bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant‐associated protein A
Authors: Phelps David S. , Floros Joanna 
Published: May 2005

AbstractStudies from several laboratories involving amino acid analysis and sequencing of the Mr 35 000 pulmonary surfactant‐associated proteins (SP‐A) have detected hydroxyproline residues. These residues are present in a region with a collagen‐like sequence that has been revealed by direct amino acid sequencing and from the deduced amino acid sequence of the cDNA clones coding for SP‐A. We treated human lung tissue with tunicamycin to block N‐glycosylation and with 2,2‐dipyridyl to inhibit the hydroxylation of proline residues. The SP‐A synthesized under these conditions showed a shift in apparent molecular weight to 27 000 and 29 000 compared to 29 000 and 31 000 for SP‐A synthesized in the presence of tunicamycin alone. Dipyridyl treatment alone caused an alteration in electrophoretic mobility similar to that seen with tunicamycin, although this was more difficult to evaluate since changes in molecular weight due to glycosylation occurred under these conditions. These results indicate that proline hydroxylation in the collagen‐like portion of SP‐A decreases its electrophoretic mobility.
Improvement and simplification of low‐background silver staining of proteins by using sodium dithionite
Authors: Rabilloud Thierry , Carpentier Gilles , Tarroux Philippe 
Published: May 2005

AbstractHigh sensitivity and low background, the attractive characteristics of the procedure of Blum et al., Electrophoresis 1987, 8, 93–99, for silver staining of proteins in polyacrylamide gels have been improved by sensitizing the gels with sodium dithionite instead of sodium thiosulfate and by equilibration in water after fixation and prior to sensitization. These modifications decrease the background and allow for a longer development period, which in turn increases sensitivity and color contrast. In addition, the colors of the spots are shifted toward colder tones when compared with the original method.
Determination of molecular mass, stokes' radius, frictional coefficient and isomer‐type of non‐denatured proteins by time‐dependent pore gradient gel electrophoresis
Authors: Rothe Gunter M. 
Published: May 2005

AbstractMolecular mass, Stokes' radius, frictional coefficient and isomer‐type of non‐denatured proteins can be obtained by time‐dependent gradient gel electrophoresis by evaluating the resulting data using a two‐step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non‐denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49) from current‐year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.1.1.42) and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current‐year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose‐6‐phosphate dehydrogenase isozyme than those from non‐injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present‐day air and soil pollutants.
N‐terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients
Authors: Aebersold Ruedi H. , Pipes Gary , Hood Leroy E. , Kent Stephen B. H. 
Published: May 2005

AbstractIsolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis or two‐dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N‐terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N‐terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically‐modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas‐phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow‐bore reversed‐phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.
Microheterogeneity of human serum transferrin: A biological phenomenon studied by isoelectric focusing in immobilized pH gradients
Authors: de Jong Gerard , van Eijk Hendrik G. 
Published: May 2005

AbstractThe heterogeneity of human transferrin results from (i) differences in iron content, (ii) genetic polymorphism and (iii) differences in the carbohydrate moiety. This article primarily deals with the last phenomenon, the microheterogeneity of human transferrin. Owing to the comparatively simple carbohydrate structure of human transferrin and the high resolving power of isoelectric focusing in immobilized pH gradients, microheterogeneous forms of transferrin can be separated. Differences between samples can be quantitated by crossed immunoelectrophoresis. Examples of the differences between the microheterogeneity patterns of transferrin in several biological fluids and the changes that can be observed in diseases such as rheumatoid arthritis, idiopathic hemochromatosis and Kahler's disease are presented. Special attention has been focused on changes occurring during pregnancy.
Two‐dimensional electrophoresis as a tool for structural and genetic studies of seed proteins from Poaceae and Fagaceae
Authors: Garcia‐Olmedo Francisco , Salcedo Gabriel , Aragoncillo Cipriano , Sánchez‐Monge Rosa , Collada Carmen , Gómez Luis 
Published: May 2005

AbstractThe application of two‐dimensional electrophoretic procedures to structural and genetic studies of seed proteins from Poaceae (including the cultivated cereals) and Fagaceae is described. The following related problems have been considered: covalent and non‐covalent association of protein subunits in multiple oligomeric structures; chromosomal locations of genes encoding seed proteins; quantitation of gene products in relation to gene expression and regulation; purification of protein components to study their homology relationships and in vitro activities; evolutionary and phylogenetic relationships; identification of genetic stocks. Isoelectric focusing, pore‐gradient electrophoresis, electrophoresis at different pH's, are among the separation procedures used in the first dimension, whereas sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and starch‐gel electrophoresis at acid pH have been the preferred second‐dimensional methods. Dissociating conditions (sodium dodecyl sulfate, Nonidet P‐40, or urea) and reducing conditions (2‐mercaptoethanol) have been used when required.
Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa
Authors: Krochko Joan E. , Bewley J. Derek 
Published: May 2005

AbstractHoloprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two‐dimensional electrophoretic techniques. The variety of two‐dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non‐seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin‐like globulin), alfin (a vicilin‐like globulin) and a family of lower molecular weight albumins (LMW1–3). These comprise 30 %, 10 %, and 20 %, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr ∼ 150 000) composed of polypeptides ranging in size from Mr 14 000 to 50 000 (α1‐α6; 50 000, 38 000, 32 000, 20 000, 16 000 and 14 000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1–A9; Mr 49 000 to 39 000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24 000, 23 000 and 20 000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1 /B2). Subunits (Mr ∼ 50 000–65 000) are assembled as trimers (8S) or larger oligomers (12S–15S) in mature seeds. The lower molecular weight albumins (LMW1–3) are acidic (pI < 6), and consist of sets of disulphide‐bonded polypeptides (Mr 15 000 and 11 000).
Improvement of electrophoretic transfer by casting acrylamide gels on a cellophane sheet
Authors: Abbal Michel , Julie Claudine 
Published: May 2005

AbstractElectrophoretic transfer of protein after isolectric focusing using a polyacrylamide gel of less than 0.5 mm is difficult if the gel is backed to an electrically nonconductive casting support, such as glass plate or plastic films. By casting the gel on a cellophane sheet, it is not necessary to remove the gel from the support prior to electrophoretic transfer. The use of a cellophane support does not alter the quality of the final pattern.
Fully automated capillary isotachophoresis of proteins
Authors: Manabe Takashi , Yamamoto Hideko , Okuyama Tsuneo 
Published: May 2005

AbstractAn apparatus for fully automated capillary isotachophoresis was constructed. A commercial apparatus (Shimadzu IP‐2A) was modified in the electrolyte pumping system and the flow lines were simplified. An automatic sampler was used for sequential sampling. The equipment, pumps, the sampler, a high‐voltage DC power supply, and a recorder, were controlled by a system controller which comprises a microcomputer and a BASIC program for time‐control of the equipments. The apparatus was successfully used for the automated sequential analysis of human serum proteins. Forty serum samples were analyzed within 17 h without manual operation and for each sample the serum proteins were resolved into about twenty UV peaks or shoulders.
Transverse alternating field electrophoresis and applications to mammalian genome mapping
Authors: Gardiner Katheleen , Patterson David 
Published: May 2005

AbstractThe transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes from S. pombe and S. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.
Gel electrophoresis method for quantitation of gamma ray induced single‐ and double‐strand breaks in DNA irradiated in vitro
Authors: Chen Chun‐Zhang , Sutherland John C. 
Published: May 2005

AbstractWe describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single‐ and double‐strand breaks formed by γ‐rays for DNA irradiated in vitro. For single‐strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double‐strand break doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single‐strand breaks is linear while the dose response function for double‐strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.
Direct N‐terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Benjamin Timothy , Niu Chien‐Hua , Parmelee David C. , Huggett Anthony C. , Yu Betty , Roller Peter P. , Thorgeirsson Snorri S. 
Published: May 2005

AbstractNine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after transblotting to Immobilon‐P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N‐terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N‐termini. Prior to electrophresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2‐D PAGE using pH 4–6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon‐P membranes and located by staining with Coomassie Brilliant Blue R‐250. Our results demonstrate that N‐terminal sequencing (gas‐phase) can be achieved on polypeptides obtained from approximately 250 μg of total glycoproteins applied to a single 2‐D gel.
Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain
Authors: Jungblut Peter , Zimny‐Arndt Ursula , Klose Joachim 
Published: May 2005

AbstractProteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two‐dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (> 30 %) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (< 20 %) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1 % vs. 0.2 %; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3 %) than in brains (0.0 %), and higher in solubilized nuclear proteins (5.3 %) than in structure‐bound nuclear proteins (2.1 %).
Human alpha‐1‐antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel
Authors: Alonso Antonio 
Published: May 2005

AbstractThe polymorphism of alpha‐1‐antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35–4.75 and 4.35–4.55, following rehydration with pH 4.2–4.9 carrier ampholytes. The use of the separator N‐(2‐acetamido)‐2‐aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M‐7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.
Microheterogeneity of orosomucoid in pathological conditions
Authors: Hansen John‐Erik S. , Bøg‐Hansen Thorkild C. , Pedersen Bo , Neland Keld 
Published: May 2005

AbstractStudies of orosomucoid (α1‐acid glycoprotein) in human serum have revealed that orosomucoid is a mixture of molecules with differences in the glycan chains. This microheterogeneity has been studied using crossed affinoimmuno‐electrophoresis with the lectin concanavalin A which binds to biantennary glycans. The relative proportions of the three orosomucoid subtypes are altered in various pathological conditions independently of the total serum orosomucoid concentration. There are reproducible differences in microheterogeneity patterns between some pathological conditions: Acute tissue injury or inflammation results in a high proportion of orosomucoid with biantennary glycans. Conditions with increased estrogen levels are associated with a high proportion of orosomucoid with tri‐ or tetraantennary glycans and a low total serum orosomucoid concentration. Chronic inflammation also seems to be associated with a high proportion of orosomucoid with tri‐ or tetraantennary glycans but with a high total serum concentration of orosomucoid. Other diseases, such as cancer, can not be associated with any specific microhetero‐geneity pattern. The microheterogeneity pattern in these conditions seems to be determined by disease activity and unspecific inflammation in surrounding tissues.
Lectin affinity electrophoresis of alkaline phosphatase for the differentiation of bone and hepatobiliary disease
Authors: Rosalki Sidney B. , Foo A. Ying 
Published: May 2005

AbstractAn affinity electrophoresis procedure is described for the separation and quantification of the bone‐ and liver‐derived fractions of alkaline phosphatase in plasma. Separation is carried out on cellulose acetate membrane pre‐soaked with buffer containing wheat germ lectin. The electrophoretic mobility of the bone enzyme is preferentially retarded by the lectin and this fraction is well separated from the liver fraction. After separation, enzyme activity is demonstrated by staining using an indigogenic alkaline phosphatase substrate incorporated in agar gel, and the stained fractions quantified by densitometry. The procedure has low imprecision, good linearity, and the activities of the bone and liver fractions correlate well with values obtained using nonelectrophoretic quantification methods. The procedure is especially suitable for use in the diagnostic laboratory.
Electrophoretic behavior and partial characterization of disease‐associated serum forms of gammaglutamyltransferase
Authors: Sacchetti Lucia , Castaldo Giuseppe , Salvatore Francesco 
Published: May 2005

AbstractWe have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma‐glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other, procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha1 and alpha2‐globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver‐involving neoplasias, we observed some disease‐specific GGT forms: an albumin comigrating enzyme (Alb‐GGT) specific of liver neoplasia; a gamma‐globulin comigrating GGT (gamma‐GGT) and a nonmigrating isoform (dep‐GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta‐gamma‐ and dep‐GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha1‐GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb‐GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins. The amount of GGT complexed with LDL + VLDL differs in various hepatobiliary diseases, and using a cutoff of 20 U/L it is possible to use this test to distinguish non‐cholestatic diseases from liver malignancies (i. e. in monitoring cirrhotics and other high risk groups of patients as to the possible evolution to liver cancer, in patients with gastrointestinal tumor which frequently metastasizes to the liver). Furthermore, treatment of GGT with neuraminidase confirms the association of sialic acid with all the GGT fractions, although Alb‐GGT is more sensitive to this treatment than is beta‐GGT. Our study, indicating the existence of clinical biochemistry correlations between isoenzyme patterns of serum GGT and diseases of hepatobiliary nature including neoplasias, opens up the possibility of enlarging the vocabulary of biochemical signals in these disorders. Furthermore, the lipoprotein‐precipitated fractions of serum GGT are an additional test for discriminating hepatocarcinoma from noncholestatic chronic hepatitis and cirrhosis.
Simultaneous phenotyping of erythrocyte acid phosphatase and esterase D by nonequilibrium agarose isoelectric focusing
Authors: Dimo‐Simonin Noëlle , Brandt‐Casadevall Conxita , Gujer Hans‐Rudolf 
Published: May 2005

AbstractNonequilibrium agarose isoelectric focusing in a pH 4.5–7 gradient with 3‐(N‐morpholinopropanesulfonic) acid (MOPS) and taurine as chemical separators presents a fast, easy and reliable method for the simultaneous determination of the common erythrocyte acid phosphatase and esterase D phenotypes in hemolysates and dried bloodstains. The application of the method to experimental and casework bloodstains is described.
Detection of human serum amyloid A protein in very low density – and high density lipoproteins of patients after acute myocardial infarction
Authors: Feussner Giso , Ziegler Reinhard 
Published: May 2005

AbstractTwo acute‐phase proteins have been identified in very low density (VLDL)‐ and high density lipoproteins (HDL) of patients after acute myocardial infarction. Both proteins have a relative molecular weight of 11 000 and isoelectric points pI 6.08 and 6.27, and do not contain cysteine or sugar residues. Polyclonal antibodies to these acute phase reactants did not cross‐react with other serum apolipoproteins. Evidence is given that both proteins are polymorphic forms of the human serum amyloid A protein.