bluuebunny/crossref_metadata_2025 huggingface dataset:
Atmospheric monitoring of volatile organic compounds using programmed temperature vaporization injection
Authors: Lewis Alastair C. , Bartle Keith D. , McQuaid James B. , Pilling Michael J. , Seakins Paul W. , Ridgeon Peter
Published: May 2005

A method has been developed for the automated determination of C5C10 and C2C6 volatile organic compounds in urban and rural air, using programmed temperature vaporization injection from a sorbent tube trap. A single activated charcoal sorbent tube was repeatedly used to collect samples of air with trapped VOCs being subsequently desorbed onto either a wide bore dimethyl polysiloxane (C5C10) or porous layer open tubular (C2C6) gas chromatography column without use of intermediate cryogenic refocussing. The high flow rates of helium used during the analysis resulted in the sample tube being cleaned and ready to reuse following the analytical separation. Examples of analysis of aromatic VOCs in urban air, and biogenic emissions in rural air, collected in a Sitka Spruce forest are presented. Using this method it is possible to quasi‐continuously monitor concentrations of VOCs in locations where high sensitivity in situ analysis is required, but where cryogenic coolants may not be readily available or desirable.
Determination of polar analytes in aqueous matrices by purge and trap
Authors: Kaufmann Anton
Published: May 2005

(not readable) fully autimated purge and trap system has been developed for (not readable) detemination of volatile wine components. The proposed system consists of sampler, a purge and trap instrument, and a GC, (not readable) steps, from the uptake of the infiltrated wine from the sampler (not readable) to the printout of the intergrated chromatograms, proceed(not readable), Typical problems occurring when analyzing polar analytes by the classical purse and trap approach have been specifically addressed. The system has been desiggned to handle such difficult (not readable) analytes. Efforts have been made to improve the reproducibility and the carry‐over of these analytes. The measures adopted include: thorough rinsing and subsequent conditioning of the P + T parger; elimination of matrix effects by dilution and high salt (not readable); tight specification of purge relevant parameters; split‐inter‐(not readable) to improve GC retention time reproducibility.
Determination of artemisinin and artemisinic acid by capillary and packed supercritical fluid chromatography
Authors: Köhler Marcel , Haerdi Werner , Christen Philippe , Veuthey Jean‐Luc
Published: May 2005

Artemisinin (an antimalarial compound) and its bioprecursor artemisinic acid, present in the plant Atemisia annua L., were analyzed by supercritical fluid chromatography (SFS) using capillary and packed columns, coupled respectively with a flame ionization detector (FID) and an evaporative light scattering detector (ELSD). Both methods were optimized and validated with columns of different polarity in order to separate artemisinin and artemisinic acid. Analytical results were comparable, but the paced SFC‐ELSD method was faster. Indeed, artemisinin and artemisinic acid were separated with an aminopropyl silica column in less than 8 minutes instead of about 25 minutes by capillary SFS. Contrary to conventional gas and liquid chromatography coupled to an UV‐visible detector, SFS methods determined both compounds directly, without degradation and/or derivatization in the concentration range expected in the plant material. Results obtained on plant extracts by capillary SFS‐FID and packed SFS‐ELSD were confirmed by GC‐MS.
Eavaluation of automatic thermal desorption–capillary GC for determination of semivolatile organic compounds (SVOCS) in indoor air
Authors: Clausen Per Axel , Wolkoff Peder
Published: May 2005

Some data on a newly developed filter/sorbent indoor air SVOC sampling device for thermal desorption analyiss are described. Thermal desorption of SVOCs spiked on Tenax had reponse factors identical to on‐column injection except for highly polar compounds like fatty acids. SVOCs spiked on quartz fiber filters had response factors that on an average were 80% of the on‐column response factors (66% for oxygen containing compounds and 87% for non‐oxygen compounds) Low nanogram on‐tube amounts of SVOCs were found generally to have lower recoveries than larger amounts from both Tenax and quart fiber filters. This appeared to be explained in part by a relatively larger “memory” effect and lower desorption efficiency. In addition, it was indicated that the “memory” effect was an important source of background contaminations that might impair analysis of low nanogram on‐tube amounts of some SVOCs. Polar SVOCs. Polar SVOCs in the gas phase appear to adsorbto the quartz fiber filters. This functions as a precleaning of the sample and thus minimizes the problem with coeluting peaks. The relative standard from nine duplicate samples appeared to be sufficiently low to distinguish a day variation.
Standard equivalent chain length values of monoenic and polyenic (methylene interrupted) fatty acids
Authors: Stránsky Karel , Jursík Tomáš , Vítek Antonín
Published: May 2005

The equivalent chain length (ECL) values of the methyl esters of 83 defined fatty acids (FAs) have been determined by gas chromatog‐raphy (GC) on three fused silica DB‐WAX and three DB‐1 columns. ECL values of further 46 FAs were calculated by different methods. Conditions of chromatography, methods of ECL values calculation and differences between ECL values on individual columns and between trans‐ and cis‐isomers of corresponding FAs are also discussed.
Enantiomer separations by capillary GC on modified gyclodextrins
Authors: Kim Byoung Eog , Lee (deceased) Sun Haing , Park Keung Shik , Lee Kwang Pill , Park Jung Hag
Published: May 2005

Three new chiral selectors, 6‐tert‐butyldimethylsilyl‐2,3‐diethyl‐a‐cyclodextrin, 6‐tert‐butyldimethylsilyl‐2,3‐diethyl‐ and dipropyl‐β‐cyclodextrin (TBDE‐α‐CD, TBDE‐β‐CD, TBDP‐β‐CD) were synthesized and tested as chiral stationary phases in capillary gas chromatography. TBDE‐β‐CD in particular showed a high enan‐tioselectivity for test chiral compounds due to good solubility in a polar polysiloxane (OV‐1701). Enantioselectivity obtained with TBDE‐β‐CD was compared with that of 6‐tert‐butyldimethylsilyl‐2,3‐di‐O‐methyl‐β‐cyclodextrin (TBDM‐β‐CD). Better enantiose‐lectivity was obtained with TBDE‐P‐CD than with TBDM‐β‐CD for the test chiral compounds studied. This is probably due to greater effect of the increased hydrophobicity of TBDE‐β‐CD which favors inclusion of the analytes than the effect of increased steric hindrance. With TBDP‐β‐CD the less polar lactones are well separated due most likely to increased hydrophobicity of the propyl groups while the more polar are not well resolved. For TBDP‐β‐CD it is likely that the unfavorable steric hindrance is predominant over the favorable hydrophobicity of the propyl groups, thus hindering the formation of inclusion complexes of the alcohols with TBDP‐β‐CD. TBDE‐α‐CD was also a valuable chiral selector for the separation of small chiral molecules such as simple secondary alcohols and nitro‐substituted alcohols.
Unusual Δ5cis‐fatty acids in seed oils of Cimicifuga species
Authors: Tsevegsüren Nanzad , Aitzetmüllei Kurt
Published: May 2005

A range of unusual fatty acids with cis‐5‐unsaturation had been reported in the seed oil of Caltha palustris. Seed oils of Cimicifuga spp. have now been found to contain the same unusual fatty acids as are present in Caltha, plus several other minor fatty acids to give a more complex and more unsaturated seed oil fatty acid pattern.

The gas chromatographic fatty acid patterns found seem to be consistent and chemotaxonomically significant, because essentially the same pattern was found in several species of the genus Cimicifuga. These findings may shed a new light on the relation of Cimicifuga to Caltha, and to other genera in the plant family Ranunculaceae.

The situation is illustrated by capillary GLC seed oil fatty acid methyl ester “fingerprints” obtained from Cimicifuga and Caltha, and is discussed in relation to other genera. The occurrence in nature of several of these unusual fatty acids, and their chemotax‐onomic significance is discussed. The close relation of GLC fatty acid patterns of Caltha and Cimicifuga could indicate monophyly and/or their belonging to the same tribe or subtribe. These observations are not in accordance with the phylogenetic systematic schemes of the genera in this plant family as published by various authors.
Simultaneous determination of nicotinamide, nicotinic acid, riboflavin, thiamin, and pyridoxine in enriched Brazilian foods by HPLC
Authors: Agostini Tania S. , Godoy Helena T.
Published: May 2005

The control of the enrichment levels in foods is difficult, due mainly to the lack of appropriate analytical methodologies. The amounts of the five B‐group vitamins (nicotinamide, nicotinic acid, ribofla‐vin, thiamin, and pyridoxine) have been determined in enriched Brazilian foods by a high‐performance liquid chromatography (HPLC) method. Fifty products, such as biscuits, liquid and dry milks, flavored milk drinks, flour, macaroni, and cereals were analyzed. Some products showed the amounts declared on the package. Although some slight quantitative variations were shown in the biscuits, one showed levels of riboflavin 35% lower than the value declared. Of five different corn cereal brands, only one showed the declared vitamin content, the others showing levels 30 % lower than that declared. No B‐group vitamins were detected in one brand of enriched macaroni, except for the nicotinic acid naturally present in the flour. On the other hand, one flavored milk drink exhibited vitamins levels 200% higher than the amounts declared and one milk drink mix presented thiamin, riboflavin, and nicotinamide levels 3 to 5 times greater than stated. These results suggest an absence of control of the amount of vitamins in enriched foods.
Application of capillary electrophoresis in migration studies of food contact materials – part 1: Substituted phenolic additives
Authors: Abrantes Shirley , Philo Mark R. , Damant Andrew P. , Castle Laurence
Published: May 2005

A method has been developed to determine 11 phenolic antioxidants in the food simulants distilled water, 3% acetie acid, and 15% ethanol, using; micellar capillary electrophoresis (MCE). All the phenols could he analyzed within 35 min. The analytical recovery from spiked simulants was 80 to 119% except for 2,6‐di‐tert‐butyl‐4hydroxytoluenc (BHT) and octyl gallate, which could not be recovered from 3% acetic acid simulant. Calibration graph correlation coefficients for the 11 phenols were 0.982 to 0.999. Limits of detection (LoDs) were from 2.8 to 8.6 mg/L. These LoDs are well below European Union migration limits for these substances. It is concluded therefore that MCE offers a rapid and reliable analysis for the control of migration from plastics intended for food contact which employ these phenols as antioxidants.
Experimental design optimization of the analysis of gasoline by capillary gas chromatography
Authors: Durand Jean Pierre , Gautier Serge , Robert Eric , Guilhem Marie Christine , Phan‐Tan‐Luu Roger
Published: May 2005

An optimization procedure for multi‐step temperature programmed capillary GC was investigated for the detailed hydrocarbon analysis of gasoline. A set of twelve responses, represented by the value of the resolution of the most difficult separations, was selected from several isothermal analyses. The variation of these responses versus the temperature programming conditions was then modeled using a Doehlert matrix for experimental design. Optimal experimental conditions for the twelve responses were then obtained from a response surface optimization. The predicted and experimental resolutions were in good agreement.
Analysis of volatile sesquiterpenoids in environmental and geological samples
Authors: Elias Valdimir O. , Simoneit Bernd R. T. , Cardoso Jari N.
Published: May 2005

In order to evaluate vloatile compounds, especiallly sesquiter‐penoids, in environmental and geological samples a method was penoids, in environmental and geological samples a method was required to characterize these components when they occur at low concentrations. After fractionation of the total sample extracts by TLC the aliphatic fractions were analyzed by HRGC AND THE kovats indices of all resolved peaks were calculated and comkpared with indices of all resolved peaks were calculated and compared with those obtained for commercial standards. As final step, the fractions were analyzed by HRGC‐MS and structures confirmed by comparison of their mass spectra with those of certified sesuiterpene standards. Additionally sesquiterpenoids present in the dissolved phase in water samples were concentrated by solid phase extraction using XAD‐2 resin. In this initial study, recovery tests with the resin were carried out in order to evaluate its performance rather than validating the methodology.
A continuous liquid‐liquid extraction system coupled on‐line with capillary gas chromatography for environmental and ecotoxicological analysis
Authors: Goosens Elise C. , de Jong Dirk , de Jong Gerhardus J. , Brinkman Udo A. Th.
Published: May 2005

The applicability of a liquid‐liquid extraction system which is coupled on‐line with a capillary gas chromatograph was studied for environmental and ecotoxicological analyses. The optimized and automated system was used for the determination of apolar and rather non‐volatile organic compounds in aqueous samples. Relevant aspects of sample introduction, phase separation and selection of extraction solvent are discussed. Three routine‐tipe applications are described, viz. an improved method for the determination of hexachlorocyclohexanes in ground water and the determination of the so‐called NCC‐ether and ACC‐ether in ecotoxicological studies. Depending on the application studied, the concentration levels varied from 0.1 to 6000 μg/I, using ECD and/or FID detection. Typical coefficients of variation obtained with the total extraction–GC procedure were 2–25%.

The system was found to be rugged, it saves time compared with set‐ups involving off‐line liquid‐liquid extraction and considerably reduces the manual work load.
Determination of benzene alkyl derivatives in heavily loaded environmental aqueous samples by means of combination of distillation and purge and trap‐gas chromatography‐mass spectrometry
Authors: Zygmunt Bogdan
Published: May 2005

Simple distillation was used to prepare aqueous environmental samples (especially those with high content of accompanying dissolved and suspended organic and inorganic matter) for determination of benzene, toluene, ethylbenzene, and xylenes (BTEX) by means of purge and trap (PT) coupled to gas chromatography‐mass spectrometry (GC‐MS). The PT step was carried out with a laboratory‐built device in which alalytes desorbed from a Primary trap (macrotrap) are focused in a microtrap (also with sorbent) and moisture is removed from purge gases by a Nafion tube (walls selectively permeable to water vapor). Recoveries, if only the first 10 ml distillate was collected, were of the order of 40% at optimum distillation parameters. At a probability level of 95% recoveries were independent of concentration in a studied concentration range of 0.50–30 ppb. Enrichment factors for distillation were of the order off 20. Real samples, i.e., raw and treated waste water were analyzedc for BTEX content by the developed medthod.
Cyclodextrin derivatives in GC separation of racemic mixtures of volatiles – part XII: Thick‐film wide‐bore columns for enantiomer GC preparation
Authors: Bicchi Carlo , D'Amato Angela , Manzin Valeria , Galli Anna , Galli Mario
Published: May 2005

Preliminary results on thick‐film wide‐bore (0.53mm i.d.) columns for GC preparation of pure enantiomers are described. In particular the loading capacity for several racemates of a 3μm, 30% 2,6‐di‐O‐methyl‐3‐O‐Pentyl‐β‐CD/OV1701 column and of a 2 μm, 30% 2,3‐di‐O‐acetyl‐6‐O‐dimethyl‐t‐butylyl‐β‐CD/PS‐086 collumn were determined. Consideration is also given to the relation‐ship between resolution values of two enantiomers on anayltical columns and their loading capacity and scaling‐up to the corresponding micropreparative columns.
Enantiomeric separation of 5‐alkyl‐5methylhydantion derivatives by capillary gas chromatography on permethylated β‐cyclodextrin and molecular modeling
Authors: Cardinaéul Pascal , Ndziéa Elias , Petit Samuel , Coquerel Géurard , Combret Yvette , Combret Jean Claude
Published: May 2005

Permethylated β‐cyclodextrin (hereafter designated perMe‐β‐CD), dililuted or not in polysiloxane, is an efficient chiral discriminant for native, 3‐methylated, and 1,3‐dimethylated series of 5‐methyl‐5‐(CnH2n + 1)hydantoins. From thermodynamic data obtained with pure perMe‐β‐CD and with the aid of molecular modeling, it is concluded that: (i) For native hydantoin derivatives having a carbon chain at the 5‐position varying from ethyl to nonyl (2⩽n⩽9), the retention time is related to their H‐bonding capability. The resolution is poor for derivatives with n⩽4. For n⩽5, the resolution is considerably improved and this increase in resolution is attributed to the long alkyl chain being capable of being buried inside the cyclodextrin cavity. (ii) For 1,3‐dimethylhydantion derivatives having a carbon chain at the 5‐position varying from ethyl to hexyl(2⩽n⩽6), the retention time is mainly determined by a steric fit between the solute and the inner volume of the macrocyclic cavity. For n = 2, a particular behavior is observed, consistent with a total inclusion of the solutes inside the cavity. By contrast with the native hydantoins, the selectively is high for light derivatives. Thus, the selectivity does not correlate with a strong docking energy (such as the presence of H‐bonds postulated with native derivatives). (iii) For 3‐methylhydantoin derivatives having a carbon chain at the 5‐position varying from ethyl to octyl (2⩽n⩽8), as expected, retension time and selectivity are intermediate between that of native hydantoins and 1,3‐dimethylhydantoins. For all three series, where n⩽5, the increase in chain length (n) does not bring about substantial changes in the chromatographic results. Therefore, for these long alkyl chain derivatives, similar dominant interactions with perMe‐β‐CD are postulated.
Nonlinear variation of sorption parameters of n‐alkane homologsl in temperature‐programmed gas chromatography (tpgc) and new equation for calculation of retention indixces
Authors: Golovnya Rimma V. , Syomina Lyuba A. , Samusenko Alexei L.
Published: May 2005

It has been demonstated that the considaerable difference in temperature increments of sorption parameters of n‐alkanes under isothermal conditions is the main reason for nonlinear dependence of sorption parameters on molecular mass of homolog in temperaturre programmed gas chromatography (TPGC). A new nonlinear 4th parameter equation has been given for calculation of the retention indices. Coefficients of the equation are calculated from n‐alkanes. The equation allows extrapolation and interpolation calculations of retention indices under TPGC conditions with experimental precision. The results obtained; for fatty acidkl methyl esters demonstrate the advantage of ovr equation in comparison with van den Dool and Kratz's equation.
Separation of native amino acids at low pH by capillary electrophoresis
Authors: Thorntion Michelle J. , Fritz James S. , Klampfl Christain W.
Published: May 2005

Amino Acids are cations at low PH and can be readily separated by capillarty electrophoresis provided an alkanesulfonic acid is added to the elecrolyte carrier. Formation of a Positive net charge on the bare fused‐silica surface at low PH was confirmed by measurement of an anodic electroosmotic flow. The addition of ethanesulfonic acid or octanesulfonic acid to the electrolyte carrier causes a reversal of the EOF. A mechanism is proposed in which the alkanesulfonic acid adsorbs to the positively‐charged capillary wall through electrostatci attraction. Adsorption of a second molecule of alkanesulfonate by hudrophobic attraction to the carbon chain forms a negatively‐charge coating on the capillary wall. The alkanesuflfonate also imparts selectivety to the system by participation in ionpairing interactions with the native amino acids to improve resolution. The CE separation of a mixture of the twenty common amino acids at PH 2.8 with direct absorabance detection at 185 nm resulted in 17 amino acid peals in 20 minutes with a 30 KV applied voltage. The effect of several variables was studied including electrolyte carrieres containing different alkanesulfonic acids, the influence of PH, applied voltage, and concentration of electrolyte carrier.
Separation of metal cations in acidic solution by capillary electrophoresis with direct and indirect UV detection
Authors: Thornton Michelle J. , Fritz James S.
Published: May 2005

Methods for the detectionn of metal cations under acidic conditions, near PH 2, in capillary electrophoresis (CE) were investigatged. Conditions for direct UV detection of UV absorbing metal cations such as Cr3+, Cu2+, Fe3+, UO22+, VO2+, and VO2+ were established With aqueous HCl or HClO4 as the electroyte carrier. The speciation of vanadium(IV) and vanadium(V) at PH 2.3 by CE was achieved with direct detection at 185 nm. With the strong absorbance at 185 nm, no complexation was needed to detect the metal cations. An indirect UV detrection scheme for acidic conditions was also investigated. Several background carrier electolytes (BCES) were studied including 4‐methylbenzylamince, nicontinamide, pyridazine, guanidine, 3‐picoline, and chromium (III) to determine their effectivencess under very acidic conditions. The efect of ioni c surfactants and the nonionic surfactant, Trition X‐100, on the peak heights and N Values was also studied.
Determination of solvent recondensation or evaporation in the GC‐precolumn by measurement of temperature changes on the column wall
Authors: Hyötyläuinen Tuulia , Grob Konrad , Riekkola Marja‐Liisa
Published: May 2005

Temperature measurements on the column outer well were used for detecting recondensation or evaporation of solvent inside the precolumn during injection or on‐line transfer of large solvent volumes. This facilitates the choice of the most critical parameter of these techniques, i.e. oven temperature. When using the vaporizer/precolumn solvent split/gas discharge system, the dew point of the solvent is determined, either to just prevent solvent recondensation or to limit it to the capacity of the precolumn to retain liquid. In concurrent eluent evaporation through the loop type LC‐GC interface, temperature measurement enables the determination of whether or not the flooded zone exceeds a given limit. Fanally, when solvent trapping is used (on‐column injector/partially concurrent solvent evaporation evaporation or vaporizer/partial recondensation), temperature measurement near the front end of the flooded zone is used as a signal for accurate closure of the vapor exit shortly before the end of solvent evaporation.
Theory of fast capillary gas chromatography part. 2: Speed of analysis
Authors: Blumberg Leonid M.
Published: May 2005

Using hold‐up time as a measure of the speed of analysis with a given column efficiency, a theory of pneumatic optimization of a column in the fast high pressure drop isothermal and temperature programmed GC is developed. Expression for the hold‐up time as a function of column efficiency, carrier gas velocity and film thickness is derived. Also found are the expressions for the minimum hold‐up time and optimum gas velocity in a speed‐optimized column. These quantities are compared with their counterparts in the efficiency‐optimized column.
Studies on a possible molecular basis for the structure of mitochondrial cristae
Authors: Sumegi Balazs , Freeman Dale A. , Inman Lindsey , Srere Paul A.
Published: May 2005

We have investigated a possible molecular basis for mitochondrial cristae formation. Proteoliposomes containing electron transport proteins, cytochrome oxidase, or complex III in their proper orientation bind to pig heart mitoplasts but not pig heart mitochondria. Using Leydig tumor cells, we have confirmed earlier reports that chloramphenicol causes a diminution in cristae content and a change in its characteristic lamellar form. We show that the proteoliposomes containing cytochrome oxidase or complex III in the proper orientation bind to mitoplasts from Leydig tumor cells but do not bind as well to mitoplasts from chloramphenicol‐treated Leydig tumor cells. These experiments provide a possible mechanism to explain cristae formation.
Cognitive features of continuous antigenic determinants
Authors: Geysen H. Mario , Mason Tom J. , Rodda Stuart J.
Published: May 2005

We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes with in 63 well‐defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each apitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined from with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.
T4 Phage deoxyribonucleoside triphosphate synthetase: Purification of an enzyme complex and identification of gene products required for integrity
Authors: Moen Laura K. , Howell Meredith L. , Lasser Gerald W. , Mathews Christopher K.
Published: May 2005

We have isolated a highly enriched preparation of the multienzyme complex which synthesize Deocyribonuleoside triphosphates (dNTPs) from bacteriophage T4‐infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparation. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exits in vivo. The purified complex catalyzes the efficient five‐step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene product encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP‐synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.
Glycolipid‐directed FH6 monoclonal antibody recognizes high molecular weight glycprotein antigen carrying sialyl LeX‐i determinant in the culture supernatant of PC‐9 cells
Authors: Yamagata Yoko , Shigeta Katsuyoshi , Murachi Takashi , Kannagi Reiji , Masuda Tohru , Imura Hiroo , Shin Sadahito , Tachikawa Tetsuya , Noda Atsunari , Ohe Yasuo
Published: May 2005

The properties of the antigen recognized by monoclonal antibody FH6 have been analyzed. FH6 was originally generated against a glycolipid, i.e. a difucoganglioside isolated from human colonic adenocarcinoma, and specifically reacts with sialyl Lex‐i detreminant. Several culture supernatants of human carcinoma cell line cells were found to have high levels of FH6‐reactive antigen, and PC‐9, a human lung carcinoma cell line was used for the analysis. A solid‐phase sandwich radioimmunoassay was performed to detect the antigen. The antigenic activity was extractable in 0.6 M PCA or 7% TCA, and was sensitive to mild alkaline treatment and to Pronase digestion. Most of the antigen was eluted in the void volume of a Sepharose CL‐2B column, which indicates that its molecular weights is greater than several million. It was eluted from a DEAE‐cellulose column at a NaCl concentration in the range of 0.2–0.25 M. The immunoaffinity‐purified antigen has a high carbohydrate content of more than 80%. These data indicate that the antigen recognized by FH6 in the culture supernatant of PC‐9 is not a glycolipid, but a high molecular weight glycoprotein which could be referred to as a mucin, or a proteoglycan, which contains keratan‐sulfate like glycosaminoglycan chains, as judged from the results of the glycosidase treatments.
Fluorescence spectroscopic studies on tryptophan at the saccharide‐binding site of castor bean hemagglutinin
Authors: Yamasaki Nobuyuki , Absar Nural , Funatsu Gunki
Published: May 2005

The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed fluorescence spectroscopy with regard to saccharide binding. Upon binding of specfic saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelenght 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide‐binding site. By analyzing the change in the fluorescence intensity at 320nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide‐binding site on each B‐chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non‐reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface‐localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accesible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue atresidu at the saccharide‐binding site on each B‐chain is localized near the surface, and present in the positively charged environment.
Sequence simplification and the intra‐ and intermolecular self‐recognition properties of vasopressin/neurophysin biosynthetic precursor
Authors: Fassina Giorgio , Chaiken Irwin M.
Published: May 2005

The self‐assembly properties of the arginine 8‐vasopression/bovine neurophysin II (AVP/BNPII) biosynthetic precursor were studied using glycopeptide‐deleted and sequence‐redesigned semisynthetic derivaties. Semisythetic precursors were prepared by chemically coupling synthetic vasopressinyl sequence domains and native protein‐derived neurophysin II domain. Measurement of precursor‐protein association by extent of affinity chromatographic retardation on agarose‐immobilizes BNPII verified that the semisynthetic precursor with native AVP sequence has an enhanced self‐association propensity similar to that predicted for native precursor. Here, the stabilizing contacts between hormone and neurophysin domains, mainly the positively charged protonated α‐amino group and tyrosyl 2 side chain of the hormone, are retained. Semisynthetic precusor variants in which the hormone domain is sequence‐simplified by introducing alanyl residues in position not considered important for neurophysin recognition show non‐reduced association to BNPII. In contrast, removal of one of the main contact element between hormone and neurophysin by acetylation of the hormone α‐amino group abolishes potentiation of precursor self‐association. The results show that the presence of the C‐termial glycopeptide sequence domain of native vaspressin precursor is not required to promote self‐assemblly of the precursor. The data verify the view proposed for the oxytocinyl precursor that intramolecular domain interaction is the triggering event which promotes the increase in affinity of precursor self‐association (intermolecular self‐recognition). The data also define some of the intramolecular self‐recognition element in the folded precursor required for the high affinity intermolecular self‐recognition.
Production of anti‐idiotypic antibodies by immunization with a pair of complementary peptides
Authors: Bost Kenneth L. , Blalock J. Edwin
Published: May 2005

Previous investigations have suggested that pairs of peptides specified by complementary RNA sequences (termed complementary peptides) can interact with one another. In the light of this finding, we hypothesized that an antibody directed against a peptide might interact with an antibody against its complementary peptide at the antigen combining site. To address this possibility, polyclonal antibodies against a peptide, Leu‐Glu‐Arg‐Ile‐Leu‐Leu (LERILL), and its complementary peptide, Glu‐Leu‐Cys‐Asp‐Asp‐Asp (ELCDDD), were made monospecific by affinity chromatography. Using radioimmunsassays, anti‐ELCDDD antibodies were shown to interact with 125I‐anti‐LERILL antibodies but not with 125I‐control antibodies. More importantly, the interaction of the two antibodies could be blocked using either peptide antigen, but not with control peptides. Furthermore, 125I‐anti‐LERILL binding to LERILL could be blocked with anti‐ELCDDD antibody and vice versa. We concluded therefore that antibody/antibody binding occurred at or near the antigen combining site, demonstrating that this interaction was an idiotypic/anti‐idiotypic one.
Molecular recognition in synthetic polymers. Enantiomeric resolution of amide derivatives of amino acids on molecularly imprinted polymers
Authors: O'Shannessy Daniel J. , Andersson Lars I. , Mosbach Klaus
Published: May 2005

Molecular imprints were prepared using L‐phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer. Methacrylic acid interacts ionically with the primary amine of the print molecule and via hydrogen bonding with the amide function. In the HPLC mode such polymers were shown to exhibit efficient enantiomeric resolution of a racemic mixture of the original print molecule. Enantiomeric resolution was shown to be dependent on the ratio of methacrylic acid to print molecule in the pre‐polymerization mixture and specific for the presence of both print molecule and functional monomer. Further analysis showed the importance of both the primary amino and amide functions in the correct stereochemistry for recognition and enantiomeric resolution of compounds on such polymers. Other amide derivatives of amino acids including p‐nitoranilides, β‐naphthylamides and amides were recognized by such polymers, and enantiomeric resolution was obtained for amide derivatives of amino acid ranging from alanine to tryptophan on a single polymer. The implications of these findings with respect to the mechanism of recognition and the ability to predict enantiomeric resolution of molecules on molecularly imprinted polymers will be discussed.
Interaction between phage G13 and its oligosaccharide receptor studied by equilibrium dialysis
Authors: Bruse Gert W. , Wollin Ralfh , Lindberg Alf A.
Published: May 2005

The reversible binding of phage G13, a ϕX174‐like single‐strand DNA phage, to a 3H‐labeled nonasaccharide from the lipopolysaccharide of its natural host Esacherichia coli C was studied with equilibrium dialysis. The binding constant (Ka) was determined to 1.3 × 107 M−1 in Scatchard and Lineweaver–Burk plots. Approximately one saccharide bound per G13 phage particle which suggests that only one of the 12 spikes in each G13 virion was engaged in the phage/receptor saccharide interaction. Equilibrium dialysis inhibition experiments with saccharides from lipopolysaccharides of an isogenic series of Salmonella typhimurium mutants showed that hepta‐ and pentasaccharides from two G13‐sensitive bacteria, i.e., with efficiencies of plating of 0.1–1.0 compared to E. coli C, were efficient inhibitors with Ka‐values ≥ 1.2 × 107 M−1. The octa‐ and hexasaccharides from two G13 resistant strains, with efficiency of plating ⩽ × 10−4, were either > 1000‐fold of > 15‐fold less efficient as inhibitors with Ka‐values ⩽8.8 − 105 M−1. The results show that phage G13 binds in a specific and reversible way to penta‐, hepta‐, and nonasaccharides from G13 sensitive bacteria with the specificity residing in the hexose and heptose region of the core lipopolysaccharide.
Molecular recognition between oligopeptides and nucleic acids. Specificity of binding of a monocationic bis‐furan lexitropsin to DNA deduced from footprinting and 1H NMR studies
Authors: Lee Moses , Krowicki Krzysztof , Shea Regan G. , Lown J. William , Pon Richard T.
Published: May 2005

MPE‐Fe(EDTA) footprinting of a novel monocationic bis‐furan lexitropsin 6 on a HindIII/EcoRI restriction fragment of pBR322 DNA revealed a series of four‐base binding sites (all 5′ → 3′) of (primary) TGTA, TGAA, AAAT, ACAA, TTAT, and (secondary) CTAA, TCGT, TGTA, GTCA, and GGTT. Thus 6 can accept a GC pair at positions 1, 2 or 3 of the binding site with a strict 3′ (4 position) AT requirement. Marked enhancement of cleavage, particularly at GC rich sequences, is observed at regions flanking or even up to 18 base pairs remote from a given binding site. The non‐exchangeable and imino 1H NMR resonances of the 1:1 complex and d‐[CATGGCCATG]2 were assigned using a combination of NOE differences, NOESY and COSY techniques. 1H NMR studies (ligand induced chemical shifts and NOE differences) of Lexitropsin 6 with d‐[CATGGCCATG]2 show unambiguously the location and orientation of the N to C termini of 6 on the sequence 5′‐G5C6C7A8‐3′, with the C terminus oriented to A8. This orientation of 6 in the minor groove of 5′‐GCCA is confirmed by an NOE observed between H1 2a of 6 and AH8(8). This preference for binding of 6 to the sequence 5′‐GCCA when challenged with d‐[CATGGCCATG]2 is in accord with the conclusions of the footprinting experiments wherein GC base pairs can be accepted in the first three positions and with a strict 3′ terminus AT reading requirement. Collectively the data support the inference of a GC recognizing capacity for a 2,5‐substituted furan moiety within a lexitropsin. The 1H NMR data indicate that the decadeoxyribonucleotide duplex exists in the B conformation in both the 1:1 complex and the free from. The apparent binding constant of 6 to calf thymus DNA is 1.68 × 105 M−1 whereas netropsin under similar conditions gives a value of 1.85 × 107 M−1. This suggests that if advantage is to be taken of the GC recognizing property of a 2,5‐substituted furan in longer lexitropsins it should be flanked by more strongly bound moieties.
Conformational analysis of blood group A‐active glycosphingolipids using HSEA‐calculations. The possible significance of the core oligosaccharide chain for the presentation and recognition of the A‐determinant
Authors: Nyholm Per‐Georg , Samuelsson Bo E. , Breimer Michaelle , Pascher Irmin
Published: May 2005

Conformational analysis of four different A‐active glycosphingolipids, A types 1–4, was carried out using HSEA‐calculations with the GESA‐program. In their minimum energy conformations the oligosaccharide chains are more or less curved; in particular the type 3 and 4 have a strongly bent shape. When the carbohydrate structures are linked to ceramide, using the conformational features predominantly observed in crystal structures of membrane lipids, rather drastic differences in the orientation of the oligosaccharide chains are obtained. For the type 1 glycosphingolipid the model study indicates that the A‐determinant extends almost perpendicularly to the membrane plane whereas for type 2, 3 and 4 the terminal part of the oligosaccharide chains is more parallel to the membrane. The fucose branch on type 3 and type 4 thereby appears directed towards the environment whereas for type 2 it would face the membrane. Due to restrictions imposed by the membrane layer this core specific orientation is largely preserved even if the flexibility of the saccharide‐ceramide linkage is taken into account. Hydrophilic and hydrophobic sites on the surface of the different oligosaccharide chains in their minimum energy conformation were located using the GRID‐program. It is suggested that the core‐dependent presentation of the A‐determinant might explain the chain type specificity observed for different monoclonal anti‐A antibodies. The results further suggest that assay systems ensuring a membrane‐like presentation of the glycolipid antigen should be used in studies of glycolipid/protein interactions.
Model‐Building of Fnr and FixK DNA‐binding domains suggests a basis for specific DNA recognition
Authors: Cherfils Jacqueline , Gibrat Jean‐François , Levin Jonathan , Batut Jacques , Kahn Daniel
Published: May 2005

The DNA‐binding C‐terminal domains of the regulatory proteins Fnr from Escherichia coli and FixK from Rhizobium meliloti have been modelled on the basis of their homologies to the CAP protein from E. coli. Residues Glu181, Thr182 and Arg185 of CAP, which are exposed residues of the DNA‐recognition helix αF, are conserved in Fnr and FixK. However, Arg180 and Gly184 are substituted by Val and Ser respectively in Fnr. We propose that this valine makes a Van der Waals' contact with the first thymine in the Fnr consensus TTGA‐N6‐TCAA, and that the serine contributes to the binding by displacing a thymine‐bound water molecule. The corresponding residues in FixK, Ile and Ser allow the same interactions with a thymine. Therefore we predict that FixK may recognize the same sites as Fnr. This is supported experimentally by showing that Fnr can substitute for FixK in activating the fixN gene in E. coli.
Binding characteristics of a series of dimeric tripeptide enkephalins for δ opiate receptors in rat brain and NG108–15 cells
Authors: Shimohigashi Yasuyuki , Kodama Hiroaki , Costa Tommaso , Lutz Rudolf A. , Chen Hao‐Chia , Rodbard David
Published: May 2005

The N‐terminal tripeptide enkephalin analogue, Tyr‐D‐Ala‐Gly, was dimerized at the C‐terminus systematically with a series of α,ω‐diaminoalkanes, NH2(CH2)nNH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for δ opiate receptors in rat brain were evaluated and compared with those for δ receptors in NG108–15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8–900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14–18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that δ receptors in rat brain and NG cells may have slight structural differences.
Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity
Authors: Rizzo Vincenzo , Battistini Carlo , Vigevani Aristide , Sacchi Nereo , Razzano Giuseppe , Arcamone Federico , Garbesi Anna , Colonna Francesco Paolo , Capobianco Massimo , Tondelli Luisa
Published: May 2005

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self‐complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two 9‐deoxydoxorubicin (3) and 3′‐deamino‐3′‐hydroxy‐4′‐epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.‐X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445–466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x‐ray structure determination of the daunorubicin intercalative complex (Wang, A. H.‐J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152–1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double stranded form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di‐intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence‐preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9OH on the aglycone and NH on daunosamine, are estimated not to exceed 2 kcal/mol.
Effect of intercalative binding compared to external binding on Z/B equilibrium of poly d(GMe5C) using fluorescent oxazolopyridocarbazoles as probes
Authors: Le Ber Pierre , Schwaller Marc‐Antoine , Auclair Christian
Published: May 2005

Using the fluorimetric determination of the binding isotherms in combination with circular dichroism, we have investigated the effect of the binding of the intercalating chromophore oxazolopyridocarbazole (OPC) to poly d(Gme5C) on B/Z equilibrium, compared to the effect of the external binder OPC derivative pentyl‐2‐OPC. The intercalating OPC appears to be very efficient in reversing left‐handed poly d(Gme5C) into the right‐handed conformation, according to a cooperative mode. For each OPC molecule intercalated into the B form, 7 base pairs were switched from the Z to B conformation. In contrast, the binding of the external binder pentyl‐OPC resulted in a limited Z to B transition, involving the switch of 1.4 base pairs from the Z to B conformation. Moreover, OPC appears much more efficient than pentyl‐OPC in inhibiting both the extent and kinetics of the salt‐induced B/Z transition. At low drug to DNA ratio (D/P = 1/50), a 7‐fold and 1.5‐fold inhibition of the B/Z transition kinetics occurs in the presence of OPC and pently‐OPC, respectively. These features are discussed in terms of the difference existing between the entropic contribution in the DNA binding of intercalating agents, compared to external binders.