bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II
Authors: Spitzner J. R. , Muller M. T. 
Published: May 2005

AbstractA consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533–5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti‐tumor drug 4′‐(9‐acridinyl‐amino)‐methanesulfon‐manisidide (m‐AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the ‘matrix mean’ method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the ‘unique score’ model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p < 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m‐AMSA using the same consensus sequence.
Compartmentation of deoxypyrimidine nucleotides for nuclear DNA replication in S phase mammalian cells
Authors: Reddy G. Prem Veer 
Published: May 2005

AbstractDNA synthesis in S phase Chinese hamster embryo fibroblast cells in the presence of exogenous 3H‐dUrd shows incorporation of the labeled precursor with very little dilution by the large unlabeled intracellular precursor pools. Full mixing would predict a specific activity 10‐fold less than that measured. This coupled with the finding that 80% of the radioactivity from the exogenous 3H‐dUrd appears in the karyoplasts implies a compartmentation where 3H‐dUMP and 3H‐dTTP derived from exogenous 3H‐dUrd do not mix freely with endogenous cytoplasmic pools.
A heme‐ and metal‐binding hexampeptide from the sequence of rabbit plasma histidine‐rich glycoprotein
Authors: Morgan William T. , Deaciuc Victor , Riehm John P. 
Published: May 2005

AbstractRabbit histidine‐rich glycorprotein (HRG) binds low‐spin heme and metal tightly at several sites that contain histidine. As part of an on‐going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2‐Gly‐His‐Phe‐Pro‐Phe‐His‐Trp‐… was found in a 16kDa heme‐binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C‐terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis‐histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not effect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 ± 25 μM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 μM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis‐histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme‐ and metal‐binding site of rabbit HRG.
Binding of lanthanum and gadolinium ions to Concanavalin A studied calorimetrically at 25°C
Authors: Barone Guido , Castronuovo Giuseppina , Del Vecchio Pompea , Elia Vittorio , Guarrata Patrizia 
Published: May 2005

AbstractThe interaction between Concanavalin A (ConA) and the lanthanide ions La3+ and Gd3+ has been studied calorimetrically at 25°C. The measurements were carried out at a pH of 4.5, where the protein exists prevailingly as a dimer. Calorimetry allows the direct determination of the binding enthalpy and the evaluation of both the apparent association constant, and the apparent free energy and entropy. Three groups of data were collected. The first concerns the interaction of the ‘native’ protein, i.e., fully metallized with Mn2+ and Ca2+, with the lanthanides. The second concerns the interaction of the completely demetallized protein with La3+ and Gd3+. Finally, the affinity of each complex was tested for the specific sugar α‐methylmannopyranoside. The analysis of the thermodynamic parameters obtained, led to the following conclusions: (1) a specific site, named S3, exists on the protein for the lanthanides, distinct from the S1 site of the transition metal and from the S2 site, specific for calcium. There is only one S3 site per protomer when the protein has Mn2+ in S1 and Ca2+ in S2. Moreover, there is no appreciable competition for S1 and S2 from the lanthanides. The ‘native’ protein, metallized with La3+ or Gd3+, is a fully functional protein. (2) The demetallized protein (ApoCon A) has at least two sites per protomer for the lanthanides. The hypothesis is that, besides the S3 site, the lanthanides, in the absence of Mn2+, can also occupy the S1, but not the S2, site. The protein metallized only with gadolinium ions is completely inactive toward the interaction with the mannoside. The same happens when, along with gadolinium, only calcium or manganese is present. Hence, in the absence of the transition metal in S1 or of calcium in S2, the protein is not in the conformation suitable to interact with its specific substrate.
Molecular recognition between oligopeptides and nucleic acids. DNA sequence specificity and binding properties of an acridine‐linked netropsin hybrid ligand
Authors: Bailly Christian , Helbecque Nicole , Hénichart Jean‐Pierre , Colson Pierre , Houssier Claude , Rao K. Ekambareswara , Shea Regan G. , Lown J. William 
Published: May 2005

AbstractThe binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA withK1= 9.1 × 105M−1at low r values (0.003–0.1), and a binding site sizen= 10, indicative of binding of both moeities. At high binding ratios (>0.1),K2= 0.9 × 105M−1andn= 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5′‐AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G‐C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurments of NETGA indicate that the oligopeptide moiety (netropsin‐like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety.
The formation of mispairs by 8‐oxyguanine as a pathway of mutations induced by irradiation and oxygen radicals
Authors: Poltev Valery I. , Shulyupina Nataly V. , Bruskov Vadim I. 
Published: May 2005

AbstractGuanine conversion to 8‐oxyguanine (OG) was induced by irradiation and oxygen radicals. The pathways of mutations caused by such DNA damage have been considered. Minima of interaction energies of OG with nucleic acid bases are revealed via classical potential function calculations. The minima exist for OG*:G and OG*:A base pairs, with syn‐conformation of OG* and OG:T wobble base pairs. The mutual positions of glycosyl bonds in these base pairs are quite close to those for Watson‐Crick pairs, and energies of OG*:G, OG*:A and OG:T mispairs are close to the energy of A:T. The results calculated suggest that these mispairs could arise as intermediates in the transversions C:G to G:C and C:G to A:T, and the transition G:C to A:T.
Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability
Authors: Caliceti P. , Schiavon O. , Veronese F. M. , Chaiken I. M. 
Published: May 2005

AbstractThe effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase‐immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 × 10−8 M was found for unmodified RNase, as compared to values of 1.3 × 10−7 and 1.2 × 10−6 M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine‐2′,3′‐cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67°C for native protein to 57°C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility. The latter is likely a strong contribution to the reduced immunogenicity observed generally with MPEG modified proteins.
The specificity of the binding of platelet activating factor (PAF) to anti‐PAF antibodies
Authors: Smal Mary A. , Baldo Brian A. , Harle David G. 
Published: May 2005

AbstractQuantitative hapten inhibition experiments employing sheep anti‐PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody binding to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16‐ and C18:1‐PAF proved to be the most potent inhibitors and more active than C18‐PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1‐acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.
Citrate synthase 1 interacts with the citrate transporter of yeast mitochondria
Authors: Grigorenko Elena V. , Small W. Curtis , Persson Lars‐Olof , Srere Paul A. 
Published: May 2005

AbstractWe have previously shown that citrate synthase binds to an intrinsic protein of the mitochondrial inner membrane (D'Souza and Srere, 1983). In this paper we present evidence that this citrate synthase binding protein is the citrate transporter. We have used citrate synthase 1 mutants of Saccharomyces cerevesiae and transformants containing citrate synthase inactivated by site‐directed mutagenesis to study the effect of the CS1 protein upon mitochondrial function (Kispal and Srere). In the present study citrate uptake and oxidation were measured during state 3 conditions (presence of 200 μM ADP) in the mitochondria of several strains of Saccharomyces cerevesiae: a parental strain containing wild‐type mitochondrial citrate synthase (CS1) and strains derived from a CS1 deficient strain in which the CS1 gene was disrupted by insertation of the LEU2 gene. These strains were generated from the CS1− cells by transformation with vectors encoding site‐specific mutants of CS1 possessing very low levels of enzymatic activity. One such strain in this study was subsequently found to have undergone reversion to produce a strain which had activity very similar to wild type. Positive correlation between citrate uptake and the rate of citrate oxidation was found, suggesting coupling of the two processes. Both mitochondrial citrate uptake and oxidation were decreased in the mutant lacking any form of CS1 protein. Reintroduction of mutagenized CS1 into yeast causes an enhancement in the rate of state 3 oxygen consumption and of citrate uptake. The observed respiratory increase produces a state 3 oxygen consumption rate which is 1.5‐ to 17‐fold greater than the rate of reintroduced mutagenized CS1 activity; conversely, wild‐type CS1 enzyme levels are about 30‐fold greater than the respiratory rate in wild‐type cells. Further evidence for uncoupling of the process of citrate utilization from CS1 activity is also seen in the revertant strain which has lower respiratory rate than the wild‐type strain. This difference is not due to decreased concentration of the citrate carrier of those cells with lower respiratory rates, a change in equilibrium for citrate across the mitochondrial membrane, or to decreased citrate saturability of such mitochondria. These results, supported by the interaction observed during column chromatography between matrix‐immobilized pig citrate synthase and the protein from yeast mitochondrial membrane preparations which exhibits citrate transport activity, strongly suggest physical interaction between CS1 and the mitochondrial citrate transport protein.
Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 Å resolution
Authors: Mattevi Andrea , Gatti Giuseppina , Coda Alessandro , Rizzi Menico , Ascenzi Paolo , Brunori Maurizio , Bolognesi Martino 
Published: May 2005

AbstractThe binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X‐ray crystallography. The three‐dimensional structure of the complex has been refined at 1.9 Å resolution to a crystallographic R‐factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co‐ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side‐chain if Arg66 (E10) which, form an outer orientation in the ‘aquo‐met’ ligand‐free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.
Production and characterization of antibodies to mouse scrapie‐amyloid protein elicited by non‐carrier linked synthetic peptide immunogens
Authors: Di Martino Alessandro , Bigon Emilio , Corona Giuseppe , Callegaro Lanfranco 
Published: May 2005

AbstractTwo polyclonal antibodies were raised by immunizing rabbits with two non carrier‐linked synthetic peptides whose amino acid sequences corresponded to codons 89–107 (peptide P1) and 219–233 (peptide P2) of the translated cDNA sequence of murine PrP protein. These free peptides, whose structural characteristics in solution were studied by circular dichroism, elicited a reasonable immunologic response in animals. Both antibodies still recognized the corresponding immunogens after affinity chromatography purification. However, only antibodies raised to the former sequence reacted by immunobolt with a purified preparation of murine scrapie amyloid protein. These findings are discussed together with their correlation to peptide structure and the effectiveness of this simplified immunization procedure.
Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous β‐galactoside‐binding lectin (galaptin)
Authors: Ahmed Hafiz , Sharma Ahsu , DiCioccio Richard A. , Allen Howard J. 
Published: May 2005

AbstractGlutaraldehyde‐polymerized human splenic galaptin, a β‐galactoside‐binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative of native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2‐, 4‐ and 12‐membered species after reductive alkylation. Both forms of galaptin bound, at 4 °C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 × 107 binding sites/cell with affinity constant Ka = 2.2 × 105 M for dimeric galaptin and 17 × 107 binding sites/cell with Ka = 3.4 × 105 M−1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic‐adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic‐adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30–50% by lactose for plastic‐adsorbed polymeric and dimeric galaptin, respectively, Native fetuin bound to the adsorbed dimeric galaptin in a lactose‐insensitive manner. These data indicate that cell surface receptor‐galaptin interaction is carbohydrate specific whereas polystyrene‐adsorbed galaptin may demonstrate protein‐protein interactions with soluble ligands.
MHC Class I/peptide interactions: Binding specificity and kinetics
Authors: Margulies David H. , Corr Maripat , Boyd Lisa F. , Khilko Sergei N. 
Published: May 2005

AbstractRecent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran‐modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three‐dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25°C are in the range of ca 10−4–10−6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid‐phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.
A novel computational tool for automated structure‐based drug design
Authors: Böhm Hans‐Joachim 
Published: May 2005

AbstractThe computer program LUDI for automated structure‐based drug design is described. The program constructs possible new ligands for a given protein of known three‐dimensional structure. This novel approach is based upon rules about energetically favourable non‐bonded contact geometries between functional groups of the protein and the ligand which are derived from a statistical analysis of crystal packings of organic molecules. In a first step small fragments are docked into the protein binding site in such a way that hydrogen bonds and ionic interactions can be formed with the protein and hydrophobic pockets are filled with lipophilic groups of the ligands. The program can then append further fragments onto a previously positioned fragments or onto an already existing ligand (e.g., a lead structure that one seeks to improve). It is also possible to link several fragments together by bridge fragments to form a complete molecule. All putative ligands retrieved or constructed by LUDI are scored. We use a simple scoring function that was fitted to experimentally determined binding constants of protein–ligand complexes. LUCI is a very fast program with typical execution times of 1–5 min on a work station and is therefore suitable for interactive usage.
From crystals to enzymes: Simple models of molecular recognition
Authors: Czugler Mátyás 
Published: May 2005

AbstractStructural models resulting from the x‐ray crystal structure determination of small molecules are shown to be closely mimicking some of the protein structure and function features. Crystals of inclusion compounds, a special type of solid molecular associates, lend special support to such a view. They are inherently suited for such modelling purpose due to easily visible association and molecular recognition effects. It is envisaged that combination of the analysis of high‐resolution small molecule crystallographic models and those of protein structures will yield to further fruitful analogies.