bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and pharmacological characterization of recombinant rat/mice N‐methyl‐	D‐aspartate receptor subtypes in the yeast Saccharomyces cerevisiae
Authors: Becker Jürgen , Li Ziyu , Noe Christian R.
Published: March 2003

The genes encoding the ionotropic N‐methyl‐
D‐aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi‐copy yeast‐Escherichia coli shuttle vectors pMB01 and pMB02. The protease‐deficient yeast Saccharomyces cerevisiae cl3‐ABYS‐86 (leu−, ura−, his−) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes.

Western‐blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains.

By immunofluorescence microscopy using the specific subunit antibodies and fluorescence‐labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells.

Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1−3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88 ± 5.38 nM (NR1a), 1365.11 ± 76 nM (NR1a/NR2D), 22.97 ± 3.37 nM for NR1a/NR2B/NR2D and 7.4 ± 1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07 ± 0.92 (NR1a), 14.63 ± 0.50 (NR1a/NR2D), 12.85 ± 1.68 (NR1a/NR2B/NR2D) and 8.3 ± 0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7‐dichlorokynurenate (DCKA), ethyl‐2‐carboxy‐4.6‐dichloro‐3‐indoleacetate (ECDI) and itself, but not by glycine,
D‐serine and 1‐amino‐cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using
L‐[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536 ± 43, 688 ± 60, and 856 ± 48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.
Two distinct forms of human mast cell chymase
Authors: McEuen Alan R. , Gaça Marianna D. A. , Buckley Mark G. , He Shaoheng , Gore Michael G. , Walls Andrew F.
Published: March 2003

Chymase, a chymotrypsin‐like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin‐agarose chromatography of high‐salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0−1.2 M NaCl (peak B) and 1.8−2.0 M NaCl (peak C), with peak B containing 75−90 % of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S‐200. Both had a molecular mass of 28−29 kDa, yielded the N‐terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.
The laminarinase from thermophilic eubacterium Rhodothermus marinus
Authors: Krah Martin , Misselwitz Rolf , Politz Oliver , Thomsen Karl Kristian , Welfle Heinz , Borriss Rainer
Published: March 2003

A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278. The enzyme purified from recombinant Escherichia coli cells hydrolyses mixed 1,3‐1,4‐β‐glucans (lichenan, barley and oat β‐glucan) and 1,3‐β‐homoglucans (laminarin, curdlan) by an endo type action pattern. The CD spectrum of laminarinase is characteristic for a protein with prevailing β secondary‐structural elements, and the fluorescence spectrum points to a surface localisation of the tryptophan residues. A half‐transition concentration of 5.4 M guanidinium chloride was measured for the denaturant‐induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence. Substitution of acidic residues Glu129, Asp131 and Gln134, which are invariant in family 16 glycosyl hydrolases, caused a severe reduction of β‐glucan−hydrolysing activity suggesting their central role in enzymatic hydrolysis. Deletion of Met133 drastically reduced catalytic activity. Met133 is invariant in family 16 laminarinases but not present in the active‐site region of bacterial 1,3‐1,4‐β‐glucanases which also belong to glycosyl hydrolase family 16. Replacement of Met133 by Ala, Cys or Trp did not affect activity against 1,3‐1,4‐β‐polyglucans and 1,3‐β‐polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1‐4G1‐3Gr) was increased about 10 times. Hydrolysis of 1,3‐β‐oligosaccharides and 1,4‐β‐oligosaccharides (DP 2‐7) demonstrated the ability of the enzyme to cleave 1,3‐β‐linkages and 1,4‐β‐linkages in low‐molecular‐mass carbohydrates independent of the structure of neighbouring linkages. The laminarinase contains five or six subsites for substrate binding according to the action pattern deduced from hydrolysis of labelled and unlabelled curdlan oligosaccharides of different chain length.
Structural information on a cecropin‐like synthetic peptide, Shiva‐3 toxic to the sporogonic development of Plasmodium berghei
Authors: Boisbouvier Jérôme , Prochnicka‐Chalufour Ada , Nieto Alejandro R. , Torres Jorge A. , Nanard Nathalie , Rodriguez Mario H. , Possani Lourival D. , Delepierre Muriel
Published: March 2003

This study is a contribution towards the understanding of the mode of action of Shiva‐3 and more generally that of cecropin‐like peptides. Structural information on Shiva‐3 (a cecropin‐like synthetic peptide) in water and in a membrane‐mimicking environment (trifluoroethanol alcohol, SDS) were obtained using analytical centrifugation, CD and NMR spectroscopies. A total of 20 converged structures were retained on the basis of 197 non‐redundant experimental constraints, including 166 distance constraints from the nuclear Overhauser effect measurements and 31 dihedral angle restraints derived from the purged COSY experiments. Some results obtained in presence of SDS are also presented.

The toxic effects of the peptides obtained by cleavage (trypsin and lysine‐C hydrolysis) of Shiva‐3 on Escherichia coli and on Plasmodium berghei sporogonic stages are reported. Biological effects are discussed in relation to the calculated structure. The antiparasite activity and the low mosquito toxicity of Shiva‐3 make this peptide a good candidate for genetic transformation of mosquito vectors which warrants further studies aimed at the improvement of the molecule.
New isoform of the neuronal Ca2+ channel α1E subunit in islets of Langerhans and kidney
Authors: Vajna Rolf , Schramm Martin , Pereverzev Alexei , Arnhold Stefan , Grabsch Heike , Klöckner Udo , Perez‐Reyes Edward , Hescheler Jürgen , Schneider Toni
Published: March 2003

The expression of Ca2+ channel α1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned α1E cDNA sequences led to the prediction of α1E splice variants. Transcripts of two cloned α1E isoforms, which are discriminated by a carboxy terminal 129‐bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter α1E isoform have been assigned to the rat cerebrum and to neuron‐like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)‐PCR and visualized on the protein level by Western blotting with common and isoform‐specific antibodies. Transcripts of the longer α1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS‐1 (rat) and βTC‐3 (mouse), in the pituitary cell line AtT‐20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of α1E in cell lines and tissues shows that the wide expression of α1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called α1Ee has been determined by RT‐PCR, while in cerebellum a set of different α1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue‐specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform α1Ee in islets of Langerhans and kidney leads to the suggestion that α1E might be involved in the modulation of the Ca2+‐mediated hormone secretion.
Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis
Authors: Sluis‐Cremer Nicolas , Wallace Louise , Burke Jonathan , Stevens Julie , Dirr Heini
Published: March 2003

The binding interactions between dimeric human class alpha glutathione S‐transferase A1‐1 (GST A1‐1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1‐1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (Kd = 8 ± 2 μM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1‐1 and protein‐bound aflatoxin B1 was calculated to be 22.7 Å by means of fluorescence resonance energy transfer. The aflatoxin‐binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two‐fold axis. Hill‐plot analyses suggest that a positive co‐operative interaction exists between BSP and the dimeric GST A1‐1 (h = 1.6 ± 0.1; K ′ = 14 ± 0.6 μM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent‐accessible properties of the enzyme's Trp20 residue. Site‐directed mutagenesis of Trp20 (Trp20←Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a co‐operative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.
Quantification of thyroid hormone receptor isoforms, 9‐cis retinoic acid receptor γ, and nuclear receptor co‐repressor by reverse‐transcriptase PCR in maturing and adult skeletal muscles of rat
Authors: Schuler Michael J. , Pette Dirk
Published: March 2003

Quantitative competitive reverse‐transcriptase polymerase chain reaction (qcRT‐PCR) was established for determining absolute molecule numbers of the thyroid hormone receptor (T3R) isoforms T3Rα1, T3Rα2, T3Rβ1, and the 9‐cis retinoic acid receptor γ (RXRγ) in developing and adult fast‐twitch extensor digitorum longus (EDL) and slow‐twitch soleus (SOL) muscles of rat. Expression levels of the nuclear receptor co‐repressor (NCoR) were measured in the same muscles because responses to thyroid hormones during muscle maturation might not only depend on the expression levels of the various receptors but might also be modulated by changes in the expression of NCoR. The qcRT‐PCR method was based on the addition of known amounts of homologous competitor RNAs to the reverse transcriptase (RT) reaction. We show that all nuclear receptors under study were expressed in fast and slow muscles. Transcript numbers of T3Rβ1, which was the most abundant isoform, were higher in SOL than in EDL during all developmental stages. The mRNAs for T3Rα1, T3Rα2, RXRγ and the NCoR displayed molecule numbers in similar ranges, but were differentially expressed. T3Rα1 mRNA increased in SOL during postnatal development, while T3Rα2 mRNA initially decreased, then increased to adult levels. Conversely, pronounced decreases were observed for T3Rα1 (10‐fold) and T3Rα2 (28‐fold) mRNAs in the EDL muscle during postnatal maturation. RXRγ mRNA was 10‐fold downregulated during EDL maturation, but unaltered in maturing SOL. NCoR transcript number displayed only minor changes in both muscles.
Structural determination of the O‐antigenic polysaccharide from Escherichia coli O35 and cross‐reactivity to Salmonella arizonae O62
Authors: Rundlöf Torgny , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigenic polysaccharide from Escherichia coli O35 has been investigated with the aid of NMR spectroscopy, sugar and methylation analyses. The sequence of the sugar residues could be determined by NOESY and heteronuclear‐multiple‐bond‐connectivity NMR experiments. The polysaccharide is composed of hexasaccharide repeating units with the following structure, where Rha and GalNAcAN represent rhamnose and 2‐acetamido‐2‐deoxy‐galacturonamide, respectively :

←3)‐β‐
D‐GlcpNAc‐(1←3)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←

2

↑

1

α‐ D‐GalpNAcAN

The O‐antigen of Escherichia coli O35 is similar to the O‐specific polysaccharide from Salmonella arizonae O62, which instead has a terminal 2‐acetamido‐2‐deoxy‐α‐
D‐galacturonic acid residue. Immunochemical analyses using a rabbit antiserum specific for the Salmonella arizonae O62 O‐antigen showed an identical reactivity with both lipopolysaccharides.
Glycan specificity of myelin‐associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides
Authors: Strenge Karen , Schauer Roland , Bovin Nicolai , Hasegawa Akira , Ishida Hideharu , Kiso Makoto , Kelm Sørge
Published: March 2003

Myelin‐associated glycoprotein (MAG) and sialoadhesin (Sn) bind to sialylated glycans on cell surfaces and are thought to be involved in cell‐cell interactions. In order to investigate how the interactions of these proteins are influenced by the glycan structure, we compared the inhibitory potencies of different synthetic monovalent oligosaccharides and polyvalent polyacrylamide derivatives. Using oligosaccharides with modifications in the sialic acid, galactose or N‐acetylglucosamine moieties, we could demonstrate that both MAG and Sn bind with high preference to α2,3‐linked sialic acid and interact at least with the three terminal monosaccharide units. For MAG, contacts with even more distant monosaccharides are likely, since pentasaccharides are bound better than trisaccharides. Also, an additional sialic acid at position six of the third‐terminal monosaccharide unit enhances binding to MAG, whereas it does not influence binding to Sn significantly. Modifications of the sialic acid glycerol side chain demonstrated that the hydroxy groups at positions 8 and 9 are required for binding to both proteins. Surprisingly, MAG binds 2‐keto‐3‐deoxy‐
D‐glycero‐
D‐galacto‐nononic acid significantly better than N‐acetylneuraminic acid, whereas Sn prefers the latter structure. These results indicate that the interactions of MAG and Sn are mainly with sialic acid and that additional contacts with the subterminal galactose and N‐acetylglucosamine residues also contribute to the binding strength, although to a lesser degree.
Cyclophilin‐D binds strongly to complexes of the voltage‐dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore
Authors: Crompton Martin , Virji Sukaina , Ward John M.
Published: March 2003

A cyclophilin‐D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore. A cDNA encoding cyclophilin‐D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S‐transferase/cyclophilin‐D fusion protein in Escherichia coli XL1 cells. The cyclophilin‐D in the fusion was functionally normal as judged by its peptidylprolyl cis‐trans‐isomerase activity and its inhibition by cyclosporin A. The fusion protein was bound to glutathione‐agarose to form the cyclophilin‐D affinity matrix. The matrix selectively bound 32‐kDa proteins of mitochondrial membrane extracts, but no H2O‐soluble proteins were bound. The 32‐kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage‐dependent anion channel (porin) and the adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus‐purified voltage‐dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. These properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage‐dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin‐D, and forms at contact sites between the two membranes.
Enhancement of phosphoinositide 3‐kinase (PI 3‐kinase) activity by membrane curvature and inositol‐phospholipid‐binding peptides
Authors: Hübner Stefan , Couvillon Anthony D. , Käs Josef A. , Bankaitis Vytas A. , Vegners Rolands , Carpenter Christopher L. , Janmey Paul A.
Published: March 2003

The phosphorylation of phosphatidylinositol (PtdIns) on the 3′ position of the inositol ring by phosphoinositide 3‐kinase (PI 3‐kinase) is shown to depend strongly on the curvature of liposomes containing a mixture of phosphatidylcholine (PtdCho) and PtdIns. Vesicles with an average diameter of 50 nm are phosphorylated 100 times faster than chemically identical vesicles with an average diameter greater than 300 nm. The low reactivity of large vesicles is not due to the difference in vesicle number for large and small vesicles at constant total lipid, nor to occlusion of lipid surfaces in multilammelar structures, and can be reversed by addition of low (< 1 : 100) molar ratios of either the PtdIns transfer protein sec14p or a ten‐residue peptide derived from the inositol‐phospholipid‐binding site of gelsolin. Similar measurements using PI 4‐kinase showed a weak dependence on vesicle size. The strong dependence of PI 3‐kinase function on membrane curvature suggests possible localization of PI 3‐kinase activity at sites where clustering of receptors, for example, may locally deform the membrane, and suggests that once PI 3‐kinase is localized and activated at surface sites, the reaction may become self‐accelerating.
Both Ets‐1 and GATA‐1 are essential for positive regulation of platelet factor 4 gene expression
Authors: Minami Takashi , Tachibana Keisuke , Imanishi Takeshi , Doi Takefumi
Published: March 2003

In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions −880, −75 and −135, −30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either −75Ets, −135GATA, or −30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel‐retardation assays revealed that Ets‐1 and GATA‐1 proteins from HEL and MEG‐01 cells bound to the Ets motifs and GATA motifs, respectively. Co‐transfection experiments showed that the overexpression of Ets‐1 and/or GATA‐1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets‐1 and GATA‐1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets‐1 increased 10‐fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets‐1 and GATA‐1 play key roles in the positive regulation of PF4 gene expression.
Elapid venom toxins: multiple recruitments of ancient scaffolds
Authors: Alape‐Girón Alberto , Persson Bengt , Cederlund Ella , Flores‐Díaz Marietta , Gutiérrez José María , Thelestam Monica , Bergman Tomas , Jörnvall Hans
Published: March 2003

Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and α‐neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+‐channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface‐exposed residues.
Analysis of a conditional degradation signal in yeast and mammalian cells
Authors: Lévy Frédéric , Johnston Jennifer A. , Varshavsky Alexander
Published: March 2003

The N‐end rule pathway is a ubiquitin‐dependent proteolytic system, the targets of which include proteins that bear destabilizing N‐terminal residues. The latter are a part of the degradation signal called the N‐degron. Arg‐DHFRts, an engineered N‐end rule substrate, bears N‐terminal arginine (a destabilizing residue) and DHFRts[a temperature‐sensitive mouse dihydrofolate reductase (DHFR) moiety]. Previous work has shown that Arg‐DHFRts is long‐lived at 23 °C but short‐lived at 37 °C in the yeast Saccharomyces cerevisiae. In the present work, we extended this analysis, and found that the degradation of Arg‐DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low‐Mr ligand of DHFR. In S. cerevisiae, Arg‐DHFRts is degraded at 37 °C exclusively by the N‐end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety. In mouse cells, MTX completely inhibits the degradation of Arg‐DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N‐degron. When the N‐terminus of Arg‐DHFRts was extended with a 42‐residue lysine‐lacking extension, termed eΔK, the resulting Arg‐eΔK‐DHFRts was rapidly degraded at both 23 °C and 37 °C. Moreover, the degradation of Arg‐eΔK‐DHFRts, in contrast with that of Arg‐DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg‐DHFRts at 23 °C results, at least in part, from steric inaccessibility of its N‐terminal arginine. The N‐degron of Arg‐DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell‐penetrating compound. We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.
Novel glycoinositolphosphosphingolipids, basidiolipids, from Agaricus
Authors: Jennemann Richard , Bauer , Bernhard L. , Bertalanffy , Helmut , Geyer , Rudolf , Gschwind , Ruth M. , Selmer , Thorsten , Wiegandt Herbert
Published: March 2003

From the edible mushroom, the basidiomycetes Agaricus bisporus and Agaricus campestris, a novel carbohydrate‐homologous series of four glyco‐inositol‐phospho‐sphingolipids, designated basidiolipids, was isolated and the constituents purified. The chemical structures of the basidiolipids were elucidated to be: Manpβ1‐2inositol1‐phospho‐ceramide, Galpα‐6[Fucpα‐2]Galpβ‐6Manpβ‐2inositol1‐phospho‐ceramide, Galpα‐6Galpα‐6[Fucpα‐2]Galpβ‐ 6Manpβ‐2inositol1‐phospho‐ceramide and Galpα‐6Galpα‐6Galpα‐6[Fucpα‐2]Galpβ‐6Manpβ‐2inositol1‐phospho‐ceramide. All four glycolipids contained a ceramide which was composed of phytosphingosine and predominantly α‐hydroxy‐behenic and α‐hydroxy‐lignoceric acid.
Stepwise building of a 115‐kDa macromolecular edifice monitored by electrospray mass spectrometry
Authors: Laprévote Olivier , Serani Laurent , Das Bhupesh C. , Halgand Frédéric , Forest Éric , Dumas Renaud
Published: March 2003

The macromolecular complexes formed by the enzyme acetohydroxy acid isomeroreductase with NADPH, magnesium ions and the competitive inhibitor N‐hydroxy‐N‐isopropyloxamate (IpOHA) were analysed by electrospray mass spectrometry. Each ligand was added successively to a protein solution, allowing the stoichiometry of the whole macromolecular edifice (115 583 Da) to be unambiguously determined. The combination of an electrospray ion source with the high mass range magnetic instrument used in the present studies proved to be a very powerful tool for characterizing, in a specific manner, the quaternary structures of proteins by single mass measurements.
The DNA helicases acting in nucleotide excision repair, XPD, CSB and XPB, are not required for PCNA‐dependent repair of abasic sites
Authors: Cappelli Enrico , Carrozzino Fabio , Abbondandolo Angelo , Frosina Guido
Published: March 2003

DNA repair of abasic sites is accomplished in mammalian cells by two distinct base excision repair (BER) pathways: a single nucleotide insertion pathway and a proliferating cell nuclear antigen (PCNA)‐dependent pathway involving a resynthesis patch of 2–10 nucleotides 3′ to the lesion. The latter pathway shares some enzymatic components with the nucleotide excision repair (NER) pathway acting on damage induced by ultraviolet light: both pathways are strictly dependent on PCNA and several observations suggest that the polymerization and ligation phases may be carried out by common enzymatic activities (DNA polymerase δ/ε and DNA ligase I). Furthermore, it has been postulated that the transcription‐NER coupling factor Cockayne syndrome B has a role in BER. We have investigated whether three NER proteins endowed with DNA helicase activities (the xeroderma pigmentosum D and B gene products and the Cockayne syndrome B gene product) may also be involved in repair of natural abasic sites, by using the Chinese hamster ovary mutant cell lines UV5, UV61 and 27‐1. No defect of either the PCNA‐dependent or the single nucleotide insertion pathways could be observed in UV5, UV61 or 27‐1 mutant cell extracts, thus showing that the partial enzymatic overlap between PCNA‐dependent BER and NER does not extend to DNA helicase activities.
Characterization of the leukotriene B4 receptor in porcine leukocytes
Authors: Igarashi Toshio , Yokomizo Takehiko , Tsutsumi Osamu , Taketani Yuji , Shimizu Takao , Izumi Takashi
Published: March 2003

Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide‐binding regulatory protein (G‐protein)‐coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G‐proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G‐proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G‐proteins, in combination with the fraction which contained endogenous phospholipids and Gβγ. Kinetic studies of LTB4 were performed to determine the association with G‐proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G‐proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G‐protein‐rich fraction (flow‐through of an RCA column) increased the affinity for LTB4 10‐fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G‐proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant α subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda‐9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin‐sensitive G‐proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G‐protein‐coupled receptors.
Homologous plasminogen N‐terminal and plasminogen‐related gene A and B peptides
Authors: Lewis Valerae O. , Gehrmann Marion , Weissbach Lawrence , Hyman Joshua E. , Rielly Al , Jones Deryk G. , Llinás Miguel , Schaller Johann
Published: March 2003

The cDNA corresponding to exons 2–4 of the processed human plasminogen (Pgn) gene, encoding the N‐terminal peptide domain (NTP), has been cloned, expressed in Escherichia coli as a recombinant protein (r‐NTP) containing a hexahistidine tag, and refolded to the native structure that contains two internal cystine bridges. RNA expression of the two Pgn‐related genes, PRG A and PRG B, that potentially encode 9‐kDa polypeptides having extensive similarity to the NTP has been investigated. Using RNA‐based PCR with liver RNA as template, we demonstrate that PRG A encodes a detectable mRNA species. PRG A and PRG B have been found to be transcribed in the liver and yield virtually identical mRNAs. Neither of the PRGs are expressed in a variety of other normal tissues, as determined by Northern blot analysis. Factor‐Xa digestion of the tagged r‐NTP yields cleavage products which indicates that the expressed r‐NTP domain of Pgn is endowed with a flexible conformation. Recombinant PRG B protein (r‐PRG B) fused to a hexahistidine tag was purified and analyzed for structural integrity. Preliminary 1H‐NMR spectroscopic data for r‐NTP and r‐PRG B indicate relatively fast amide 1H–2H exchange in 2H2O and close conformational characteristics for the two homologous polypeptides. Far ultraviolet‐CD spectra for r‐NTP and r‐PRG B at pH 7.0 indicate similar defined secondary structure content for both domains, with 13–17% α‐helix and 24–27% antiparallel β‐sheet. The fact that two transcriptionally active genes encode almost identical polypeptides supports the hypothesis that the Pgn NTP, together with the putative polypeptides encoded by the PRGs, may serve an important function, such as controlling the conformation of Pgn and thus its susceptibility to tissue activators.
Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin
Authors: Kunugi Shigeru , Yanagi Yuichi , Oda Kohei
Published: March 2003

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/Km of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and ln (Ki–1) was almost linear with respect to pressure. The volume of the reaction (ΔVcomp), which is the volume change accompanying enzyme–inhibitor complex formation, was calculated as + 8.1 ± 0.3 mL·mol–1, which has a sign opposite to ΔVcomp for neutral peptide inhibitors and acyl‐peptide substrates. The temperature dependence of Ki–1 gave the reaction enthalpy (ΔHcomp) and reaction entropy (ΔScomp) of the complex formation as 34.6 ± 1.4 kJ·mol–1 and 298 ± 5 J·mol–1·K–1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation.

 Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz‐Phe‐Gly‐NH2 and Cbz‐Phe also positively affected the pressure‐tolerance of thermolysin, in the following order: Cbz‐Gly‐Phe‐NH2 < Cbz‐Phe < < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.
Substrate‐specific selenoprotein B of glycine reductase from  Eubacterium acidaminophilum
Authors: Wagner Matthias , Sonntag Denise , Grimm Rudolf , Pich Andreas , Eckerskorn Christoph , Söhling Brigitte , Andreesen Jan R.
Published: March 2003

The substrate‐specific selenoprotein B of glycine reductase (PBglycine) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (α), 25 (β) and 47 kDa (γ), probably in an α2β2γ2 composition. PBglycine purified from cells grown in the presence of [75Se]selenite was labeled in the 47‐kDa subunit. The 22‐kDa and 47‐kDa subunits both reacted with fluorescein thiosemicarbazide, indicating the presence of a carbonyl compound. This carbonyl residue prevented N‐terminal sequencing of the 22‐kDa (α) subunit, but it could be removed for Edman degradation by incubation with o‐phenylenediamine.

 A DNA fragment was isolated and sequenced which encoded β and α subunits of PBglycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the γ subunit of PBglycine (grdB2). The cloned DNA fragment represented a second GrdB‐encoding gene slightly different from a previously identified partial grdB1‐containing fragment. Both grdB genes contained an in‐frame UGA codon which confirmed the observed selenium content of the 47‐kDa (γ) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N‐terminal 25‐kDa (β) subunit and a 22‐kDa (α) subunit of PBglycine. Cleavage most probably occurred at an ‐Asn‐Cys‐ site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the α subunit.
Stopped‐flow studies of the binding of 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide to fumarate reductase of Escherichia coli
Authors: Zhao Zhongwei , Rothery Richard A. , Weiner Joel H.
Published: March 2003

We have studied the kinetics of binding of the menaquinol analog 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide (HOQNO) by fumarate reductase (FrdABCD) using the stopped‐flow method. The results show that the fluorescence of HOQNO is quenched when HOQNO binds to FrdABCD. The observed quenching of HOQNO fluorescence has two phases and it can be best fitted to a double exponential equation. A two‐step equilibrium model is applied to describe the binding process in which HOQNO associates with FrdABCD by a fast bimolecular step to form a loosely bound complex; this is subsequently converted into a tightly bound complex by a slow unimolecular step. The rates of the forward and the reverse reactions for the first equilibrium (k1 and k2) are determined to be k1 = (1.1 ± 0.1) × 107 m−1·s−1, and k2 = 6.0 ± 0.6 s−1, respectively. The dissociation constants of the first equilibrium (Kd1 = k2/k1) is calculated to be about 550 n
m. The overall dissociation constant for the two‐step equilibrium, Kd overall = Kd1/[1 + (1/Kd2)], is estimated to be ≤ 7 n
m. Comparison of the kinetic parameters of HOQNO binding by FrdABCD and by dimethyl sulfoxide reductase provides important information on menaquinol binding by these two enzymes.
Regulation of cyclooxygenase‐2 expression in human mesangial  cells – transcriptional inhibition by IL‐13
Authors: Díaz‐Cazorla Manuela , Pérez‐Sala Dolores , Ros Josefa , Jiménez Wladimiro , Fresno Manuel , Lamas Santiago
Published: March 2003

Activated mesangial cells may play an important part in glomerulonephritis. Cytokines can modulate the release of prostanoids by human mesangial cells (HMC). We have investigated the effects of pro‐inflammatory stimuli on COX‐2 expression in HMC and its potential modulation by interleukin (IL)‐13. HMC released increased amounts of prostaglandin E2 (PGE2) after treatment with several combinations of IL‐1β, tumor necrosis factor (TNF)‐α and/or lipopolysaccharide. Increases in PGE2 correlated with the induction of COX‐2 protein expression. The accumulation of PGE2 elicited by a combination of IL‐1β/TNF‐α correlated closely with the temporal pattern of COX‐2 protein expression, which reflected the induction of COX‐2 mRNA. IL‐13 inhibited IL‐1β/TNF‐α‐elicited PGE2 production, as well as COX‐2 protein and mRNA expression in a concentration‐dependent fashion. With 50 ng·mL–1 IL‐13 these parameters were inhibited by 90, 80 and 84%, respectively. In HMC transfected with the 5′ regulatory region of the COX‐2 gene, IL‐13 suppressed cytokine‐induced promoter activation. Our results suggest that COX‐2 expression is a major target for IL‐13‐mediated abrogation of prostaglandin release by HMC and support that this process takes place by transcriptional inhibition of the COX‐2 gene.
Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins
Authors: Köster Mario , Hauser Hansjörg
Published: March 2003

STAT proteins (signal transducers and activators of transcription) are a family of transcription factors which are used by many cytokines and cell growth factors for initiating gene expression. They are activated by tyrosine phosphorylation through the cytoplasmic domain of stimulated receptors. Upon phosphorylation STAT proteins dimerize, translocate to the nucleus and activate transcription by binding to specific recognition sites. Different cytokines activate different subsets of STATs and other signaling proteins.

 We have made use of green fluoresencent protein (GFP) fusion proteins to visualize the subcellular localization and trafficking of STAT1, STAT2 and p48 during interferon (IFN) stimulation and have analysed in detail STAT1‐GFP trafficking in living cells. Analysis of GFP fusion proteins allowed the determination of time kinetics of subcellular trafficking in individual living cells. STAT1‐GFP is indistinguishable from its wild‐type protein displaying strong activity as transcriptional activator as well as the same time kinetics of transport to the nucleus and retreat to the cytoplasm. After prolonged exposure to IFN, STAT1‐GFP is no longer retained in the nucleus and relocation to the cytoplasm is observed. Restimulation with the same type of IFN does not lead to repeated nuclear translocation of STAT1‐GFP. STAT1 is not subject of inhibition, as restimulation with another type of IFN allows immediate reuse of previously activated STAT1‐GFP. However, restimulation with the same type of IFN can be achieved when the primary stimulus is removed after a short induction period. This method of visualizing signal transduction reveals a considerable inhomogeneity with respect to the extent of STAT1‐GFP shuttling within a clonal cell population, indicating that competence for full‐blasted IFN response is restricted to a cellular subpopulation whereas other cells respond incompletely, retarded or not at all.
The tricarboxylic acid cycle of Helicobacter pylori
Authors: Pitson Stuart M. , Mendz George L. , Srinivasan Sujatha , Hazell Stuart L.
Published: March 2003

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]‐ and [13C]‐NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, α‐ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including α‐ketoglutarate dehydrogenase, succinyl‐CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and α‐ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of α‐ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a γ‐aminobutyrate shunt, owing to the absence of both γ‐aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
The epidermal growth factor precursor
Authors: Valcarce Carmen , Björk Ingemar , Stenflo Johan
Published: March 2003

Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor.The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N‐terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post‐traslational β‐hydroxylation of Asp/Asn residues. We identified the presence of erythro‐β‐hydroxy‐aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 m·m protein−1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro‐β‐hydroxyasparagine (Hyn). The Hyn‐containing modules have a consensus calcium‐binding motif immediately N‐terminal of the first Cys residue. The synthetic EGF module 2 (residues 356–395) of the EGF precursor was found to bind calcium with low affinity, Κd ≈ 3.5 m
m, i.e. similar to the affinity of other isolated calcium‐binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd ≈ 0.2 µm, i.e. ≈104‐fold higher than that of isolated EGF modules presumably due to the influence of neigboring modules. We have detected EGF precursor in platelet‐rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30–160 EGF molecules each.
Isolation of the CXC chemokines ENA‐78, GROα and GROγ from tumor cells and leukocytes reveals NH2‐terminal heterogeneity
Authors: Wuyts Anja , Govaerts Cindy , Struyf Sofie , Lenaerts Jean‐Pierre , Put Willy , Conings René , Proost Paul , Van Damme Jo
Published: March 2003

Chemokines are a family of chemotactic peptides affecting leukocyte migration during the inflammatory response. Post‐translational modification of chemokines has been shown to affect their biological potency. Here, the isolation and identification of natural isoforms of the neutrophil chemoattractants GROα and GROγ and the epithelial‐cell‐derived neutrophil attractant‐78 (ENA‐78), is reported. Cultured tumor cells produced predominantly intact chemokine forms, whereas peripheral blood monocytes secreted mainly NH2‐terminally truncated forms. The order of neutrophil chemotactic potency of these CXC chemokines was GROα > GROγ > ENA‐78 both for intact and truncated forms. However, truncated GROα(4,5,6‐73), GROγ(5‐73) and ENA‐78(8,9‐78) were 30‐fold, fivefold and threefold more active than the corresponding intact chemokine. As a consequence, truncated GROα(4,5,6‐73) was 300‐fold more potent than intact ENA‐78 indicating that both the type of chemokine and its mode of processing determine the chemotactic potency. Similar observations were made when intact and truncated GROα, GROγ and ENA‐78 were compared for their capacity to induce an increase in the intracellular calcium concentration in neutrophilic granulocytes, and to desensitize the calcium response towards the CXC chemokine granulocyte chemotactic protein‐2 (GCP‐2). It must be concluded that physiological proteolytic cleavage of CXC chemokines in general enhances the inflammatory response, whereas for CC chemokines NH2‐terminal processing mostly results in reduced chemotactic potency.
Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae
Authors: Jørgensen Marianne U. , Emr Scott D. , Winther Jakob R.
Published: March 2003

Vps10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)‐invertase or PrA(137)‐invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p‐dependent sorting of CPY‐invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY‐invertase and PrA‐invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.