bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular modeling of acrylamide derivatives: The case of N‐acryloylaminoethoxyethanol versus acrylamide and trisacryl
Authors: Miertus Stanislav , Righetti Pier Giorgio , Chiari Marcella 
Published: May 2005

AbstractMolecular mechanics (for evaluation of total energies of individual structures of monomers and oligomers) and molecular dynamics (for evaluating dynamic dependencies of structural features) were used for obtaining information on some unique chemical behavior of a novel N‐substituted acrylamide (N‐acryloylaminoethoxyethanol; AAEE) vs. conventional acrylamide and trisacryl (N‐acryloyl‐2‐amino‐2‐hydroxymethyl‐1, 3‐propane diol, an extremely hydrophilic derivative; Tris‐A). As free monomers, Tris‐A degrades with zero‐order and acrylamide with first‐order kinetics, whereas AAEE is highly resistant to hydrolysis. It is found that Tris‐A (and its dideoxy derivative) is constantly forming hydrogen bonds between the OH groups and the carbonyl of the amido group (bond distances of 1.64 to 1.70 Å); this activates a mechanism of “NO acyl transfer” which leads to quick degradation of the amido bond even under mildly alkaline conditions. Conversely, AAEE (which also contains an ω‐OH group increasing its hydrophilicity) has no tendency to form H‐bonds with the amido carbonyl, thus being resistant to the above degradation mechanism. In fact, the oxygen in the ethoxy moiety of the N‐substituent chain acts as a preferential partner for H‐bond formation with the ω‐OH group. In the oligomeric state, it is found that Tris‐A (tetrameric and dodecameric structures were simulated) tends to form inter‐residue H‐bonds (approximately parallel to the growing chain) competing with the intra‐residue H‐bonds (folding onto the amido carbonyl and approximately perpendicular to the oligomer chain), thus greatly increasing its stability. However, none of these polymeric structures can compete with a poly(AAEE) matrix, which still shows a 500‐fold greater resistance to alkaline hydrolysis.
Fractionation of human serum proteins by capillary and recycling isotachophoresis
Authors: Caslavska Jitka , Gebauer Petr , Thormann Wolfgang 
Published: May 2005

AbstractThe fractionation of human serum proteins using capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) in presence of low molecular mass spacer compounds is reported. Anionic CITP was performed in an instrument equipped with a Teflon capillary of 0.5 mm ID as well as in an apparatus which features an open‐tubular fused‐silica capillary of 75 μm ID. RITP was performed in a recycling fast flow focusing apparatus in which fluid flows rapidly through a narrow, rectangular cell and the effluent from each outlet port is reinjected into the electrophoresis chamber through the corresponding input port. Typically, 1 mL serum was processed batchwise within about 2.5 h prior to collection of 30 fractions of about 4 mL each. The fractions were analyzed separately for conductivity, pH and UV absorbance and selected fractions were characterized by an immunoassay for transferrin, as well as by gel isoelectric focusing, two‐dimensional gel electrophoresis, CITP and capillary zone electrophoresis. The search for suitable electrolyte systems and spacers was executed by CITP and by computer simulation. For simple configurations, separations predicted by simulation are shown to qualitatively agree with fractionation performed by CITP and RITP. Configurations producing three protein subgroups, the first containing mainly albumin, the second transferrin and the third the globulins, are discussed.
The use of protein synthesis elongation factor EF‐Tu as internal calibration standard in two‐dimensional electrophoretic studies of differentiation in Streptomyces
Authors: Xin ‐ming Li , Vohradský Jiři , Weiser Jaroslav 
Published: May 2005

AbstractProtein synthesis elongation factor EF‐Tu is presented as an internal calibration standard for quantitative analysis of two‐dimensional (2‐D) protein electrophoresis gels. EF‐Tu was selected on the basis of concentration measurements in cell‐free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one‐dimensional (1‐D) and 2‐D electrophoresis gels. The results demonstrated that the amount of EF‐Tu synthesised in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF‐Tu an ideal internal protein standard for quantitation of protein spots on 2‐D electrophoresis gels. For such quantitative analysis we developed a computer‐aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.
Computer simulation of pulsed field gel runs allows the quantitation of radiation‐induced double‐strand breaks in yeast
Authors: Kraxenberger Alfred , Friedl Anna A. , Kellerer Albrecht M. 
Published: May 2005

AbstractA procedure for the quantification of double‐strand breaks in yeast is presented that utilizes pulsed field gel electrophoresis (PFGE) and a comparison of the observed DNA mass distribution in the gel lanes with calculated distributions. Calculation of profiles is performed as follows. If double‐strand breaks are produced by sparsely ionizing radiation, one can assume that they are distributed randomly in the genome, and the resulting DNA mass distribution in molecular length can be predicted by means of a random breakage model. The input data for the computation of molecular length profiles are the breakage frequency per unit length, α, as adjustable parameter, and the molecular lengths of the intact chromosomes. The obtained DNA mass distributions in molecular length must then be transformed into distributions of DNA mass in migration distance. This requires a calibration of molecular length vs. migration distance that is specific for the gel lane in question. The computed profiles are then folded with a Lorentz distribution with adjusted spread parameter Γ to account for and broadening. The DNA profiles are calculated for different breakage frequencies α and for different values of Γ, and the parameters resulting in the best fit of the calculated to the observed profile are determined.
Focusing of alkaline proteases (subtilisins) in pH 10–12 immobilized gradients
Authors: Bossi Alessandra , Righetti Pier Giorgio , Vecchio Giuseppe , Severinsen Steen 
Published: May 2005

AbstractIsoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9–11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10–12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 ± as 0.15; that of Durazym as 10.95 ± 0.20 and that of the pI marker cytochrome c as 10.6 ± 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10°C, instead of the tabulated values at 25°C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a pI of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10°C) of 11.15.
Sample application by in‐gel rehydration improves the resolution of two‐dimensional electrophoresis with immobilized pH gradients in the first dimension
Authors: Rabilloud Thierry , Valette Cecyl , Lawrence Jean Jacques 
Published: May 2005

AbstractWe describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5%T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 μL for a standard 3 × 160 × 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design.
Restriction fragment length polymorphism: Molecular weight analysis and calculation with a scanner‐based computer system
Authors: Luckenbach Christine , Luckenbach Alexander , Ritter Horst 
Published: May 2005

AbstractFor an exact and reproducible molecular weight calculation, an essential requirement in all applications of DNA profiling, we present a new system, called RFLP‐MAC (restriction fragment length polymorphism – molecular weight analysis and calculation). The system is composed of a sensitive digital gray‐scale scanner unit and an Apple Macintosh computer supported by a powerful combination of a shareware and self‐developed software package. Molecular weights are calculated by profiling the optical density and band particle processing parameters to identify the unknown DNA fragments. Mathematical models were used to interpolate the transverse and horizontal migration directions of the molecular weight standards, insuring consistent results from gel to gel. All data are stored in a relational multi‐user database system. Descriptive statistics are performed and allele frequencies are analyzed. Procedures for image‐analysis, molecular weight calculation, statistical evaluations and database management were developed and run within the multitasking environment of 4th Dimension. Two sets of data, intragel and intergel measurements, are investigated in order to check the precision of the RFLP‐MAC system. All these data are subjected to a standard analysis of variance resulting in a maximal 3 SD value of 0.005 (0.288%) for the intragel and of 0.091 (1.654%) for the intergel variation.
Multilocus DNA fingerprinting: The independence problem in quantitative paternity testing
Authors: Krawczak Michael 
Published: May 2005

AbstractA simulation study was performed in order to determine whether or not the assumption of independence, made in the quantitative analysis of multilocus DNA fingerprints, represents an inadmissible over‐simplification. A total of 10000 cases of true and false paternity, respectively, were simulated in twenty replicas of various genetic models. Log‐likelihood ratios (paternity vs. nonpaternity; LR) were calculated using published likelihood formulae and assuming position‐wise independence. The resulting LR distributions were compared to (i) the results of a classical analysis of the underlying genotype data, and (ii) the distributions expected from the likelihood model employed in the LR calculations. Although considerable discrepancies were observed between these distributions, decision making about paternity appeared to be only marginally affected, especially when only a fraction of each multilocus DNA fingerprint was analyzed.
Poly(ethylene glycol)methacrylate‐acrylamide copolymer media for hydrophobic protein and low temperature electrophoresis
Authors: Harrington Michael G. , Zewert Thomas E. 
Published: May 2005

AbstractThe properties of poly(ethylene glycol)methacrylate‐acrylamide copolymer media (PEGMACs) were exploited in two ways: (i) in the first‐dimensional gel of two‐dimensional electrophoresis (2‐DE) of hydrophobic proteins and (ii) for high speed, high‐resolution electrophoresis at low temperatures. In the first application, improved resolution and yield of isoelectric focusing (IEF) separations for the hydrophobic protein zein was achieved compared to IEF in standard polyacrylamide gels. This appears promising as a candidate approach for higher resolution 2‐DE mapping of uncharacterized hydrophobic proteins. In the second application, PEGMACs compatible with hydroorganic antifreeze buffer systems bellow cooling of the gels to low temperatures (−20°C), which allowed greater current to be tolerated during electrophoresis. PEGMAC gels enabled us to perform sixfold faster electrophoretic separations and achieve threefold improved resolution of six standard proteins at the lower temperatures in a direct comparison with the normal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) system. If this approach is coupled with more precise instrumentation to control low temperatures during electrophoresis, greater separation speeds and resolution may be anticipated.
Separation of long DNA fragments by capillary gel electrophoresis with laser‐induced fluorescence detection
Authors: Nishikawa Tetsuo , Kambara Hideki 
Published: May 2005

AbstractLong DNA fragments were separated by capillary gel electrophoresis with laser‐induced fluorescence detection, and two approaches to obtaining high‐resolution separation of DNA fragments were examined. One method achieved high resolution at the expense of detection time, while the other achieved both high resolution and fast detection. The former approach used a 300 cm long capillary with a gel concentration of 4% T and an electric field strength of 70 V/cm. The resolution limit in this electrophoresis was 800 bases with a resolution value of 0.5 for adjacent peaks. Under these conditions, the measurement time was 44 h. The latter approach used a 200 cm long capillary with a gel concentration of 3% T and an electric field strength of 170 V/cm. The resolution limit in this electrophoresis was 680 bases with a resolution value of 0.5 for adjacent peaks, and the measurement time was only 10 h. With both approaches, fragments with less than 600 bases were efficiently separated. The resolution values for adjacent peaks with less than 500 bases are greater than 1.0, and those for peaks with less than 100 bases are greater than 2.7. These approaches thus improve the accuracy of the base sequence determination.
Effect of operational variables on the separation of proteins by capillary sodium dodecyl sulfate‐gel electrophoresis
Authors: Guttman András , Shieh Paul , Hoang Dao , Horváth Judith , Cooke N. 
Published: May 2005

AbstractCapillary sodium dodecyl sulfate (SDS)‐gel electrophoresis, for fast and high‐resolution separations of protein molecules based on their molecular masses was investigated, employing a polymeric sieving network of polyethylene oxide (PEO). A standard mixture of five proteins ranging from 14.2‐66.0 kDa in molecular mass was used to evaluate the sieving matrix. A general migration velocity equation was derived for capillary SDS‐gel electrophoresis of proteins and supported by the experimental data. This equation gives a better understanding of SDS‐protein separations via capillary gel electrophoresis. Results are presented regarding the effects of different operational variables such as gel concentration, electric field strength, molecular mass, and temperature on the electrophoretic migration properties of the different size protein molecules.
Capillary electrophoresis monitoring of the competitive adsorption of albumin onto the orosomucoid‐coated polyisobutylcyanoacrylate nanoparticles
Authors: Olivier Jean‐Christophe , Taverna Myriam , Vauthier Christine , Couvreur Patrick , Baylocq‐Ferrier Danielle 
Published: May 2005

AbstractThe simultaneous and rapid quantitation of a glycoprotein, human orosomucoid, and a protein, human serum albumin (HSA), by micellar electrokinetic capillary electrophoresis was developed and optimized (pH and sodium dodecyl sulfate concentration) in order to study the competitive adsorption of HSA onto orosomucoid‐coated nanoparticles of polyisobutylcyanoacrylate. This method provided satisfactory repeatability and the response was linear for concentrations of orosomucoid and HSA ranging from 0.025 to 0.500 mg/mL (r > 0.994 for both proteins). To study the competitive adsorption of HSA, the amounts of free orosomucoid and free HSA were determined in the supernatant obtained after centrifugation of the orosomucoid‐coated nanoparticle suspension incubated with HSA. Furthermore, the quantities of both orosomucoid and HSA adsorbed onto nanoparticles were also determined after desorption of these molecules from the nanoparticle surface, using sodium dodecyl sulfate. No analytical interference was observed with this surfactant. The results showed that HSA did not at all adsorb onto the orosomucoidcoated nanoparticles and, thus, that this protein did not displace the layer of adsorbed orosomucoid from the nanoparticle surface.
Analysis of the proteins of aging Caenorhabditis elegans by high resolution two‐dimensional gel electrophoresis
Authors: Vanfleteren Jacques R. , De Vreese Annemie 
Published: May 2005

AbstractThe metabolic rate decreases dramatically as a function of age in the nematode worm Caenorhabditis elegans. Superoxide anion production, which is tightly linked to oxygen consumption, and thus to metabolic rate, drops to a 20‐fold lower level in 10‐day‐old, senescent worms, as compared to 4‐day‐old young adults. In a long‐lived mutant strain of the same species metabolic activity is much better preserved. High resolution two‐dimensional gel electrophoresis was employed to study alterations in the protein profile, correlating with changes of metabolic activity. Surprisingly, few proteins show age‐ or age‐ and strain‐specific variations of spot intensity. The abundance of the huge majority of proteins displayed on these gels remains unaltered, irrespective of age and strain differences. These results imply that there are no major age‐related alterations of proteins due to faulty protein synthesis or free radical attack, and that age‐related changes in the rate of protein synthesis and breakdown must be strictly coordinated throughout the aging process.
Two‐dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: Application of mass spectrometry to peptide‐mass fingerprinting
Authors: Ji Hong , Whitehead Robert H. , Reid Gavin E. , Moritz Robert L. , Ward Larry D. , Simpson Richard J. 
Published: May 2005

AbstractProtein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two‐dimensional electrophoresis gels (2‐DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75–80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt‐like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (Mr ∼ 18 000, pI 6.7–6.9) and b (Mr ∼ 24 000, pI 5.9–6.0), were detected in the 2‐DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide‐mass fingerprinting, utilizing capillary reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (Mr ∼ 66 000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide‐mass database.
Identification of transformation sensitive proteins recorded in human two‐dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing
Authors: Rasmussen Hanne H. , Mørtz Ejvind , Mann Matthias , Roepstorff Peter , Celis Julio E. 
Published: May 2005

AbstractA comprehensive human keratinocyte two‐dimensional (2‐D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2‐D gel patterns as well as to reveal hitherto unknown proteins. To date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up‐ or down‐regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis of high performance liquid chromatography (HPLC) purified peptides or by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification allowing fast screening of proteins recorded in 2‐D gel databases. The mass spectrometric approach when combined with microse‐quencing strengthened identification, and added the possibility of full characterization of post‐translational modifications and sequence variations.