bluuebunny/crossref_metadata_2025_split huggingface dataset:
Denaturation of apolipoprotein A‐I and the monomer form of apolipoprotein A‐IMilano
Authors: Suurkuusk Malin , Hallén Dan 
Published: March 2003

In this study the thermal and denaturant induced unfolding of apolipoprotein A‐I (apo A‐I) and the monomer form of apolipoprotein A‐IMilano (apo A‐IM) was followed. Dimer apo A‐IM was reduced with dithiothreitol, which was present in the protein solutions in all experiments. Thermal denaturation is followed by differential scanning calorimetry (DSC) and far‐UV and near‐UV CD. Both apo A‐I and monomer apo A‐IM have a broad asymmetric DSC peak that could be deconvoluted into three non two‐state transitions, apo A‐I being more stable than the monomer apo A‐IM. Estimation of melting of tertiary structure by near‐UV CD is lower than that for secondary structure determined from far‐UV. This together with the non two‐state unfolding of the proteins observed with DSC is indicative of unfolding via a molten globular‐like state. Apo A‐I and monomer apo A‐IM are equally susceptible to guanidinum chloride, half‐unfolded at 1.2 m denaturant. The presence of 0.5 and 1.0 m denaturant, lower and equalize the denaturation temperatures of the proteins, respectively.
Autoantibodies interacting with purified native thyrotropin receptor
Authors: Atger Michel , Misrahi Micheline , Young Jacques , Jolivet André , Orgiazzi Jacques , Schaison Gilbert , Milgrom Edwin 
Published: March 2003

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti‐TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (α‐subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves’ disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25–1 × 10−10 m) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves’ disease patients (3.50 ± 0.36 mg·L−1) than in control subjects (1.76 ± 0.21) (mean ± SEM), there was an overlap between the groups. Receptor‐stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of α‐subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves’ disease. The ELISA test measures total anti‐TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves’ disease is related to the appearance of anti‐TSHR antibodies not present in normal subjects. Actually, anti‐TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves’ disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.
The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms
Authors: van der Veen Bart A. , van Alebeek Gert‐Jan W. M. , Uitdehaag Joost C. M. , Dijkstra Bauke W. , Dijkhuizen Lubbert 
Published: March 2003

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme–intermediate complex (substituted‐enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an α‐glycosidic bond of a linear malto‐oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping‐pong mechanism. Cyclization (cleavage of an α‐glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single‐substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis–Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an α‐glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto‐oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism.In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.
Structure of the O‐specific polysaccharide of Proteus penneri 71 and classification of cross‐reactive P. penneri strains to a new proposed serogroup O64
Authors: Zych Krystyna , Kocharova Nina A. , Kowalczyk Marcin , Toukach Filip V. , Kaminska Dominika , Shashkov Alexander S. , Knirel Yuriy A. , Sidorczyk Zygmunt 
Published: March 2003

A neutral O‐specific polysaccharide (O‐antigen) was isolated from the lipopolysaccharide (LPS) of the bacterium Proteus penneri 71. On the basis of sugar analysis and 1H‐ and 13C‐NMR spectroscopic studies, including two‐dimensional COSY, 13C,1H heteronuclear COSY and ROESY, the following structure of the trisaccharide repeating unit of the polysaccharide was established:         →3)‐β‐d‐GlcpNAc‐(1→4)‐β‐d‐GlcpNAc‐(1→3)‐α‐d‐Galp‐(1→The polysaccharide has the same carbohydrate backbone as the O‐specific polysaccharide of P. penneri 19 and both are similar to that of P. penneri 62 studied by us previously. A cross‐reactivity of anti‐P. penneri 71, 19 and 62 O‐antisera with 11 P. penneri strains was revealed and substantiated at the level of the O‐antigen structures. These strains could be divided into three subgroups within a new proposed Proteus O64 serogroup containing P. penneri strains only.
Structure of a 2‐aminoethyl phosphate‐containing O‐specificpolysaccharide of Proteus penneri 8 from a new serogroup O67
Authors: Knirel Yuriy A. , Zych Krystyna , Vinogradov Eugeny V. , Shashkov Alexander S. , Sidorczyk Zygmunt 
Published: March 2003

An acidic O‐specific polysaccharide was obtained by mild acid degradation of the Proteus penneri 8 lipopolysaccharide and found to contain d‐glucose, d‐galacturonic acid, 2‐acetamido‐2‐deoxy‐d‐glucose, 2‐acetamido‐2‐deoxy‐d‐galactose, 2‐acetamido‐2,6‐dideoxy‐l‐galactose (l‐FucNAc) and 2‐aminoethyl phosphate (PEtn) in the ratios 2 : 1 : 1 : 1 : 1 : 1. 1H and 13C NMR spectroscopy was applied to the intact and dephosphorylated polysaccharides, and the following structure of the hexasaccharide repeating unit was established:The O‐specific polysaccharide has a unique structure, and, accordingly, we propose for P. penneri 8 a new Proteus O67 serogroup, in which this strain is at present the single representative. The nature of epitopes on LPS of P. penneri 34, P. mirabilis O16, P. mirabilis O23 and P. vulgaris O22, which cross‐react with O‐antiserum against P. penneri 8, is discussed.
The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase
Authors: Lambeir Anne‐Marie , Backmann Jan , Ruiz‐Sanz Javier , Filimonov Vladimir , Nielsen Jens Erik , Kursula Inari , Norledge Brian V. , Wierenga Rik K. 
Published: March 2003

The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride‐induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild‐type; and (c) the stability of the wild‐type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH‐dependent decrease in overall stability is due to weakening of the monomer–monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (ΔΔG ° of 2 kcal·mol−1 per monomer) and the elimination of a charged group in the dimer interface (ΔΔG ° of at least 9 kcal·mol−1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results.The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.
The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251
Authors: van der Veen Bart A. , Uitdehaag Joost C. M. , Dijkstra Bauke W. , Dijkhuizen Lubbert 
Published: March 2003

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active‐site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products.Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins.Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47→Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47→Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.
Immunohistochemical evidence for the myeloperoxidase/H2O2/halide system in human atherosclerotic lesions
Authors: Malle Ernst , Waeg Georg , Schreiber Renate , Gröne Elisabeth F. , Sattler Wolfgang , Gröne Hermann‐Josef 
Published: March 2003

The ‘oxidation theory’ of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol‐engorged foam cells. To identify HOCl‐modified‐epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl‐modified LDL) that do not cross‐react with other LDL modifications, i.e. peroxynitrite‐LDL, hemin‐LDL, Cu2+‐oxidized LDL, 4‐hydroxynonenal‐LDL, malondialdehyde‐LDL, glycated‐LDL, and acetylated‐LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl− added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl‐modified‐epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion‐prone location (type I). HOCl‐oxidation‐specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl‐modified‐epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system‐mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.
Structure/thermodynamics relationships of lectin–saccharide complexes
Authors: Bradbrook Gail M. , Forshaw Jeffrey R. , Pérez Serge 
Published: March 2003

Molecular dynamics (MD) simulations of Erythrina corallodendron lectin binding to a monosaccharide, α‐galactose, and a disaccharide, N‐acetyl lactosamine, have been performed in order to investigate the relationship between structure and thermodynamics.A simulated annealing protocol has been used to generate ensembles of structures for the two complexes, from which both qualitative and quantitative information on binding dynamics have been extracted. The ensembled averaged lectin–saccharide interaction enthalpy is equivalent for both sugars, whereas the calculation based on the X‐ray structures does show a difference. Within large statistical errors, the calculated ‘binding enthalpy’ is also the same for the two systems. These errors arise largely from terms involving solvent and are a typical limitation of current MD simulations.Significant qualitative differences in binding between the two complexes are, however, observed over the ensembles. These could be important for unraveling the structure/thermodynamic relationship. Stated simply, there are a greater number of binding options available to the disaccharide compared to the monosaccharide. The implications of alternative binding states on thermodynamic parameters and the ‘breaking of enthalpy–entropy compensation’ are discussed. The role of solvent in lectin–saccharide complex formation is suggested to be significant.
Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin‐A can be produced in human kidney 293 cellsbut not in Chinese hamster ovary cells
Authors: Van den Nieuwenhof Ingrid M. , Koistinen Hannu , Easton Richard L. , Koistinen Riitta , Kämäräinen Meerit , Morris Howard R. , van Die Irma , Seppälä Markku , Dell Anne , Van den Eijnden Dirk H. 
Published: March 2003

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO‐type glycans and is devoid of any of the N,N′‐diacetyllactosediamine (lacdiNAc)‐based chains previously identified in glycodelin‐A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a β1→4‐N‐acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc‐based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc‐based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic‐type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.
Flavin–protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C‐ and 15N‐labelled flavin
Authors: Fleischmann Garrit , Lederer Florence , Müller Franz , Bacher Adelbert , Rüterjans Heinz 
Published: March 2003

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C‐ and 15N‐labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen‐bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the π‐electron distribution.The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N‐chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C‐NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N‐H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins.With respect to N(10), it is more sp3‐hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3‐hybridized than in free FMN, but N(5) exhibits a higher degree of sp3‐hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane.In addition, two‐electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N‐chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins.31P‐NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.
Sterol‐induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double‐bond position in the sterol tetracyclic ring structure
Authors: Leppimäki Petra , Mattinen Jorma , Slotte J. Peter 
Published: March 2003

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7‐dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 µm cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol·mg−1 protein (from 90 nmol·mg−1 to 190 nmol·mg−1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol‐enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7‐dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane‐bound CTP:phosphocholine cytidylyltransferase, the rate‐limiting enzyme, was increased by 47 ± 4% in cholesterol‐enriched cells whereas its activity was unchanged in lathosterol‐enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down‐regulated acetate‐incorporation into cholesterol, and upregulated sterol esterification in the sterol‐enriched fibroblasts. Using 31P‐NMR to measure the lamellar‐to‐hexagonal (Lα–HII) phase transition in multilamellar lipid dispersions, lathosterol‐containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine and either cholesterol or lathosterol (70 : 30 mol/mol), differential scanning calorimetry also revealed that the Lα–HII‐transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lα–HII‐transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.