bluuebunny/crossref_metadata_2025_split huggingface dataset:
Theory of fast capillary gas chromatography. Part 1: Column efficiency
Authors: Blumberg Leonid M. 
Published: May 2005

AbstractTypically, fast GC requires high column pressure drop. Under that condition, known relations do not provide a complete description of dependence of [apparent] plate height, ĥ, on average velocity, u, of carrier gas.A complete descriptions for ĥ vs. u is derived and analyzed. It is shown that, under the high pressure drop, ĥ = B/u2 + C1 u2 + C2 u. This is substantially different from the Van Deemter equation ĥ = B/u + Cu for the low pressure drop. Analytical expressions for the minimum plate height and for the respective optimum u under the high pressure drop are derived and analyzed.
Investigations on wine bouquet components by solid‐phase microextration ‐capillary gas chromatography (SMPE‐CGC) using different fibers
Authors: De la Calle García Demetrio , Reichenbächer Manfred , Danzer Klaus , Hurlbeck Christian , Bartzsch Christine , Feller Karl‐Heinz 
Published: May 2005

AbstractThe extraction of wine bouquet components with solid‐phase microextraction (SPME) using different fibers was studied. Polyacrylate fibers (PA‐85) achieve the most complete bouquet profiles. About ninety substances were identified by capillary gas chromatography‐MS and their retention factors were calculated to allow peak identification in capillary‐GC‐FID chromatograms. The terpene profiles of different wines were recorded by SPME (PA‐85)‐CGC‐FID.
Analysis and use of the serum albumin binding domains of streptococcal protein G
Authors: Nygren Per‐Åke , Eliasson Margareta , Abrahmsén Lars , Uhlén Mathias , Palmcrantz Elisabeth 
Published: May 2005

AbstractStreptococcal protein G is an IgG‐binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG‐binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG‐ and albumin‐binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity Chromatography, using IgG or HSA immobilized on Sepharose, Showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin‐binding. The structure of this part of the proteins suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one‐step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.
Conformational homologies among cytokines: Interleukins and colony stimulating factors
Authors: Parry David A. D. , Minasian Elizabeth , Leach Sydney J. 
Published: May 2005

AbstractSome 30 cytokine amino acid sequences (mainly interleukins, colony stimulating factors and tumor necrosis factors) have been examined for evidence of secondary structure as well as longer‐range interactions of a type likely to lead to stable α‐helical bundles. Most, though not all, of the cytokines examined have a high predicted α‐helical content (40–60%) and quasi‐repeating heptads containing i/i+3 apolar periodicities. This major subset of the cytokines is predicted to be characterized by molecules in which 4‐α‐helical bundles with an average length of 25Å are the most marked conformational features. Based on these conclusions, we suggest structures for huG‐CSF, huGM‐CSF and muIL‐5 in which defined loop segments at the ends of helical bundles are the most likely sites for binding and recognition by specific cell receptors. As such, they provide a means for testing or refining the three working models we have defined, using currently available methods of site‐directed substitution and deletion mutagenesis, as well as synthetic peptides corresponding to the proposed loop sequences and the use of monoclonal antibodies of defined epitopic specificity. The structure arrived at for huGM‐CSF is consistent with the limited data currently available concerning the residues which are important for binding and activity.
Molecular basis for potentiation of bleomycin‐mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies
Authors: Strekowski Lucjan , Harden Donald B. , Wydra Roman L. , Stewart Kent D. , Wilson W. David 
Published: May 2005

AbstractThe bleomycin‐mediated degradation of DNA is stimulated (amplified) by certain DNA binding compounds, such as polyamines, that distort the double helix. Computer modelling studies suggest that putrescine (1), spermidine (2), and spermine (3) bind preferentially on the floor of the major groove of (dGdC)5·(dGdC)5. This interaction results in a bend of the oligomer helix toward the major groove and enlargement of the minor groove, both effects being in the order 1 < 2 < 3. These polyamine‐induced distoritions, as obtained from theoretical studies, parallel the experimental values of the amplification activites of 1–3 in the bleomycin‐mediated degradation of poly(dGdC)·poly(dGdC). The amplification mechanism of non‐competitive binding of amplifier molecules in the major groove, and bleomycin in the minor groove, is proposed. It is suggested that the amplifier‐induced conformational changes of the DNA helix increase affinity of the activated bleomycin complex toward the DNA minor groove and, consequently, result in an increased efficiency of the bleomycin‐mediated degradation of the helix.
Preparation of a functional, highly selective polymer by molecular imprinting. A demonstration with L‐p‐aminophenylalanine anilide as a template molecule allowing multiple points of attachment
Authors: Moradian Aydin , Mosbach Klaus 
Published: May 2005

AbstractThe technique of molecular imprinting was tested using various phenylalanine derivatives. It could be shown that polymers obtained by imprinting with L‐p‐aminophenylalanine anilide gave best recognition of the original L‐form, with separation factors (α) above 8, on applying racemic mixtures to the polymer obtained and present in a HPLC column. We believe that these results are due to multiple points of attachment that are possible between print or template (guest) molecules and the formed polymer (host).
Sequence‐selective, pH‐dependent binding to DNA of benzophenanthridine alkaloids
Authors: Bajaj Narinder P. S. , McLean Michael J. , Waring Michael J. , Smekal Emil 
Published: May 2005

AbstractThe sequence selectivity associated with binding to DNA of three alkaloids belonging to the benzophenanthridine family has been analysed by DNase I footprinting, and the results were compared with those obtained from an analysis of the behaviour of the standard intercalator, ethidium bromide. Like the ethidium, the benzophenanthridine compunds appear to bind best to regions of mixed nucleotide sequence, especially those containing alternating purines and pyrimidines, although there are some notable differences in behaviour. There is also a marked lack of binding to sequences such as (AT)n, where n⩾3. The binding to DNA of the benzophenanthridines is specifically related to the hydrogen ion concentration of the medium, in that the DNase I footprints are considerably enhanced when the reaction is performed at a pH below 7.0. We discuss these results in terms of a greater preponderance of the intercalating species being present at lower pH.
Conformational constraints: Nonpeptide β‐turn mimics
Authors: Ball Jonathan B. , Alewood Paul F. 
Published: May 2005

AbstractThe β‐turn, which has also been referred to as the β‐bend, β‐loop or reverse turn, has been implicated as an important site for molecular recognition in many biologically active peptides and in globular proteins. This small secondary structure therefore makes an attractive target for mimicry by a conformational constraint, because a peptide which is constrained in a biologically active conformation can display a number of advantages over the parent substrate. The less peptide‐like such a constraint is, the more potential there is to maximize these advantages. A decade has passed since the first (and highly successful) attempt to mimic the β‐turn with a nonpeptide conformational constraint was disclosed by Freidinger et al. (1980). Since this report, rapidly growing interest in the field of nonpeptide β‐turn mimics has seen a variety of experimental approaches and a mixed bag of results. It is attempted in this review, not only to summarize and critically analyse these approaches, but also to touch on the compexities associated with the conformational mimicry of such a diverse structure as the β‐turn.
27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity
Authors: Nishikawa Satoshi , Matsuo Noriyuki , Isaka Yoshitaka , Uesugi Seiichi , Miura Kazunobu , Ohtsuka Eiko , Takeda Ken , Konno Kunio , Ikehara Morio 
Published: May 2005

AbstractIn order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N‐terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LTδ37N, in which 37 residues were absent from the N‐terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LTδ28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LTδ27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.
Antibody affinity measurements
Authors: Azimzadeh A. , Van Regenmortel M. H. V. 
Published: May 2005

AbstractThe use of antibodies in immunoaffinity separations represents one of the most specific methods for purifying substances of biological interest. Since the binding affinity of antibody greatly influences its behavior in such separations, it is often important to know the value of the antibody affinity expressed as an equilibrium constant K. The present review discussed the equations used in the quantitative analysis of antigen/antibody interactions and describes currently used experimental methods for measuring K values. Advantages and shortcomings of the solution phase and solid phase approaches used for measuring antibody affinity are discussed.
New developments in affinity chromatography
Authors: Lowe Christopher R. , Burton Steven J. , Burton Nicolas , Stewart David J. , Purvis Duncan R. , Pitfield Ian , Eapen Saji 
Published: May 2005

AbstractThe design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.
Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver
Authors: Spacciapoli Peter , Thurlow David L. 
Published: May 2005

AbstractRecognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidylransferase from rabbit liver was studied using 12 tRNAs, previously treated with the chemical modifier diethylpyrocarbonate (DEP). Such chemically modified tRNAs were labeled with 32P by nucleotidyltransferase, using α‐[32P]ATP as a cosubstrate. A carbethoxylated purine at position 57 in the Ψ‐loop interfered with recognition of the tRNA in all instances. DEP‐modified purines at other positions (58 in the Ψ‐loop, 52 or 53 in the Ψ‐stem, and 71–73 in the acceptor stem), also interfered with the interaction, but in only a few tRNAs. The mammalian enzyme was more similar to the homologous enzyme from yeast than that from bacteria, in its requirements for chemically unmodified purines. The extent of exclusion of modified bases from 32P‐labeled material diminished as the concentration of enzyme increased, demonstrating that interference was not due to the inability of the chemically altered tRNA to refold into a recognizable conformation. The degree of purification of the enzyme did not affect the identity of bases that inhibited the reaction when modified.
X‐ray crystal structure of the bovine α‐chymotrypsin/eglin c complex at 2.6 Å resolution
Authors: Bolognesi Martino , Pugliese Luisa , Gatti Giuseppina , Frigerio Francesco , Coda Alessandro , Antolini Luciano , Schnebli Hans Peter , Menegatti Enea , Amiconi Gino , Ascenzi Paolo 
Published: May 2005

AbstractThe crystal structure of the molecular complex formed by bovine α‐chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 Å resolution were employed to determine the structure by molecular replacement techniques. Bovine α‐chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 Å resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine α‐chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine α‐chymotrypsin between subsites P3 and P′2; Leu 45I acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic‐ and secretory‐ inhibitors are found around the scissile peptide bond.
Binding of the neuroleptic drug haloperidol to a monoclonal antibody: Refinement of the binding site molecular model using canonical structures
Authors: Anchin Jerry M. , Subramaniam Shankar , Linthicum D. Scott 
Published: May 2005

AbstractThe ligand binding site of a monoclonal antibody (185), which binds the neuroleptic drug haloperidol, has been modelled using canonical structures and energy minimization techniques. This refined modelling protocol has allowed us to predict the variable region loop conformation. Three key residues, H:50(W), H:100a(D) and L:96(Y) appear to create the basis of the electrostatic, π–π stacking interactions and hydrogen bonding required for the high affinity binding site characteristics present in this antibody. The use of computer‐aided graphics techniques and appropriate three‐dimensional modelling permits inspection of the predicted molecular recognition features of the ligand binding site.
Cell interactions of enkephalin/polypeptide conjugates
Authors: Tsukada Motohiko , Sasaki‐Yagi Yoshiko , Kimura Shunsaku , Imanishi Yukio 
Published: May 2005

AbstractEnkephalin molecules were bound to poly(Lys) (poly‐K) or poly(Ala‐Lys‐Ala‐Leu) (poly‐A) and their interactions with NG108‐15 cells, platelets, erythrocytes and fibroblast cells were investigated. A fluorescent probe, rhodamine, also was bound to the conjugates for monitoring interactions with these cells. Observations by fluorescence microscopy revealed that NG108‐15 cells, and fibroblast cells were labelled by the conjugates, whereas erythrocytes were not. Since polypeptides without enkephalin moieties were only weakly adsorbed on the cells, it was concluded that the enkephalin/polypeptide conjugates were bound specifically to receptors on the cells membrane. Interestingly, when the enkephalin/poly‐K conjugate was bound to NG108‐15 and fibroblast cells, fluorescent patches appeared on the membrane. Such patch formation was not clearly observed with an enkephalin/rhodamine or enkephalin/poly‐A conjugate. In the case of fibroblast cells, the fluorescence converged to a large cluster, which was ultimately internalized. The results suggest that clustering of the receptors in cell membranes in influenced by the carrier polymer presumably due to cross‐linking of the receptors and/or the effect of the cationic polypeptides.
A 500 MHz proton NMR study of stacking interactions: Binding of tripeptide Lys‐Tyr‐Lys to tetradeoxynucleotide d‐GpCpGpC
Authors: Barthwal R. , Mujeeb A. , Kukreti S. , Gupta A. , Govil G. 
Published: May 2005

AbstractThe complete sequential assignment and conformation of d‐GpCpGpC in D2O has been determined from ID NMR spectra at 285–320 K and room temperature 2D‐COSY and NOESY spectra. The tetradeoxynucleotide exists primarily as a right handed double helix at 285 K, having Tm as 314 K. On binding to a tripeptide Lys‐Tyr‐Lys in a concentration equimolar to tetranucleotide duplex, the Tyr ring protons shift uplifted by 0.14 ppm at 285 K. The increase in Tm on binding suggests stabilization of duplex. The existence of intermolecular NOEs between C4 sugar protons and Tyr αC and Lys αC protons give direct evidence of proximity of Tyr residue to the C4 base of d‐GpCpGpC. The conformation of d‐GpCpGpC remains unchanged on binding. The observed results are interpreted in terms of preferential stacking of aromatic ring of Tyr residue with proximal base‐pair of d‐GpCpGpC, stabilized by electostatic interaction of Lysine side chains with backbone phosphates. This is in contrast to intercalculation of aromatic dyes within base‐pairs resulting in a change in sugar conformation at the binding site.
‘Hot spot’ amino acid distribution in Ha‐ras oncogene product p21: Relationship to guanine binding site
Authors: Cosic Irena , Hearn Milton T. W. 
Published: May 2005

AbstractAlthough the rules which describes the atomic basis of structure–function relationships of proteins have yet to be deciphered, they are nevertheless coded within the framework of the amino acid and nucleotide sequence. The objectives of the present investigations were to document a composite, new approach for the evaluation of the structure–function dependencies of proteins based on the analysis of the informational content of the primary amino acid sequence as well as the topological and functional regions of a protein. This approach is validated with the example of the p21 Ha‐ras oncogene family of proteins. Using this approach is validated with the example of the p21 Ha‐ras oncogene family of proteins. Using this approach, amino acids crucial for p21 transforming activity have been identified and these amino acid residue assignments compared with experimental data.
A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium
Authors: Hsu Carol S. , Minton Allen P. 
Published: May 2005

AbstractA method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alternations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loadings compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i. e. without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from ‘sensitive’ regions of the discriminator map.
Operational aspects of antibody affinity constants measured by liquid‐phase and solid‐phase assays
Authors: Azimzadeh A. , Pellequer J. L. , Van Regenmortel M. H. V. 
Published: May 2005

AbstractThe association constant of monoclonal antibodies (MAbs) to tobacco mosaic virus has been determined in solution and solid‐phase binding assays. The ElISA equilibrium titration method developed by Friguet et al. (1985) was found to be suitable for large antigens such as viruses. In the case of intact IgG antibody, it gave equilibrium constant (K) values ca 30% lower than those obtained by classical solution‐phase assay while in the case of Fab', the same values were obtained in both assays. Solid‐phase binding assays gave higher K values than solution‐phase assays by a factor which varied with the Mab tested (1.5‐ to 5.4‐fold higher). Furhtermore, in solution‐phase assay, K values were found to depend on the antibody concentration used in the assay. These results confirm the operational nature of antibody affinity constants and indicate that in order to compare the affinity of different Mabs in a meaningful way, it is necessary to use a single technique under standardized conditions.
Design of novel cationic ligands for the purification of trypsin‐like proteases by affinity chromatography
Authors: Burton Nicolas P. , Lowe Christopher R. 
Published: May 2005

AbstractA number on new cationic ligands have been designed and synthesized for the selective resolution an purification of the trypszin‐like proteases. A series of ligands based on 4‐[2′‐methyl‐4′‐(2″,4″‐dichloro‐1″,3″,5″‐triazin‐6‐ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin‐like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4‐aminophenyltrimethylammonium or 4‐aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis‐cationic ligands bound pancreatic kallikrein in solution ad following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis‐cationic ligands was used to purify pancreatic kallikrein 9.5‐fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.