bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequencing by hybridization: Towards an automated sequencing of one million M13 clones arrayed on membranes
Authors: Drmanac Radoje , Drmanac Snezana , Labat Ivan , Crkvenjakov Radomir , Vicentic Aleksandra , Gemmell Anne 
Published: May 2005

AbstractAn immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864‐well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96‐well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13 824 samples on an 8 × 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200–2000 32P‐labeled probes comprising 16–256 10‐mers having a common 8‐mer, 7‐mer, or 6‐mer in the middle (20 probes per day, each hybridized with 250 000 dots); (v) scoring hybridization signals of 5 million sampleprobe pairs per day using storage phosphor plates; and (vi) computing clone order and partial‐to‐complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.
High‐throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory
Authors: Ansorge Wilhelm , Voss Hartmut , Wiemann Stefan , Schwager Christian , Sproat Brian , Zimmermann Jürgen , Stegemann Josef , Erfle Holger , Hewitt Neil , Rupp Thomas 
Published: May 2005

AbstractOne of the aims of the facility is to develop and push the automated on‐line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10–20, up to 1500 bases per hour per clone on ultrathin (about 100 μm) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein‐15‐*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with “walking” primers and F‐15‐*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.
Towards a fingerprint method covering the immunological genome
Authors: Gomolka Maria , Schwaiger Werner , Weyers Eva , Epplen Cornelia , Buitkamp Johannes , Epplen Jörg T. 
Published: May 2005

AbstractThe principle of a novel typing techniques is described for screening with high efficiency the “immunological genome” by using various intronic and exonic oligonucleotide hybridization probes simultaneously or subsequently.
Chromosomal localization of the HYP2‐gene in Saccharomyces cerevisiae and use of pulsed‐field gel electrophoresis for detection of irregular recombination events in gene disruption experiments
Authors: Wöhl Thorsten , Baur Monika , Friedl Anna A. , Lottspeich Friedrich 
Published: May 2005

AbstractIn the hypusine‐containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4‐amino‐2‐hydroxybutyl‐lysine). The HP has been designated as an eucaryotic translation initiation factor—eIF‐5A—because of its stimulating effect in the methionyl‐puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1‐gene is identical to the AN B1‐gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2‐gene has not been elucidated. By using pulsed‐field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2‐gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.
Plasma protein map: An update by microsequencing
Authors: Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Ravier Florence , Pasquali Christian , Sanchez Jean‐Charles , James Richard , Tissot Jean‐Daniel , Bjellqvist Bengt , Hochstrasser Denis F. 
Published: May 2005

AbstractThe reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two‐dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two‐dimensional gel database.
Using two‐dimensional gel electrophoresis to study immune response against intracellular bacterial infection
Authors: Kovarova Hana , Stulik Jiri , Macela Ales , Lefkovits Ivan , Skrabkova Zuzana 
Published: May 2005

AbstractPrincipal component analysis was applied to two‐dimensional (2‐D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so‐called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2‐D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.
Two‐dimensional electrophoresis analysis of human serum proteins during the acute‐phase response
Authors: Bini Luca , Magi Barbara , Cellesi Carla , Rossolini Aldo , Pallini Vitaliano 
Published: May 2005

AbstractThe serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute‐phase proteins were separated by two‐dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.
Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel
Authors: Chiari Marcella , Righetti Pier Giorgio , Negri Armando , Ceciliani Fabrizio , Ronchi Severino 
Published: May 2005

AbstractIt has been found that the double bond of free, unreacted acrylamide in a gel can react with a free ‐SH group of proteins, forming a cysteinyl‐S‐propionamide adduct. When β‐lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu‐149 to lle‐162. Residue No. 160 was found to be a cysteinyl‐S‐propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including ‐NH2 terminus and free ‐NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the ‐SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.
Chicory seed lot variety identification by leucine‐aminopeptidase and esterase zymogram analysis
Authors: Baes Peter , Van Cutsem Pierre 
Published: May 2005

AbstractA method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility‐density scheme. This quick, easy and low‐cost method can be a useful tool for chicory breeders.
Mouse liver protein database: A catalog of proteins detected by two‐dimensional gel electrophoresis
Authors: Giometti Carol S. , Taylor John , Tollaksen Sandra L. 
Published: May 2005

AbstractAlterations in the abundance or structure of mouse liver proteins are being studied using two‐dimensional gel electrophoresis (2‐DE) to build a database of protein changes correlating with exposure to ionizing radiation or toxic chemicals. Thus far, studies have included the analysis of proteins from the offspring of exposed parents or from the exposed individuals themselves. In order to characterize and identify proteins found altered by such exposures, sex‐ and strain‐related differences in protein patterns have been analyzed, and the subcellular locations of a large portion of the mapped proteins have been determined. As part of these studies, data are collected and stored using a variety of computer hardware and software tools that allow the accumulation of information on the origin of samples, gel identification, experiment description, and protein similarities and differences. This accumulation of information constitutes the mouse liver protein database. Relational database software is used to tie the different facets of the data‐base together so that the results of a variety of experiments can be compared and interrelated. The database optimizes the information obtained from 2‐DE gel sets by allowing use of the data for many purposes, including monitoring of gel resolution to ensure the collection of high quality data and correlation of protein effects induced by different agents. This first edition of the Argonne National Laboratory mouse liver protein database lays the foundation for future work and communication that should elucidate the significance of observed protein effects as possible markers of exposure to toxic agents.
Comparison of different staining methods for polyvinylidene difluoride membranes
Authors: Christiansen Jesper , Houen Gunnar 
Published: May 2005

AbstractSeveral new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl‐containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.
Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients
Authors: Bischoff Rainer , Roecklin Dominique , Roitsch Carolyn 
Published: May 2005

AbstractIsoelectric focusing in immobilized pH gradients (IEF‐IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion‐exchange and reversed‐phase chromatography proved to be homogeneous with regard to its isoelectric point (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI of 4.30. IEF‐IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human α1‐antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion‐exchange, metal chelate and hydrophobic‐interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF‐IPG between pH 5–6. The measured pI values of 5.43–5.58 were slightly lower than the calculated pI based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF‐IPG in a pH 7.3–8.3 gradient. It appeared to be heterogeneous with regard to its pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17. N‐Terminal amino acid sequencing as well as amino acid composition analysis were performed on the separated forms of p28 after electroblotting showing the feasibility of combining IEF‐IPG with subsequent analytical methods to obtain structural information.
Production of mitogen‐contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis
Authors: Zimmermann Ulrich , Klöck Gerd , Federlin Konrad , Hannig Kurt , Kowalski Matthias , Bretzel Reinhard G. , Horcher Andrea , Entenmann Heike , Sieber Ulrike , Zekorn Tobias 
Published: May 2005

AbstractCommercial alginates consisting of variable homopolymeric regions of β‐D‐mannuronic acid and α‐L‐guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross‐linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10–20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross‐linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.
High resolution two‐dimensional mapping of tissue‐specific polypeptides in the desert locust, Schistocerca gregaria
Authors: Annadurai Ramasamy Sambasivam , Oberthür Walter , Remboldm Heinz 
Published: May 2005

AbstractHigh resolution two‐dimensional gel electrophoresis (2‐DE) using immobilized pH gradients was used to map the tissue‐specific polypeptides of the desert locust, Schisiocerca gregaria. Highly specific comprehensive 2‐DE reference maps (“master gels”) were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4–7 range in the first dimension and within the 14–94 kDa molecular mass range in the second dimension.
Photopolymerization of polyacrylamide gels with methylene blue
Authors: Lyubimova Tatyana , Caglio Silvia , Gelfi Cecilia , Righetti Pier Giorgio , Rabilloud Thierry 
Published: May 2005

AbstractPhotopolymerization of polyacrylamide gels in the presence of methylene blue (100 μM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 μM dipheriyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross‐linker). It is found that methylene blue‐activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate‐activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.
Theoretical studies on the mobility‐shift behavior of binary protein‐DNA complexes
Authors: Cann John R. 
Published: May 2005

AbstractThe theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility‐shift behavior of binary protein‐DNA complexes. Several systems have been modeled in terms of either (i) specific binding of a protein molecule to a single site on the DNA molecule; (ii) cooperative binding to two or three sites; (iii) noncooperative binding to two sites, both of which bind protein with equal affinity; (iv) statistical binding to multiple sites having identical intrinsic binding constants; or (v) protein‐induced DNA loop formation. Both models (iii) and (v) embody the concept of reversible isomerization of protein‐DNA complexes. The resulting simulations have provided fundamental information concerning (i) the factors governing the electrophoretic persistence and separation of protein‐DNA complexes; (ii) the shape of experimental mobility‐shift patterns; (iii) the generation of the protein‐DNA ladder upon titration, for example, of the 203‐base pair operator with lac repressor; and (iv) the theoretical bases for quantitative interpretation of the patterns in terms of thermodynamic and kinetic parameters. The practical implications of these findings are discussed.
Peptide dot immunoassay and immunoblotting: Electroblotting from aluminum thin‐layer chromatography plates and isoelectric focusing gels to activated nitrocellulose
Authors: Lauritzen Edgar , Másson Már , Rubin Inger , Bjerrum Ole J. , Holm Arne 
Published: May 2005

AbstractNitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6‐diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6–10 amino acid residues were dotted directly onto the matrix at 45°C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase‐coupled anti‐rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin‐layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid. The versatility of dot immunobinding on activated nitrocellulose was shown with two sera from patients infected with human immunodeficiency virus, HIV‐1 and HIV‐2, by using pH 10.2 in incubation media, resulting in a low background. These sera were bound specifically to either one of the closely related HIV‐1 and HIV‐2 peptide antigens from the two viruses.
Chemical activation of nitrocellulose membranes for peptide antigen‐antibody binding studies: Direct substitution of the nitrate group with diaminoalkane
Authors: Másson Már , Lauritzen Edgar , Holm Arne 
Published: May 2005

AbstractA method to covalently link peptide and proteins, through a diaminoalkane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by covalent incorporation of diaminoalkane spacers without any prior activation. The incorporation was shown primarily to involve α‐elimination of the nitrate groups, and an imine was formed between the carbonyl group on the membrane and the diaminoalkane. The rate of incorporation increased exponentially with the length of the diaminoalkane as determined by a ninhydrin colorometric reaction, which was developed for the study. More than 200 nmole diamine per mg nitrocellulose could be incorporated, but less than 11 nmol/mg (63 nmol/cm2) was chosen in order to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptide, angiotensin II, was covalently bound. A dot immunoassay was performed where specific anti‐angiotensin II antibodies reacted with the peptide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 μg All per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased retention of radiolabeled [125I]angiotensin II.
Western blot as a tool in the diagnosis of Lyme borreliosis
Authors: Zöller Lothar , Cremer Josef , Faulde Michael 
Published: May 2005

AbstractBorrelia burgdorferi is the causative agent of Lyme borreliosis, a multisystem disorder, which can mimic numerous immune disorders and inflammatory diseases. Laboratory diagnosis of Borrelia infection relies on immunodiagnostic assays, which, however, are hampered by unsatisfactory specificity. The Western blot technique has been employed to analyze the humoral immune response in Lyme borreliosis and is used as a serodiagnostic confirmation test. The most important immunodominant proteins of Borrelia burgdorferi are the 94 kDa, 60 kDa, 41 kDa (flagellin), 34 kDa (Osp B), 31 kDa (Osp A), 30 kDa, 21 kDa (Osp C), and 17/18 kDa proteins. Whereas the 60 kDa, 41 kDa, and 34 kDa constituents reveal a marked cross‐antigenicity with other spirochetes and even more distantly related bacteria, antibodies against the 94 kDa, 31 kDa and 21 kDa proteins are largely species‐specific. The early immune response in Lyme borreliosis is triggered mainly by the flagellin. In the later stage a wide range of immunogenic proteins is involved, with the 94 kDa antigen being the best marker for late immune response. If the Western blot is used for diagnostic purposes the differences between early and late‐stage immunogenicity of Borrelia proteins must be taken into account. Interpretation criteria for blot positivity in early‐stage borreliosis are primarily based on the presence of the 21 kDa band and the semiquantitatively recorded intensity of the 41 kDa band. In the diagnosis of late‐stage infection, blot positivity relies on the presence of the 94 kDa, 39 kDa, 31 kDa, 30 kDa and 21 kDa bands.