bluuebunny/crossref_metadata_2025 huggingface dataset:
The antimicrobial peptide trichogin and its interaction with phospholipid membranes
Authors: Epand Raquel F. , Epand Richard M. , Monaco Vania , Stoia Simona , Formaggio Fernando , Crisma Marco , Toniolo Claudio
Published: March 2003

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4‐amino‐4‐carboxy‐2,2,6,6‐tetramethylpiperidino‐1‐oxyl (TOAC), replaced one of the three α‐aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐S‐indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin‐promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.
Identification and characterization of potential new therapeutic targets in inflammatory and autoimmune diseases
Authors: Mangold Ursula , Dax Claudia I. , Saar Karin , Schwab Wilfried , Kirschbaum Bernhard , Müllner Stefan
Published: March 2003

The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti‐inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel® matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3‐phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore® analysis. Kd values of glyceraldehyde 3‐phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT‐4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.
Characterization of adhesive epitopes with the OmpS display system
Authors: Lång Hannu , Mäki Minna , Rantakari Anssi , Korhonen Timo K
Published: March 2003

OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins. Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed. According to this model, OmpS contains 18 transmembrane β‐strands and nine surface‐accessible loops. Adhesive epitopes can, when inserted into surface‐accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics. We inserted three d‐repeats from the Staphylococcus aureus fibronectin‐binding protein FnBPA into L4 of OmpS and showed that E. coli cells expressing these hybrids bind fibronectin. DNA fragments covering the N‐terminal half of the globoside‐binding P‐fimbrial adhesin class II PapG of E. coli were cloned into the same surface accessible loop (L4) of OmpS. Fragments of papG encoding 53 or 186 amino acids from the N‐terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside. Removal of 23 amino acids from the N‐terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it. The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins. We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG. This antiserum recognized the P‐fimbriae on E. coli cells. These data provide evidence that the N‐terminal first 53 amino acids of class II PapG contain the receptor‐binding domain.
Assignment of heme methyl 1H‐NMR resonances of high‐spin and low‐spin ferric complexes of cytochrome P450cam using one‐dimensional and two‐dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments
Authors: Mouro Corinne , Bondon Arnaud , Jung Christiane , De Certaines Jacques D. , Simonneaux Gérard
Published: March 2003

An 1H‐NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low‐spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett.455, 302–306]. In this study, heme methyl protons of cytochrome P450 in the native high‐spin and low‐spin states were assigned through one‐dimensional and two‐dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high‐spin and low‐spin states. The methyl order observed in the ferric low‐spin isocyanide complexes is related to the orientation of the cysteinate ligand.
The structure of the carbohydrate backbone of core‐lipid A region ofthe lipopolysaccharides from Proteus mirabilis wild‐type strain S1959 (serotype O3) and its Ra mutant R110/1959
Authors: Vinogradov Evgeny , Radziejewska‐Lebrecht Joanna , Kaca Wieslaw
Published: March 2003

The following structure of core‐lipid A region of the lipopolysaccharide (LPS) from Proteus mirabilis strain 1959 (serotype O3) and its rough mutant R110/1959 (Proteus type II core) was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS:

Incomplete substitutions are indicated by italics. All sugars are in pyranose form, α‐Hep is the residue lglycero‐α‐dmanno‐Hep, α‐dd‐Hep is the residue dglycero‐α‐dmanno‐Hep. The differences with the previously reported structures are discussed.
Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase
Authors: Espín Juan C. , Varón Ramón , Fenoll Lorena G. , Gilabert M. Angeles , García‐Ruíz Pedro A. , Tudela José , García‐Cánovas Francisco
Published: March 2003

This paper reports a quantitative study of the effect of ring substituents in the 1‐position of the aromatic ring on the rate of monophenol hydroxylation and o‐diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V Mmax values for each monophenol was found. In the case of o‐diphenols the same effect was observed but the size of the side‐chain became very important. NMR studies on the monophenols justified the sequence of the V Mmax values obtained. As regards the o‐diphenols, on the other hand, only a fair correlation between NMR and V Dmax values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step (k33) is not the rate‐determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o‐diphenols. The rate‐limiting step of the monophenolase activity could be the nucleophilic attack (k51) of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k52) of the oxygen atom of the active site of oxytyrosinase on the C‐3 of the monophenolic ring. However, the rate‐limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3‐position (k32) and to the size of the substituent side‐chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed.
Permeation of tetracyclines through membranes of liposomes and Escherichia coli
Authors: Sigler Albrecht , Schubert Peter , Hillen Wolfgang , Niederweis Michael
Published: March 2003

Uptake of tetracycline (tc), 2‐tetracyclinonitrile (CN‐tc), and 9‐(N,N‐dimethylglycylamido)‐6‐demethyl‐6‐deoxytetracycline (DMG‐DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG‐DMDOT into liposomes yielded permeation coefficients of (2.4 ± 0.6) × 10−9 cm·s−1 and (3.3 ± 0.8) × 10−9 cm·s−1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate‐limiting step. The permeation coefficients translate into half‐equilibration times of approximately 35 ± 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN‐tc into liposomes was at least 400‐fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN‐tc.
Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress
Authors: Tavazzi Barbara , Di Pierro Donato , Amorini Angela Maria , Fazzina Giovanna , Tuttobene Michele , Giardina Bruno , Lazzarino Giuseppe
Published: March 2003

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5–10 mm) of hydrogen peroxide for 1 h at 37 °C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP‐deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mm H2O2), oxidative stress produced, starting from 1 mm H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20‐fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mm). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP‐deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mm H2O2‐treated erythrocytes (10‐fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mm H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP‐deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.
Production of recombinant human β2‐microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis
Authors: Linke Reinhold P. , Schäeffer Jürgen , Gielow Peter , Lindner Peter , Lottspeich Friedrich , Plückthun Andreas , Weiss Elisabeth H.
Published: March 2003

Amyloid of β2‐microglobulin (β2m) origin can be diagnosed using 131I‐radiolabelled‐β2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer β2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products.

In order to exclude these risks we have produced recombinant human β2m (rhβ2m) in Escherichia coli. The expression vector pASK40ΔLβ2m(His)5 contains a C‐terminal (His)5‐tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification.

The isolated rhβ2mH5 reacted in an immunochemically identical manner to native human β2m, and showed a single band of ≈ 11.8 kDa in Western blot analysis and revealed a single spot in two‐dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N‐terminal amino‐acid sequence analysis.

The rhβ2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium‐111‐labelled rhβ2mH5 was monitored in hemodialysed patients with and without known β2m‐amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid.

Thus, this rhβ2mH5 preparation is suitable for detecting amyloid‐containing organs of the β2m‐class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium‐111 instead of iodine‐131 has reduced the radioactive load and resulted in higher resolution.
Electron transfer rates and equilibrium within cytochrome c oxidase
Authors: Farver Ole , Einarsdóttir Ólöf , Pecht Israel
Published: March 2003

Intramolecular electron transfer (ET) between the CuA center and heme a in bovine cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1‐methyl nicotinamide radicals in a diffusion‐controlled reaction, as monitored by absorption changes at 830 nm. After the initial reduction phase, the 830 nm absorption was partially restored, corresponding to reoxidation of the CuA center. Concomitantly, the absorption at 445 nm and 605 nm increased, indicating reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration. This demonstrates that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA → heme a ET and the reverse (heme a → CuA) process were found to be 13 000 s−1 and 3700 s−1, respectively, at 25 °C and pH 7.4. This corresponds to an equilibrium constant of 3.4 under these conditions. Thermodynamic and activation parameters of the ET reactions were determined. The significance of these results, particularly the observed low activation barriers, are discussed within the framework of the known three‐dimensional structure, ET pathways and reorganization energies.
Inhibition of phosphatidylserine synthesis during Jurkat T cell activation
Authors: Pelassy Claudette , Breittmayer Jean P. , Aussel Claude
Published: March 2003

Sodium ortho‐vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 µm. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3‐induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3‐induced T cell activation pathway such as p56lck, p72syk and ZAP‐70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.
A change in reaction specificity of sheep liver serine hydroxymethyltransferase
Authors: Talwar Rashmi , Rao N. Appaji , Savithri H. S.
Published: March 2003

Both serine hydroxymethyltransferase and aspartate aminotransferase belong to the α‐class of pyridoxal‐5′‐phosphate (pyridoxalP)‐dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X‐ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of serine hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing l‐aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and d‐mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 µm and 5.2 × 10−3 s−1, respectively. This oxidation was not observed with either the wild type serine hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver serine hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.
Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae
Authors: Zweytick Dagmar , Leitner Erich , Kohlwein Sepp D. , Yu Chunjiang , Rothblatt Jonathan , Daum Günther
Published: March 2003

In the yeast Saccharomyces cerevisiae, two acyl‐CoA:sterol acyltransferases (ASATs) that catalyze the synthesis of steryl esters have been identified, namely Are2p (Sat1p) and Are1p (Sat2p). Deletion of either ARE1 or ARE2 has no effect on cell viability, and are1are2 double mutants grow in a similar manner to wild‐type despite the complete lack of cellular ASAT activity and steryl ester formation [Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T. M., Rothstein, R. & Sturley, S. L. (1996) Science272, 1353–1356; Yu, C., Kennedy, J., Chang, C. C. Y. & Rothblatt, J. A. (1996) J. Biol. Chem.271, 24157–24163]. Here we show that both Are2p and Are1p reside in the endoplasmic reticulum as demonstrated by measuring ASAT activity in subcellular fractions of are1 and are2 deletion strains. This localization was confirmed by fluorescence microscopy using hybrid proteins of Are2p and Are1p fused to green fluorescent protein (GFP). Lipid analysis of are1 and are2 deletion strains revealed that Are2p and Are1p utilize sterol substrates in vivo with different efficiency; Are2p has a significant preference for ergosterol as a substrate, whereas Are1p esterifies sterol precursors, mainly lanosterol, as well as ergosterol. The specificity towards fatty acids is similar for both isoenzymes. The lack of steryl esters in are1are2 mutant cells is largely compensated by an increased level of free sterols. Nevertheless, terbinafine, an inhibitor of ergosterol biosynthesis, inhibits growth of are1are2 cells more efficiently than growth of wild‐type. In a growth competition experiment are1are2 cells grow more slowly than wild‐type after several rounds of cultivation, suggesting that Are1p and Are2p or steryl esters, the product formed by these two enzymes, are more important in the natural environment than under laboratory conditions.
Inactivation of creatine kinase by high pressure may precede dimer dissociation
Authors: Zhou Jun‐Mei , Zhu Li , Balny Claude
Published: March 2003

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high‐pressure stopped‐flow method in the pressure range 0.1–200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8‐anilinonaphthalene‐1‐sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.
Structural investigations of the CuA centre of nitrous oxide reductase from Pseudomonas stutzeri by site‐directed mutagenesis and X‐ray absorption spectroscopy
Authors: Charnock John M. , Dreusch Andreas , Körner Heinz , Neese Frank , Nelson Jane , Kannt Aimo , Michel Hartmut , Garner C. David , Kroneck Peter M. H. , Zumft Walter G.
Published: March 2003

Nitrous oxide reductase is the terminal component of a respiratory chain that utilizes N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate‐reducing catalytic centre, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed‐valence metal site. To provide further structural information on the CuA centre of N2O reductase, site directed mutagenesis and Cu K‐edge X‐ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA centre of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild‐type residues. The mutations identified the amino acids His583, Cys618, Cys622 and Met629 as ligands of Cu1, and Cys618, Cys622 and His626 as the minimal set of ligands for Cu2 of the CuA centre. Other amino acid substitutions indicated His494 as a likely ligand of CuZ, and an indirect role for Asp580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K‐edge X‐ray absorption spectra have been recorded to investigate the local environment of the Cu centres in N2O reductase. Cu K‐edge Extended X‐ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at ≈ 2.5 Å. The Cu K‐edge EXAFS of the CuA centre of N2O reductase is very similar to that of the CuA centre of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu–N of 1.92 Å, two sulfur atoms at 2.24 Å and a Cu atom at 2.43 Å, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 Å. The interpretation of the CuA EXAFS is in line with ligands assigned by site‐directed mutagenesis. By a difference spectrum approach, using the Cu K‐edge EXAFS of the holoenzyme and that of the CuA‐only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA centre of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.
Characterization of a novel type VII β‐turn conformation for a bio‐active tetrapeptide rigin
Authors: Ashish  , Grover Arvinder , Kishore Raghuvansh
Published: March 2003

The conformational analysis of an immunomodulating tetrapeptide rigin (H‐Gly‐Gln‐Pro‐Arg‐OH), shown to possess diverse immunological activity, has been investigated both theoretically and experimentally for its conformational preferences. Unrestrained molecular dynamics simulation studies in implicit dimethylsulfoxide provide strong support for the existence of a significant population of ordered reverse turn structures for the major trans isomer. Of the three different energy minimized families, generated from computer molecular modelling, only one could be complemented by most of the 1D and 2D 1H NMR parameters obtained in dimethylsulfoxide‐d6. A variable temperature NMR experiment in dimethylsulfoxide‐d6 revealed that the preferred conformation is not stabilized by an intramolecular hydrogen bonding interaction. An analysis of the 2D ROESY experiment provides evidence in favour of an uncommonly observed, rather ill‐defined type VII β‐turn structure. A survey of the observed specific inter‐and intra‐residue NOE connectivities and their comparison with one of the predicted low‐energy conformations, demonstrates synergy between the theoretical molecular modelling and experimentally determined NMR spectral data. The primary structure, rather than long‐range interactions, appears to be critical in determining the folding behaviour of the bio‐active rigin. The present structural attributes may be valuable in peptide drug design and development of the rigin analogs having more effective stimulating activity.
Contribution of C‐tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A
Authors: Marino‐Buslje Cristina , Venhudová Gabriela , Molina Miguel A. , Oliva Baldomero , Jorba Xavier , Canals Francesc , Avilés Francesc X. , Querol Enrique
Published: March 2003

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C‐terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site‐directed mutants: the point substitution of each C‐terminal residue by glycine and the sequential deletions of the C‐terminal residues. The mutant PCI–CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteady‐state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C‐tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI–CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C‐terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI–CPA complex.
Pyridoxal 5′‐phoshate Schiff base in Citrobacter freundii tyrosinephenol‐lyase
Authors: Bazhulina Natalia P. , Morozov Yurii V. , Papisova Anastasia I. , Demidkina Tatyana V.
Published: March 2003

Spectral properties of the internal Schiff base in tyrosine phenol‐lyase have been investigated in the presence of an activating cation K+ and a cation‐inhibitor Na+. The holoenzyme absorption spectra in the pH range 6.5–8.7 were recorded in the presence of K+. No apparent pKa value of the coenzyme chromophore was found in this pH range, indicating that the internal Schiff base does not change its ionic form on going from pH 6.5 to 8.7. To determine the ionic state and tautomeric composition of the Schiff base in tyrosine phenol‐lyase, the absorption and circular dichroism spectra were analyzed using lognormal distribution curves. The predominant form of the internal Schiff base is that with protonated pyridinium and aldimine nitrogen atoms and deprotonated 3′‐hydroxy group, i.e. the ketoenamine. This form is in prototropic equilibrium with its enolimine tautomer. The internal aldimine ionic form is changed upon replacement of K+ with Na+. This replacement leads to a significant decrease in the pKa value of pyridinium nitrogen of the pyridoxal‐P.
Expression, stability and performance of the three‐component alkane mono‐oxygenase of Pseudomonas oleovorans in Escherichia coli
Authors: Staijen Ivo E. , van Beilen Jan B. , Witholt Bernard
Published: March 2003

We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants. The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E. coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P. oleovorans. The enzyme components were less stable in recombinant E. coli hosts than in P. oleovorans. In addition, the specific activity of the alkane mono‐oxygenase component AlkB was five or six times lower in E. coli than in P. oleovorans. Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E. coli. These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins.
The structure of the linkage between the O‐specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited
Authors: Olsthoorn Maurien M. A. , Petersen Bent O. , Duus Jens , Haverkamp Johan , Thomas‐Oates Jane E. , Bock Klaus , Holst Otto
Published: March 2003

Salmonella enterica sv. Typhimurium strain 1135 possesses smooth(S)‐form lipopolysaccharide (LPS). Although the structures of the core region and the O‐specific polysaccharide were investigated intensively between the 1960s and the 1980s, the structure of the linkage region between the O‐chain and the core was not elucidated unequivocally. By using modern MS and high‐field NMR spectroscopy for analysis of the isolated carbohydrate backbone of the LPS, it has been shown that it is a β‐d‐Galp residue that links the first repeating unit of the O‐specific polysaccharide to O‐4 of the last d‐Glcp residue of the core region. Interestingly, this particular d‐Galp residue is α‐linked in all following repeating units. The data are discussed with regard to the ligation of O‐specific polysaccharide and core region during LPS biosynthesis.
Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella
Authors: Fröbius Andreas C. , Kanost Michael R , Götz Peter , Vilcinskas Andreas
Published: March 2003

Three inducible serine protease inhibitors (ISPI‐1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI‐1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell‐free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI‐1), 6.3 kDa (ISPI‐2) and 8.2 kDa (ISPI‐3) with isoelectric points ranging between 7.2 and 8.3. The N‐terminal amino‐acid sequences of ISPI‐1 and ISPI‐3 are not similar to other known proteins, whereas that of ISPI‐2 exhibits extensive similarity to known Kunitz‐type protease inhibitors.
Gene cloning, nucleotide sequencing, and purification and characterization of the D‐stereospecific amino‐acid amidase from Ochrobactrum anthropi SV3
Authors: Komeda Hidenobu , Asano Yasuhisa
Published: March 2003

The gene encoding the d‐stereospecific amino‐acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the d‐amino‐acid amidase. This enzyme, DaaA, is composed of 363 amino‐acid residues (molecular mass 40 082 Da), and the deduced amino‐acid sequence exhibits homology to alkaline d‐peptidase from Bacillus cereus DF4‐B (32% identity), dd‐peptidase from Streptomyces R61 (29% identity), and other penicillin‐recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active‐site motifs identified in the penicillin‐binding proteins and β‐lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell‐free extracts of E. coli was 33.6 U·mg−1 with d‐phenylalaninamide as substrate, which is about 350‐fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel‐filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 °C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward d‐amino‐acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the dd‐peptidase and β‐lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.
Determination of glycosylation sites, disulfide bridges, and the C‐terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry
Authors: Shimizu Tetsuya , Miyairi Kazuo , Okuno Toshikatsu
Published: March 2003

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver‐leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI‐MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N‐binding sugar chains matches that of a high‐mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C‐terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography‐electrospray mass spectrometry (LC‐ESI‐MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3–Cys17, Cys175–Cys191, and Cys300–Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.
Overexpression of enzymes that repair endogenous damage to DNA
Authors: Frosina Guido
Published: March 2003

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6‐alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6‐alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6‐alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N‐purine glycosylase or DNA polymerase β) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.
The HIV‐1Lai RNA dimerization
Authors: Theilleux‐Delalande Valérie , Girard Frédéric , Huynh‐Dinh Tam , Lancelot Gérard , Paoletti Jacques
Published: March 2003

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5′ ends. A region upstream from the splice donor comprising an auto‐complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV‐1Lai RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem–loop structure. It has already been shown that this stem–loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop–loop dimer or ‘kissing complex’ and an extended dimer.

We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248–270 of the HIV‐1Lai SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop–loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to – 29.4 ± 5 kcal·mol−1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don’t indicate a dissociation of the GCGCGC sequence engaged in the loop–loop interaction during the rearrangement of the loop–loop complex into the extended dimer. Our data suggest that the loop–loop interaction is maintained during the temperature dependent conformational change while the intramolecular base‐pairing of the stems is disrupted and then reconstituted to form an intermolecular base‐pairing leading to an extended dimer.
Curvature properties of novel forms of phosphatidylcholine with branched acyl chains
Authors: Epand Richard M. , Epand Raquel F. , Decicco Anthony , Schwarz Dieter
Published: March 2003

We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side‐chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2‐position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X‐ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7–75 °C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain.
Formation of a native‐like β‐hairpin finger structure of a peptide from the extended PDZ domain of neuronal nitric oxide synthase in aqueous solution
Authors: Wang Ping , Zhang Qiang , Tochio Hidehito , Fan Jing‐Song , Zhang Mingjie
Published: March 2003

Neuronal nitric oxide synthase (nNOS) is targeted to the cell membrane via interactions of its extended PDZ domain with PDZ domains of membrane‐associated proteins including PSD‐95 and α1‐syntrophin. The formation of heterodimers between the nNOS PDZ domain and the PDZ domains of nNOS‐binding proteins requires a stretch of continuous amino‐acid residues C‐terminal to the canonical nNOS PDZ domain. In this work, we show that a 27‐residue peptide comprising the C‐terminal extension of the extended nNOS PDZ domain is capable of binding to PSD‐95. The structure of the 27‐residue peptide in aqueous solution was determined using multidimensional NMR‐spectroscopic techniques. The free peptide adopts a native‐like β‐hairpin finger structure in aqueous solution. The results indicate that the C‐terminal extension peptide of the nNOS PDZ domain may represent a relatively independent structural unit in the mediation of the interaction between nNOS and PDZ domain‐containing proteins including PSD‐95 and α1‐syntrophin.
Differential gene expression by endothelial cells in distinct angiogenic states
Authors: Glienke Jens , Schmitt Armin O. , Pilarsky Christian , Hinzmann Bernd , Weiß Bertram , Rosenthal Andre , Thierauch Karl‐Heinz
Published: March 2003

Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT‐PCR we confirmed the differential expression and showed differences up to 14‐fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP‐6, PEA‐15 and PINCH. With ADAMTS4 and hADAMTS1/METH‐1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.
Structural and functional divergence of a nuclear receptor of the RXR family from the trematode parasite Schistosoma mansoni
Authors: de Mendonça Ricardo L. , Escriva Hector , Bouton Didier , Zelus Dominique , Vanacker Jean‐Marc , Bonnelye Edith , Cornette Jocelyne , Pierce Raymond J. , Laudet Vincent
Published: March 2003

We describe the cloning and functional characterization of Schistosoma mansoni retinoid‐X‐receptor (SmRXR; NR2B4‐B), a novel member of the nuclear receptor superfamily from S. mansoni, a homologue of vertebrate retinoid‐X‐receptor. The DNA‐binding C domain of SmRXR shows 80% sequence identity to both human RXRα and Drosophila ultraspiracle (USP), but a much lower level of conservation of the ligand‐binding E domain (22–25% identity). Phylogenetic analysis places SmRXR within the RXR group as an early offshoot of this clade. SmRXR mRNA is expressed at all life‐cycle stages but at higher levels in the free‐living larval stages. However, the SmRXR protein is expressed at markedly different levels, being almost absent from eggs while present at the highest concentration in schistosomula. Recombinant SmRXR fails to bind to the consensus direct repeat response elements, either alone, or as a heterodimer with mouse retinoic acid receptor α or the Drosophila ecdysone receptor. However, the use of chimaeric constructions shows that the C domain of SmRXR will bind to conventional response elements as a heterodimer, and that its specificity is modified by the presence of the D and E domains. In accordance with these results, native SmRXR failed to transactivate the transcription of a reporter gene after cotransfection of mammalian cell lines.
Arginine‐based structures are specific inhibitors of cathepsin C
Authors: Horn Martin , Pavlík Manfred , Dolečková Lucie , Baudyš Miroslav , Mareš Michael
Published: March 2003

Novel synthetic peptide inhibitors of lysosomal cysteine proteinase cathepsin C have been designed through the use of soluble peptide combinatorial libraries. The uncovered structural inhibitory module consists of the N‐terminal cluster of l‐arginine residues. Its modification with d‐amino acids or arginine derivatives did not increase the inhibition strength. Inhibitory potency of oligoarginines improves with the elongation of peptide chain reaching a maximum for octa‐l‐arginine. The oligoarginines specifically interact with the cathepsin C active site as shown by competitive‐type inhibition kinetics (Ki ≈ 10−5 m) and intrinsic fluorescence measurements. The inhibitory interaction of oligoarginines is established through the specific spatial contact of a net of guanidino groups in the arginine side‐chains, as indicated by comparison with inhibitory action of low molecular mass guanidine derivatives (Ki ≈ 10−3 m). Nonarginine polyionic compounds cannot mimic the inhibitory effect of oligoarginines. The arginine‐based peptide inhibitors were selective towards cathepsin C among other cysteine proteinases tested.