bluuebunny/crossref_metadata_2025 huggingface dataset:
A Target of Phosphatidylinositol 3,4,5‐Trisphosphate with a Zinc Finger Motif Similar to that of the ADP‐Ribosylation‐Factor GTPase‐Activating Protein and Two Pleckstrin Homology Domains
Authors: Tanaka Kenichi , Imajoh‐Ohmi Shinobu , Sawada Takayuki , Shirai Ryuichi , Hashimoto Yuichi , Iwasaki Shigeo , Kaibuchi Kouzou , Kanaho Yasunori , Shirai Toshiyuki , Terada Yoh , Kimura Koutarou , Nagata Satoshi , Fukui Yasuhisa
Published: July 2004

We have purified a protein that binds phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3] using beads bearing a PtdIns(3,4,5)P3 analogue. This protein, with a molecular mass of 43 kDa, was termed PtdIns(3,4,5)P3‐binding protein. The partial amino acid sequences were determined and a full‐length cDNA encoding the protein was isolated from bovine brain cDNA library. The clone harbored an open reading frame of 373 amino acids which contained one zinc finger motif similar to that of ADP‐ribosylation‐factor GTPase‐activating protein and two pleckstrin homology domains. The entire sequence was 83% similar to centaurin a, another PtdIns(3,4,5)P3‐binding protein. The protein bound PtdIns(3,4,5)P3 with a higher affinity than it did inositol 1,3,4,5‐tetrakisphosphate, phosphatidylinositol 4,5‐bisphosphate, phosphatidylinositol 3,4‐bisphosphate, and phosphatidylinositol 3‐phosphate suggesting that the binding to PtdIns(3,4,5)P3 was specific. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each pleckstrin homology domain. Introduction of both mutations abolished the activity. These results suggest that this new binding protein binds PtdIns(3,4,5)P3 through two pleckstrin domains present in the molecule.
Processing of Proendothelin‐1 at the C‐Terminus of Big Endothelin‐1 is Essential for Proteolysis by Endothelin‐Converting Enzyme‐1 in vivo
Authors: Kido Tsuneo , Sawamura Tatsuya , Hoshikawa Hajime , D'orléans‐Juste Pedro , Denault Jean‐Bernard , Leduc Richard , Kimura Jun , Masaki Tomoh
Published: July 2004

Production of endothelin‐1 is thought to be a three‐step process consisting of an initial proteolytic cleavage of the proendothelin‐1 precursor to big endothelin‐1‐Lys‐Arg, C‐terminal trimming by a carboxypeptidase and further processing of the big endothelin‐1 peptide to endothelin‐1 by endothelin‐converting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin‐1, we introduced a point mutation into endothelin‐1 cDNA to replace the Arg in the ‐4 position of the recognition motifs of furin‐like convertase in human preproendothelin‐1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CHO)‐K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg‐Ser‐Lys‐Arg motifs of preproendothelin‐1 are recognized by CHO‐K1 furin‐like convertase. Co‐transfection with ECE‐1 cDNA revealed that cleavage at Arg52 is not essential for cleavage by ECE‐1, but that cleavage at Arg92 is critical. Although a high‐molecular‐mass form of endothelin‐1 is produced by processing by ECE‐1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelinA‐receptor‐expressing cells. In conclusion, prior cleavage at Arg92 by furin‐like convertase is absolutely necessary for cleavage by ECE‐1 at Trp73 to produce mature endothelin‐1.
Identification of a Short Sequence Highly Divergent Between β‐Adrenergic‐Receptor Kinases 1 and 2 that Determines the Affinity of Binding to βγ Subunits of Heterotrimeric Guanine‐Nucleotide‐Binding Regulatory Proteins
Authors: Chuang Tsu Tshen , Pompili Elena , Paolucci Lina , Sallese Michele , DE Gioia Luca , Salmona Mario , DE Blasi Antonio
Published: July 2004

A 28‐residue peptide (peptide G), derived from the C‐terminal (W643‐S670) of the β‐adrenergic receptor kinase UARK), was previously identified as the critical domain for binding to the βγ subunits fo the heterotrimeric guanine‐nucleotide‐binding regulatory protein (Gβγ). We observed that the 18‐amino‐acid core of this domain is poorly conserved between βARK1 and βARK2 and so may provide the basis for differences in Gβγ‐binding properties. Specific antibodies raised against 18‐residue peptides derived from the divergent sequences (peptides P1 and P2 for βARK1 and βARK2, respectively) competitively inhibited Gβγ‐activation of the related βARK subtype, confirming the involvement of this region in binding to Gβγ. Peptides PI and P2 inhibited Gβγ‐stimulated activity of both βARK1 and βARK2, with P2 being significantly more potent than PI (IC50 of 179±μM for P2 and >500 pM for Pl). The 28‐residue peptides G showed the same relative inhibitory activities (IC50= 48± 5 pM for G2 and 146± 8 pM for G1). This relative order of potency G2 > G1 ≈ P2 > P1 was confirmed in a direct Gβγ‐binding assay. No binding selectivity for the β1, βZ, β3 and β4 GP subtypes was observed. The EC50 value for Gβγ‐activation of βARK1 was about double of that for βARK2, indicating a higher affinity between Gβγ and βARK2, which is the expected result based on the findings with the peptides. These findings show that the 18‐residue peptides P represent the shortest sequence of βARK that can bind to Gβγ and provide a demonstration of a functional difference between the Gβγ binding domains of βARK1 and βARK2.
NADH Oxidase Activity of Human Xanthine Oxidoreductase
Authors: Sanders Stephen A. , Eisenthal Robert , Harrison Roger
Published: July 2004

Human xanthine oxidase was purified from breast milk. The dehydrogenase form of the enzyme, which predominates in most mammalian tissues, catalyses the oxidation of NADH by oxygen, generating superoxide anion significantly faster than does the oxidase form. The corresponding forms of bovine enzyme behave very similarly. The steady‐state kinetics of NADH oxidation and superoxide production, including inhibition by NAD, by the dehydrogenase forms of both enzymes, are analysed in terms of a model involving two‐stage recycling of oxidised enzyme.

Established inhibitors of xanthine oxidoreductases (allopurinol oxypurinol, amflutizole and BOF 4272), which block all other reducing substrates, were ineffective in the case of NADH. Diphenyleneiodo‐nium, on the other hand, was a powerful inhibitor of NADH oxidation.

The potential involvement of reactive oxygen species arising from NADH oxidation by xanthine oxidoreductase in ischaemia‐reperfusion injury and other disease states, as well as in normal signal trans‐duction, is discusssed.
Sequence‐Specific Antiproliferative Effects of Antisense and End‐Capping‐Modified Antisense Oligodeoxynucleotides Targeted against the 5′‐Terminus of Basic‐Fibroblast‐Growth‐Factor mRNA in Coronary Smooth Muscle Cells
Authors: Schmidt Annette , Sindermann Jürgen , Peyman Anusch , Uhlmann Eugen , Will David W. , Müller Joachim Georg , Breithardt Günter , Buddecke Eckhart
Published: July 2004

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate‐modified 15‐residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1–2.0 μM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose‐dependent manner. The effect is sequence specific at low (0.1–0.5 μM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48–72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligo‐nucleotide‐free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15‐residue antisense oligodeoxynucleotide is markedly enhanced by adding 3–4 nonbase‐pairing guanosine residues at the 5′‐ and 3′‐termini of the 15‐residue antisense oligonucleotide.

The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF‐specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post‐angioplasty restenosis.
Biochemical Characterization of Purified, Human Recombinant Lys304→Glu Medium‐Chain Acyl‐Coa Dehydrogenase Containing the Common Disease‐Causing Mutation and Comparison with the Normal Enzyme
Authors: Kieweg Volker , Krautle Franz‐Georg , Nandy Andreas , Engst Stefan , Vock Petra , Abdel‐Ghany Abdel‐Ghany , Bross Peter , Gregersen Niels , Rasched Ihab , Strauss Arnold , Ghisla Sandro
Published: July 2004

Recombinant, normal human medium‐chain acyl‐CoA dehydrogenase (MCADH) and the common, human disease‐causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28 °C). Expression in the same system at 37 °C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl‐CoA and the same Vmax as human MCADH with the best substrate for the latter, octanoyl‐CoA, the Km in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, Vmax obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The Vmax/Km versus chain‐length profile of the mutant shows a maximum with dodecanoyl‐CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl‐CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long‐chain acyl‐CoA dehydrogenase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild‐type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purification and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.
Human Ribosomal Protein L7 Binds RNA with an α‐Helical Arginine‐Rich and Lysine‐Rich Domain
Authors: Hemmerich Peter , Bosbach Stefan , Von Mikecz Anna , Krawinkel Ulrich
Published: July 2004

In this study we mapped the RNA‐binding domain of human ribosomal protein L7 and characterized its conformation‐dependent RNA‐binding specificity. Binding competition assays demonstrated preferential binding of L7 to mRNAs and rRNA, but not to tRNA. The ribohomopolymer poly(G) is bound with high affinity whereas poly(U), poly(C), or poly(A) show low affinity to L7. Furthermore, L7 binds to double‐stranded but not to single‐stranded DNA. Deletion mapping showed that the RNA‐binding domain of L7 is represented by an arginine‐rich and lysine‐rich oligopeptide (ELKIKRLRKKFAQKMLRK‐ARRK), which is reminiscent of the arginine‐rich motif (ARM) found in one family of RNA‐binding proteins. The isolated RNA‐binding domain is capable of high‐affinity binding to the Rev‐responsive element (RRE) of human immunodeficiency virus type 1 in vitro. Circular dichroic studies demonstrated a concentration‐dependent and ligand‐induced α‐helical transition of a synthetic peptide carrying the arginine‐lysine‐rich RNA‐binding domain of protein L7. Peptides carrying a mutation that destroys the α‐helical conformation do not bind RNA.
Molecular Cloning and Expression of Bothrojaracin, A Potent Thrombin Inhibitor from Snake Venom
Authors: Arocas Véronique , Castro Helena C. , Zingalf Russolina B. , Guillin Marie‐Claude , Jandrot‐Perrus Marline , Bon Cassian , Wisner Anne
Published: July 2004

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion‐binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot‐Perrus, M., Guillin, M. C. & Bon, C. (1993)Biochemistry 32, 10794–10802]. Bothrojaracin is a 27‐kDa protein composed of two disulfide‐linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin‐like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C‐terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin‐related protein was detected in conditioned media from cells transfected with the A chain.
Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus
Authors: Glasemacher Jürgen , Bock Anne‐Katrin , Schmid Roland , Schönheit Peter
Published: July 2004

Acetyl‐CoA synthetase (ADP‐forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl‐CoA and couples this reaction with the synthesis of ATP from ADP and Pi (acetyl‐CoA + ADP + Pi→ acetate + ATP + CoA) [Schäfer, T., Selig, M. & Schonheit, P. (1993)Arch. Microbiol. 159, 72–83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96‐fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen‐stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an α2β2 structure. The N‐terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl‐CoA, ADP and Pi to acetate, ATP and CoA. The apparent Vmax value in the direction of acetate formation was 18 U/mg (55°C), the apparent Km values for acetyl‐CoA, ADP and P, were 17 μM, 60 μM and 200 μM, respectively. ADP and Pi could not be replaced by AMP and PPiv defining the enzyme as an ADP‐forming rather than an AMP‐forming acetyl‐CoA synthetase. The apparent Vmax value in the direction of acetyl‐CoA formation was about 40 U/mg (55°C), and the apparent Km values for acetate, ATP and CoA were 660 μM, 80 μM and 30 μM, respectively. The purified enzyme was not specific for acetyl‐CoA or acetate, in addition to acetyl‐CoA (100%), the enzyme accepts propionyl‐CoA (110%) and butyryl‐CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (100%), butyrate (92%), isobutyrate (79%), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl‐CoA synthetase (ADP‐forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (<3%). In addition to ADP (100%), GDP (220%) and IDP (250%) were used, and in addition to ATP (100%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg2+, which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH7. The enzyme has a temperature optimum at 90°C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KC1 (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100°C.
Structural Studies of the O‐Antigen Polysaccharide from Escherichia Coli 0138
Authors: Linnerborg Malin , Weintraub Andrej , Widmalm Goran
Published: July 2004

The structure of the O‐antigen polysaccharide from Escherichia coli O138 has been determined. NMR spectroscopy, together with component and methylation analyses, of native and reduced polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity (HMBC) NMR experiments. It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure:
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Thermodynamic Significance of the Lactate Gradient
Authors: Hockings Paul D. , Rogers Peter J.
Published: July 2004

The theory that some bacteria can save energy by an energy‐recycling process, in which protons are excreted with metabolic end‐products with variable stoichiometry, has been examined by1H‐NMR. A method has been developed that utilises observed differences in the HahnT2relaxation of metabolites in the intracellular and extracellular compartments to distinguish and quantify metabolite signals originating from both compartments. It was found that the lactate electrochemical‐potential gradient calculated from the fraction of lactate that is sufficiently mobile to contribute to the NMR signal was in exact balance with the proton electrochemical‐potential gradient over a wide range of pH values. The conclusion was reached that previous reports of variable stoichiometry were due to ‘bound’ lactate at high intracellular pH that could neither contribute neither to the NMR signal nor to the lactate electrochemical‐potential gradient.
Mediation by Arachidonic Acid Metabolites of the H2O2‐Induced Stimulation of Mitogen‐Activated Protein Kinases (Extracellular‐Signal‐Regulated Kinase and c‐Jun NH2‐Terminal Kinase)
Authors: Tournier Cathy , Thomas Ginette , Pierre Josiane , Jacquemin Claude , Pierre Michel , Saunier Bertrand
Published: July 2004

Reactive oxygen species modulate major cellular functions by mechanisms which are still poorly understood. Recently, H2O2 has been reported to stimulate the activity of the mitogen‐activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto‐oncogenes c‐fos and c‐jun. As their expression is enhanced by H2O2 in astrocytes, we studied whether these MAPKs were stimulated by H2O2 in primary cultured astrocytes. The result was positive, a maximum of stimulation being reached with 200 μM H2O2 (0.3 pmol H2O2/cell) for both ERK and JNK. ERK was previously reported to stimulate cytosolic phospholipase A2 phosphorylation and activity. H2O2 stimulated the release of arachidonic acid in astrocytes, as already reported in other cell types. We found also that cPLA2 phosphorylation was increased by H2O2. Moreover, the stimulation by H2O2 of ERK and JNK was decreased by phospholipase A2 activity inhibitors. When astrocytes were incubated first with eicosatetraynoic acid, a structural analogue competing in arachidonic acid metabolism, the stimulation of JNK by H2O2 was also inhibited, suggesting the involvement of arachidonic acid metabolites. Cyclooxygenase or cytochrome P450 monooxygenase inhibitors failed in decreasing the MAPK stimulation by H2O2, whereas lipoxygenase inhibitors completely abolished that of JNK. Mitogenicity has been reported to be stimulated by H2O2 in other cell types. Although ERK was strongly and durably stimulated by 200 μM H2O2 in astrocytes, at the same extent as by mitogenic growth factors, basal thymidine incorporation rate was decreased by more than 80% after 12–15 h. Moreover, the stimulation of thymidine incorporation induced by basic fibro‐blast growth factor was transiently abolished by H2O2. Furthermore, H2O2 likely induced the expression of CL100/PAC1/MKP‐1, a dual specificity phosphatase which has been implicated in ERK and JNK inactivation in the nucleus. Finally, the prior treatment of astrocytes with MK886, a 5‐lipoxygenase‐activating protein inhibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H2O2. These results strongly suggest an involvement of arachidonic acid and/or its metabolites in the stimulation of both ERK and JNK following the oxidative stress evoked by H2O2, which induced a cell cycle arrest probably independent of the stimulation of JNK.
Addition of Acetaldehyde to the N‐Terminus of a Recombinant Schistosoma Mansoni Glutathione S‐Transferase Upon High‐Level Expression in Saccharomyces Cerevisiae
Authors: Roecklin Dominique , Klarskov Klaus , Cavallini Bruno , Sabatié Jean , Bouchon Bernadette , Loew Damarys , Van Dorsselaer Alain , Bischoff Rainer
Published: July 2004

Intracellular expression of recombinant Schistosoma mansoni protein p28 (Smp28) in soluble form to a concentration of more than 6 g/l culture in Saccharomyces cerevisiae was accompanied by a post‐translational modification, which occurred during the late stage of the culture. The modified protein, which had a reduced isoelectric point, was isolated by anion‐exchange HPLC and characterized by tryptic mapping by means of on‐line reversed‐phase HPLC/electrospray mass spectrometry. Comparison with non‐modified recombinant Smp28 allowed us to localize the modification to the N‐terminal hexapeptide AGEHIK, which had an increased mass of 26 Da. Reversed‐phase HPLC of the modified peptide with a shallow acetonitrile gradient revealed the presence of two components of identical mass and amino acid composition. Both peptides were inaccessible to N‐terminal Edman sequencing, indicating that a rearrangement of the N‐terminal region of recombinant Smp28 had taken place during tryptic digestion leading to two isomeric, N‐terminally blocked peptides. Deuterium‐exchange mass spectrometry showed that the modified peptides lacked two exchangeable protons, suggesting cyclic modifications implying the N‐terminal amino group. Tandem mass spectrometry by means of the nano‐electrospray technique and collision‐induced dissociation allowed us to identify the modified sites as Alal, His4 and Lys6 based on a characteristic modified a1, ion of Alal (70.0 Da), a modified immonium ion of His4 (136.0 Da) and a modified y1” ion (173.2 Da) of Lys6. Combination of all the above results led to the conclusion that recombinant Smp28 was initially modified at its N‐terminus by addition of acetaldehyde to form an aldimine which rearranged during tryptic digestion to two different cyclic peptides.
Lipopolysaccharides of Helicobacter Pylori Serogroups O:3 and O:6 Structures of a Class of Lipopolysaccharides with Reference to the Location of Oligomeric Units of D‐Glycero‐α‐D‐Manno‐Heptose Residues
Authors: Aspinall Gerald O. , Monteiro Mario A. , Shaver Robert T. , Kurjanczyk Linda A. , Penner John L.
Published: July 2004

Lipopolysaccharides (LPS) from antigenically different strains assigned to serogroups O:3 and O:6 of Helicobacter pylori were isolated as water‐soluble material of high Mr and as water‐insoluble gels of low Mr. Chemical and spectroscopic analyses of the soluble LPS and oligosaccharides liberated from the water‐insoluble gels led to proposed structures with Lewis (Le) antigen determinants terminating regular repeating units of different types, linked in turn to inner core regions of invariable structure. The O:6 LPS has two populations of related molecules with chains of 3‐linked D‐glycero‐α‐D‐manno‐heptose residues similar to those in the MO19 strain, one with and the other without a single terminal Lewis (Ley) epitope. In contrast, in the O:3 LPS, Lewis (Lex and Ley) epitopes terminate a partially fucosylated N‐acetyllactosaminoglycan, but a heptan chain similar to that in the O:6 LPS was shown to connect the outer chains to the inner core. These LPS provide examples of the molecular mimicry of cell‐surface glycoconjugates. Structural variations of LPS between strains, and differences in some aspects of structure within strains, between high Mr and low Mr LPS indicate a class of LPS whose mechanisms of biosynthesis lead to overall architectures different from those characteristic of most LPS from enteric bacteria.
Differences in Metabolism and Isomerization of All‐trans‐Retinoic Acid and 9‐cis‐Retinoic Acid between Human Endothelial Cells and Hepatocytes
Authors: Lansink Mirian , Bennekum Ariëtte M. Van , Blaner William S. , Kooistra Teake
Published: July 2004

Retinoic acid stimulates the expression of tissue‐type plasminogen activator (t‐PA) in vascular endothelial cells in vitro and enhances t‐PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t‐PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that All‐trans‐retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All‐trans‐retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9‐cis‐Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9‐cis‐Retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise All‐trans‐retinoic acid to 9‐cis‐Retinoic acid and vice versa. In both endothelial cells and hepatocytes All‐trans‐retinoic acid metabolism was inhibitable by the cytochrome P‐450 inhibitors liarozole (10 μM) and ketoconazole (10 μM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10‐fold lower All‐trans‐retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t‐PA. In conclusion, our results clearly demonstrate that All‐trans‐retinoic acid and 9‐Cis retinoic acid are actively but differently metabolized and isomerised by human endothelid cells and hepatocytes. The rapid metabolism of retinoic acid explains the relatively high concentrations of retinoic acid required to induce t‐PA in cultured endothelial cells.
Characterization of Human and Pig Kidney Long‐Chain‐Acyl‐CoA Dehydrogenases and their Role in β‐Oxidation
Authors: Eder Michael , Kräutle Franz , Dong Yu , Vock Petra , Kieweg Volker , Kim Jung‐Ja P. , Strauss Arnold W. , Ghisla Sandro
Published: July 2004

Long‐chain‐acyl‐CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli. Pig kidney LCADH was purified using an optimized method which also produces apparently pure short‐chain‐acyl‐CoA dehydrogenase (SCADH) and medium‐chain‐acyl‐CoA dehydrogenase (MCADH) in good yields. LCADH from both sources has a maximal turnover rate (Vmax, of 650‐700min−1 at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously. The human enzyme has an approximately fivefold higher Km, compared with the pig kidney enzyme with substrates of chain length from C10, to C18, and a significantly different dependence of Vmax on the chain length. Pig kidney LCADH has a similar Vmax/Km with C10, to C18, substrates as MCADH does with C6, to C10, substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C,J than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in β‐oxidation than in the pig, or that post‐translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acyl‐CoA dehydrogenases in fatty acid oxidation at physiological, mitochondrial pH with optimal substrates of chain length C10–C14. The extent of the LCADH‐flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl‐CoA dehydrogenase. Both LCADH are inactivated by the substrate analogue 2‐octynoyl‐CoA, possibly via covalent modification of GIu261, the active‐site residue involved in deprotonation of the substrate (α)C‐H.
Mass Determination, Subunit Organization and Control of Oligomerization States of Keyhole Limpet Hemocyanin (KLH)
Authors: Söhngen Sabine M. , Stahlmann Alexandra , Harris J. Robin , Müller Shirley A. , Engel Andreas , Markl Jürgen
Published: July 2004

Analytical dark‐field scanning transmission electron microscopy (STEM) of freeze‐dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400‐kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N‐terminal sequencing, eight distinct 45–60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C‐terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.
Disruption of Three Acid Proteases in Aspergillus Niger— Effects on Protease Spectrum, Intracellular Proteolysis, and Degradation of Target Proteins
Authors: Van Den Hombergh Johannes P. T. W. , Gelpke Maarten D. Sollewijn , Van De Vondervoort Peter J. I. , Buxton Frank P. , Visser Jaap
Published: July 2004

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern‐blot analysis showed the absence of wild‐type protease mRNAs in the disruptants while western‐blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the DpepA and the DpepB disruptants, repectively. In the DpepE disruptant, the total intracellular proteolytic activity was reduced to 32%. Apart from the reduced intracellular pepstatin‐inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the DpepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non‐disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed DpepADpepB, DpepBDpepE and DpepADpepE double disruptants as well as a DpepADpepB DpepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity was the highest in the triple disruptant and the DpepADpepB recombinant.
Structural and Functional Domains of the Troponin Complex Revealed by Limited Digestion
Authors: Takeda Soichi , Kobayashi Tomoyoshi , Taniguchi Hisaaki , Hayashi Hiroshi , Maéda Yuichiro
Published: July 2004

Troponin (Tn), consisting of three subunits, TnT, TnC, and TnI, plays a crucial role in the calcium‐dependent regulation of vertebrate striated muscle contraction. In the present study, we have applied limited proteolysis to the Tn complex in order to study domain structures and to detect conformational differences of Tn under different conditions. We found that both TnT and TnI were susceptible to chymotryptic digestion: while TnT was cleaved into TnT‐(1–158)‐peptide and TnT‐(159–259)‐peptide irrespective of Ca2+ concentration, the cleavage sites of TnI were dependent on the Ca2+ occupancy of TnC. In addition, we characterized the effects of depletion of the C‐terminal part of TnI on acto‐S1 ATPase activity. The TnT–(159–259)‐peptide–TnC–TnICa‐frag complex [TnICa‐frag= (TnI–(1–134 and 1–140)‐peptide], which was produced in the presence of CaCl2 and MgCl2, retains both the activating and inhibitory capabilities of whole Tn on the acto‐S1 ATPase activity, while TnT‐(159–259)‐peptide–TnC–TnIMg‐frag complex [TnIMg‐frag= (TnI‐(1–116)‐peptide], which was obtained in the presence of MgCl2 and EGTA, lost its ability to activate acto‐S1 ATPase activity. Our results indicate that residues 117–134 or 117–140 of TnI undergo structural changes upon Ca2+‐binding to the regulatory sites of TnC and are necessary for the Ca2+‐dependent inhibitory action of the Tn complex on acto‐S1 ATPase activity. We also showed that residues 135–181 or 141–181 of TnI are involved in the interaction of Tn with the tropomyosinactin filament.
Mutations in the 1.1 Subdomain of Escherichia coli Sigma Factor σ70 and Disruption of its Overall Structure
Authors: Gopal Vijaya , Chatterji Dipankar
Published: July 2004

Among various group I sigma factors, two amino acids, Val55 and Ala59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]σ70 and [Gly59]σ70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild‐type σ70 has an apparent molecular mass of 87 kDa. However, [Gly434]σ70, which contains a mutation at the DNA‐binding rpoD box region, also migrates as a 70‐kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]σ70 and [Gly59]σ70 have reduced helicity (20%) compared to wild‐type σ70 (50%). Binding of sigma factors with the hydrophobic, surface active probe 1‐anilinonapthalene‐8‐sulphonate, has shown that more hydrophobic surfaces are available/exposed in [Gly55]σ70, [Gly59]σ70 as well as in [Gly434]σ70 in comparison to wild‐type σ70. Time‐resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (α2ββ′) have shown reduced transcriptional activity in comparison to the enzyme containing wild‐type sigma factor. However, another mutation (Val→Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83]σ70, has similar properties as the wild‐type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in σ70 may interact hydrophobically with the 2.3/2.4 DNA‐binding region.
Gloverin, an Antibacterial Protein from the Immune Hemolymph of Hyalophora Pupae
Authors: Axén Andreas , Carlsson Anette , Engström Åke , Bennich Hans
Published: July 2004

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (PI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins.

Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1–3 μM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100°C, 10 min) but is inhibited by Mg2+and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an α‐helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell‐bound LPS.

The activity of gloverin is in many respects similar to that of attach, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.‐Y. & Lindholm, D. A. (1991) Eur. J. Biochem. 201, 23–31].
The Structure, Regulation and Function of the Janus Kinases (JAKs) and the Signal Transducers and Activators of Transcription (STATs)
Authors: Pellegrini Sandra , Dusanter‐Fourt Isabelle
Published: July 2004

Since the discovery of their physiological roles in cytokine signalling, the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs) have attracted considerable attention, to the point that the concept of a intracellular signalling pathway, named JAWSTAT, has emerged. As originally defined, this pathway involves ligand‐dependent activation of a particular class of receptor‐associated tyrosine kinases, the JAK proteins, which phosphorylate themselves and receptor components, creating recruitment sites for STAT transcription factors. The STATs are phosphorylated, they dissociate from the receptor. JAK complex and translocate to the nucleus where they participate in transcriptional gene activation. Although this pathway was found initially to be activated by interferons, it is now known that a large number of cytokines, growth factors and hormonal factors activate JAK and/or STAT proteins. Recent findings have suggested that the interdependence of JAKs and STATs might not be absolute as originally thought.
Incomplete Processing of Type II Procollagen by a Rat Chondrosarcoma Cell Line
Authors: Fernandes Russell J. , Schmid Thomas M. , Harkey Michael A. , Eyre David R.
Published: July 2004

The Swarm rat chondrosarcoma cell line, RCS‐LTC, deposits an extracellular matrix that contains the typical type II, IX, and XI collagen phenotype of hyaline cartilage, but the fibrils appear abnormally thin. By N‐terminal sequence analysis, the type TI collagen from the matrix was shown to have retained its N‐propeptides with no evidence of normal processing to type 11 collagen. Amplification and sequencing of cDNA prepared from the proα1(II) mRNA of these cells showed a normal N‐propeptide cleavage site. Furthermore, the type II N‐procollagen could be processed to type I1 collagen by incubation with culture medium from normal chondrocytes. The findings indicate that the RCS‐LTC cell line fails to express an active type I1 procollagen N‐proteinase and, therefore, offers a useful culture system in which to study the role of N‐propeptide removal in fibrillogenesis.
Expression of CYP3A in the Human Liver — Evidence that the Shift between CYP3A7 and CYP3A4 Occurs Immediately After Birth
Authors: Lacroix Dan , Sonnier Michelle , Moncion Arlette , Cheron Gérard , Cresteil Thierry
Published: July 2004

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P‐450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16α hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6β derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30–40% of the adult activity after one month.

CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age.

These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.
Identification of Amino Acid Residues at Nucleotide‐Binding Sites of Chaperonin GroEL/GroES and cpn10 by Photoaffinity Labeling with 2‐azido‐adenosine 5′‐triphosphate
Authors: Bramhall Elizabeth A. , Cross Richard L. , Rospert Sabine , Steede N. Kalaya , Landry Samuel J.
Published: July 2004

Although the chaperonin GroEL/GroES complex binds and hydrolyzes ATP, its structure is unlike other known ATPases. In order to better characterize its nucleotide binding sites, we have photolabeled the complex with the affinity analog 2‐azido‐ATP. Three residues of GroEL, Prol37, Cysl38 and Thr468, are labeled by the probe. The location of these residues in the GroEL crystal structure [Braig, K., Otwinowski, Z., Hedge, R., Boisvert, D., Joachimiak, A., Horwich, A. & Sigler, P. (1994)Nature 371, 578–586; Boisvert, D. C., Wang, J., Otwinowski, Z., Horwich, A. L. & Sigler, P. B. (1996)Nat. Struct. Biol. 3, 170–177] suggests that 2‐azido‐ATP binds to an alternative conformer of GroEL in the presence of GroES. The labeled site appears to be located at the GroEL/GroEL subunit interface since modification of Pro 137 and Cysl38 is most readily explained by attack of a probe molecule bound to the adjacent GroEL subunit. Labeling of the co‐chaperonin, GroES, is clearly demonstrated on gels and the covalent tethering of nucleotide allows detection of a GroES dimer in the presence of SDS. However, no stable peptide derivative of GroES could be purified for sequencing. In contrast, the GroES homolog, yeast cpnlO, does give a stable derivative. The modified amino acid is identified as the conserved Prol3, which corresponds to Pro5 in Escherichia coli GroES.
Identification of Phosphopeptide Ligands for the Src‐Homology 2 (SH2) Domain of Grb2 by Phage Display
Authors: Gram Hermann , Schmitz Rita , Zuber Jean François , Baumann Götz
Published: July 2004

We report here on the identification of phosphopeptide ligands which interact with the Src‐homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c‐Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S‐transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST‐Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand She. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.
Effect of Complexes of ADP and Phosphate Analogs on the Conformation of the Cys707‐Cys697 Region of Myosin Subfragment 1
Authors: Phan Brigitte C. , Peyser Y. Michael , Reisler Emil , Muhlrad Andras
Published: July 2004

Recent crystallographic studies have suggested structural differences between the complexes of S1 ˙ Mg ˙ ADP with the phosphate analogs aluminium fluoride (AlF−4), vanadate (VO3‐4) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960–8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404–54171. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707 – Cys697 (SH1‐SH2) cross‐linking, were used to compare the S1 ˙ ADP ˙ BeFx, S1 ˙ ADP ˙ AlF−4 and S1 ˙ ADP ˙ VO3‐4 complexes with specific states of the myosin‐ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin and N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20°C lead to the conclusion that S1˙ ADP ˙ BeFx is similar to S1*˙ ATP, while S1 ˙ ADP ˙ AIF−4 and S1 ˙ ADP ˙ VO43‐ are more similar to S1**˙ ADP ˙ Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1‐N6‐ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707‐modified (by N‐ethylmaleimide) and Cys707‐Cys697‐cross‐linked (by N,N′‐p‐phenylene dimaleimide) S1 ˙ ADP[C2H2] ˙ AlF−4 and S1 ˙ ADP[C2H2] ˙ BeFx. Our results suggest that the conformations of the S1 ˙ ADP ˙ AlF−4, S1 ˙ ADP ˙ VO3‐4 and S1 ˙ ADP ˙ BeFx, complexes in the Cys707–Cys697 region are distinct from each other, with the former two at least partially resembling the S1**˙ ADP ˙ Pi state, while the latter is similar to the prehydrolyzed S1*˙ ATP state.