bluuebunny/crossref_metadata_2025 huggingface dataset:
Color management of liquid crystal display placed under light environment
Authors: Yoshida Yasuhiro , Yamamoto Yoichi
Published: March 2003

We studied the color management of liquid crystal displays (LCDs) placed in a light environment. In this paper, we first explained LCD problems related to color management and proposed an appropriate color management system when using an LCD. We also applied this system and examined the color management of an LCD placed in a light environment. Based on the proposed method, we showed that favorable color reproduction properties are obtained for LCDs in a light environment as well as a dark room. We reevaluated the LCD from the perspective of compatibility with the ICC profile. LCDs have superior γ and brightness characteristics, but have the problem of the chromaticity coordinates of reference white and the primary colors changing in response to the input signal value. This indicated a problem related to the contrast ratio of the LCD and showed that the changes in the chromaticity coordinates of the primary colors can be adequately described if the leakage light is considered. Based on this, we proposed a suitable device profile and color management system for LCDs and demonstrated excellent color reproduction in a dark room. Next, we examined the LCD placed in a light environment. We showed that if the LCD is designed so that the surface reflected light produced in a light environment is handled in the same manner as leakage light and suitable surface reflected light is produced, fluctuations in the chromaticity coordinates of reference white could be avoided. We placed a prototype of this type of LCD in an actual light environment and applied and evaluated the proposed color management system. This result showed that a low color reproduction error could be maintained even when a dark image was displayed on an LCD placed in a light environment and confirmed a subjective improvement in the image quality. © 2003 Wiley Periodicals, Inc. Electron Comm Jpn Pt 3, 86(7): 1–14, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjc.10074
Minimum constituents of chaos neural network composed of conventional neurons
Authors: Kawamura Satoshi , Yoshida Hitoaki , Miura Mamoru
Published: March 2003

Many studies of the neural network with chaotic output have been presented. In most of these, however, a neuron model or a network model is specially prepared to realize the chaotic response, and few studies have been based on the conventional model. This paper considers a neural network based on the conventional neuron model, such as is used in the Hopfield and other neural networks, and discusses the oscillation phenomenon. It is shown theoretically and experimentally that the behavior of the network composed of two neurons is either convergence or periodic oscillation. Then the network composed of three neurons is examined experimentally, and it is shown that convergence, periodic oscillation, quasi‐periodic oscillation, or chaos can occur. In other words, it is shown that the minimum configuration of a neural network based on the conventional neuron model that can generate chaos is a network composed of three neurons. © 2003 Wiley Periodicals, Inc. Electron Comm Jpn Pt 3, 86(7): 62–71, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjc.1152
Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus
Authors: Langlade‐Demoyen Pierre , Garcia‐Pons Francisco , Castiglioni Paola , Garcia Zacharias , Cardinaud Sylvain , Xiong Sidong , Gerloni Mara , Zanetti Maurizio
Published: March 2003

We report on the induction of primary and long‐term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence‐competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell‐deficient mice suggests that B lymphocytes function as antigen‐presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti‐viral CTL responses and to assess the parameters critical for long‐lasting and protective CTL responses in vivo.
All‐trans retinoic acid down‐regulates expression and function of β2 integrins by human monocytes: opposite effects on monocytic cell lines
Authors: Babina Magda , Henz Beate M.
Published: March 2003

All‐trans retinoic acid (ATRA) plays an important role in the differentiation of malignant myeloid cells but its effects on primary leukocytes have been poorly investigated. We report here that ATRA negatively affects expression and function of leukocyte integrins that play a key role in monocyte adhesive interactions. As evidenced by flow cytometry, ATRA (at 1 μM) clearly and donor‐independently suppressed the expression of all integrin chains investigated (CD11a, CD11b, CD11c, and CD18), most strikingly of CD11a. Down‐regulation was detectable after 24 and maximal after 72–96 h. Reverse transcription‐PCR analysis revealed diminished steady‐state concentrations of α specific transcripts but not of the common β chain, suggesting that heterodimer expression was predominantly regulated through α chains. Results obtained with blood‐derived monocytes were in sharp contrast to those for the leukemic cell lines THP‐1 and U937, both of which showed marked increase in all integrin subunits in response to ATRA. ATRA‐pretreated monocytes displayed significantly diminished β2 integrin‐dependent homotypic aggregation, and adhesion to stimulated endothelial cells (EC), while ATRA‐pretreated monocytic cell lines showed the opposite behavior displaying markedly enhanced aggregation and CD18‐mediated adhesion to EC. Therefore, the level of leukocyte integrins was obviously a decisive factor for these adhesive interactions irrespective of the cellular source. Collectively, our data indicate a striking difference between leukemic cell lines and normal hematopoietic cells with regard to ATRA responsiveness. By acting on key adhesive structures of normal leukocytes, ATRA mediates processes that may be of substantially broad range applying to inflammation and immunity in addition to differentiation and proliferation.
Phosphatidylinositol 3‐kinase is involved in Toll‐like receptor 4‐mediated cytokine expression in mouse macrophages
Authors: Ojaniemi Marja , Glumoff Virpi , Harju Kirsi , Liljeroos Mari , Vuori Kristiina , Hallman Mikko
Published: March 2003

Recent evidence suggests a role for phosphatidylinositol (PI) 3‐kinase in various inflammatory responses. In this study, the consequences of LPS‐induced PI 3‐kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3‐kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL‐1β, tumor necrosis factor‐α (TNF‐α) and macrophage inflammatory protein (MIP)‐2. The induction of IL‐1β, but not of MIP‐2 or TNF‐α, was blocked by the PI 3‐kinase inhibitors LY294002 and wortmannin. The nuclear factor‐κB (NF‐κB) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL‐1β and TNF‐α, but had no effect on MIP‐2 expression. Inhibition of PI 3‐kinase decreased the LPS‐induced transcriptional activity of NF‐κB, but it had no effect on the nuclear DNA binding activity of NF‐κB. These findings suggest that, while NF‐κB nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL‐1β gene in the absence of PI 3‐kinase activity. Taken together, our results demonstrate that activation of Toll‐like receptor (TLR)‐4 results in PI 3‐kinase‐MyD88 complex formation, and that PI 3‐kinase activity selectively leads to cytokine induction downstream of TLR4.
Extensive proliferation of T cell lineage‐restricted progenitors in the thymus: an essential process for clonal expression of diverse T cell receptor β chains
Authors: Kawamoto Hiroshi , Ohmura Koichiro , Fujimoto Shinji , Lu Min , Ikawa Tomokatsu , Katsura Yoshimoto
Published: March 2003

For clonal diversification of TCR, a large number of T cell progenitors are required in which highly diverse TCRβ chains are accommodated individually. In the present study, we examined the proliferative potential of thymic progenitors that have been defined to be T cell lineage restricted. We show that the earliest fetal thymus (FT) cells from Rag2–/– mice, when cultured individually in a thymic organ culture system, produced 150–1,800 CD25+ cells. Since differentiation and proliferation of Rag2–/– thymocytes are arrested at the stage of TCRβ chain gene rearrangement, the observed proliferation was considered to represent the proliferative potential of progenitors prior to the TCRβ rearrangement. A comparable level of proliferation was revealed to occur by analyzing the Dβ–Jβ rearrangement profiles of T cells generated from single progenitors in the earliest population of FT from normal mice. The proliferative potential of progenitors declined along with the progression of developmental stages. Such an extensive proliferation of progenitors after the restriction to the T cell lineage may be an essential process ensuring the clonal diversification of TCRβ chains.
Heterozygous null mutation of myelin P0 protein enhances susceptibility to autoimmune neuritis targeting P0 peptide
Authors: Miyamoto Katsuichi , Miyake Sachiko , Schachner Melitta , Yamamura Takashi
Published: March 2003

Mice with a heterozygous null mutation in myelin protein zero (P0+/–) develop late‐onset clinical paralysis associated with inflammatory pathology in the peripheral nerves. Although the development of this illness is known to require T cells and macrophages, little is understood regarding the immunological defect in the mice. Here we report that young P0+/– mice, free from clinical manifestations, have a defect in central tolerance to P0, and are more prone to induction of experimental autoimmune neuritis (EAN) by sensitization against P0180–199 peptide. Notably, we found that the P0 gene is transcribed in the thymus of wild‐type and the P0+/– mice in an amount proportional to the gene dosage. We then replaced the thymus of wild‐type mice with that of the P0‐deficient mice and vice versa. Immunization of these mice with P0180–199 revealed that a lower thymic P0 transcript would be associated with the higher recall T cell response to P0180–199, thus accounting for the higher susceptibility of the P0+/– mice to P0‐induced EAN. These results imply that a heterozygous mutation in an autoantigen could cause defective central tolerance to the autoantigen. As such, autoimmune T cells may play some role in "genetic" diseases caused by a heterozygous gene defect.
CD4 T cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25+ T cells
Authors: Wing Kajsa , Lindgren Susanne , Kollberg Gittan , Lundgren Anna , Harris Robert A. , Rudin Anna , Lundin Samuel , Suri‐Payer Elisabeth
Published: March 2003

Regulatory T cells expressing CD25 have been shown to protect rodents from organ‐specific autoimmune diseases. Similar CD25+ cells with a memory phenotype exerting suppressive function after polyclonal or allogeneic stimulation are also present in adult human blood. We demonstrate that adult human CD25+ cells regulate the response to myelin oligodendrocyte glycoprotein (MOG), as depletion of CD25+ cells increases responses of PBMC and the addition of purified CD25+ cells suppresses MOG‐specific proliferation and IFN‐γ production of CD4+CD25– T cells. In contrast, cord blood CD25+ cells do not inhibit responses to self antigens, and only a small subpopulation of cord CD25+ cells expresses the typical phenotype of adult regulatory T cells (CD45RA– and GITR+) enabling suppression of polyclonal responses. We conclude that activation of self‐reactive T cells in normal healthy individuals is prevented by the presence of self‐antigen‐specific CD25+ regulatory T cells and that the majority of these cells mature after birth.
Recognition of HLA‐G by the NK cell receptor KIR2DL4 is not essential for human reproduction
Authors: Gómez‐Lozano Natalia , de Pablo Rosario , Puente Sabino , Vilches Carlos
Published: March 2003

A central issue of reproductive immunology in mammals is why a semi‐allogeneic embryo is not rejected by the pregnant mother. This is particularly intriguing since, in different species, the early pregnant uterus is infiltrated by numerous maternal lymphocytes, predominantly NK cells. The human NK cell receptor KIR2DL4 has been implicated in the maternal tolerance to the embryo due to its recognition of HLA‐G, a non‐classical MHC molecule expressed preferentially in the placenta. Killer cell Ig‐like receptors (KIR) are believed to participate in the natural immunity to infection and tumors, but KIR2DL4 has unique structural, functional and genetic features that could confer it a different role. However, we demonstrate here that the KIR2DL4:HLA‐G interaction is not essential for human reproduction by showing that a multiparous woman lacks a KIR2DL4 gene.
Visualizing the course of antigen‐specific CD8 and CD4 T cell responses to a growing tumor
Authors: Klein Ludger , Trautman Lydie , Psarras Stelios , Schnell Silke , Siermann Anja , Liblau Roland , Boehmer Harald von , Khazaie Khashayarsha
Published: March 2003

Spontaneous tumors frequently express antigens that can be recognized by the immune system but nevertheless manage to evade immune surveillance. To better understand the mechanism of evasion, we followed CD8 and CD4 T cells reacting against a subcutaneously growing tumor, modified to express influenza hemagglutinin (HA) as surrogate tumor antigen. Adoptive transfer of 8,000 antigen‐specific CD8 T cells was sufficient to protect against challenge with 1x106 tumor cells, while larger numbers of T cells rejected established tumors. HA‐specific CD4 T cells could not reject tumors on their own but helped rejection by CD8 T cells. Rejection of the tumor coincided with prolonged survival of expanded antigen‐specific CD8 and CD4 T cells, while a failing anti‐tumor response was accompanied by transient expansion followed by rapid elimination of antigen‐specific T cells. Thus, a highly immunogenic tumor can evade immune surveillance because of an insufficient number of tumor‐specific T cells and antigen overload, resulting in exhaustion of the immune response. In this scenario, adoptive immunotherapy rather than vaccination promises successful treatment.
The same endothelial receptor controls lymphocyte traffic both in vascular and lymphatic vessels
Authors: Irjala Heikki , Elima Kati , Johansson Eva‐Liz , Merinen Marika , Kontula Keiju , Alanen Kalle , Grenman Reidar , Salmi Marko , Jalkanen Sirpa
Published: March 2003

The mechanisms controlling the exit of lymphocytes from tissues via lymphatics are practically unknown. We have now identified a 270–300‐kDa molecule designated common lymphatic endothelial and vascular endothelial receptor‐1 (CLEVER‐1) on human lymphatic endothelium and high endothelial venules. We show that it mediates binding of lymphocytes both to high endothelial venules and to lymphatic vessels. Moreover, blocking of the function of CLEVER‐1 results in significant reduction of lymphocyte traffic in vivo. Notably, CLEVER‐1 is also an inducible vascular adhesion molecule for other classes of leukocytes at sites of inflammation in peripheral tissues. These findings suggest that CLEVER‐1 is involved in regulation of lymphocyte recirculation and migration of leukocytes to sites of inflammation and is a potential new target to control inflammation.
Structure of a New Neuritogenic‐Active Ganglioside from the Sea Cucumber Stichopus japonicus
Authors: Kaneko Masafumi , Kisa Fumiaki , Yamada Koji , Miyamoto Tomofumi , Higuchi Ryuichi
Published: March 2003

A new ganglioside molecular species SJG‐2 has been obtained from the n‐hexane‐soluble lipid fraction of the chloroform/methanol extract of the sea cucumber Stichopus japonicus. On the basis of chemical and spectroscopic evidence, the structure of SJG‐2 has been determined, as NeuAcα2⇄4(NeuAcα2⇄3)Galβ1⇄8NeuAcα2⇄3GalNAcβ1⇄3Galβ1⇄4Glcβ1⇄1Cer. The new ganglioside SJG‐2, possessing a unique carbohydrate moiety, is the first ganglioside containing either a branched sugar chain moiety or an N‐acetylgalactosamine residue to be isolated from a sea cucumber. Partial hydrolysis with hot water and dilute acetic acid has proved useful for the structure elucidation of the complex oligosaccharide moieties. Moreover, the ganglioside SJG‐2 exhibits neuritogenic activity toward the rat pheochromocytoma cell line PC12 cells in the presence of NGF. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis of Epothilone 16,17‐Alkyne Analogs by Replacement of the C13−C15(O)‐Ring Segment of Natural Epothilone C
Authors: Karama Usama , Höfle Gerhard
Published: March 2003

Ring‐opening cross metathesis of epothilone C (4a) with ethylene, followed by silyl protection and ester hydrolysis, yielded an eastern ring segment C1−C12 as the carboxylic acid 10. Separately, a western ring segment 12 carrying a C16−C17 triple bond was synthesized and coupled with 10 to form the ester 13. Ring closure by olefin metathesis, deprotection, and then epoxidation, gave the 16,17‐alkyne analogs (14b, 3b) of epothilone C and epothilone A. The identity of 3b was proven by hydrogenation to (16Z)‐epothilone A8 (17) and comparison with an authentic sample prepared from natural epothilone A8 (18). The biological activity of the new epothilones was determined. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Fast, Solid‐Phase Synthesis of Chiral Peptide Nucleic Acids with a High Optical Purity by a Submonomeric Strategy
Authors: Sforza Stefano , Tedeschi Tullia , Corradini Roberto , Ciavardelli Domenico , Dossena Arnaldo , Marchelli Rosangela
Published: March 2003

The solid‐phase synthesis of chiral peptide nucleic acids (PNAs) usually results in partial epimerization of the products, since the α‐nitrogen atom of the amino acid is involved in an amidic bond. It is also time‐consuming, since all the chiral monomers bearing different nucleobases have to be independently synthesized. In order to prevent racemization and to speed up the synthetic procedure we adopted a submonomeric approach by using a solid‐phase, Boc‐based PNA synthesis in which the chiral backbone orthogonally Nα‐Fmoc‐protected (submonomer) was first linked to the growing chain on the resin, followed by Fmoc‐deprotection and derivatization with the carboxymethylnucleobase. The submonomer bearing the D‐lysine residue was designed by protecting the Nα‐(aminoethyl)amino acid moiety with an Fmoc protecting group, compatible with standard Boc chemistry, and with the use of an MBHA‐PS resin, normally employed for PNA synthesis. Different synthetic pathways towards the desired submonomer were studied by using the amino acid D‐lysine as a chiral synthon, obtaining a fast method leading to a high yield and an excellent enantiomeric excess of the submonomer. The solid‐phase submonomeric reaction conditions were optimized for the synthesis of a thyminyl PNA dimer and then used to synthesize two different chiral PNAs. In this way two advantages were obtained: a lower degree of racemization in the coupling step during the solid‐phase synthesis and the possibility of using the same submonomer for every different nucleobase. All the D‐lysine‐based chiral PNAs were obtained in good yields and, as compared with PNAs synthesized by other coupling methods, showed the highest optical purity reported so far. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Controlled Stepwise Release of Fragrance Alcohols from Dendrimer‐Based 2‐Carbamoylbenzoates by Neighbouring Group Participation
Authors: Frérot Eric , Herbal Karim , Herrmann Andreas
Published: March 2003

2‐Carbamoylbenzoates which are chemically bound to the surface of dendrimers were found to release tertiary fragrance alcohols by neighbouring group assisted alkaline hydrolysis under mild reaction conditions. Owing to the excellent separation of the intermediate reaction products by analytical HPLC, the kinetic rate constants of the first two consecutive reaction steps of fragrance release could be determined for a series of modified dendrimers with increasing size. Because of the intramolecular neighbouring group effect, the kinetic rate constants were found to be independent of the dendrimer generation and are not influenced by steric effects on the surface of the macromolecules. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Asymmetric IrI‐Catalysed Allylic Alkylation Of Monosubstituted Allylic Acetates With Phosphorus Amidites As Ligands
Authors: Bartels Björn , García‐Yebra Cristina , Helmchen Günter
Published: March 2003

Monodentate phosphorus amidites derived from 2,2′‐binaphthol and a variety of chiral amines were employed as ligands in IrI‐catalysed allylic alkylations of unsymmetrically substituted allylic acetates. The enantio‐ and regioselectivities of these reactions were investigated. Phosphorus amidites of bulky secondary chiral amines induced enantioselectivities of up to 94% ee in reactions of linear substrates. Phosphorus amidites derived from chiral primary amines, which have not been previously employed in asymmetric catalysis, furnished improved regioselectivities. The use of LiCl as additive led to improved regio‐ and enantioselectivities. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
An Aza Analogue of iso‐Levoglucosenone: Synthesis and Application of a New Building Block for Imino Sugars
Authors: Ostrowski Jens , Altenbach Hans‐Josef , Wischnat Ralf , Brauer David J.
Published: March 2003

The aza analogue 3 of iso‐levoglucosenone, which is easily accessible from furylglycine, was used as a flexible building block for the synthesis of various imino sugars. Enantiomerically pure products are available starting from optically active furylglycine derivatives. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Cover Picture: Eur. J. Org. Chem. 6/2003
Authors: Brotin Thierry , Dutasta Jean‐Pierre
Published: March 2003

The cover picture shows the different NMR spectra of xenon atoms encapsulated in cryptophane‐A and C2‐symmetrical analogs. The high sensitivity of the xenon chemical shift to its environment allows the detection of small structural perturbations of the hosts. A slight modification of the internal volume of the cage compounds leads to an appreciable change of the 129Xe NMR chemical shifts. There exists a direct correlation between the stability constant Ka and the van der Waals volume of the cavity. Details of the synthesis of the new cryptophane hosts and complexation dynamics are presented in the article by T. Brotin and J.‐P. Dutasta on p. 973 ff.
Attributions for the negative historical actions of a group
Authors: Doosje Bertjan , Branscombe Nyla R.
Published: March 2003

Two field studies examined the attributions made for the historically negative behaviour of a group as a whole, depending on whether the actions were committed by the ingroup or an outgroup. In the first study, Jewish people assigned more internal responsibility to Germans for their treatment of Jewish people during the Second World War than Germans assigned to their own group. In the second study, people attributed the negative historical actions of another nation more internally (and less externally) than similar negative historical actions committed by their own nation. This pattern of intergroup attributional bias was more pronounced among people who highly identified with their national ingroup. Outgroup homogeneity and perceptions of differences between the groups were also significantly predicted by ingroup identification. Links between social identity theory and the intergroup attribution bias are considered. Copyright © 2002 John Wiley & Sons, Ltd.
Poly(vinyl alcohol) hydrogels: Their synthesis and steps towards control of electroendosmosis
Authors: Purss Helen K. , Caulfield Marcus J. , Solomon David H. , Sommer‐Knudsen Jens
Published: March 2003

Poly(vinyl alcohol) (PVAl) hydrogel networks cross‐linked with glutaraldehyde were prepared and their properties as membranes examined using a variety of techniques including preparative electrophoresis. Electroendosmosis (EEO) was observed and shown to be the result of charges on the membrane and of complexation with borate buffer ions. Investigation of “glutaraldehyde” solutions showed acid entities in, or formed in “glutaraldehyde” were responsible for EEO. Techniques for using “glutaraldehyde” which minimize EEO are described.
A family of single‐isomer, sulfated γ‐cyclodextrin chiral resolving agents for capillary electrophoresis: Octa(6‐O‐sulfo)‐γ‐cyclodextrin
Authors: Zhu Wenhong , Vigh Gyula
Published: March 2003

The second member of the single‐isomer, sulfated γ‐cyclodextrin family, the sodium salt of octa(6‐O‐sulfo)‐γ‐cyclodextrin (OS) has been synthesized, characterized and used to separate the enantiomers of nonelectrolyte, acidic, basic, and ampholytic analytes by capillary electrophoresis in acidic aqueous background electrolytes. The anionic effective mobilities of the nonelectrolyte and anionic analytes increased with increasing concentration of OS. The effective mobilities of strongly complexing cationic analytes became anionic with very low OS concentrations and passed local anionic effective mobility maxima as the OS concentration, and along with it, the ionic strength, of the background electrolyte increased. The effective mobilities of the weakly binding cationic analytes became only slightly anionic at high OS concentration values and did not show the local anionic effective mobility maxima. For nonelectrolyte analytes, separation selectivities decreased with increasing OS concentration. For cationic analytes, separation selectivities were highest where the effective mobilities of the less mobile enantiomers approached zero. OS proved to be a broadly applicable chiral resolving agent.
Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis
Authors: Rodriguez Isabel , Lesaicherre Marie , Tie Yan , Zou Quanbo , Yu Chen , Singh Janak , Meng Lim T. , Uppili Sridhar , Li Sam F. Y. , Gopalakrishnakone Ponnampalam , Selvanayagam Zachariah E.
Published: March 2003

An integrated system of a silicon‐based microfabricated polymerase chain reaction (μPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was ±0.3°C using an improved novel “joint‐heating” scheme. Thermal cycling was digitally controlled with a temperature accuracy of ± 0.2°C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by μPCR was achieved in microchip, and application of the integrated μPCR‐μchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.
Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis
Authors: Li Yan , DeVoe Donald L. , Lee Cheng S.
Published: March 2003

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics‐based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10–100 fold in comparison with conventional IEF.
Extraction of proteins from plant tissues for two‐dimensional electrophoresis analysis
Authors: Giavalisco Patrick , Nordhoff Eckhard , Lehrach Hans , Gobom Johan , Klose Joachim
Published: March 2003

To increase the number of proteins detectable by two‐dimensional electrophoresis (2‐DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2‐DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.
Proteomic analysis of immunostained, laser‐capture microdissected brain samples
Authors: Moulédous Lionel , Hunt Sybille , Harcourt Rebecca , Harry Jenny L. , Williams Keith L. , Gutstein Howard B.
Published: March 2003

Proteomic analysis is often performed on homogenized preparations of whole tissues, which does not provide any information about relevant biochemical changes in specific cell types. Laser‐capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section. Phenotypically defined cells of interest may be visualized by immunostaining prior to microdissection. Previously published immunostaining protocols adapted to LCM require the use of very high antibody titers and very short incubation times. This raises the concern that low‐abundance antigens would not be detected and that antisera would be rapidly depleted. In addition, protein recovery from samples was not evaluated in most of these studies. Here, we describe an optimized immunostaining method based on immunofluorescence. By comparing two‐dimensional electrophoresis (2‐DE) results obtained from immunostained LCM brain tissue samples to those obtained from unstained, manually dissected samples, we demonstrated that immunofluorescent staining gave comparable protein recovery and similar resolution of protein spots on 2‐DE gels. Moreover, matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry analysis of selected spots from gels derived from control and immunostained LCM samples revealed that the immunostaining process had minimal effect on protein identification. LCM of immunofluorescently labeled tissue sections is a practical and powerful method to perform proteomic studies on specifically defined cell groups.
Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases
Authors: Dürrschmid Karin , Marzban Gorji , Dürrschmid Eberhard , Striedner Gerald , Clementschitsch Franz , Cserjan‐Puschmann Monika , Bayer Karl
Published: March 2003

The expression of human superoxide dismutase in fed‐batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two‐dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide‐range Immobiline Dry Strips pH 3–10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time‐consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress‐relevant. Out of these, 24 proteins could be identified by using the SWISS‐2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.
Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection
Authors: Sulzbacher Caruso Célia , Lanças Fernando Mauro , Carrilho Emanuel
Published: March 2003

We developed a method for the analysis of multiplexed double‐stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single‐column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A X174HinfI and a pGEM® DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe4+) as the only cation in the buffer.
Monitoring folding transitions of synthetic, branched‐chain polypeptides by capillary zone electrophoresis
Authors: Verzola Barbara , Perduca Massimiliano , Mezô Gábor , Hudecz Ferenc , Righetti Pier Giorgio
Published: March 2003

The coil/helix transition of a synthetic, branched‐chain polymeric polypeptide (poly (Lys(Glu1‐DL‐Ala3)EAK), 50‐Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mMNaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid‐point at 60% v/v methanol concentration, plateauing atca.80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in α‐helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a “rugby ball”.
Failure of immunocompetitive capillary electrophoresis assay to detect disease‐specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt‐Jakob disease
Authors: Cervenakova Larisa , Brown Paul , Soukharev Serguei , Yakovleva Oksana , Diringer Heino , Saenko Evgueni L. , Drohan William N.
Published: March 2003

The emergence of a new environmentally caused variant of Creutzfeldt‐Jakob disease (vCJD), the result of food‐born infection by the causative agent of bovine spongiform encephalopathy (BSE), has stimulated research on a practical diagnostic screening test. The immunocompetitive capillary electrophoresis (ICCE) assay has been reported to detect disease‐specific, proteinase‐resistant prion protein (PrPres) in the blood of scrapie‐infected sheep. We have applied this method to blood from CJD‐infected chimpanzees and humans. The threshold of detection achieved with our ICCE was 0.6 nM of synthetic peptide corresponding to the prion protein (PrP) C‐terminus, and 2 nM of recombinant human PrP at the optimized conditions. However, the test was unable to distinguish between extracts of leucocytes from healthy and CJD‐infected chimpanzees, and from healthy human donors and patients affected with various forms of CJD. Thus, the ICCE assay as presently performed is not suitable for use as a screening test in human transmissible spongiform encephalopathies (TSEs).
Determination of egg white lysozyme by on‐line coupled capillary isotachophoresis with capillary zone electrophoresis
Authors: Kvasnička František
Published: March 2003

An on‐line coupled capillary isotachophoresis – capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH4OH + 20 mM acetic acid (leading electrolyte), 5 mM ε‐aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM ε‐aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0–50 νg/mL), accuracy (recovery 96±5%), intra‐assay (3.8%), quantification limit (1 νg/ml), and detection limit (0.25 νg/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.
A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography
Authors: Fanali Salvatore , Catarcini Paolo , Presutti Carla , Quaglia Maria G. , Righetti Pier‐Giorgio
Published: March 2003

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta‐Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 νm diol‐modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 νm ID fused‐silica capillary. The CEC experiments were carried out by using an aqueous reversed‐phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m‐hydroxymandelic acid (m‐OH‐MA), p‐OH‐MA, and 3‐hydroxy‐4‐methoxymandelic acid (3‐OH‐4‐MeO‐MA). The CEC system was less enantioselective towards 2‐phenyllactic acid (2‐PhL) and 3‐PhL while mandelic acid methyl ester (MA‐Et‐Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.
Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe
Authors: Attia S.M. , Kliesch U. , Schriever‐Schwemmer G. , Badary O.A. , Hamada F.M. , Adler I.‐D.
Published: March 2003

The topoisomerase II (topo II) inhibitors etoposide (VP‐16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP‐16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5–60 mg/kg) increased the MNPCE frequencies in a dose‐dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP‐16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition. Environ. Mol. Mutagen. 41:99–103, 2003. © 2003 Wiley‐Liss, Inc.
Photomutagenicity of thiabendazole, a postharvest fungicide, in bacterial assays
Authors: Watanabe‐Akanuma Mie , Ohta Toshihiro , Yamagata Hideo
Published: March 2003

We investigated the photomutagenicity of thiabendazole (TBZ), a postharvest fungicide commonly used on imported citrus fruits. Using UVA light (320–400 nm), we irradiated bacterial cultures with or without TBZ in a 24‐well multiplate. UVA‐irradiation without TBZ was not mutagenic to the tester strains, nor was unirradiated TBZ. TBZ was strongly photomutagenic in Escherichia coli WP2uvrA and WP2uvrA/pKM101 strains, weakly photomutagenic in Salmonella typhimurium TA100 and TA98, and not photomutagenic in S. typhimurium TA1535 and TA1538. The photomutagenicity of TBZ was more evident in WP2uvrA/pKM101, which carries the trpE65 ochre mutation (TAA), than in TA100, which carries the hisG46 missense mutation (CCC). In E. coli WP3101–WP3106 and the corresponding pKM101‐containing strains, photoactivated TBZ induced predominantly G:C→A:T transitions and A:T→T:A transversions. In the plasmid‐containing strains only, TBZ induced a moderate number of A:T→G:C transitions and a few A:T→C:G and G:C→T:A transversions. The observation that UVA‐irradiated TBZ mutated both G:C and A:T basepairs may explain why WP2uvrA/pKM101 was more sensitive to its mutagenicity than TA100. TBZ that was irradiated before it was added to the WP2uvrA/pKM101 cells was not photomutagenic, which suggests that the photomutagenic products of TBZ were unstable or rapidly reacted with other molecules before being incorporated into cells. Environ. Mol. Mutagen. 41:92–98, 2003. © 2003 Wiley‐Liss, Inc.
Mouse bone marrow micronucleus test results do not predict the germ cell mutagenicity of N‐hydroxymethylacrylamide in the mouse dominant lethal assay
Authors: Witt Kristine L. , Hughes Lori A. , Burka Leo T. , McFee Alfred F. , Mathews James M. , Black Sherry L. , Bishop Jack B.
Published: March 2003

N‐Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31‐day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow‐up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180–720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose‐related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice. Environ. Mol. Mutagen. 41:111–120, 2003. Published 2003 Wiley‐Liss, Inc.
Biomonitoring with the comet assay of Greek greenhouse workers exposed to pesticides
Authors: Piperakis S.M. , Petrakou E. , Tsilimigaki S. , Sagnou M. , Monogiudis E. , Haniotakis G. , Karkaseli H. , Sarikaki E.
Published: March 2003

The pesticides in use in Greek greenhouses include a number of agents known to be mutagens and carcinogens. In the present study, we evaluated whether occupational exposure of agricultural workers to a complex mixture of pesticides resulted in a significant increase in DNA damage in human peripheral blood lymphocytes (PBLs). A total of 116 healthy individuals were divided into groups based on exposure to pesticides, smoking status, and gender. Alkaline comet assays performed on PBLs from these individuals indicated no statistically significant differences in basal DNA damage between the study groups. In addition, exposure of PBLs to a dose of hydrogen peroxide led to a similar degree of DNA damage and subsequent repair for all the study populations. The results of the study indicate that the agricultural workers who participated in this study had no detectable increase in DNA damage or alteration in the cellular response to DNA damage. Environ. Mol. Mutagen. Environ. Mol. Mutagen. 41:104–110, 2003. © 2003 Wiley‐Liss, Inc.