyperosmolarity delays MKP-1 accumulation by insulin and this corresponds to impaired MKP-1 synthesis, whereas MKP-1 degradation remains unaffected by hyperosmolarity. Rapamycin, which inhibits signalling downstream from the mammalian target of rapamycin (mTOR) and a peptide inhibiting protein kinase C (PKC) ζ/λ abolish insulin-induced MKP-1 protein but not mRNA expression, suggesting the involvement of the p70 ribosomal S6 protein kinase (p70S6-kinase) and/or the eukaryotic initiation factor 4E-binding proteins (4E-BPs) as well as atypical PKCs in MKP-1 translation. Hyperosmolarity induces sustained suppression of p70S6-kinase and 4E-BP1 hyperphosphorylation by insulin, whereas insulin-induced tyrosine phosphorylation of the insulin receptor (IR) β subunit and the IR substrates IRS1 and IRS2, recruitment of the phosphoinositide 3-kinase (PI 3-kinase) regulatory subunit p85 to the receptor substrates as well as PI 3-kinase activation, and Ser-473 phosphorylation of protein kinase B and Thr-410/403 phosphorylation of PKC ζ/λ are largely unaffected under hyperosmotic conditions. The hyperosmotic impairment of both, MKP-1 expression and p70S6-kinase hyperphosphorylation by insulin is insensitive to K2CrO4, calyculin A and vanadate, and inhibition of the Erk-1/Erk-2 and p38 pathways. The suppression of MKP-1 may further contribute to insulin resistance under dehydrating conditions by allowing unbalanced MAP kinase activation.
Effect of polyvalencies of glycotopes on the binding of a lectin from the edible mushroom, Agaricus bisporus
Authors: WU Albert M. , WU June H. , HERP Anthony , LIU Jia-Hau
Published: April 2003

Agaricus bisporus agglutinin (ABA) isolated from edible mushroom has a potent anti-proliferative effect on malignant colon cells with considerable therapeutic potential as an anti-neoplastic agent. Since previous studies on the structural requirement for binding were limited to molecular or submolecular levels of Galβ1-3GalNAc (T; Thomsen–Friedenreich disaccharide glycotope; where Gal represents d-galactopyranose and GalNAc represents 2-acetamido-2-deoxy-d-galactopyranose) and its derivatives, the binding properties of ABA were further investigated using our collection of glycans by enzyme-linked lectinosorbent assay and lectin–glycan inhibition assay. The results indicate that polyvalent Galβ1-related glycotopes, GalNAcα1-Ser/Thr (Tn), and their cryptoforms, are the most potent factor for ABA binding. They were up to 5.5×105 and 4.7×106 times more active than monomeric T and GalNAc respectively. The affinity of ABA for ligands can be ranked as: multivalent Tα (Galβ1-3GalNAcα1-), Tn and I/II (Galβ1-3GlcNac/Galβ1-4GlcNAc, where GlcNAc represents 2-acetamido-2-deoxy-d-glucopyranose)>>>>monomeric Tα and Tn>I>>GalNAc>>>II, L (Galβ1-4Glc, where Glc represents d-glucopyranose) and Gal (inactive). These specific binding features of ABA establish the importance of affinity enhancement by high-density polyvalent (versus multiantennary I/II) glycotopes and facilitate our understanding of the lectin receptor recognition events relevant to its biological activities.
Biochemical filtering of a protein-protein docking simulation identifies the structure of a complex between a recombinant antibody fragment and alpha-bungarotoxin
Authors: BRACCI Luisa , PINI Alessandro , BERNINI Andrea , LELLI Barbara , RICCI Claudia , SCARSELLI Maria , NICCOLAI Neri , NERI Paolo
Published: April 2003

The structural characterization of a complex of α-bungarotoxin with a recombinant antibody fragment that mimics the acetylcholine receptor was achieved using docking simulation procedures. To drive the computer simulation towards a limited set of solutions with biological significance, a filter, incorporating general considerations of antigen–antibody interactions, specificity of the selected antibody fragment and results from α-bungarotoxin epitope mapping, was adopted. Two similar structures were obtained for the complex, both of them stabilized by cation-π and hydrophobic interactions due to tyrosilyl residues of the antibody fragment. Site-directed mutagenesis studies, removing each of the latter aromatic residues and causing full inactivation of the interaction process between the antibody fragment and the neurotoxin, support the validity of the calculated structure of the complex.
Discrimination of intracellular calcium store subcompartments using TRPV1 (transient receptor potential channel, vanilloid subfamily member 1) release channel activity
Authors: TURNER Helen , FLEIG Andrea , STOKES Alexander , KINET Jean-Pierre , PENNER Reinhold
Published: April 2003

The store-operated calcium-release-activated calcium current, ICRAC, is a major mechanism for calcium entry into non-excitable cells. ICRAC refills calcium stores and permits sustained calcium signalling. The relationship between inositol 1,4,5-trisphosphate receptor (InsP3R)-containing stores and ICRAC is not understood. A model of global InsP3R store depletion coupling with ICRAC activation may be simplistic, since intracellular stores are heterogeneous in their release and refilling activities. Here we use a ligand-gated calcium channel, TRPV1 (transient receptor potential channel, vanilloid subfamily member 1), as a new tool to probe store heterogeneity and define intracellular calcium compartments in a mast cell line. TRPV1 has activity as an intracellular release channel but does not mediate global calcium store depletion and does not invade a store coupled with ICRAC. Intracellular TRPV1 localizes to a subset of the InsP3R-containing stores. TRPV1 sensitivity functionally subdivides the InsP3-sensitive store, as does heterogeneity in the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase isoforms responsible for store refilling. These results provide unequivocal evidence that a specific ‘CRAC store’ exists within the InsP3-releasable calcium stores and describe a novel methodology for manipulation of intracellular free calcium.
Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803
Authors: SHEPHERD Mark , REID James D. , HUNTER C. Neil
Published: April 2003

Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C6 propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-l-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded β-sheet, common among small-molecule methyltransferases. Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP. Initial rate studies showed that the reaction proceeds via a ternary complex. Product (S-adenosyl-l-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further. SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD. This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme or a ChlM–MgD complex. Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.
Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase
Authors: SHELTON Shary N. , BARYLKO Barbara , BINNS Derk D. , HORAZDOVSKY Bruce F. , ALBANESI Joseph P. , GOODMAN Joel M.
Published: April 2003

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100min−1 were obtained at the PI/Triton X-100 ratio of 1:5. The Km value for ATP was 266μM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p–green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.