bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes
Authors: Olsen Eydfinnur , Rasmussen Hanne Holm , Celis Julio E. 
Published: May 2005

AbstractComparison of the protein expression patterns of proliferating normal primary human keratinocytes plated in serum‐free medium (SFKM), supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin‐binding proteins (gelsolin and tropomyosin 9220), annexins (annexins IV and VIII), hsp28, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14‐3‐3 family, involucrin, E‐cadherin, cystatin A, desmoglein and integrins α2 and β1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (124.0 times), MRP8 (42.4 times), MRP14 (14.9 times), tropomyosin 9220 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp28, and tropomyosin 9220 were also highly upregulated in quiescent keratinocytes indicating that their increased levels in the differentiated cell may be due to loss of proliferative activity. Highly downregulated proteins included PAI‐2, tropomyosins 9213, 9121 and 9122, keratin 5, calnexin, 14‐3‐3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C2, α‐enolase, eIF‐4D, thioredoxin, annexins III and V, moesin, nucleolar protein B23, GST π and PCNA/cyclin. Both the high expression of keratin 6 and 16 – which are markers for an alternative pathway of keratinocyte differentiation – as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have differentiated via an abnormal pathway.
Gaining insight into a complex organelle, the phagosome, using two‐dimensional gel electrophoresis
Authors: Burkhardt Janis , Huber Lukas A. , Dieplinger Hans , Blocker Ariel , Griffiths Gareth , Desjardins Michel 
Published: May 2005

AbstractPhagosomes are the organelles formed de novo in a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two‐dimensional (2‐D) gel electrophoresis was performed to generate a map of the overall [35S]methionine‐labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X‐114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2‐D gel approach in the mapping of organelle proteins is discussed.
Accessibility to tissue‐specific genes from methylation profiles of mouse brain genomic DNA
Authors: Watanabe Sachihiko , Kawai Jun , Hirotsune Shinji , Suzuki Harukazu , Hirose Kenji , Taga Chiharu , Ozawa Naoki , Fushiki Shinji , Hayashizaki Yoshihide 
Published: May 2005

AbstractThe DNA methylation status of a large number of genomic loci is visualized simultaneously and quantitatively as two‐dimensional gel spots in the newly developed restriction landmark genomic scanning with a methylation‐sensitive restriction enzyme (RLGS‐M). Here, we demonstrate that RLGS‐M using NotI as a methylation‐sensitive enzyme could also scan gene loci of mammalian genomes, since almost all of the NotI loci corresponding to randomly chosen RLGS‐M spots were located near or in transcriptional units (6 out of 7 NotI‐linking clones) when mouse brain genomic DNA was used. This supports the previous prediction that most NotI sites are located in CpG islands (Lindsay and Bird, Nature 1987, 327, 336–338). Furthermore, beginning with RLGS‐M spots we examined how to approach their corresponding RNA messages, whose expression may be associated with methylation. We compared RLGS‐M patterns among various developmental stages of the mouse brain from embryonic day 9.5 to postnatal 8 weeks or among in vitro cell lines, and detected alterations of RLGS‐M spots which were due to methylation of NotI sites. Two experiments using NotI‐linking clones or polymerase chain reaction (PCR) were carried out to approach to their corresponding RNA messages. Consequently, we isolated two PCR‐amplified clones (# 15 and # 91) which corresponded to methylatable loci and gave positive signals to mRNA from the adult brain. Furthermore, we identified two NotI‐linking clones (C211 and C198) whose corresponding NotI loci localized near or at transcriptional units and were methylated in cell lines. Thus, RLGS‐M is expected to be widely applicable for the isolation of tissue‐specific genes whose genomic loci are associated with DNA methylation.
Quantitative and qualitative genetic variation in two‐dimensional DNA gels of human lymphocytoid cell lines
Authors: Asakawa Jun‐Ichi , Kuick Rork , Neel James V. , Kodaira Mieko , Satoh Chiyoko , Hanash Samir M. 
Published: May 2005

AbstractThere is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutation rates. To this end, we have explored the potential usefulness of two‐dimensional (2‐D) electrophoresis of human DNA fragments obtained from restriction‐enzyme‐digested genomic DNA, using samples from father/mother/child trios. On a single 2‐D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimension and 0.3 to 2.0 kbp in the second dimension are visualized. To enter into a genetic analysis of quantitative variation, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately ⩽ 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variations that segregated within families according to Mendelian principles. Additionally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variation. A total of 142 variable spots were identified; the heterozygosity index for these DNA fragments was 4.4%. The genetic nature of the additional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. This technology should be highly efficient in monitoring for mutation resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
High resolution microscopic mapping of DNA using multicolor fluorescent hybridization
Authors: Windle Bradford , Silvas Emilio , Parra Irma 
Published: May 2005

AbstractWe describe a procedure for microscopically mapping the relative positions of DNA probes along extended strands of DNA. The procedure referred to as direct visual hybridization (DIRVISH) DNA mapping involves the simultaneous hybridization of multiple probes and the fluorescent colors, red, green and blue to produce images that convey high‐resolution mapping information. The images appear as long strings of fluorescent signals positioned as they are in the genome. A visual multi‐color map is generated within 2 days. Cosmid probes span a distance of 10 μm or more and have been observed to contain patterns within the strings of signals. We have developed computer imaging programs to scan through the strings of signals and plot the intensities. Scans through multiple signal strings for one cosmid probe revealed consistent patterns. We have interpreted the patterns as the result of suppression of repetitive DNA sequence hybridization. These patterns may prove useful as fingerprints for regions of DNA.
Molecular epidemiology of respiratory syncytial virus: A review of the use of reverse transcription‐polymerase chain reaction in the analysis of genetic variability
Authors: Cane Patricia A. , Pringle Craig R. 
Published: May 2005

AbstractRespiratory syncytial virus (RSV) is the major viral cause of lower respiratory tract disease (bronchiolitis and pneumonia) in babies and infants. Infections with the virus occur as annual winter epidemics in temperate climates, placing considerable pressure on the provision of hospital beds. The virus is unusual in that it can reinfect individuals and it can infect babies despite the presence of maternal antibody. RSV has a negative sense nonsegmented RNA genome and as such is liable to high levels of mutation. This paper describes methods developed to determine the degree of genetic variability of the virus both during individual epidemics and worldwide. It is necessary for these methods to be quick, easy and cheap so that large numbers of samples can be analysed readily. They are based on extraction of viral RNA directly from clinical samples or from viral cultures, reverse transcription of the viral RNA, and then amplification of selected regions of the genome by the polymerase chain reaction (PCR). PCR products are then analysed by restriction mapping, or, if necessary, direct nucleotide sequencing. In this way isolates of RSV have been shown to fall into a number of genotypes, with epidemics being made up of cocirculating genotypes whose relative proportions vary with each epidemic. An understanding of the molecular epidemiology of this important human pathogen will be of significance in the search for an effective vaccine.
An improvement of restriction analysis of bacteriophage DNA using capillary electrophoresis in agarose solution
Authors: Klepárník Karel , Malá Zdeňka , Doškař Jiři , Rosypal Stanislav , Boček Petr 
Published: May 2005

AbstractSeven representatives of the serogroup B Staphylococcus aureus bacteriophages, 29, 53, 55, 83A, 85, ϕ11 and 80α, were examined by capillary electrophoresis (CE) for genomic homology using DNA restriction analysis. Genomic DNA of individual bacteriophages was cleaved by HindIII restriction endonuclease, and the resulting restriction fragments were separated by standard horizontal agarose slab gel electrophoresis (SGE) as well as by CE in low‐melting‐point agarose solutions. The number and size of restriction fragments identified by both methods were compared. The high separation power of CE makes it possible to extend the restriction fragment patterns. In most of the restriction patterns, some additional restriction fragments as small as 150 bp, not identified by SGE, were detected. With respect to speed, high separation efficiency, low sample consumption and automation, CE offers a simple procedure for processing of multiple samples cost‐effectively in a reasonable time. The comparison of the complemented restriction patterns of the different phage strains and the subsequent identification of their common fragments leads to a deeper understanding of their phylogenetic relationships. The genome homologies expressed for individual phage pairs in terms of coefficient F values ranged from 15 to 69%. These values are in good accordance with the degree of DNA homology of these phages as determined by DNA hybridization studies and thermal denaturation analysis of DNA by other authors. The total size of each phage genome was estimated by adding the sizes of individual restriction fragments.
Hydrolytically stable cellulose‐derivative coatings for capillary electrophoresis of peptides, proteins and glycoconjugates
Authors: Huang Mingxian , Plocek Jiri , Novotny Milos V. 
Published: May 2005

AbstractA method for the preparation of versatile, hydrolytically stable coatings of fused silica capillaries is described. First, the HNO3‐pretreated silica surface is reacted with 7‐oct‐1‐enyltrimethoxysilane to form a stable self‐assembled alkylsilane monolayer. Secondly, the copolymer of hydroxypropylcellulose and 2‐hydroxyethyl methacrylate is statically coated onto a hydrophobized wall. This allows the formation of a highly crosslinked copolymer layer which is tightly bound to the wall surface. Hydrolytical stability in the pH range of 2–10 for several weeks has been demonstrated. The coating generates a low electroosmotic flow at alkaline pH, which can be easily masked by using special buffers. The versatility of this column technology is documented by separations of peptides, glycoproteins and derivatized oligosaccharides.
Electrophoretic analysis of bile proteins from patients with and without gallstones
Authors: Ota Yukiko , Ido Kenichi , Kimura Ken , Fukui Emiko , Ikemoto Shigenori 
Published: May 2005

AbstractBile proteins from 82 cases with various types of gallstones (pure cholesterol stones, cholesterol stones with a small amount of other substances, mixed stones and pigment stones) and 9 controls without gallstones were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A unique bile protein with a molecular mass of 32 kDa was identified. The 32 kDa protein could be stained on immunoblotting membranes using immunoperoxidase with concanavalin A. The 32 kDa protein was found in all controls but only in a portion (22.2–36.4%) of the patients with gallstones of various types. Following electrophoretic elution from the SDS‐PAGE gels, the 32 kDa protein was analyzed by reversed phase – high‐performance liquid chromatography, yielding three peaks for controls and only two peaks for patients with gallstones.
High resolution, standard format two‐dimensional protein electrophoresis using disposable glass micropipettes and non‐dedicated equipment
Authors: Sarmiento Marion 
Published: May 2005

AbstractA method for high resolution, two‐dimensional polyacrylamide gel electrophoresis (PAGE) of proteins on a standard format that utilizes nondedicated electrophoretic equipment is described. This method combines key features of various previous protocols into an improved approach which can readily be applied by persons experienced only in sodium dodecyl sulfate (SDS)‐PAGE. The method yields high‐quality two‐dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focusing (IEF) dimension and streamlining the initiation of the second (SDS‐PAGE) dimension. Important features of this method include the use of piperazine diacrylate cross‐linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equilibration of the first‐dimensional gels is accomplished at the same time the gels are loaded onto the second dimension. Loss of protein resolution due to diffusion after the end of the first dimension is reduced by minimizing the time required to load the SDS‐PAGE dimension. The method is versatile and adaptable to the analysis of one or many samples. The high degree of resolution, reproducibility and uniformity attained by this method are sufficient to abrogate the need for dedicated equipment in most applications.
Cross‐species identification of proteins separated by two‐dimensional gel electrophoresis using matrix‐assisted laser desorption ionisation/time‐of‐flight mass spectrometry and amino acid composition
Authors: Cordwell Stuart J. , Wilkins Marc R. , Cerpa‐Poljak Anne , Gooley Andrew A. , Duncan Mark , Williams Keith L. , Humphery‐Smith Ian 
Published: May 2005

AbstractAmino acid analysis and matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two‐dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS‐PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer‐generated rankings. Results were verified using N‐terminal protein microsequencing. The combined use of amino acid composition and MALDI‐TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4–10 tryptic digest fragments). A further two parameters, estimated Mr and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.
Microchip electrophoresis with sample stacking
Authors: Jacobson Stephen C. , Ramsey J. Michael 
Published: May 2005

AbstractA fused quartz microchip with a serpentine column geometry is fabricated to perform rapid microchip electrophoresis of dansylated amino acids. A 67 mm separation column is constructed in a 7 × 10 mm area on a quartz substrate using standard photolithographic, etching and deposition techniques. Buffer and sample flows within the channel manifold are precisely controlled through potentials applied to the reservoirs. To enhance the detection limits, a stacking injection technique is used to concentrate the sample at the inlet of the separation column. The stacked injections exhibit high reproducibility (2.1% relative standard deviation in peak area). Using a separation length of 67 mm and a separation field strength of 1100 V/cm, separations are performed in ⩽ 15 s generating ≈︁ 40 000 theoretical plates.
Solid‐phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis
Authors: Pinto Devanand M. , Arriaga Edgar A. , Sia Samuel , Li Zhong , Dovichi Norman J. 
Published: May 2005

AbstractThe fluorescent labeling of peptides at concentrations as low as 10−8 M can be achieved by using a solid‐phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post‐column laser‐induced fluorescence detection (mass limit of detection of 2.4 × 10−21 moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid‐phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.
Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries
Authors: Tadey Tanya , Purdy William C. 
Published: May 2005

AbstractThe addition of sodium dodecyl sulfate (SDS) micelles in the running buffer can be used to resolve mono‐ and diphosphorylated isomers of the insulin receptor peptide by capillary electrophoresis. The effect of SDS on peptide resolution is very dependent on pH. Complete resolution of three monophosphorylated isomers is achieved in uncoated capillaries filled with phosphate buffer containing 25 mM SDS and buffered at pH 6.1. Resolution of the diphosphorylated isomers can be significantly improved by using polyacryla‐mide coated capillaries. In coated capillaries electroosmotic flow is negligible and the migration order of the isomers is reversed. This allows for a longer period of interaction between the diphosphorylated isomers and the micelle and therefore selectivity is improved. Efficiency of all peptide isomers was also improved in coated capillaries due to reduced adsorption to the capillary wall.
Fused‐silica capillaries with surface‐bound dextran layer crosslinked with diepoxypolyethylene glycol for capillary electrophoresis of biological substances at reduced electroosmotic flow
Authors: Mechref Yehia , Rassi Ziad El 
Published: May 2005

AbstractThis report is concerned with the introduction of novel surface modification involving the covalent attachment of branched, high molecular weight dextrans and subsequent crosslinking with polyether chains on the inner surface of fused silica capillaries with the aim of producing hydrophilic capillaries with reduced electroosmotic flow (EOF). Three different molecular mass dextrans, namely 45, 71 or 150 kDa were covalently attached to the capillary surface, and subsequently crosslinked with diepoxypolyethylene glycol (PEG). This chemistry produced stable coating over a wide range of conditions including high and low pH aqueous solutions. Moreover, the various dextran‐PEG‐coated capillaries exhibited reduced electroosmotic flow. These features of the dextran‐PEG coatings were useful for the separations of basic and acidic proteins, and provided high resolution separation for closely related acidic monosaccharides and sialooligosaccharidesPresented as a poster at Electrophoresis Forum '94, Munich, Germany, October 24–26, 1994
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Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin
Authors: Wongkham Sopit , Taketa Kazuhisa , Liu Miao , Taga Hiroko 
Published: May 2005

AbstractThe oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two‐dimensional lectin affinity electrophoresis of α‐fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex‐type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man α→3 arm, monosialyl residue of the Man α1→6 arm, monosialyl residue of the Man α1→3 arm and bisecting GlcNAc, and disialyl residues. BMA did not recognize Neu5Ac α2→6 and Neu5Ac α2→3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy‐associated alteration of AFP sugar chains.