bluuebunny/crossref_metadata_2025 huggingface dataset:
Agonist derived molecular probes for A2 adenosine receptors
Authors: Jacobson Kenneth A. , Pannell Lewis K. , Ji Xiao‐Duo , Jarvis Michael F. , Williams Michael , Hutchison Alan J. , Barrington William W. , Stiles Gary L.
Published: May 2005

The adenosine agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)‐5′‐N‐ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2 adenosine receptor subtype, which mediates its hypotensive action. To investigate structure/activity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p‐hydroxyphenylpropionic, 2‐thiophenylacetic, and p‐aminophenylacetic acids were prepared. The latter derivative (PAPA‐APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A2 adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350‐fold selective for A2 receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl‐1‐piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta‐ and para‐phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2 receptors is relatively insensitive to distal structural changes at the 2‐position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity lebelling methodology.
Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography
Authors: Bayer Edward A. , Wilchek Meir
Published: May 2005

The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin‐coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti‐transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr‐activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.
Interaction between immobilized and soluble protein subunits. Analysis and applications
Authors: Chiancone Emilia , Gattoni Maurizio
Published: May 2005

A distinctive property of oligomeric and self‐associating proteins is the high specificity of the subunit recognition process. Protein subunits immobilized covalently on a solid matrix maintain this characteristic and are able to bind soluble subunits of the same or a closely related protein under conditions that allow the establishment of a finite association/dissociation equilibrium. The basic theory for studying the immobilized‐soluble subunit interaction is presented together with the methodology for a proper protein immobilization. Specific examples are discussed to illustrate on the one hand benefits and caveats of using immobilized protein subunits to measure interaction constants, and on the other preparative applications of subunit affinity columns.
Enzymatic semisynthesis of human insulin: An update
Authors: Morihara Kazuyuki
Published: May 2005

Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1–29)‐A(1–21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr‐X (X = blocking residue) (two‐step reaction) or (2) subjecting Ala‐B30 of procine insulin or Gly‐A1 of the single chain insulin precursor to transpeptidation with Thr‐X (one‐step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two‐step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two‐step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.
Purification and characterization of fab fragments from anti‐mouse NGF polyclonal antibodies
Authors: Callegaro L. , Skaper S. D. , Vantini G. , Benvegnù D. , Di Martino A. , Schiavo N. , Triban C. , Minozzi C. , Leon A.
Published: May 2005

A functional role for Nerve Growth Factor (NGF) in the peripheral nervous system is well‐documented, but a similar case for NGF in the central nervous system remains to be established. One approach to answering this question would be the availability of high‐affinity monospecific Fab fragments obtained against NGF. In the present studies we describe the preparation and characterization of such Fab fragments from anti‐mouse NGF polyclonal antibodies. Following their purification by the use of a NGF Sepharose‐coupled affinity column, the Fab fragments were examined for biological competence in several ways. In vitro, the anti‐Fab fragments blcoked the neuronotrophic activity of NGF, as measured by the survival of chicken embryonic day 8 dorsal root ganglion neurons. In vivo, these Fab fragments, when administered systemically to neonatal rats, produced a decrease of noradrenaline levels in two sympathetically innervated organs, the heart and the spleen. These findings suggest that affinity purified Fab fragments of anti‐NGF antibodies can be a useful tool for studying the physiological function of NGF in the nervous system.
Binding of the soybean Bowman‐Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine α‐chymotrypsin and bovine β‐trypsin. A thermodynamic study
Authors: Ascenzi Paolo , Amiconi Gino , Bolognesi Martino , Menegatti Enea , Guarneri Mario
Published: May 2005

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman‐Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F‐C(p), F‐T(p) and F‐T(t), respectively) to bovine α‐chymotrypsin (α‐chymotrypsin) and bovine β‐trypsin (β‐trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: β‐chymotrypsin: BBI ≈ β‐trypsin:BBI ≈ β‐trypsin:F‐T(t) ≈ β‐trypsin:F‐T(p) ≫ α‐chymotrypsin:F‐C(p), F‐C(p), F‐T(p) and F‐T(t) do not inhibit β‐trypsin and α‐chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F‐C(p), F‐T(p) and/or F‐T(t) binding to α‐chymotrypsin and β‐trypsin decrease, thus reflecting the acid‐pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F‐C(p), F‐T(p) and F‐T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.
Effect of modification of enkephalin C‐terminal functions on affinity selection of opioid receptors
Authors: Kodama Hiroaki , Uchida Hiroaki , Yasunaga Teruo , Kondo Michio , Costa Tommaso , Shimohigashi Yasuyuki
Published: May 2005

Enkephalin analogs with various C‐terminal modifications have been synthesized to evaluate the corresponding structural elements in the opioid receptors. The carboxyl group of the C‐terminal leucine5 or glycine5 was converted into the mercaptomethyl (–CH2SH) and hydroxymethyl (–CH2OH) groups, starting from leucinthiol or leucinol for Leu5‐derivatives and from cysteamine or ethanolamine for Gly5‐derivatives. Interactions of synthetic peptides with the opioid receptors were examined by the radioligand receptor binding assays using rat brain and tritiated enkephalin analogs. The data suggest that the C‐terminal carboxyl group in enkephalins is important, but not electrostatically, for interaction with delta‐opioid receptors. With leucinthiol–enkephalin in biological assays which examine its inhibitory activity for electrically stimulated contractions of isolated smooth muscle, it was found that the reactive thiol group exists in the mu receptors present in the guinea pig ileum. Leucinthiol‐enkephalin became bound covalently to this receptor‐thiol group via disulfide formation after prolonged incubation.
Proposed loci of interaction between bombesin and its receptor
Authors: Tritsch G. L.
Published: May 2005

The non‐coding strand of the bombesin receptor gene, when ‘translated’ 5′ 3′, contains and interrupted sequence of the 10 amino acids of bombesin. This finding forms the basis for proposing the points of contact between bombesin and its receptor as well as a partial conformation of the binding region of the receptor as well as partial conformation of the binding region of the receptor.
Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII
Authors: Gromova Elizabeth S. , Kubareva Elena A. , Vinogradova Marina N. , Oretskaya Tatjana S. , Shabarova Zoe A.
Published: May 2005

To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar–phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neo‐schizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.
Evaluation of peptide/metal ion interactions by UV laser desorption time‐of‐flight mass spectrometry
Authors: Hutchens T. William , Nelson Randall W. , Yip Tai‐Tung
Published: May 2005

A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix‐assisted UV laser desorption time‐of‐flight mass spectrometry (LDTOF‐MS), has been evaluated as a new means to investigate the metal ion‐binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDA human plasma metal‐binding transport protein histidine‐rich glycoprotein (HRG) has been identified as a bioactive metal‐binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF‐MS before and after the addition of Cu(II) in solution with 2,5‐dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)‐binding capacity observed for the 26‐residue peptide (5 g‐atoms/mol) suggest that up 1 Cu(II) may be bound per 5‐residue internal repeat unit (GHHPH) within this peptide: several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)‐binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution‐phase binding capacity of metal‐binding peptides by LDTOC‐MS.
Design of novel affinity adsorbents for the purification of trypsin‐like proteases
Authors: Burton Nicolas P. , Lowe Christopher R.
Published: May 2005

A number of ligands for the selective purification by affinity chromatography of the trypsin‐like protease, porcine pancreatic kallikrein, were designedde novoby computer‐aided molecular design. The ligands were designed to mimic the side‐chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, and analogue of a Phe‐Arg dipeptide, when immobilized to Sepharose CL‐4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110‐fold in one step from a crude pancreatic acetone extract.
Complex carbohydrate–lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar‐binding domain of a glycoprotein lectin
Authors: Chakrabarti Abhijit , Podder Sunil K.
Published: May 2005

We describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell–cell adhesion mediated by multiple protein–carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide‐induced disaggregation of the two types of vesicles were followed by monitoring the time‐dependent change in turbidity. Depending on the surface density of the asialoGM1, 40–60% of the resulting precipitin complex was dissociable only in the presence of both RCAI‐specific galactose and Con A‐specific α‐methyl‐D‐mannoside. Results indicate simultaneous participation of both the saccharide‐binding domain and carbohydrage sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell–cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell‐surface membranes.
Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions
Authors: Sakamoto Hiroshi , Shimohigashi Yasuyuki , Maeda Iori , Nose Takeru , Nakashima Kin‐ichi , Nakamura Ichiro , Ogawa Tomoshisa , Ohno Motonori , Kawano Keiichi
Published: May 2005

A complete series of configurationally isomers (L‐L, L‐D, D‐L AND D‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz 1H NMR, the L‐D and D‐L isomers, but not hte L‐L and D‐D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH2 and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH2, the NOE enhancement observed between Leu‐δCH3 and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated that L‐D and D‐L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, the L‐L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D‐L isomer was strongest with Ki of 2.2 × 10−5 M. It was found that the D‐L isomer was only slowly hydrolysed but the L(or D)‐D isomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (Ki = 6.3 × 10−6 M). Since the free acid analogue of the D‐L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S1 site of chymotrypsin, while the side chain‐side chain complexing hydrophobic core fits the S2 site.
PLIM: A protein–ligand interaction modeller
Authors: Harris Mark R. , Kihlen Mats , Bywater Robert P.
Published: May 2005

A Computer program is presented that models the binding of selected chemical groups to a protein surface. The groups are successively incorporated at energetically favourable positions to build up a pharmacophore pattern that may be used as the basis for a database search for possible ligands. The ability to predict known binding points in a trypsin–inhibitor complex is demonstrated, and the results from a run on dihydrofolate reductase are shown to be usable as a pharmacophore pattern for a database search.
Influence of fractal kinetics on molecular recognition
Authors: Savageau Michael A.
Published: May 2005

Molecular recognition is a central issue for nearly every biological mechanism. The analysis of molecular recognition to has been conducted within the framework of classical chemical kinetics, in which the kinetic orders of a reaction have positive integer values. However, recent theoretical and experimental advances have shown that the assumption inherent in this classical framework are invalid under a variety of conditions in shown that the assumptions inherent in this classical framework are invalid under a variety of condition in which the reaction environment may be considered nonideal. A good example is provided by reactions that are spatially constrainal and diffusion limited. Bio molecular reactions confined within two‐dimensional membranes, one‐dimensional channels or fractal surfaces in general exhibit kinetic orders that are noninteger. An appropriate framework for the study of these nonideal phenomena is provided by the Power‐Law formalism, which includes as special cases the Mass‐Action formalism of chemical kinetics and the Michaelis–Menten formalism of enzyme kinetics. The Power‐Law formalism is an appropriate representation not only for fractal kinetics per se, but also for other nonideal kinetic phenomena, provided the range of variation in concentration is not too large. After defining some elementary concepts of molecular recognition, and showing how these are manifested in classical kinetic terms, this paper contrasts the implications of classical and fractal kinetics in a few simple cases. The principal distinction lies in the ability of fractal kinetics to nonlinearly transform, rather than proportionally transmit, the input S/N ratio. As a consequence, fractal kinetics create a threshold for the input signal below which no recognition occurs and above which amplified recognition takes place. Thus, fractal kinetics implies an intimate relationship between design of the physiological mechanisms regulating the environment of the process and design of the molecular process itself. These results also suggest that recognition in the presence of a favorable input ration would emphasize rapid reactions, while recognition in the presence of an unfavorable input ratio would emphasize slow reactions.
Temporal analysis of metabolic systems and its application to metabolite channelling
Authors: Easterby John S.
Published: May 2005

When a metabolic system undergoes a transition between steady states, the lag or transition time of the system is determined by the aggregated lifetimes of the metabolite pools. This allows the transition time, and hence the temporal responsiveness of the system, to be estimated from a knowledge of the starting and finishing steady states and obviates the need for dynamic measurements. The analysis of temporal response in metabolic systems may be integrated with the general field of metabolic control analysis by the definition of a temporal control coefficient (C) in terms of flux and concentration control coefficients. The temporal control coefficient exhibits summation and other properties analogous to the flux and concentration control coefficients. For systems in which static metabolite channels exits, the major kinetic advantage of channelling is a reduction in pool sizes and, as a result, a more rapid system response reflected in a reduced transition time. The extent of the channelling advantage may therefore be assessed from a knowledge of the system transition time. This reveals that no channelling advantage is achieved at high enzyme concentration (i.e., comparable to Km) or, in the case of ‘leaky’ channels, where rapid equilibrium kinetic mechanisms obtain. In the case of a perfect channel with no leakage and direct transfer of metabolite between adjacent enzyme active sites, the transition time is minimized and equal to the lifetime of the enzyme–substrate complex.
Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview
Authors: Beeckmans Sonia , Van Driessche Edilbert , Kanarek Louis
Published: May 2005

In recent years it has become clear that a cell cannot be visualized as a ‘bag’ filled with enzymes dissolved in bulk water. The aqueous‐phase properties in the interior of a cell are, indeed, essentially different from those of an ordianry queous solutin. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as ‘metabolons’. Very often interactions between enzymes of a pathway can exclusively be evidenced in Vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glucolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate–malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques we also discussed.
Kinetic analysis of engineered antibody‐antigen interactions
Authors: Malmqvist Magnus
Published: May 2005

Molecular 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

The 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

Consideration 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

The 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

We 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

(+)‐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

The 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

Oligodeoxyribonucleotide 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

The 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

Recent 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

Antigen‐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

Compared 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

A 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

A 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

Various 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

The 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.
Plasma‐membrane‐Bound mcromoleculas are dynamically aggregated to form non‐random codistribution patterns of selected functional elements. Do pattern recognition processes govern antigen presentation and intercellular interactions?
Authors: Vereb György , Mátyus László , Bene László , Panyi György , Bacsó Zsolt , Balázs Margit , Matkó János , Szöllősi János , Gáspár Rezső , Damjanovich Sándor , Dale Robert E. , Pieri Carlo , Ameloot Marcel
Published: May 2005

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane‐bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin‐2 receptor, the T‐cell receptor–CD3 system, the intercellular adhesion molecule ICAM‐1, and the major histocompatilibilty complex class I and II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of icon channel activities, adduced as one of the major regulatory mechanisms of cell–cell communications. A general role in the mediation and regulation of intercellular interactions is suggested for cell‐surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accomodation and recognition processes at the cellular level.