bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell detection based on protein array using modified glass slides
Authors: Zhang Chun Xiu , Liu He Ping , Tang Zu Ming , He Nong Yue , Lu Zu Hong
Published: September 2003

A protein array for cell detection was fabricated by spotting different antibodies on modified glass slides. Glass slides were modified to allow antibodies to be immobilized on it and to selectively bind antigens. Antibodies were specially selected with the cells to be detected as targets, which permitted target cells in samples to bind specifically to the array with little nonspecific binding. Results can be obtained by directly putting the samples onto the array for 1 h or a little longer to let the cells specifically interact with the antibodies. After washing the unbound samples away, images were observed with a microscope and captured with a CCD camera. The assessment of antibody‐cell binding was evaluated by capturing red blood cells (RBCs) in human blood with blood group antibodies (anti‐A and anti‐B). Blood group antibodies were spotted on the modified glass slide and kept at 4<.deg>°C overnight for immobilization. Human blood samples diluted to different concentrations were used to examine the sensitivity and specificity of the method.
Capillary electrophoresis‐ion trap mass spectrometry analysis of Ziagen® and its phosphorylated metabolites
Authors: Cai Zongwei , Fung Eliza N. , Sinhababu Achintya K.
Published: September 2003

A capillary electrophoresis‐ion trap mass spectrometry method with a time‐segment program was developed to simultaneously analyze Ziagen® and its phosphorylated metabolites such as carbovir monophosphate, carbovir diphosphate, and carbovir triphosphate. By using the time‐segment program, the positively charged nucleoside analog and negatively charged nucleotides were separated and detected in a single electrophoretic run. The limits of detection were less than 2 μM for all of the analytes. Calibration curves of the compounds showed excellent linearity over the range of 2–100 μM. The capability of the method was demonstrated by analyzing Ziagen® and its phosphorylated metabolites that were spiked in cellular extracts of human peripheral blood mononuclear cells at 20 μM levels. Some endogenous nucleotides such as adenosine 5'‐triphosphate, adenosine 5'‐diphosphate, and adenosine 5'‐monophosphate, were also detected in the cellular extracts.
High‐speed chiral separations on a microchip with UV detection
Authors: Ludwig Martin , Kohler Frank , Belder Detlev
Published: September 2003

Fast chiral separations of a variety of basic and acidic compounds could be realized on microfluidic quartz chips. A microchip electrophoresis instrument equipped with a linear imaging UV‐detector was used. The usually applied but troublesome fluorescence tagging in order to enable fluorescence detection could be omitted. Using sulfated cyclodextrins as chiral selectors baseline separation of 19 compounds could be achieved in less than 1 min with high reproducibility. The relative standard deviation of migration time was below 7%. The fastest separation could be performed in 2.5 s which is to date the fastest separation of enantiomers reported. It was possible to apply microchip electrophoresis (MCE) for the determination of high enantiomeric excess (ee) values, as exemplarily shown for pseudoephedrin where 2% of the minor enantiomer could reliably be determined beside high amount of the other isomer. Successful separation of a mixture of 3 chiral drugs could be performed in a single run in less than 11 s utilizing a separation length of only 12 mm. These results show that MCE has great potential for fast chiral analysis and high‐throughput screening.
Effect of lipid composition on phospholipase A2‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column
Authors: Liu Xue‐Ying , Nakamura Chikashi , Hasegawa Miki , Miyake Jun
Published: September 2003

Phospholipase A2 (PLA2)‐catalyzed membrane leakage can be detected by immobilized liposomes containing a self‐quenching fluorescent dye, calcein, on an open column using off‐line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA2 was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4‐dichloroaniline, to PLA2. The competitive immunoreaction between the 3,4‐dichloroaniline‐PLA2 conjugate and PCB samples on the anti‐PCB antibody column caused the release of the bound PLA2 conjugates in proportion to the PCB concentration. The released PLA2 conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.
Trypsin activity assay in substrate‐specific one‐ and two‐dimensional gels: A powerful method to separate and characterize novel proteases in active form in biological samples
Authors: Zhao Zhenjun , Russell Pamela J.
Published: September 2003

To separate and identify the proteases, a substrate‐specific, sensitive assay in sodium dodecyl sulfate (SDS)‐polyacrylamide gels after two‐dimensional (2‐D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6–11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc‐Gln‐Ala‐Arg‐MCA (4‐methyl‐coumaryl‐7‐amide). The gels were washed in cold 2.5% Triton X‐100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide‐MCA generated fluorescent 7‐amino‐4‐methyl‐coumarin. In 1‐D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R2 = 0.999). In 2‐D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R2 = 0.974). To optimize renaturation conditions, 5×5 min washes with 2.5% Triton X‐100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37°C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.
On‐line biosensors for simultaneous determination of glucose, choline, and glutamate integrated with a microseparation system
Authors: Shi Guoyue , Yamamoto Katsunobu , Zhou Tianshu , Xu Fan , Kato Takeshi , Ji‐ye Jin , Jin Litong
Published: September 2003

An effective microseparation system integrated with ring‐disc electrodes and two microfluidic devices was fabricated for in vivo determination using a microdialysis pump. The major interference of ascorbic acid (AA) was excluded by direct oxidation with ascorbate oxidase. Glucose, glutamate, and choline were successfully determined simultaneously through the biosensors modified with a bilayer of osmium‐poly(4‐vinylpyridine)gel‐horseradish peroxidase (Os‐gel‐HRP)/glucose oxidase (GOD), glutamate oxidase (GlutaOD) or choline oxidase (ChOD). To stabilize the biosensors, 0.2% polyethylenimine (PEI) was mixed with the oxidases. The cathodic currents of glucose, glutamate, and choline biosensors started to increase after the standard solutions were injected into the microseparation system. The on‐line biosensors show a wide calibration range (10−7–10−5 mol/L) with a detection limit of 10−8 mol/L at the working potential of −50 mV. The variations of glucose, glutamate, and choline were determined simultaneously in a free moving rat when we perfused the medial frontal cortex with 100 μmol/L N‐methyl‐D‐aspartate (NMDA) solution, which is the agonist of the NMDA receptor.
Fabrication and characterization of poly(methyl methacrylate) microchannels by in situ polymerization with a novel metal template
Authors: Chen Zhifeng , Gao Yunhua , Su Rongguo , Li Chengwu , Lin Jinming
Published: September 2003

A stainless steel template for the fabrication of plastic microfluidic devices has been developed by photolithography and chemical etching technique. The preparation process of the template is simple, rapid, and low‐cost. The cross sectional profiles of raised microchannels on the template are trapezoidal. The surface roughness of the templates was controlled down to 190 nm. The template can be used repeatedly to generate devices reproducibly. The microfluidic devices of poly(methyl methacrylate) (PMMA) were fabricated by in situ polymerization using the templates. The reproducibility of the fabricated microchannel is high and the relative standard deviation is 0.7% by the in situ polymerization approach. Some physical properties of the polymerized microchannels were characterized including the transparency, the thermal deformation temperature, and the dimensional information. Current monitoring was used to evaluate the electroosmotic flow within the microchannels under the electric field strength of 300 V/cm.
Analysis of organic acids and inorganic anions in beverage drinks by capillary electrophoresis
Authors: Fung Ying Sing , Lau Kap Man
Published: September 2003

To meet the need for a new and validated analytical method for simultaneous determination of inorganic and organic acid anions in beverage drinks, a capillary zone electrophoresis (CZE) procedure had been developed based on a new background electrolyte (BGE) system containing 3 mmol/L 1,3,5‐benzenetricarboxylic acid (BTA), 15 mmol/L tris(hydroxymethyl)aminomethane and 1.5 mmol/L tetraethylenepentamine (TEPA) at pH 8.4. Baseline separation of anions commonly found in beverage drinks could be achieved in less than 14 min with indirect UV detection at 240 nm. Comigration problems for hydroxycarboxylic acids could be solved using TEPA as BGE additive. The results indicate excellent repeatability for migration time (RSD, 0.27–0.67%, n = 5) and good precision for both peak height (RSD, 3.2–4.2%, n = 5) and peak area (RSD, 3.1–4.5%, n = 5). Under the optimized conditions and using corrected peak area for quantitation, an excellent linear dynamic range (with correlation coefficient > 0.997 in a concentration range from 0.005 to 2.0 mmol/L) and low detection limit (1–4 μmol/L) were obtained for all the anions investigated. The applicability and reliability of the CE procedure developed were established by parallel method determination using established ion chromatography procedure for the analysis of inorganic and organic acid anions in orange juice and wine samples. Our CZE procedure provided a sensitive and economic technique for simultaneous determination of inorganic and organic acid anions in orange juice, red and white wine samples.
Signal denoising and baseline correction by discrete wavelet transform for microchip capillary electrophoresis
Authors: Liu Bi‐Feng , Sera Yoichi , Matsubara Norio , Otsuka Koji , Terabe Shigeru
Published: September 2003

Signal denoising and baseline correction using discrete wavelet transform (DWT) are described for microchip capillary electrophoresis (MCE). DWT was performed on an electropherogram describing a separation of nine tetramethylrohodamine‐5‐isothiocyanate labeled amino acids, following MCE with laser‐induced fluorescence detection, using Daubechies 5 wavelet at a decomposition level of 6. The denoising efficiency was compared with, and proved to be superior to, other commonly used denoising techniques such as Fourier transform, Savitzky‐Golay smoothing and moving average, in terms of noise removal and peak preservation by directly visual inspection. Novel strategies for baseline correction were proposed, with a special interest in baseline drift that frequently occurred in chromatographic and electrophoretic separations.
Stacking and separation of enantiomers by acetonitrile‐salt mixtures in micellar electrokinetic chromatography
Authors: Choy Tom M. H. , Chan Wing‐Hong , Lee Albert W. M. , Huie Carmen W.
Published: September 2003

The feasibility of employing the “acetonitrile stacking” method in micellar electrokinetic chromatography (MEKC) for the on‐line preconcentration and separation of enantiomers is demonstrated for the first time. The effects of various experimental parameters on the stacking and separation of three different pairs of optical isomers, i.e., two substituted naphthyl enantiomers and one dansylated‐DL‐amino acid, were examined. In particular, the effectiveness of the addition of acetonitrile and salt in the sample matrix to induce narrowing of the analyte bands was investigated in the presence of sodium cholate as the chiral surfactant micelle in the separation buffer. For example, it was found that the presence of both acetonitrile and 1% NaCl in the sample matrix (volume ratio = 2:1) led to a significant improvement of the peak height and resolution for the MEKC separation of a pair of R(−)/S(+)‐1,1'‐binaphthyl diyl hydrogen phosphate enantiomers when the injection sample size was relatively large (e.g., 12% capillary volume). Furthermore, the feasibility of combining salting‐out solvent extraction (off‐line) and acetonitrile stacking (on‐line) as a novel approach for sample preconcentration in capillary electrophoresis was also demonstrated.
Discrete wavelets transform for signal denoising in capillary electrophoresis with electrochemiluminescence detection
Authors: Cao Weidong , Chen Xiaoyan , Yang Xiurong , Wang Erkang
Published: September 2003

Discrete wavelets transform (DWT) was applied to noise on removal capillary electrophoresis‐electrochemiluminescence (CE‐ECL) electropherograms. Several typical wavelet transforms, including Haar, Daublets, Coiflets, and Symmlets, were evaluated. Four types of determining threshold methods, fixed form threshold, rigorous Stein's unbiased estimate of risk (rigorous SURE), heuristic SURE and minimax, combined with hard and soft thresholding methods were compared. The denoising study on synthetic signals showed that wave Symmlet 4 with a level decomposition of 5 and the thresholding method of heuristic SURE‐hard provide the optimum denoising strategy. Using this strategy, the noise on CE‐ECL electropherograms could be removed adequately. Compared with the Savitzky‐Golay and Fourier transform denoising methods, DWT is an efficient method for noise removal with a better preservation of the shape of peaks.
Enantioseparation of phenothiazines in cyclodextrin‐modified capillary zone electrophoresis: Reversal of migration order
Authors: Lin Ching‐Erh , Liao Wei‐Ssu , Chen Kuo‐Hsing
Published: September 2003

Enantioseparations of phenothiazines with γ‐cyclodextrin (γ‐CD) as a chiral selector were investigated using citrate and phosphate buffer electrolytes at pH 3.0. Reversal of the enantiomer migration order of promethazine, ethopropazine, and trimeprazine was observed by varying γ‐CD concentration in the range of 5–9 mM, 2.5–4.5 mM and 1.5–2.8 mM, respectively, using 100 mM citrate buffer at pH 3.0. As in the case of β‐CD, the (+)‐enantiomers of phenothiazines possess greater binding strength to γ‐CD than the (−)‐enantiomers. The evaluation of the binding constants and limiting mobility of the complexes formed between the enantiomers of phenothiazines and γ‐CD reveals that the binding strength of phenothiazines to γ‐CD and the differences in the binding constants and limiting mobility of the complexes are responsible for the enantiomer migration reversal. Both the binding constants and limiting mobility of the complexes between the (+)‐enantiomers of phenothiazine and γ‐CD are greater than those of the corresponding (−)‐enantiomers in a citrate buffer, while the binding constants of the complexes primarily determined the migration order of the enantiomers in a phosphate buffer. Compared with the results obtained using a phosphate buffer, we may conclude that citrate buffer which involves competitive complexation with chiral selector plays a significant role in the enantiomer migration reversal.
In vivo simultaneous monitoring of γ‐aminobutyric acid, glutamate, and L‐aspartate using brain microdialysis and capillary electrophoresis with laser‐induced fluorescence detection: Analytical developments and in vitro/in vivo validations
Authors: Sauvinet Valérie , Parrot Sandrine , Benturquia Nadia , Bravo‐Moratón Eva , Renaud Bernard , Denoroy Luc
Published: September 2003

γ‐Aminobutyric acid (GABA), glutamate (Glu), and L‐aspartate (L‐Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene‐2,3‐dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser‐induced fluorescence using a 442 nm helium‐cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L‐Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl‐β‐cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L‐Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.
Capillary electrochromatography and preconcentration of neutral compounds on poly(dimethylsiloxane) microchips
Authors: Ro Kyung Won , Chang Woo‐Jin , Kim Ho , Koo Yoon‐Mo , Hahn Jong Hoon
Published: September 2003

Capillary electrochromatography (CEC) and preconcentration of neutral compounds have been realized on poly(dimethylsiloxane) (PDMS) microchips. The channels are coated with polyelectrolyte multilayers to avoid absorption of hydrophobic analytes into PDMS. The structures of a microchip include an injector and a bead chamber with integrated frits, where the particles of the stationary phase are completely retained. Dimensions of the frit structures are 25 μm×20 μm, and the space between the structures is 3 μm. A neutral compound, BODIPY, that is strongly absorbed into native PDMS, is successfully and selectively retained on octadecylsilane‐coated silica beads in the bead chamber with a concentration enhancement of up to 100 times and eluted with elution buffer solution containing 70% acetonitrile. Preconcentrations and CEC separations of coumarins have been conducted with the same device and achieved complete separations in less than 50 s.
Rapid separation and laser‐induced fluorescence detection of mutated DNA by capillary electrophoresis in a self‐coating, low‐viscosity polymer matrix
Authors: Du Ming , Flanagan James H. , Lin Bingcheng , Ma Yinfa
Published: September 2003

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low‐viscosity polymer sieving buffer for DNA separation with on‐line coating has never been reported. In this paper, a new method using capillary electrophoresis with on‐line coating and laser‐induced fluorescence detection (CE‐LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on‐line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low‐viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on‐line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen‐bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6‐ to 7‐fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL‐I endonuclease. The technique is very sensitive for the size‐separation of low‐range, middle‐range, and high‐range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.
Optimized preparation of poly(styrene‐co‐ divinylbenzene‐co‐methacrylic acid) monolithic capillary column for capillary electrochromatography
Authors: Jin Wenhai , Fu Hongjing , Huang Xiadong , Xiao Hua , Zou Hanfa
Published: September 2003

Preparation of a poly(styrene‐co‐divinylbenzene‐co‐methacrylic acid) monolithic stationary phase for the use in capillary electrochromatography (CEC) has been improved by optimizing the polymerization conditions. It is observed that the reaction time strongly affects column efficiency, while the proportion of isooctane in porogen influences peak symmetry of some solutes seriously. The lifetime of the monolithic columns prepared mainly depends on the pH of buffers used. Reproducibility of electroosmotic flow (EOF) from batch to batch columns are lower than 2.8% relative standard deviation. Unlike other types of capillary electrochromatographic monoliths, a pH‐dependent EOF was observed on this type of column. Separation of various types of compounds including aromatic hydrocarbons, hormones, anilines, basic pharmaceuticals, and peptides was achieved. The facile preparation and wide application of this monolithic column may make styrene‐based polymer a potential stationary phase in CEC.
Surface grafting of glycidyl methacrylate on silica gel and polyethylene beads
Authors: Choi Seong‐Ho , Hwang Young‐Mi , Ryoo Jae Jeong , Lee Kwang‐Pill , Ohta Kazutoku , Takeuchi Toyohide , Jin Ji‐Ye , Fujimoto Chuzo
Published: September 2003

Surface grafting of glycidyl methacrylate (GMA) on silica gel and a polyethylene bead was performed by radical polymerization and radiation‐induced polymerization, respectively, in order to improve softness. Subsequently, diethylene triamine (DETA), triethylene tetraamine (TETA), and iminodiacetic acid (IDA) were introduced to the grafted GMA for use as affinity columns. The efficiency of the affinity column was investigated by use of bovine serum albumin (BSA) and hemoglobin (Hb) as model proteins. The affinity degree of BSA was higher than Hb for the DETA and TETA column, whereas the affinity degree of Hb was higher than BSA for the IDA column supported by silica gel. The affinity degree of BSA was higher than Hb for the DETA and TTA column supported by polyethylene (PE) beads.
Formation of S‐[2‐carboxy‐1‐(1H‐imidazol‐4‐yl) ethyl]glutathione, a new metabolite of L‐histidine, from cis‐urocanic acid and glutathione by the action of glutathione S‐transferase
Authors: Kinuta Masahiro , Kinuta Keiko , Yamada Hiroshi , Abe Tadashi , Yoshida Yumi , Araki Kenta , Li Shun‐Ai , Otsuka Atsushi , Nakanishi Akira , Moriyama Yoshinori , Takei Kohji
Published: September 2003

Exposure of the skin to sunlight results in an increase of the content of epidermal trans‐urocanic acid, a key metabolite of L‐histidine, and also in occurrence of the isomerization of trans‐urocanic acid to the cis isomer. S‐[2‐Carboxy‐1‐(1H‐imidazol‐4‐yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S‐[2‐carboxy‐1‐(1H‐imidazol‐4‐yl)ethyl]‐L‐cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis‐urocanic acid by incubation with rat liver extract that contained glutathione S‐transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with γ ‐glutamyl transpeptidase resulted in the formation of N‐{S‐[2‐carboxy‐1‐(1H‐imidazol‐4‐yl)ethyl]‐L‐cysteinyl}glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high‐voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis‐urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.
Mixed C18 and C1 modification on an optical fiber for chromatographic sensing
Authors: Zhou Leiji , Wang Kemin , Zuo Xinbing , Choi Martin M. F. , Chen Yunqing , Huang Shasheng
Published: September 2003

An optical fiber‐chromatographic sensor, aiming at simultaneous and selective response to multiple components following a chromatographic separation, is described. We report an improved approach for immobilization of octadecyl (C18) and methyl (C1) moieties as stationary phase on an optical fiber suitable as a sensing phase for organic solutes. By this approach, the stability and lifetime of the sensing layer as well as the detectability and retention behavior of the chromatographic sensor could be improved. Infrared spectroscopy was employed to confirm the presence of C18 and C1 moieties on the modified surface of the optical fiber. The chromatographic sensor was applied, with good sensitivity and chemical selectivity, to the simultaneous separation and detection of bromobenzene and toluene, using water as the mobile phase.
Analysis of chicken and turkey ovalbumins by microchip electrophoresis combined with exoglycosidase digestion
Authors: Mao Xiuli , Wang Keyi , Du Yuguang , Lin Bingcheng
Published: September 2003

The polypeptide and carbohydrate patterns of two glycoproteins, chicken ovalbumin (CO) and turkey ovalbumin (TO), were analyzed by microchip electrophoresis (ME), following digestion with proteases and exoglycosidases. Glycopeptides derived from ovalbumin were obtained by digestion with Pronase, followed by dialysis, and then separated by ME. Using CO as model, the method was developed to deduce the structure of glycans from glycoproteins by comparing the electropherograms of glycopeptides with and without digestion of exolycosidases. Applying the same approach, the structure of oligosaccharides linked to TO was determined. TO was found to contain high‐mannose type oligosaccharides and oligosaccharides with terminal N‐acetylglucosamine residues. The complete primary analysis of CO and TO by ME described in this paper provides a basis for an analysis of glycoproteins with an integrated microfluidic chip.
New species of Astragalus L. (Fabaceae) from Iran, mainly of sects. Incani and Malacothrix
Authors: Podlech D. , Maassoumi A. A.
Published: September 2003

During the work on Astragalus for “Flora Iranica” 39 new species of Astragalus, mainly from sects. Incani and Malacothrix could be discovered among the rich material collected in recent times. They are described here: Sect. Erioceras : A. pakravaniae ; Sect. Hololeuce : A. chaldoranicus ; Sect. Incani : A. abharensis, A. bukanensis, A. estahbanensis, A. fagh‐soleimanensis, A. homandicus, A. insularis, A. khadem‐kandicus, A. kiamaky‐daghensis, A. lacus‐valashti, A. maarofii, A. pseudorobustus, A. sabetii, A. safavii, A. sarabensis, A. semiromensis, A. siahcheshmehensis, A. subglaberrimus, A. zanjanensis ; Sect. Malacothrix : A. ashtianensis, A. aznaicus, A. bejourensis, A. belgheisicoides, A. chichesticus, A. clivicola, A. hajiabadensis, A. lasiocalycinus, A. markasicus, A. mehrizianus, A. pileh‐khasehensis, A. reconditus, A. runemarkii, A. shabilensis, A. shahinii, A. sisakhtianus, A. sivendicus, A. spachianiformis ; Sect. Stereothrix : A. altimontanus.
Die Häufigkeit von Oenothera‐Arten im westlichen Mitteleuropa
Authors: Wittig R. , Tokhtar V. K.
Published: September 2003

Auf ausgewählten Industrie‐ und Verkehrstandorten in drei Ballungsgebieten des westlichen Mitteleuropas (Rhein‐Ruhr‐Gebiet: Nordrhein‐Westfalen/ Deutschland; Frankfurt: Hessen/Deutschland; Oberrheingebiet: Baden‐Württemberg/Deutschland und Elsass/ Frankreich) wurde in den Jahren 1999 bis 2002 an ausgewählten, Oenothera ‐reichen Standorten (Industriegelände, Bahngelände, Hafenanlagen) eine quantitative Bestandsaufnahme der Oenothera ‐Arten vorgenommen. Hierbei belegten O. biennis und O. fallax nahezu gleichrangig den ersten Platz in der Häufigkeitsliste. Mit teilweise recht deut‐lichem Abstand folgen O. pycnocarpa und O. glazioviana auf den Plätzen 3 und 4. Bemerkenswer‐terweise ergab eine gleichzeitig in Frankfurt durchgeführte Rasterkartierung der Oenothera ‐Arten die gleiche Reihenfolge dieser vier Spezies im Hinblick auf die Zahl der von ihnen besiedelten Rasterfelder. Wie der Vergleich mit älteren Arbeiten zeigt, in denen O. biennis stets als die eindeutig häufigste Art genannt wird, hat sich O. fallax offensichtlich erst in jüngster Zeit stark ausgebreitet.
Carotenoids in certain lichens from the Colombian Andes
Authors: Czeczuga B. , Czeczuga‐Semeniuk E. , Sipman H.
Published: September 2003

Reverse‐phase high‐performance liquid chromato‐graphy, column and thin‐layer chromatography re‐vealed the presence of following carotenoids in the thalli of 10 species (14 specimens) of lichens from the Colombian Andes: β‐carotene, β‐cryptoxanthin, zeaxanthin, lutein, calthaxanthin, caloxanthin, 3′‐hydroxyechinenone, canthaxanthin, fritschiellaxanthin, astaxanthin, antheraxanthin, violaxanthin, neoxanthin, mutatoxanthin and capsochrome. The total content of carotenoids ranged from 18.41 μg g−1 (Stereocaulon ramulosum ) to 45.78 μg g−1 (Thamnolia vermicularis, no. 3) dry mass.
Upregulation of the truncated basic hair keratin 1(hHb1‐ΔN) in carcinoma cells by Epstein‐Barr virus (EBV)
Authors: Nishikawa Jun , Kiss Csaba , Imai Shousuke , Takada Kenzo , Okita Kiwamu , Klein George , Szekely Laszlo
Published: September 2003

To investigate the role of Epstein‐Barr virus (EBV) in epithelial tumors, we compared the expression pattern of cellular genes in the EBV‐infected gastric carcinoma cell line, NU‐GC‐3, and its uninfected control. Subtractive suppression hybridization (SSH) was combined with high‐density DNA array screening to identify differentially expressed genes. We have discovered that EBV infection upregulated a truncated variant of human basic hair keratin 1 (hHb1‐ΔN), a gene that had previously been identified in metastatic breast carcinoma. We verified the differential expression of hHb1‐ΔN in 3 independent EBV‐positive and ‐negative NU‐GC‐3 clones by Northern blotting. We further verified the EBV‐dependent upregulation of hHb1‐ΔN in 3 other carcinoma cell lines (AGS, TWO3 and DLD1) by RT‐PCR. Inhibition of CpG methylation by 5‐Aza‐CdR induced hHb1‐ΔN mRNA expression in the EBV‐negative clones but did not alter the expression in the EBV‐positive clones. The expression of hHb1‐ΔN protein was detectable by immunofluorescence and Western blotting in EBV‐positive but not in EBV‐negative NU‐GC‐3 clones after proteasome inhibitor (MG132) treatment. hHb1‐ΔN protein formed fibrous structures in the cytoplasm and accumulated in distinct nuclear bodies in the euchromatic areas of the cell nucleus. We suggest that the unstable hHb1‐ΔN protein may inhibit some of the functions of the keratin cytoskeleton and/or interfere with transcription regulation. It also may establish a link between EBV and the low differentiated or anaplastic status of the carcinomas that carry the virus. © 2003 Wiley‐Liss, Inc.
Human T‐cell leukemia virus type 1 Tax activates cyclin‐dependent kinase inhibitor p21/Waf1/Cip1 expression through a p53‐independent mechanism: Inhibition of cdk2
Authors: Chowdhury Iqbal H. , Farhadi Arash , Wang Xiao‐Fan , Robb Merlin L. , Birx Deborah L. , Kim Jerome H.
Published: September 2003

We investigated the possible involvement of HTLV‐1 Tax in the transcriptional activation of p21/Waf1/Cip1 (hereafter p21), a potent inhibitor of cyclin‐dependent kinases and cell growth. Tax transfection resulted in enhanced expression of p21 protein in T and fibroblastoid cells. Similarly, Tax‐expressing cells have higher amounts of endogenous p21 protein and RNA. However, neither Tax‐negative, HTLV‐1 transformed cells or HTLV‐1‐negative T cell lines had detectable levels of p21 protein and RNA. Cotransfection of Tax strongly activated the p21 promoter. CREB/ATF defective Tax mutant (M47) activated the p21 promoter significantly less efficiently. Tax activated wild type (wt) p21 promoter in p53‐negative Jurkat and p53‐positive A301cells, irrespective of endogenous p53 status, and activated a mutant p21 promoter containing a p53 responsive element (p53RE) deletion as strongly as wt promoter. Of importance, cdk2 activity was almost completely abolished in Tax‐induced p21‐expressing MT‐2 cells, suggesting that Tax‐induced p21 predominantly affects the activity of cdk2, a late G1 and S phase kinase. Taken together, these findings suggest that HTLV‐1 Tax activates p21/Waf1/Cip1, a cell growth inhibitor, in a p53‐independent mechanism through CREB/ATF‐related transcription factors, and inhibits cdk2. Tax induction of p21 may balance the T‐cell proliferation function of Tax and may contribute to the long clinical latency of HTLV‐1 infection and the delayed development of adult T‐cell leukemia. © 2003 Wiley‐Liss, Inc.
Inhibition of JNK signaling diminishes early but not late cellular stress‐induced apoptosis
Authors: Krilleke Dominik , Ucur Esat , Pulte Dianne , Schulze‐Osthoff Klaus , Debatin Klaus‐Michael , Herr Ingrid
Published: September 2003

The human leukemic T‐cell line Jurkat was used to define the role of the cellular stress pathway with its key player kinase JNK in cancer therapy‐induced apoptosis. JNK activity was inhibited by stable transfection with a dominant negative mutant of the upstream kinase JNKK/MKK4 or with the novel, potent and selective JNK1, ‐2 and ‐3 inhibitor SP600125. Inhibition of JNK activity delayed the onset of apoptosis induced by cisplatin, doxorubicin, γ‐irradiation and CD95‐L but did not prevent apoptosis per se. Early events during apoptosis such as induction of CD95‐L, activation of caspase‐8 and exposure of phosphatidylserine on the cell surface were strongly inhibited. Also, at early time points of apoptosis, loss of the mitochondrial membrane potential and release of cytochrome c were markedly impaired. However, late signaling events during apoptosis such as cleavage of PARP and DNA fragmentation apoptosis were only marginally affected. These findings are in accordance with the activity of initiator and effector caspases. Whereas activity of the initiator caspase‐8 was strongly inhibited early and late after induction, an inhibition of caspase‐3 activity was only observed early after induction of apoptosis. We therefore suggest that cellular stress signaling contributes to the initiation of apoptosis, whereas it might be dispensable for the progression of apoptosis. Dysfunction of this pathway under pathological conditions might contribute to therapy resistance of cancer cells. © 2003 Wiley‐Liss, Inc.
MSH6 germline mutations are rare in colorectal cancer families
Authors: Peterlongo Paolo , Nafa Khedoudja , Lerman Gabriel S. , Glogowski Emily , Shia Jinru , Ye Tian Z. , Markowitz Arnold J. , Guillem José G. , Kolachana Prema , Boyd Jeffrey A. , Offit Kenneth , Ellis Nathan A.
Published: September 2003

Germline mutations in MSH6 can cause HNPCC, which is associated with a tumor phenotype featuring MSI. However, tumors arising in persons with disease‐causing mutations of MSH6 may or may not exhibit MSI. We used D‐HPLC to screen for germline mutations in the promoter region, the coding region and the 3′‐UTR of MSH6. Eighty‐four families, enrolled on the basis of Amsterdam I and II criteria (HNPCC families) and less stringent criteria (HNPCC‐like families), were tested for MMR gene mutations; 27 families had a disease‐causing mutation in MLH1 or MSH2, and the remaining 57 families were tested for mutations in MSH6. Two protein‐truncating mutations were identified in each of 2 families fulfilling the Amsterdam I criteria, being present in persons affected with early‐onset colorectal cancers exhibiting MSI. Immunohistochemical analysis showed that expression of both MSH2 and MSH6 proteins was lost in the cancer cells of the 2 mutation carriers but only MSH6 protein expression was lost in 2 adenomatous polyps. A third possibly disease‐causing mutation was found in a person affected with a tumor that did not exhibit MSI. In addition, we found 4 new polymorphisms and determined that neither of the 2 studied by association analysis conferred susceptibility to colorectal or endometrial cancer. Altogether, our results indicate that disease‐causing germline mutations of MSH6 are rare in HNPCC and HNPCC‐like families. © 2003 Wiley‐Liss, Inc.
High tumor tissue concentration of urokinase plasminogen activator receptor is associated with good prognosis in patients with ovarian cancer
Authors: Borgfeldt Christer , Bendahl Pär‐Ola , Gustavsson Barbro , Långström Eva , Fernö Mårten , Willén Roger , Grenman Seija , Casslén Bertil
Published: September 2003

The urokinase plasminogen activator (uPA) system is involved in tumor growth and metastasis. We assayed the components of the uPA system in homogenates of 64 primary epithelial ovarian tumors and 5 metastases and evaluated the association of these parameters to prognosis in the 51 malignant cases. The levels of uPA, PAI‐2 and the uPA:PAI‐1 complex increased with progressive loss of histological differentiation (ptrend <0.001, <0.05 and <0.001). The level of PAI‐1 was higher in poorly than in well/moderately differentiated tumors (p = 0.03). The content of uPAR was lower in benign tumors as compared to borderline malignancies (p = 0.002), invasive primary tumors (p < 0.001), and metastases (p = 0.002). Surprisingly, the level of uPAR was lower in poorly differentiated as compared to both borderline (p = 0.01) and well differentiated malignant tumors (p = 0.005). Also, the level of uPAR was lower in advanced as compared to early stages of the disease (ptrend = 0.002). The median follow‐up time for patients was 5.8 years. High tumor tissue levels of uPAR were associated with longer postoperative survival (HR = 0.4, 95% CI = 0.2–0.8, p = 0.01). In contrast, shorter survival was evident in patients with high tumor levels of uPA from 2 years on after operation (HR = 4.6, 95% CI = 1.2–17, p = 0.02). High tPA levels tended to be associated with shorter overall survival after 2 years (HR = 2.9, 95% 95% CI = 0.9–9.8, p = 0.08). Although high tumor tissue content of uPAR was associated with a less aggressive phenotype characterized by well differentiated histology and longer survival, low content of uPAR in the poorly differentiated tumors and metastases presumably results from increased elimination of uPAR. © 2003 Wiley‐Liss, Inc.
Expression of microsomal prostaglandin E synthase‐1 in intestinal type gastric adenocarcinoma and in gastric cancer cell lines
Authors: van Rees Bastiaan P. , Sivula Anna , Thorén Staffan , Yokozaki Hiroshi , Jakobsson Per‐Johan , Offerhaus G. Johan A. , Ristimäki Ari
Published: September 2003

Gastrointestinal carcinomas synthesize elevated levels of prostaglandin E2 (PGE2), which has been mechanistically linked to carcinogenesis. Recently, microsomal prostaglandin E synthase‐1 (mPGES‐1) was cloned, which seems to be inducible and linked to cyclooxygenase‐2 (Cox‐2) in the biosynthesis of PGE2. We examined expression of mPGES‐1 in intestinal type gastric adenocarcinomas and in gastric cancer cell lines. The transcript for mPGES‐1 was elevated in 57% (4/7) of gastric carcinomas as detected by Northern blot analysis. Moderate to strong mPGES‐1 immunoreactivity was observed in 56% (5/9) of the carcinomas as detected by immunohistochemistry. Furthermore, mPGES‐1 mRNA, protein and microsomal PGES activity were detected in gastric adenocarcinoma cell lines that originated from intestinal type tumors (MKN‐7 and MKN‐28). In contrast to Cox‐2, however, expression of mPGES‐1 mRNA or protein were not induced by phorbol 12‐myristate 13‐acetate (PMA) or interleukin‐1β (IL‐1β) in any of the gastric cancer cell lines tested (MKN‐1, ‐7, ‐28, ‐45 and ‐74). Two gastric cancer cell lines (MKN‐45 and MKN‐74) did not express mPGES‐1 and lacked microsomal PGES activity, but were still able to synthesize PGE2. Because all gastric cell lines expressed cPGES as detected by immunoblotting, it is possible that Cox‐2 can interact with cPGES or with some other yet unidentified PGES in gastric cancer cells. Furthermore, our data show that regulatory mechanisms that drive expression of mPGES‐1 and Cox‐2 dissociate in gastric cancer cell lines. © 2003 Wiley‐Liss, Inc.
Relaxin‐like peptides in cancer
Authors: Silvertown Josh D. , Summerlee Alastair J.S. , Klonisch Thomas
Published: September 2003

The members of the relaxin‐like hormone family, relaxin and INSL3, also known as relaxin‐like factor (RLF) or Leydig cell‐derived insulin‐like factor (LEY‐I‐L), are implicated in various mechanisms associated with tumor cell growth, differentiation, invasion and neovascularization. The recent discovery of the relaxin receptor LGR7 and the INSL3/relaxin receptor LGR8 has provided evidence of an auto/paracrine relaxin‐like action in tumor tissues and enables the elucidation of the cellular pathways involved in the proposed functions of relaxin in tumor biology. Our review summarizes our current knowledge of the expression of relaxin and INSL3 in human neoplastic tissues and discusses the etiological roles of these heterodimeric peptide hormones in cancer. Discussion of possible cellular cascades involved in actions linking relaxin‐like peptides and neoplasia include the role of relaxin‐like peptides in tumor cell growth and differentiation; the effect of relaxin in stimulating the synthesis of the vasodilatory and tumor cell cytostatic and antiapoptotic molecule, nitric oxide; the potential ability of relaxin to upregulate vascular endothelial growth factor to promote angiogenesis and neovascularization and the concerted fine‐tuned action of relaxin on the matrix metalloproteinases on the extracellular matrix to facilitate tumor cell attachment, migration and invasion. © 2003 Wiley‐Liss, Inc.
Smoking and the risk of gastric cancer in the European Prospective Investigation Into Cancer and Nutrition (EPIC)
Authors: González Carlos A. , Pera Guillem , Agudo Antonio , Palli Domenico , Krogh Vittorio , Vineis Paolo , Tumino Rosario , Panico Salvatore , Berglund Göran , Simán Henrik , Nyrén Olof , Agren Asa , Martinez Carmen , Dorronsoro Miren , Barricarte Aurelio , Tormo María J. , Quiros Jose R. , Allen Naomi , Bingham Sheila , Day Nicholas , Miller Antony , Nagel Gabriele , Boeing Heiner , Overvad Kim , Tjonneland Anne , Bueno‐de‐Mesquita H. Bas , Boshuizen Hendriek C. , Peeters Petra , Numans Mattijs , Clavel‐Chapelon François , Helen Ishaki , Agapitos Emmanuel , Lund Eiliv , Fahey Michael , Saracci Rodolfo , Kaaks Rudolf , Riboli Elio
Published: September 2003

Smoking has recently been recognised as causally associated with the development of gastric cancer (GC). However, evidence on the effect by sex, duration and intensity of smoking, anatomic subsite and cessation of smoking is limited. Our objective was to assess the relation between tobacco use and GC incidence in the European Prospective Investigation into Cancer and Nutrition (EPIC). We studied data from 521,468 individuals recruited from 10 European countries taking part in the EPIC study. Participants completed lifestyle questionnaires that included questions on lifetime consumption of tobacco and diet in 1991–1998. Participants were followed until September 2002, and during that period 305 cases of stomach cancer were identified. After exclusions, 274 were eligible for the analysis, using the Cox proportional hazard model. After adjustment for educational level, consumption of fresh fruit, vegetables and preserved meat, alcohol intake and body mass index (BMI), there was a significant association between cigarette smoking and gastric cancer risk: the hazard ratio (HR) for ever smokers was 1.45 (95% confidence interval [CI] = 1.08–1.94). The HR of current cigarette smoking was 1.73 (95% CI = 1.06–2.83) in males and 1.87 (95% CI = 1.12–3.12) in females. Hazard ratios increased with intensity and duration of cigarette smoked. A significant decrease of risk was observed after 10 years of quitting smoking. A preliminary analysis of 121 cases with identified anatomic site showed that current cigarette smokers had a higher HR of GC in the cardia (HR = 4.10) than in the distal part of the stomach (HR = 1.94). In this cohort, 17.6 % (95% CI = 10.5–29.5 %) of GC cases may be attributable to smoking. Findings from this large study support the causal relation between smoking and gastric cancer in this European population. Stomach cancer should be added to the burden of diseases caused by smoking. © 2003 Wiley‐Liss, Inc.
Physical activity and risk of renal cell carcinoma
Authors: Menezes Ravi J. , Tomlinson George , Kreiger Nancy
Published: September 2003

The role of physical activity in the development of renal cell carcinoma was examined using a population‐based case‐control study conducted in the province of Ontario. Physical activity was examined in several ways, including by intensity and during various life periods. Frequency‐matching by 5‐year age groups and sex produced sets of 486 females (133 cases and 353 controls) and 447 males (172 cases, 275 controls). Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). After adjusting for age, body mass index and smoking, women who performed relatively high amounts of recent recreational activity (OR = 0.41, 95% CI 0.21–0.82 for the highest vs. lowest quartile of activity, 2 years ago), as well as strenuous recreational activity (OR = 0.40, 95% CI 0.22–0.75), had a reduced risk of developing the disease. In general, moderate recreational activity did not appear to be associated with risk in females. In males, a relatively high frequency of total and moderate recreational activity was associated with reduced risk (total, OR = 0.49, 95% CI 0.27–0.87; moderate, OR = 0.49, 95% CI 0.27–0.89), especially during the teen years. Recreational activity during one's 30s was not associated with cancer risk. There was also no association between occupational activity and cancer risk in females or males. The results of our study, in general, suggest that physical activity may be associated with a decreased risk of developing renal cell carcinoma. © 2003 Wiley‐Liss, Inc.