bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of an Artificial Receptor Protein (“Anticalin”) Based on the Human Apolipoprotein D
Authors: Vogt Martin , Skerra Arne
Published: January 2004

Human apolipoprotein D (ApoD) is a prominent member of the lipocalin family of proteins and transports arachidonic acid and progesterone in various body fluids. Lipocalins share a structurally conserved β barrel as their central folding unit, which supports a set of four hypervariable loops that form the entrance to the ligand pocket. Based on this structural pattern ApoD was employed as a scaffold for the combinatorial design of artificial receptor proteins termed anticalins. After randomization of 24 amino acids located within the loop region, several ApoD variants were selected against hemoglobin, a biochemically well‐characterized model target, by using bacterial phagemid display and colony screening. One variant, dubbed HbgA, was further investigated by surface plasmon resonance interaction analysis and found to complex hemoglobin specifically and with a dissociation constant of about 2 μM. While our previous work on the structurally related insect bilin‐binding protein was focused on the generation of binding activity towards low‐molecular‐weight ligands, this study demonstrates for the first time that a lipocalin can also be tailored to recognize a protein target. The fact that even a human member of this protein family has now been successfully recruited for anticalin construction opens the possibility for future application of such engineered lipocalins as target‐recognition vehicles in medical therapy.
Addressing the Challenge of Changing the Specificity of RNase T1 with Rational and Evolutionary Approaches
Authors: Struhalla Marc , Czaja Rico , Hahn Ulrich
Published: January 2004

Although ribonuclease T1 (RNase T1) is one of the best‐characterized proteins with respect to structure and enzymatic action, numerous attempts at altering the specificity of the enzyme to cleave single‐stranded RNA at the 3′‐side of adenylic instead of guanylic residues by rational approaches have failed so far. Recently we generated and characterized the RNase T1 variant RV with a 7200‐fold increase in adenylyl‐3′,5′‐cytidine (ApC)/guanylyl‐3′,5′‐cytidine (GpC) preference, with the guanine‐binding loop changed from 41‐KYNNYE‐46 (wt) to 41‐EFRNWN‐46. Now we have introduced the asparagine residue at position 46 of the wild‐type enzyme as a single‐point mutation in variant E46N and in combination with the Y45W exchange also occurring in RV. Both variants show an improved ApC/GpC preference with a 1450‐fold increase for E46N and a 2100‐fold increase for Y45W/E46N in comparison to wild‐type activity. We also addressed the challenge of altering enzyme specificity with an evolutionary approach. We have randomly introduced point mutations into the RNase T1 wild‐type gene and into the gene of the variant RV with different mutation rates. Altogether we have screened about 100 000 individual clones for activity on RNase indicator plates; 533 of these clones were active. A significant change in substrate specificity towards an ApC preference could not be observed for any of these active variants; this demonstrated the magnitude of the challenge to alter the specificity of this evolutionary perfected enzyme.
Learning from Directed Evolution: Theoretical Investigations into Cooperative Mutations in Lipase Enantioselectivity
Authors: Bocola Marco , Otte Nikolaj , Jaeger Karl‐Erich , Reetz Manfred T. , Thiel Walter
Published: January 2004

Molecular modeling with classical force‐fields has been used to study the reactant complex and the tetrahedral intermediate in lipase‐catalyzed ester hydrolysis in 20 enzyme/substrate combinations. The R and S enantiomers of α‐methyldecanoic acid ester served as substrates for the wild‐type lipase from Pseudomonas aeruginosa and nine selected mutants. After suitable preparation of initial structures from an available wild‐type crystal structure, each system was subjected to 1 ns CHARMM force‐field molecular dynamics simulations. The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the “hot spots” at residues 155 and 162. The replacement S155F enhances S enantiopreference through a steric relay involving Leu162. The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83. The simulations provide insight into remote and cooperative effects of mutations.
Modulating Functional Loop Movements: The Role of Highly Conserved Residues in the Correlated Loop Motions
Authors: Gunasekaran Kannan , Nussinov Ruth
Published: January 2004

Loop flexibility in enzymes plays a vital role in correctly positioning catalytically important residues. This strong relationship between enzyme flexibility and function provides an opportunity to engineer new substrates and inhibitors. It further allows the design of site‐directed mutagenesis experiments to explore enzymatic activity through the control of flexibility of a functional loop. Earlier, we described a novel mechanism in which a small loop triggers the motions of a functional loop in three enzymes (β‐1,4‐galactosyltransferase, lipase, and enolase) unrelated in sequence, structure, or function. Here, we further address the question of how the interactions between various flexible loops modulate the movements of the functional loop. We examine β‐1,4‐galactosyltransferase as a model system in which a Long loop undergoes a large conformational change (moves in space up to 20 Å) upon substrate binding in addition to a small loop (Trp loop) that shows a considerably smaller conformational change. Our molecular‐dynamics simulations carried out in implicit and explicit solvent show that, in addition to these two loops, two other neighboring loops are also highly flexible. These loops are in contact with either the Long loop or the Trp loop. Analysis of the covariance of the spatial displacement of the residues reveals that coupled motions occur only in one of these two loops. Sequence analysis indicates that loops correlated in their motions also have highly conserved residues involved in the loop–loop interactions. Further, analysis of crystal structures and simulations in explicit water open the possibility that the Trp loop that triggers the movement of the Long loop in the unbound conformation may also play the same role in the substrate‐bound conformation through its contact with the conserved and correlated third loop. Our proposition is supported by the observation that four of the five conserved positions in the third loop are at the interface with the Trp loop. Evolution appears to select residues that drive the functional Long loop to a large conformational change. These observations suggest that altering selected loop–loop interactions might modulate the movements of the functional loop.
Adaptation of Class‐13 α‐Amylases to Diverse Living Conditions
Authors: Linden Anni , Wilmanns Matthias
Published: January 2004

There are currently 35 available nonredundant molecular structures of class‐13 α‐amylases (EC 3.2.1.1), mostly from microbial organisms living under a wide range of environmental conditions. One of the most recent additions has been the first α‐amylase structure of a hyperthermophilic archaeon [Linden et al., J. Biol. Chem. 2003, 278, 9875–9884]. The structure has been used for comparative analyses with a representative set of three α‐amylases from thermophilic, mesophilic and psychrophilic sources to identify molecular parameters for environmental adaptation. Our analysis supports generally observed trends such as an increase in structural compactness as well as an increase in salt bridges in order to cope with high‐temperature conditions. The two representative thermophilic structures used in this comparative study have independently evolved di‐metal centres—not present in the mesophilic and psychrophilic structures—in the vicinity of the active site. These observations may provide impetus for the design of α‐amylases with improved molecular properties to enhance their utility in biotechnological processes.
Lipase‐Specific Foldases
Authors: Rosenau Frank , Tommassen Jan , Jaeger Karl‐Erich
Published: January 2004

Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase‐specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so‐called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.
Consensus Design of Repeat Proteins
Authors: Forrer Patrik , Binz H. Kaspar , Stumpp Michael T. , Plückthun Andreas
Published: January 2004

Consensus design is a valuable protein‐engineering method that is based on statistical information derived from sequence alignments of homologous proteins. Recently, consensus design was adapted to repeat proteins. We discuss the potential of this novel repeat‐based approach for the design of consensus repeat proteins and repeat protein libraries and summarize recent results from such experiments.
A new method for computing optimum weights for calculating CIE tristimulus values
Authors: Li C. J. , Luo M. R. , Rigg B.
Published: January 2004

Weighing tables are widely used for calculating CIE tristimulus values. In this article, a direct method for computing optimum weighing tables for any illuminant and observer combination is developed. A comprehensive set of 1‐nm reflectance functions based upon Munsell samples is used to test various methods. Four types of weighing tables are compared. They are ASTM E308 Tables 5 and 6, ASTM E2022, and new tables proposed by this study. The results clearly show that the newly developed optimum tables outperform the other three types of tables. The new method is simple and avoids the iterative process used by Venable and adopted by ASTM for some of its tables. It may be used for calculating weighing tables for any combination of illuminant and observer. A detailed procedure and a worked example are given in the article. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 91–103, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10229
The principal components of reflectances
Authors: Fairman Hugh S. , Brill Michael H.
Published: January 2004

There are practical reasons to represent a set of reflectance spectra by a limited basis‐function expansion. Such an expansion is most tractable in color technology applications when it is homogenous. However, more often a true principal‐component analysis (PCA) is performed, which involves an offset of the expansion by a mean spectrum. In deference to custom, we discuss and exercise the traditional PCA on reflectances. We review the method of calculation and present tables of the principal components of 3,534 object‐color samples from three color‐order systems. Next, we describe two kinds of principal‐component coordinates of a measured reflectance spectrum, one obtained by least‐square best fit and the other by tristimulus match under one or more specified lights. Finally, we show illustrative examples, and propose some possible new uses for principal‐component analysis of reflectances. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 104–110, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10230
Museum lighting: Optimizing the illuminant
Authors: Scuello Michael , Abramov Israel , Gordon James , Weintraub Steven
Published: January 2004

Museums must choose illumination that will optimize observers' aesthetic experiences without compromising preservation of the artworks. Typical lighting is 3000 K at 200 lux. We examined the effects of color temperature by comparing the same “artwork” under several different illuminants (in side‐by‐side miniature museum rooms); for each painting, observers rated the preference for seeing it under one of the two illuminants; this was repeated for all pairings of illuminants. Our observers preferred higher color temperatures at about 3600 K. Between viewing trials observers' visual systems were adapted to a specific illuminant. This had no effect on preferred illumination of an artwork. To explore a wider range of artworks, we developed a set of numerical scales that could be applied directly to individual pictures; these were viewed, one at a time, in a single simulated room illuminated by one of a series of illuminants. Again, observers preferred a color temperature of about 3600 K. In both studies there was a small interaction between illuminant and color gamut of a given painting. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 121–127, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10231
Quantifying variations in personal color spaces: Are there sex differences in color vision?
Authors: Bimler David L. , Kirkland John , Jameson Kimberly A.
Published: January 2004

We report a search for group differences in color experience between male and female subjects, focusing on the relative prominence of the axes of color space. Dissimilarity data were collected in the form of triadic (odd‐one‐out) judgments, made with the caps of the D‐15 color deficiency test, with lighting conditions controlled. Multidimensional scaling reduced these judgments to a small number of dimensional‐weight parameters, describing each subject's sensitivity to color axes, i.e., how much each axis contributes to the inter‐color dissimilarities perceived by each subject. Normal trichromatic subjects in two age bands were examined, teenagers and university students, and in both cases males placed significantly less weight on a ‘red‐green’ axis, and more on ‘lightness’. We consider the implications and possible explanations. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 128–134, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10232
Spectrophotometric error in colour coordinates introduced by fluorescence of white calibration tile
Authors: Pons Alicia , Campos Joaquín
Published: January 2004

Fluorescence has been detected in some white standard tiles intended for the calibration of spectrophotometers for colour measurements. The problem is that usually no warning is given to the user about it, giving raise to errors if they are calibrated using conventional spectrophotometric techniques. This work analyzes the differences on the colour coordinates of one of these standards measured with a polychromatic illumination spectrophotometer and with a monochromatic illumination one. This work also analyzes the colour coordinates error obtained when other coloured standards are calibrated against one of these white fluorescent standards, when reflectance measurement is done with a polychromatic illumination spectrophotometer as well as with a monochromatic illumination instrument. These are the two most typical situations found in practice where the use of fluorescent standards may produce errors. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 111–114, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10233
Proposal of an opponent‐colors system based on color‐appearance and color‐vision studies
Authors: Nayatani Yoshinobu
Published: January 2004

A new theoretical color order system is proposed on the basis of various studies on color appearance and color vision. It has three orthogonal opponent‐colors axes and an improved chromatic strength of each hue. The system has color attributes whiteness w, blackness bk, grayness gr, chroma C, and hue H. A method is given for determining Munsell notations of any colors on any equi‐hue planes in the system. A method is also given for determining grayness regions and grayness values on hue‐chroma planes in the system. It is concluded that colors with the same color attributes [w, gr, bk, C] but with different hues in the theoretical space have approximately the same perceived lightness, the same degree of vividness (“azayakasa” in Japanese), and also the same color tone. The tone concept, for example used in the Practical Color Coordinate System (PCCS), is clarified perceptually. The proposed system is a basic and latent color‐order system to PCCS. In addition, the concept of veiling grayness by a pure color with any hue is introduced. Further, relationships are clarified between generalized chroma c(gen) and grayness. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 135–150, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.10234
Growth of Lanthanum Oxide Thin Films by Liquid Injection MOCVD Using a Novel Lathanum Alkoxide Precursor
Authors: Aspinall H.C. , Gaskell J. , Williams P.A. , Jones A.C. , Chalker P.R. , Marshall P.A. , Smith L.M. , Crichlow G.W.
Published: January 2004

Carbon‐free lanthanum oxide is deposited by liquid injection MOCVD over a wide temperature range (300–600 °C) in the presence of oxygen using a new volatile lanthanum alkoxide precursor [La(mmp)3]. SEM of a typical lanthanum oxide film deposited at 450°C from a [La(mmp)3]/tetraglyme mixture in toluene reveals a columnar growth structure with XRD reflection pattern and intensity approximately consistent with that of La2O3 powder. The paper suggests the suitability of this low temperature process for the deposition of high‐κ lanthanum oxide films as gate dielectric for Si‐based TFTs.
Vapor Pressures of Precursors for the Chemical Vapor Deposition of Silicon‐Based Films
Authors: Alcott G.R. , van de Sanden R.M.C.M. , Kondic S. , Linden J.L.
Published: January 2004

Thermal stability and vapor pressure measurement for a number of SiO2 precursors are studied. For the first time results from1,2‐bis(trimethyl)siloxyethane (TMSE), (3‐glycidoxypropyl) trimethylethoxysilane (Glymol) and 1H,1H, 2H, 2H‐perfluorooctyltriethoxysilane (PTES) are reported. Results from tetraethoxysilane (TEOS), tripropylsilane (TPS) are found to agree with previously published values. Measurements are made in the temperatures range of 20 to 200 °C and values are fitted using the integrated Clausius–Claperon equation. Enthalpies of vaporization are calculated.
Growth of Thin Films of Molybdenum and Tungsten Oxides by Combustion CVD Using Aqueous Precursor Solutions
Authors: Davis M.J. , Benito G. , Sheel D.W. , Pemble M.E.
Published: January 2004

Using combustion (C) CVD, layers of molybdenum and tungsten oxides have been deposited on glass and silicon at low temperatures. Inexpensive ammonium salts of molybdate and metatungstate ions were used as precursors and were delivered to the coating flame as an aqueous solution using a nebulizer. The resulting films were analyzed by scanning electron microscopy (SEM), energy‐dispersive analysis of X‐rays (EDAX), Rutherford backscattering (RBS), X‐ray photoelectron spectroscopy (XPS), and X‐ray diffraction (XRD). These indicated that the films were continuous, moderately smooth, and consisted of amorphous, disordered molybdenum and tungsten trioxides.
Deposition of Antimony and Antimony Oxides by MOCVD
Authors: Myers C.P. , Haycock P.W. , Pichot M. , Horley G.A. , Molloy K.C. , Rushworth S.A. , Smith L.M.
Published: January 2004

Thin films of antimony and antimony oxides have been deposited by metal–organic (MO) CVD from a variety of metal–organic precursors at temperatures in the range 150–650 °C under both atmospheric and reduced pressure. Below 400 °C, uniform films of pure senarmontite (Sb2O3) with a microstructure and crystal texture that was strongly dependent on the deposition temperature, were deposited. Above 400 °C, mixed phase material was produced, with isolated crystallites of pure antimony becoming the dominant phase as the temperature was increased. The maximum temperature at which pure senarmontite could be formed was higher for larger precursor ligands. Addition of oxygen to the precursor flow led to the production of higher antimony oxidation states.
Developments with CVD
Authors: Hitchman M.L.
Published: January 2004

Read how CVD is growing and developing to meet the needs of authors and readers alike.
Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser‐induced fluorescence detection
Authors: Zhang Hua , Jin Wenrui
Published: January 2004

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 μm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser‐induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8–32 amol/single cell.
Determination of carbohydrates in juices by capillary electrophoresis, high‐performance liquid chromatography, and matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry
Authors: Cabálková Jana , Žídková Jitka , Přibyla Lubomír , Chmelík Josef
Published: January 2004

The objective of this work was to develop a sample preparation procedure for determination of the carbohydrate profiles in commercial juice samples by three principally different analytical methods: capillary electrophoresis (CE) with indirect detection, high‐performance liquid chromatography (HPLC), and matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). The preparation and purification of juice samples prior to analysis is described. The method using Carrez reagents was found to be an efficient preparation tool for all three methods. The addition of Carrez reagents to the samples for mass analysis improved the quality of the mass spectra of oligosaccharides. The amounts of glucose, fructose, and sucrose as major carbohydrates in fruit juices measured by CE using a simple instrument are in good agreement with the HPLC values and the data declared by the producers of the juices. The results from both methods are critically evaluated and their impact for studies of authenticity is discussed. The decrease of sucrose amount during the storage of samples was explained by acid hydrolysis of this disaccharide.
Effect of matrix chain length on the electrophoretic mobility of large linear and branched DNA in polymer solutions
Authors: Saha Sourav , Heuer Daniel M. , Archer Lynden A.
Published: January 2004

We study the effect of matrix chain molecular weight Mw and concentration c on the electrophoretic mobility μ of large linear and star‐like, branched DNA in polymer solutions. Polyethylene oxide (PEO) with narrow molecular weight distributions form the main focus of this study. For PEO concentrations ranging from one half the overlap concentration, c*, to 3c*, the effective drag coefficient, ζ $\equiv {{\mu _0 } \over \mu } - 1$, satisfies the following approximate scaling relationship, ζ $\sim cM_w^{0.7}$ . Here, μ0 is the electrophoretic mobility in free solution. While the concentration dependence is consistent with predictions from the transient entanglement coupling (TEC) model, the molecular weight dependence is significantly weaker. Although a similar dependence of mobility on Mw can be predicted when nonentangling collisions are the dominant source of drag, a model based on these collisions alone cannot reproduce the experimental observations. We also find that the architecture of large DNA does not affect either the concentration dependence or molecular weight dependence of the electrophoretic mobility.
Two‐dimensional protein database of human pancreas
Authors: Hu Liping , Evers Stefan , Lu Zhao Hui , Shen Yan , Chen Jie
Published: January 2004

We report here the two‐dimensional protein database of human pancreas. The proteins were analyzed by two‐dimensional electrophoresis followed by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Totally, 302 proteins were identified, of which about 27% were enzymes with a broad range of catalytic activities. Several of these are specifically expressed in pancreas, such as pancreatic amylase, pancreatic stone protein, pancreatitis‐associated protein, pancreatic lipase, pancreatic elastase, etc. Structural and cytoskeletal proteins are also strongly represented on the gels. Thus, the pancreatic proteome reflects the organ's function. This work paves the way for further studies on pancreatic protein expression in health and disease, such as diabetes and pancreatic cancer.
Sodium dodecyl sulfate‐capillary gel electrophoresis of polyethylene glycolylated interferon alpha
Authors: Na Dong H. , Park Eun J. , Youn Yu S. , Moon Byung W. , Jo Yeong W. , Lee Sung H. , Kim Won‐Bae , Sohn Yeowon , Lee Kang C.
Published: January 2004

Sodium dodecyl sulfate‐capillary gel electrophoresis (SDS‐CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG‐IFN). The SDS‐CGE method resulted in a clearer resolution in both the PEG‐IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS‐CGE was consistent with that obtained by SDS‐polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry. SDS‐CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS‐CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.
Development and validation method for determination of Paroxetine and its metabolites by nonaqueous capillary electrophoresis in human urine. Experimental design for evaluating the ruggedness of the method
Authors: Rodríguez Flores Juana , Barzas Nevado Juan J. , Contento Salcedo Ana M. , Cabello Díaz Maria P.
Published: January 2004

A simple, rapid, and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure Paroxetine (one of the mostly used antidepressants for mental diseases treatment) and three metabolites has been developed and validated. Optimum separation of paroxetine and metabolites was obtained on a 57 cm×75 μm capillary using a nonaqueous buffer system of 9:1 methanol‐acetonitrile containing 25 mM ammonium acetate and 1% acetic acid, with temperature and voltage of 25°C and 15 kV, respectively, and hydrodynamic injection. Fluoxetine was used as an internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy, and precision. Detection limits between 9.3 and 23.1 μg·L−1 were obtained for paroxetine and its metabolites. A ruggedness test of the method was carried out using the Plackett‐Burman fractional factorial model with a matrix of 15 experiments. This method has been used to determine paroxetine and its main metabolite B at clinically relevant levels in human urine. Prior to NACE determination, the samples were purified and enriched by means of an extraction‐preconcentration step with a preconditioned C18 cartridge and eluting the compounds with methanol.
Creatinine determination in serum by capillary electrophoresis
Authors: Paroni Rita , Fermo Isabella , Cighetti Giuliana , Ferrero Carlo Alberto , Carobene Anna , Ceriotti Ferruccio
Published: January 2004

Creatinine in human serum was separated in a fused‐silica capillary with H3PO4 (75 mmol/L, pH 2.5) as BGE, followed by UV detection at 200 nm. Serum with methylimidazole added as internal standard was deproteinized with acetonitrile and the supernatant, after dilution with water was injected at pressure mode. Creatinine and methylimidazole were baseline‐resolved in 6.5 min. Linearity in the 0–880 μmol/L range gave an r2 ≥ 0.998, recovery was 102 ± 2.8% (n = 6). Enzymatic breakdown with creatininase confirmed that serum does not interfere. The within‐day and between‐days coefficient of variation (CV) were ≤ 2.16 and 2.7%, respectively. The accuracy, determined for lyophilized samples by isotope dilution gas chromatography‐mass spectrometry was ≤ ± 2.0%. The results were compared with HPLC for 32 lyophilized samples and on 27 serum pools. Capillary electrophoresis, rapid and inexpensive, seems a promising alternative to high‐performance liquid chromatography (HPLC) for creatinine determination in human serum.
Capillary zone electrophoresis for determination of carbohydrate‐deficient transferrin in human serum
Authors: Fermo Isabella , Germagnoli Luca , Soldarini Armando , Dorigatti Fernanda , Paroni Rita
Published: January 2004

Carbohydrate‐deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di‐, mono‐, and asialo‐Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused‐silica capillary (50 μm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40°C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo‐ to pentasialo‐Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo‐Tf isoform related to the area of tetrasialo‐Tf in 50 healthy donors (24 males and 26 females; aged 25–50 years) was 3.15 ± 0.76% (mean ± SD). The comparison between CDT values obtained by this CZE procedure and the “Axis‐Shield %CDT” kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.
Development of a fully buffered molybdate electrolyte for capillary electrophoresis with indirect detection and its use for analysis of anions in Bayer liquor
Authors: Chovancek Milan , Choo Peter , Macka Miroslav
Published: January 2004

A new counterion‐buffered molybdate electrolyte was developed and optimized for simultaneous quantitative determination of up to eight anions (chloride, sulphate, oxalate, fluoride, formate, malonate, succinate, and acetate) in Bayer liquor by capillary electrophoresis with indirect detection at 214 nm. The separation parameters were optimized in respect to separation of the critical analyte group fluoride – formate – malonate, with the optimal electrolyte prepared from molybdic trioxide containing 5.0 mmol/L MoO3, 1.3 mmol/L cetyltrimethylammonium bromide (CTAB), and buffered with diethanolamine (DEA) to pH 9.2 (ca. 20 mM DEA). Total length of separation capillary was 80 cm, resulting in run time of under 4 min. The method is suitable for a wide concentration range of the analytes (1–50 mg/L) with linear calibration plots (R2 = 0.9983–0.9999). Relative standard deviations were 0.05%–0.07% for migration times and 0.67%–2.04% for peak areas. The detection limits were in the range of 0.17–0.51 mg/L or 2–10 μmol/L (hydrostatic injection of 30 s of 1000× diluted sample). Due to its good buffering capacity, the electrolyte exhibited an excellent ruggedness and good tolerance to the alkaline samples. Consequently, Bayer liquor samples could be diluted as little as 100× which allows more sensitive determination of minor components over previous methods. The method was successfully applied to analysis of Bayer liquor samples with recoveries in the range of 95–105%.
Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated β‐cyclodextrins
Authors: Lipka Emmanuelle , Daniel Cecile , Vaccher Marie‐Pierre , Glaçon Virginie , Ewing David , Mackenzie Graham , Len Christophe , Bonte Jean‐Paul , Vaccher Claude
Published: January 2004

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD‐CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated‐CD or highly S‐CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S‐CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S‐α‐CD, S‐β‐CD or S‐γ‐CD at 30°C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng·mL−1 for the first enantiomer.
Application of capillary electrophoresis with different sample stacking strategies for the determination of a group of nonsteroidal anti‐inflammatory drugs in the low μg ∵ L−1 concentration range
Authors: Macià Alba , Borrull Francesc , Aguilar Carme , Calull Marta
Published: January 2004

Several on‐column sample preconcentration modes – large‐volume sample stacking using the EOF pump (LVSEP), LVSEP with anion‐selective exhaustive injection (LVSEP‐ASEI) and field‐amplified sample injection with sample matrix removal using the electroosmotic flow (EOF) pump (FAEP) – were used to analyze some nonsteroidal anti‐inflammatory drugs (NSAIDs) by capillary electrophoresis, and then compared. Methanol was the background electrolyte solvent to suppress the EOF. The effect of the type and length of the solvent plug, and the sample injection time were investigated in FAEP to determine the conditions that provided the best response. LVSEP, LVSEP‐ASEI, and FAEP improved the sensitivity of the peak area by 100‐, 1200‐, and 1800‐fold, respectively. The methodology developed, in combination with solid‐phase extraction (SPE), was applied to the analysis of water samples.
Explorative data analysis of two‐dimensional electrophoresis gels
Authors: Schultz Jakob , Gottlieb David Mark , Petersen Marianne , Nesic Ljiljana , Jacobsen Susanne , Søndergaard Ib
Published: January 2004

Methods for classification of two‐dimensional (2‐DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two‐dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots on the gels for further investigation. The explorative approach in analysis of 2‐D gels makes it possible, in a fast and convenient way, to screen many gels in order to determine the protein patterns that form clusters and could be selected for further examination.
Verrucous carcinoma of the paranasal sinuses: Case report and clinical update
Authors: Paleri Vinidh , Orvidas Laura J. , Wight Richard G. , Bradley Patrick J.
Published: January 2004

Background.

Verrucous carcinoma is a low‐grade malignancy that has been reported to occur in all anatomic sites of the head and neck. Fourteen cases of verrucous carcinoma of paranasal sinus origin have been reported to date in the English literature.

Methods.

Case report and retrospective review of all cases of verrucous carcinoma of the paranasal sinuses in the English literature. All authors were contacted to provide missing data and long‐term follow‐up.

Results.

Five of the eight authors contacted responded, and the most current data from all 15 cases was compiled. Eleven of the 15 patients (73%) were men and ranged in age from 35 to 81 years (median, 68 years). The maxillary sinus is the most common paranasal sinus involved (93%). Presentation often occurred late, with 12 of 15 (80%) initially being seen at stage T3 or higher. Surgical excision was the treatment of choice, and median disease‐free survival was 54 months.

Conclusions.

Verrucous carcinoma of the paranasal sinuses is a rare but potentially curable disease. Treatment is surgical, and prognosis is good with early intervention. © 2003 Wiley Periodicals, Inc. Head Neck 26: 184–189, 2004
Parapharyngeal lymph node metastasis: An unusual presentation of papillary thyroid carcinoma
Authors: Lombardi Davide , Nicolai Piero , Antonelli Antonino R. , Maroldi Roberto , Farina Davide , Shaha Ashok R.
Published: January 2004

Background.

Parapharyngeal space nodal metastases are usually secondary to malignancies of the pharynx and sinonasal tract, although localization of lymphomas is also possible. Parapharyngeal metastases arising from thyroid papillary carcinoma are instead an exceedingly rare event, with only 10 cases reported up to now in the literature.

Methods.

We describe two cases of parapharyngeal metastasis from thyroid papillary carcinoma in a man and a woman, aged 40 and 52 years, respectively.

Results.

Both patients had a lesion that clinically appeared to be located in the parapharyngeal space; they underwent CT and MRI, which detected a cystic mass in the poststyloid compartment. In the first patient, fine‐needle aspiration cytology failed in identifying the histologic nature of the lesion, which was excised through a transcervical approach. A diagnosis of metastatic thyroid papillary carcinoma was rendered and therefore the patient underwent total thyroidectomy. In the second patient, a total thyroidectomy, previously scheduled for multinodular goiter, was performed along with the removal of the parapharyngeal mass. Definitive histologic findings revealed that the two parapharyngeal masses were cystic metastases from a thyroid papillary carcinoma. Both patients received postoperative 131I treatment. Twenty‐four months after surgery, the first patient is free of disease, whereas the second one has clear signs of abnormal 131I uptake in the lungs.

Conclusions.

The differential diagnosis of a parapharyngeal poststyloid mass should also include metastasis from thyroid papillary carcinoma. When the lesion displays a cystic appearance on imaging, it is advisable to rule out a thyroid primary by ultrasonographic examination. The occurrence of a metastasis in such unusual site, even though rarely reported, does not seem to significantly affect the prognosis of the disease. © 2003 Wiley Periodicals, Inc. Head Neck 26: 190–196, 2004
Tailored endoscopic surgery for the treatment of sinonasal inverted papilloma
Authors: Lee Ta‐Jen , Huang Shiang‐Fu , Huang Chi‐Che
Published: January 2004

Background.

This retrospective study was designed to evaluate the efficacy of tailored endoscopic surgery. Tailored endoscopic surgery aims at resecting the inverted papilloma completely with a customized surgical approach, especially when an en‐bloc excision cannot be comprehensively or routinely achieved because of the immense extent of the tumor.

Methods.

Between November 1991 and March 2002, 43 patients with sinonasal inverted papillomas were treated by tailored endoscopic surgery. The average duration of follow‐up for this population was 25.3 months (range, 9–150 months). A staging system developed by Krouse was adopted for tumor grading. On the basis of tailored endoscopic surgery, 15 localized lesions and 12 smaller extensive lesions (Krouse stages 1 and 2) were treated by ordinary endoscopic resection, whereas 16 larger extensive lesions (Krouse stages 3 and 4) in which the tumors were immense were subjected to sequential segmental endoscopic surgery (SSES). Seven of these 16 larger extensive lesions combined with endoscopic medial maxillectomy because of extensive encroachment of maxillary sinus antrum.

Results.

Four patients (9.3%) had residual disease, each requiring one revision surgery. All tumors were successfully resected. No patient required lateral rhinotomy or midfacial degloving procedure. No major complications were encountered in any of the patients. None of the patients had residual disease at the time of this writing.

Conclusions.

Tailored endoscopic surgery is a safe and effective treatment that obviates the need for more extensive surgery for the management of inverted papilloma. Proper preoperative evaluations, intraoperative determination of extent and attachment of the tumor, close endoscopic follow‐up, and expert application of endoscopic techniques are the keys to the successful use of tailored endoscopic surgery. © 2004 Wiley Periodicals, Inc. Head Neck 26: 145–153, 2004
Prognostic factors in sinonasal tumors involving the anterior skull base
Authors: Suarez Carlos , Llorente Jose L. , Fernandez De Leon Ramon , Maseda Eduardo , Lopez Aurora
Published: January 2004

Background.

Anterior craniofacial resection is a standardized procedure for the treatment of ethmoid and frontal orbital tumors with intracranial invasion.

Methods.

A retrospective review of 100 patients with sinonasal tumors involving the anterior skull base who underwent combined craniofacial surgery at the Hospital Central de Asturias.

Results.

The most frequent pathologic entity was adenocarcinoma (53 cases) and other epithelial tumors (29 cases). Five‐year actuarial survival according to the Kaplan–Meier method was 40%. Factors such as involvement of surgical margins, orbital periosteum involvement, frontal sinus invasion, or spread into the dura had no significant effect on survival. Survival, however, was affected by the histologic findings of the tumor (p = .03), brain involvement (p = .04), deep soft tissue involvement of the orbit (p = .003), involvement of the sphenoid sinus (p = .001), previous treatment (p = .05), and postoperative recurrence (p = .0000). Neither the INT staging system nor the UICC system showed statistical prognostic significance. After multivariate analysis and Cox regression analysis, only recurrence after craniofacial resection, involvement of soft tissues of the orbit, and invasion of the sphenoid sinus significantly influenced survival.

Conclusions.

Standard staging systems did not show statistical prognostic significance. Only involvement of some critical areas was reliable as predictor of an unfavorable outcome. © 2003 Wiley Periodicals, Inc. Head Neck 26: 136–144, 2004
The treatment of early laryngeal cancers (T1–T2 N0): surgery or irradiation?
Authors: Jones Andrew Simpson , Fish Brian , Fenton John Eugene , Husband David John
Published: January 2004

Background.

Carcinoma of the larynx is the most common cancer affecting the head and neck region. In Northern Europe, early laryngeal cancer is almost universally treated by irradiation, but elsewhere it is treated by surgery. The main aim of this study was to determine whether there was any difference in survival between the two main therapeutic options. The secondary aim was to assess speech and voice quality in a small, randomized sample of patients from each treatment group.

Methods.

The subjects investigated were 488 patients with T1–2, N0 squamous cell carcinoma of the larynx. The patients form an unselected sequential group of our institution's experience with treating this disease over three decades. Four hundred nineteen patients were treated by irradiation, and 69 were treated with surgery. Most surgical patients were treated earlier in the series, whereas radiotherapy later became the treatment of choice. The primary outcome measures were recurrence at the primary site, recurrence in the neck, and tumor‐specific survival. The secondary outcome measure was speech and voice quality. Statistical analysis was by univariate and multivariate analysis of association and survival. Surgery included horizontal or vertical partial laryngectomy and various minor procedures on the glottis, including cordectomy. Over a 30‐year period, radiotherapy was administered to a dose of 60–66 Gy given over 30–33 daily fractions.

Results.

Surgery tended to be performed early on in the series and radiotherapy thereafter. Surgery was more likely to be carried out for supraglottic disease. These differences apart, the radiotherapy and surgery groups of patients were well matched. The 5‐year tumor‐specific survival for those treated by irradiation was 87% and for surgery it was 77% (p= .1022). Glottic cancer and T1 disease were associated with high 5‐year survivals: 90% and 91%, respectively. Supraglottic site and T2 disease both had a poorer prognoses: 79% and 69%, respectively. The differences for both sets of data were significant. There was no significant difference in primary site recurrence rates for the two treatment modalities, but regional recurrence was higher in the surgery group. Further analysis demonstrated that this was not a function of surgery per se but rather of the unit's policy toward the N0 neck at the time surgery was carried out. Regarding speech and voice quality, radiotherapy was far superior to surgery. All patients in the radiotherapy group but only 3 of 10 in the surgery group were judged to have a good or normal voice (p= .0017).

Conclusions.

Both surgery and irradiation are equally effective at treating early laryngeal carcinoma. Speech and voice were highly significantly better in patients treated by irradiation than in those treated by surgery. © 2004 Wiley Periodicals, Inc.Head Neck26:127–135, 2004