bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic analysis of engineered antibody‐antigen interactions
Authors: Malmqvist Magnus 
Published: May 2005

AbstractMolecular engineering antibodies has made it possible to produce specific domains of the antibody molecule and combine them with other protein domains to achieve new properties. Using site directed mutagenesis, amino acid residues can be exchanged within the binding site; and, by analysis of crystal structures, the positions of these amino acids can be determined in three dimensions at atomic resolution. In addition, gene libraries and phage selection technology can be used to generate new antibody fragments directly from a gene pool. Both mutagenesis and selection from libraries offer opportunities to identify antibody‐derived molecules with altered and useful antigen recognition properties. The detailed analysis both kinetic and equilibrium binding affinity are therefore essential to understand the activity of the molecules resulting from antibody engineering and to guide the progress of their further design. The paper reviews recently evolving techniques for the binging analysis of antibodies, their functional domains and antibody chimerae.
Molecular recognition of DNA structure by proteins that mediate genetic recombination
Authors: Lilley David M. J. 
Published: May 2005

AbstractThe latter half of genetic recombination is mediated by proteins that recognise the structure of the four‐way DNA junction, and manipulate this structure. In solution the four‐way junction adopts a stacked X‐structure in the presence of metal ions. The folding is brought about by the pairwise coaxial stacking of helices in a right‐handed antiparallel X‐shaped structure. The four‐way junction is cleaved by structure‐selective resolving enzymes that have been isolated from a wide variety of sources, from eubacteria and their phages through to mammals. In addition, another class of proteins accelerate the branch migration of the junction. These proteins all appear to be divisible into a component that recognises structure and another that carries out a reaction on the junction. Thus the ability of structure‐selective binding to the four‐way DNA junction is a key feature of enzymes important in genetic recombination.
DNA recognition by lexitropsins, minor groove binding agents
Authors: Lown J. William 
Published: May 2005

AbstractConsideration is given to alternative approaches to the development of DNA sequences selective binding agents because of their potential applications in diagnosis and treatment of cancer as well as in molecular biology. The concept of lexitropsins, or information‐reading molecules, is introduced within the antigene strategy as an alternative to, and complementary with, the antigene approach for cellular intervention and gene control The chemical, physical and paharmacological factors involved in the design of effective lexitropsins are discussed and illustrated with experimental results. Among the factors contributing to the molecular recognition processes are: the presence and disposition of hydrogen bond accepting and donating groups, ligand shape, chirality, stereochemistry, flexibility and charge. For longer ligands, such as are required to target unique sequences in biological systems (14–16 base pairs), the critical feature is the phasing or spatial corresponding between repeat units in the ligand and receptor. The recently discovered 2:1 lexitropsin‐DNA binding motif provides a further refinement in molecular recognition in permitting discrimination between GC and CG base pairs. The application of these factors in the design and synthesis of novel agents which exhibits anticancer, antiviral and antitretroviral properties, and inhibition of critical cellular enzymes including topoisomerases is discussed. The emerging evidence of a relationship between sequence selectivity of the new agents and the biological responses they invoke is also described.
The interaction of intercalators and groove‐binding agents with DNA triple‐helical structures: The influence of ligand structure, DNA backbone modifications and sequence
Authors: Wilson W. D. , Mizan Shaikh , Taniuos Farial A. , Yao Shijie , Zon Gerald 
Published: May 2005

AbstractThe effect of ligand structure and properties, DNA backbone modifications and DNA sequence on the interaction of a variety of well‐known groove‐binding agents and intercalates with DNA duplexes and triplexes have been evaluated by thermal melting experiments and molecular modeling. Both methyphosphonate and phosphorothioate substitutions generally destabilize DNA duplexes and triplexes. Modified duplexes can be strongly stabilized by both groove‐binding agents and intercalators whereas triplexes are primarily stabilized by intercalators. Of the compounds tested, the intercalators coralyne and quinacrine provide the largest stabilization of the triplex dT19 · dA19 · dT19. Molecular modeling studies suggests that the large intercalating ring systems of coralyne stacks well with the triplex bases whereas the alkylamino side chain of quinacrine fits snugly into the remaining space of the major groove of dT19 · dA19 · dT19 triplex and forms extensive van der Waals contacts with the thymine methyl groups that line the groove. Converting some of the T · A · T base triples to C+ · G · C (e.g. dT19 · dA19 · dT19 to d(T4C+)3T4 · d(A4G)3A4) causes very significant decreases in observed Tm increases for compounds such as quinacrine and coralyne. Although removal of thymine methyl groups and addition of positive charge on substitution of C+ · G · C for T · A · T should reduce binding of cationic intercalates, the large difference observed between the pure AT and the mixed sequence triplexes suggest that they may also have differences in structure and properties.
Design of molecules that specifically recognize and cleave apurinic sites in DNA
Authors: Berthet N. , Boudali A. , Constant J. F. , Decout J. L. , Demeunynck M. , Fkyerat A. , Garcia J. , Laayoun A. , Michon P. , Lhomme J. 
Published: May 2005

AbstractWe have prepared a series of a tailor‐made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, an nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 104 to 106 M−1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP‐sites per DNA‐molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleaved plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP‐DNA suggest a strand break induced by a β‐elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable along (tetrahydrofuran) of the abasic site for high field 1H NMR spectrometry and footprinting experiments. All results are consistent with a β‐elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site nucleases and can be used advantageously as substitutes for the natural enzyme for in vitro cleavage of AP‐sites containing DNA.
(+)‐CC‐1065 as a probe for intrinsic and protein‐induced bending of DNA
Authors: Hurley Laurence H. , Sun Daekyu 
Published: May 2005

Abstract(+)‐CC ‐1065 is biologically potent DNA‐reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N‐3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug‐induced bending of DNA has similarities to intrinsic. A‐tract bending and the 3′ adenine of A‐tracts shows a unique reactivity to alkylation by (+) ‐CC‐1065. Upon covalent modification of A‐tracts, the magnitude of bending is increased and helix is stiffened. Using high‐field NMR, hydroxyl‐radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ ‐ AGTTA* (an asterisk indicates the covalent modification site) to (+)‐CC‐1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug‐induced bending is focused over the TT region. By analogy with the junction bend model for A‐tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)‐CC‐1065 entrapped/induced bending of DNA as a probe for the Sp1‐induced bending of the 21‐base‐pair repeat an Mu transpose bending of the att L3 sequence is described.
Electron microscopic studies of sequence‐specific recognition of duplex DNA by different ligands
Authors: Cherney Dmitry I. , Kurakin Alexey V. , Lyamichev Victor I. , Frank‐Kamenetskii Maxim D. , Zinkevich Vitaly E. , Firman Keith , Nielsen Peter E. 
Published: May 2005

AbstractThe following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5′‐CTCTCTCTCTCTCT‐3′ capable of forming triplex DNA, and PNA oligomer H‐T10‐LysNH2. For each ligand the best conditions for electron microscopic (EM)detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15–20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence‐specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.
Ethidium and azidoethidium oligonucleotide derivatives: Synthesis, complementary complex formation and sequence‐specific photomodification of the single‐stranded and double‐stranded target oligo‐ and polynucleotides
Authors: Koshkin A. A. , Kropachev K. Yu. , Mamaev S. V. , Bulychev N. V. , Lokhov S. G. , Vlassov V. V. , Lebedev A. V. 
Published: May 2005

AbstractOligodeoxyribonucleotide derivatives containing ethidium or azidoethidium residues attached to 3′ and/or 5′ end were prepared. These derivatives formed tight specific complexes with complementary oligodeoxyribonucleotides where each attached ethidium residue led to an increase of complex Tm by 20–30°C. Tandem complexes of two oligodeoxyribonucleotides containing ethidium residues with an oligodeoxyribonucleotide having two adjacent complementary sequences for these Oligonucleotide were investigated. Photoinduced reactions of a number of ethidium and azidoethidium oligodeoxyribonucleotide derivatives with target complementary single‐stranded and double‐stranded oligo‐ and polydeoxyribonucleotides were investigated. The irradiation led to direct photocleavage of the target oligo‐ or polynucleotide, to formation of hidden (piperidine cleavable) modifications of the target of formation of covalent adducts between ethidium oligodeoxyribonucleotide derivative and the target. In a number of experiments, azidoethidium dyes were demonstrated to be considerably stronger photosensitizers than ethidium ones. Depending on the nature of the target (single‐ or double‐stranded DNA) and on the irradiation conditions, the total damages to the target oligo‐ or polydeoxyribonucleotides ranged from 10–70% (for ethidium dyes) to 30–80% (for azidoethidium dyes).
Properties of RecA‐oligonucleotide complexes
Authors: Simonson T. , Kubista M. , Sjöback R. , Ryberg H. , Takahashi M. 
Published: May 2005

AbstractThe interaction of RecA protein with short single‐stranded oligonucleotides is characterised by flow linear dichroism (LD), isoelectric focusing (IEF) and electron microscopy (EM). From LD and EM it is evident that RecA forms long filaments with at least some 50 oligonucleotides in a ‘train formation’. The tendency to form trains is substantially lower when an amino group is attached to the 5′ end of the oligonucleotide, suggesting that the modification impairs protein‐protein interactions at the interface between two oligomers. From LD it is also evident that no bridging occurs between RecA–Oligonucleotide complexes containing more than one oligomer strand per RecA filament. This property make them manageable in polyacrylamide gels, hence allowing characterisation by IEF. RecA was found acidic with a pI of 5.0. The pI was not dependent on the presence of bound cofactor (ATPγS) and oligonucleotides suggesting that protonation of the protein readily occurs to compensate for the negative charges provided by bound cofactor and DNA.
Predicting the conformation of proteins from sequences. Progress and future progress
Authors: Benner Steven A. 
Published: May 2005

AbstractRecent progress in structure prediction has allowed bona fide predictions, those made and published before an experimental structure is determined, to be remarkably accurate. The most successful methods rely on an analysis of patterns of conservation and variation within homologues protein sequences, extract tertiary structural information before secondary structure is predicted, and a avoid ‘three state per residue scores’ as a tool for evaluating a prediction, focusing instead on efforts to understand why a prediction is successful when it is successful, and why it fails when it fails.
Fate of intercellular MHC–peptide–T‐cell receptor complexes during T‐cell activation
Authors: Clark Brian R. 
Published: May 2005

AbstractAntigen‐specific T‐cell activation requires the formation of a transient cell‐cell conjugate between a T cell and an appropriate antigen presenting cell (APC). Focal aggregation of T‐cell receptor (TCR) molecules at the T‐cell–APC membrane interface accompanies formation of multiple non‐covalent intercellular bridges consisting of TCRs on the T cell and cognate MHC–peptide complexes on the APC. Enhanced adhesiveness and T‐cell activation follow the T‐cell signalling that results from crosslinking of T‐cell receptors (TCR). Models of AT‐cell activation propose that the APC and activated T cell separate following a decline in the enhanced adhesiveness. The rate of intercellular TCR‐(MHC‐peptide) complexes formed during T‐cell activation is unknown. Based on the reported CD4‐positive T‐cell internalization of the peptide moiety of preformed cognate MHC II–peptide complexes, it is proposed here that translocation of the peptide moiety leads to destabilization and decomposition of intercellular trimolecular TCR–(MHC–peptide) complexes in the T‐cell–APC interface. This decomposition accompanies or results in the decline in enhanced adhesiveness leading to separation of the APC and activated T cell.
Optimization of protein G chromatography for bio pharmaceutical monoclonal antibodies
Authors: Bill Eberhard , Lutz Ulrike , Karlsson Britt‐Marie , Sparrman Marianne , Allgaier Hermann 
Published: May 2005

AbstractCompared to more stable chromatographic media the use of affinity media with biological ligands such as Protein G Sepharose® 4 Fast Flow poses special challenges regarding regeneration and sanitization. This is especially critical for the purification of pharmaceutical proteins, where complete regeneration of the column between runs is of paramount importance. Here, the problems encountered during process development and up scaling of regeneration methods for a protein G Sepharose Fast Flow column intended for the large‐scale purification of pharmaceutical monoclonal anitbiodies are reported. The initially chosen alkaline regeneration buffer led to an increase in the affinity of Protein G towards antibodies which made elution increasingly difficult. A combination of urea and acetic acid was selected to ensure efficient cleaning of the matrix without affecting ligand properties.Validation experiments were done to demonstrate the functional integrity of the matrix after repeated cycles of use and regeneration, as well as the efficiency of the cleaning process.
Determination of the interactions between lectins and glycoproteins by surface plasmon resonance
Authors: Okazaki Issay , Hasegawa Yukio , Shinohara Yasuro , Kamasaki Tadayoshi , Bhikhabhai Rama 
Published: May 2005

AbstractA simple and rapid analytical method for detecting interaction between loigosaccharides of glycoproteins and different lectins was studies by surface plasmon resonance using a biosensor (BIA coreTM). The interactions of three lectins, Smambucus Sieboldiana agglutinin (SSA), Ricinus communis agglutinin I (RCA I) and Concanavalin A (Con A) for fetuni and digested fetuins wiht glycosidases, asialo‐, agalacto‐, and aglucosamino‐fetuin, were investigated as a model system. These fetuins were immobilized to the matrix of the sensor chip and the lectins were injected into the sensor chip cartridge. The association and dissociation reactions could be monitored as resonance signals in real time. The interactions with lectins significantly changed as the oligosaccharides of fetuins were trimmed. The interactions between fetuin and SSA, asialofetuin and RCAI, and aglucosaminofetuin and Con A show the highest affinity properties, respectively. The association constants of these lectins were estimated to be 1.4 × 107, 1.9 × 108 and 5.3 × 107 (M−1), respectively. These results suggested that the interactions between lectins and glycoproteins could be well defined in real time and kinetically, and that the partial structure of oligosaccharides of glycoproteins can be estimated by determination of the interactions with various lectins after glycosidase digestions using the biosensor.
A novel class of embryonic cell adhesion glycan epitopes is expressed in human colon carcinomas
Authors: Misevic Gradimir N. , Popescu Octavian 
Published: May 2005

AbstractA new class of acidic glycans isolated from marine sponges and sea urchin embryos was shown to mediate cell adhesion via carbohydrate–carbohydrate interactions. Cell aggregation blocking monoclonal antibodies (Block 1 and Block 2 mAbs) directed against these polysaccharides localized functional epitopes in embryonic sea urchin gut. Immunofluorescence light microscopy of human colon carcinomas and healthy colon samples with Block 1 and Block 2 mAbs established that the carbohydrate structures similar to the invertebrate acidic glycan adhesion molecules are also expressed in humans. The Block 1 mAb labeled basal and apical lamina of tumor cells, whereas the Block 2 bound exclusively to the apical part of the epithelium. In normal tissue whole goblet cell membrane was stained with both antibodies indicating that transformation leads to spatial rearrangement of glycan antigens. Acidic glycans from human colon carcinomas and normal colon were isolated after delipidation, and complete pronase and DNase digestion, by gel filtration and adsorption to anion exchange membranes. Immunodot assay with Block 1 and Block 2 mAbs revealed that tumor cells have elevated expression of both carbohydrate structures. These results suggest that the acidic glycan adhesion molecules, originally found in sponges and sea urchin embryos, may represent a new class of carbohydrate carcino‐embryonal antigens involved in cellular interactions associated with morphogenesis, metastasis and renewal of adult tissue.
Importance of the matrix and its chemical activation on the stability of immuno‐adsorbents
Authors: Ubrich N. , Hubert P. , Dellacherie E. , Rivat C. 
Published: May 2005

AbstractVarious matrices were reacted with four different activation reagents, in order to prepare immuno‐adsorbents for the selective removal of low‐density lipoproteins from blood plasma of patients affected by hypercholesterolaemia. The resulting immuno‐adsorbents were compared to that obtained earlier with cyanogen bromide activation, in terms of coupling yield, adsorption capacity and moreover stability towards the various chemical and biochemical conditions to which they are submitted during their handling. Tresyl chloride‐activated Sepharose 6 Fast Flow turns out to afford optimal stability in the whole pH range tested.
Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies
Authors: Johnsson Bo , Löfås Stefan , Lindquist Gabrielle , Edström Åsa , Hillgren Rose‐Marie Müller , Hansson Anna 
Published: May 2005

AbstractThe authors have recently described the development of a carboxymethyl dextran‐based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N‐hydroxysuccinimide and N‐ethyl‐N′‐(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin–avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e. g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a ‘site directed’ immobilization strategy for monoclonal antibodies has no advantage. In general the specific activity for optimized systems was approximately 75% for the binding of β2μ‐globulin to an immobilized monoclonal antibody directed against β2μ‐globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioraton of the active site of the antibody.