bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of natural and recombinant isoforms of grass pollen allergens
Authors: Petersen Arnd , Grobe Kay , Lindner Buko , Schlaak Max , Becker Wolf‐Meinhard 
Published: May 2005

AbstractMore than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2‐DE blotting using patients' IgE and monoclonal antibodies, we detected IgE‐reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol 1 1): 32 and 34 kDa, respectively. By N‐terminal microsequencing we determined single amino acid substitutions in different‐sized group I allergens. The post‐translational modifications (one N‐glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross‐reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2‐DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post‐translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate contentPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996
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Capillary zone electrophoresis at subzero temperatures II: Chiral separation of biogenic amines
Authors: Ma Stacey , Horváth Csaba 
Published: May 2005

AbstractThe separation of enantiomer pairs of eight biogenic amines by capillary zone electrophoresis (CZE) was investigated with heptakis (2,6‐di‐O‐methyl)‐β‐cyclodextrin as the chiral selector at temperatures ranging from −20° to 40°C by using a commercial electrophoresis unit retrofitted with an external thermostated refrigerated circulating bath in order to assist the original cooling system. Sodium phosphate in both neat aqueous and methanolic media at pH 2.5, as measured by the glass pH electrode, were used with the fused silica capillary from 1.5° to 40°C and −20° to 40°C, respectively. The effect of temperature on enantioselectivity was found to depend on the number of phenolic hydroxyl groups in the molecule. Upon lowering the temperature from 40° to −20°C, the chiral selectivity of the system, as measured by the relative mobility difference, increased tenfold for the amines with two vicinal phenolic hydroxyls, whereas the increase was insignificant for those having no phenolic hydroxyl groups. The complex formation constants of three amines which have the same molecular structure but the number of phenolic hydroxyl groups were determined at different temperatures and the thermodynamic parameters as well as compensation temperatures for the process were evaluated. Whereas the compensation temperature was 690 K for the amine without phenolic hydroxyl group, it was < 400 K for the amines with one or two phenolic hydroxyl groups. The difference in the compensation temperatures indicates that the intrinsic mechanisms of their complexation with the chiral selector are not the same; this may account for the discrepancies observed in the temperature dependency of the chiral selectivity. The enthalpy change per phenolic hydroxyl group was 2.5 kcal mol−1, which compares favorably with the typical value for a single hydrogen bond. Therefore, when the amines have phenolic hydroxyl groups, the strong increase in chiral selectivity with decreasing temperature may be due to enhanced H‐bonding between the cyclodextrin and the phenolic hydroxyls under the conditions employed in this study.
Electrokinetic chromatography employing an anionic and a cationic β‐cyclodextrin derivative
Authors: Jakubetz Hansjörg , Juza Markus , Schurig Volker 
Published: May 2005

Abstractβ‐Cyclodextrin, 1, can act as a chiral buffer additive in capillary zone electrophoresis (CZE) owing to its ability to form diastereomeric complexes with the enantiomers of ionic compounds. Reaction of 1 with 2,3‐epoxy‐propyltrimethylammonium chloride gave preponderantly 6‐O‐(2‐hydroxy‐3‐trimethyl‐ammoniopropyl) derivatives (2) of varying degrees of substitution (d.s.). Analogous reaction of 1 with 1,3‐propanesultone yielded 6‐O‐(sulfo‐n‐propyl) derivatives (SPE‐β‐CD, 3) of varying d.s. The purity and the d.s. of 2 and 3 were determined by 1H NMR and electrospray ionization mass spectrometry (ESI‐MS). These charged β‐cyclodextrin derivatives were used for the chromatographic enantiomer separation of a series of neutral barbiturates, of chlorthalidone, terbutaline, warfarin, salbutamol and brompheniramine by cyclodextrin electrokinetic chromatography (CD‐EKC). During the separation of chlorthalidone with 3 as chiral additive, a reversible interconversion of the enantiomers (enantiomerization) was observed at temperatures below 20°C.
Enantioseparation of nonsteroidal anti‐inflammatory drugs by capillary electrophoresis using mixtures of anionic and uncharged β‐cyclodextrins as chiral additives
Authors: Fillet Marianne , Hubert Philippe , Crommen Jacques 
Published: May 2005

AbstractNine nonsteroidal anti‐inflammatory drugs (NSAIDs) were enantioseparated by capillary electrophoresis using an anionic cyclodextrin derivative (sulfobutyl ether β‐cyclodextrin or carboxymethyl‐β‐cyclodextrin) in combination with a neutral cyclodextrin as chiral additives to a pH 3 phosphoric acid‐triethanolamine buffer. In the presence of a negatively charged cyclodextrin, the analytes were given an appropriate mobility but relatively low enantioselectivities were generally obtained when such a cyclodextrin was the only selector added to the buffer. The addition of an uncharged cyclodextrin, such as the native β‐cyclodextrin or one of its derivatives (dimethyl‐, trimethyl‐ and hydroxypropyl‐β‐cyclodextrin), to this kind of buffer containing an anionic cyclodextrin, was found to give rise to considerable improvement in chiral resolution for all compounds studied. Resolution and analysis time were optimized by varying the nature and concentration of the two cyclodextrins. The best compromise was usually achieved by the simultaneous addition of sulfobutyl ether β‐cyclodextrin and trimethyl‐β‐cyclodextrin. Under optimum conditions, the enantiomers of all NSAIDs examined could be completely separated (most often with resolution values higher than 5) in short analysis times (generally lower than 15 min).
Analysis of the binding of deoxyribonuclease I to G‐actin by capillary electrophoresis
Authors: Carter Laura K. , Christopherson Richard I. , dos Remedios Cristobal G. 
Published: May 2005

AbstractDeoxyribonuclease I (DNase I) is an actin monomer‐sequestering actin binding protein (ABP) that inhibits the rate and extent of actin polymerisation in vitro by forming a high affinity, stoichiometric 1:1 complex. Using capillary zone electrophoresis (CZE), we have studied the interaction between G‐actin and DNase I to evaluate the capability of CZE to determine the dissociation constant (Kd value) for this interaction. We used (i) an uncoated fused‐silica capillary and ultraviolet (UV) detection at 214 nm; (ii) a hydrophilic‐coated capillary with UV detection at 214 nm; and (iii) a hydrophilic‐coated capillary with laser‐induced fluorescence (LIF) detection. Using procedure (ii), a Kd value of approximately 0.03 μM was obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination of Kd values from the micromolar range to the nanomolar range. Subsequent determination of Kd values for other actin‐binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatial relationship.
Escherichia coliproteome analysis using the gene‐protein database
Authors: VanBogelen Ruth A. , Abshire Kelly Z. , Moldover Brian , Olson Eric R. , Neidhardt Frederick C. 
Published: May 2005

AbstractThe gene‐protein database ofEscherichia coliis a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate theE. coliproteome including a project to localize on 2‐D gels all of the open reading framed encoded by theE. colichromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.
Towards a proteome project of cyanobacterium Synechocystis sp. strain PCC6803: Linking 130 protein spots with their respective genes
Authors: Sazuka Takashi , Ohara Osamu 
Published: May 2005

AbstractFollowing the complete sequencing of the genome of the univellular cyanobacterium, Synechocystis sp. strain PCC6803, within our institute, a protein‐gene linkage map of this photosynthetic microorganism was successfully constructed for 130 high abundance proteins present on two‐dimensional gels. An additional six proteins were analyzed, but were probably encoded extrachromosomally. In order to demonstrate the usefulness of this protein‐gene linkage map, we analyzed the changes that occur in cellular proteins after illumination of PCC6803 cells. The results indicate that this protein‐gene linkage map greatly simplifies the identification process of such modulated genes. After illumination, at least three distinctive spots with reduced intensity were detected on two‐dimensional gels and the corresponding genes of two of these were successfully identified as chaperonin 2 and a Tortula ruralis rehydrin‐related gene. Thus, the combination of the protein‐gene linkage map and two‐dimensional gel electrophoresis should permit a comprehensive analyses of the proteins encoded by the genome (i.e., “proteome”) of this photosynthetic autotroph. This post‐genome project represents a productive way of exploiting the information obtained from the sequencing of the cyanobacterium genome.
Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients
Authors: Cordwell Stuart J. , Basseal David J. , Bjellqvist Bengt , Shaw Denis C. , Humphery‐Smith Ian 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3–10 and narrow range pH 4–7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6–11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2‐D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well‐resolved or visible using wide‐range pH 3–10 IPG gel strips. Twenty‐seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N‐terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5–10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI ≈︁ 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6–11) IPGs has allowed the visualisation of a significantly greater percentage of the ‘functional proteome’, that portion of the total protein complement of a genome actively translated within a specific time frame, on 2‐D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.
Global analysis of physiological responses in marine bacteria
Authors: Östling Jörgen , McDougald Diane , Marouga Rita , Kjelleberg Staffan 
Published: May 2005

AbstractIn this paper, we present results from studies on marine Vibrio species, in which complex adaptive responses have been investigated. The results of two‐dimensional polyacrylamide gel electrophoresis serve to illustrate the usefulness of a global approach, and how it can be combined with other methodologies in order to achieve an improved understanding of the means by which bacteria adapt to alterations in environmental conditions. The overall strategies described in this paper are particularly useful for studies of bacteria for which efficient genetic tools, background genotypes and in depth physiological data are not yet available.
Development of a Haemophilus two‐dimensional protein database
Authors: Cash Phillip , Argo Evelyn , Langford Paul R. , Kroll John S. 
Published: May 2005

AbstractMembers of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135–148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2‐D protein profile of the reference nontypable strain of H. influenzae (HI‐64443) used as the basis for the 2‐D protein database. A composite reference 2‐D protein profile of HI‐64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI‐64443 proteins from the reference 2‐D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI‐64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2‐D protein profile of the Rd strains of H. influenzae was compared to that of HI‐64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI‐64443 when analysed in parallel. The capacity of 2‐D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A “knock‐out” mutant in the sodA gene which encodes [Mn]‐SOD was characterised with respect to proteins synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25 500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2‐D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.
Cloning of minisatellite‐containing sequences from two‐dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low‐grade gliomas of different patients
Authors: Marczinek Karola , Sugiyama Akio , Hampe Jochen , Thiel Gundula , Lehmann Karin , Neumann Regina , de Leeuw Wilio J. F. , Nürnberg Peter 
Published: May 2005

AbstractTwo‐dimensional (2‐D) DNA fingerprinting was used to investigate genomic changes in human low‐grade gliomas of different subtypes. DNA variations were identified in the 2‐D hybridization patterns as spot losses or gains. Computer‐aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2‐D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)‐amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus‐specific probe for the rehybridization of the 2‐D blots. One of these probes detected a spot loss in 7 of 28 low‐grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus‐specific probes that may be instrumental in the discovery of the ciritical early events of glioma pathogenesis.
Optimization of a hexaplex DNA amplification from short tandem repeat and amelogenin loci
Authors: Miścicka‐Śliwka Danuta , Grzybowski Tomasz , Woźniak Marcin 
Published: May 2005

AbstractAn automated DNA profiling system based on the multiplex amplification of highly polymorphic short tandem repeat (STR) markers and the amelogenin locus was developed. Five STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplifications, including HUMD1S103, HUMTH01, HUMD21S11, HUMD18S51, and HUMFIBRA. One primer for each locus was labeled with a fluorescent dye (fluorescein) which allows detection on the single wavelength ALF DNA Sequencer (Pharmacia Biotech). As part of the detailed evaluation of the suitability of the hexaplex system for routine forensic use, the effect of variation in amplification parameters on the efficiency of the system was examined. Polymerase chain reaction amplification conditions were optimized to provide specific, robust amplification of forensic samples.
Molecular sequences of two minisatellites in blacklip abalone, Haliotis rubra
Authors: Huang Bixing , Chai Zhonglin , Hanna Peter J. , Gough Keith H. 
Published: May 2005

AbstractIn the cloning and sequencing of growth‐promoting genes of the blacklip abalone, Huliotis rubra (Leach, 1814), two DNA variable number of tandem repeats (VNTRs) were identified in abalone cDNA libraries. One contained a 33 bp repeat unit (5′‐CCCAAGGTCCCCAAGGTCAGGGAGGCGAAGGCT‐3′) located in the 3′ untranslated region of a putative growth hormone (GH) gene, and the repeat was designated as GHR. The other contained an 18 bp repeat unit (5′‐ACCCGGCGCTTATTAGAG‐3′) located in the 3′ untranslated region of a putative molluscan insulin‐related peptides (MIP) gene, and was designated as MIPR. Primers flanking the two VNTR repeat regions were derived from sequence information. One hundred blacklip abalones were collected along the Victorian coastline and used in a preliminary population study. The range of GHR alleles containing the 33 bp basic unit repeat motif included 7 to 20 repeats, with allele GHR 8 not being identified. The most frequent alleles contained GHR 16 and 17 repeats (56.0% and 16.5%, respectively). Four types of alleles were identified in MIPR, viz 4, 5, 6 and 7 repeats. The alleles containing 6 and 5 repeats were the most frequent (50.0% and 41.5%, respectively). Overall, the results indicate that these two DNA minisatellites have use in abalone studies, including paternity resting, triploid testing, population genetic structure, and gene flow.
Capillary electrophoretic detection of metabolites in the urine of patients receiving hypoglycemic drug therapy
Authors: Roche Matthew E. , Oda Robert P. , Lawson George M. , Landers James P. 
Published: May 2005

AbstractMicellar electrokinetic chromatography (MEKC) in tandem with diode array detection (DAD) has been exploited as an analytical method for the separation and detection of sulfonylurea drugs. The ultimate goal is the development of an assay to detect these drugs or their metabolites in urine as a means of diagnosing sulfonylurea drug abuse. Using a spearation buffer consisting of 5 mM borate/5 mM phosphate/75 mM sodium cholate, separation of both the second and third generation sulfonylurea drugs can be achieved. The characteristic absorbance spectra associated with each of the third generation drugs, glipizide and glyburide, allow for their identification in mixtures. Coinjection of glyburide, its primary metabolite, hydroxy glyburide, and glipizide demonstrated that the metabolite was resolved from the parent drug but shared its absorbance spectral properties. MEKC analysis of a series of solid phase‐extracted urine samples from patients prescribed glipizide or glyburide, as well as from control patients not ingesting the drug, showed that the parent compounds were difficult to detect in the urine. However, the use of DAD allowed for detection of metabolites in the urine of these patients. With glyburide patients, only primary metabolites were detected, while urine from patients on glipizide showed a series of peaks whose absorbance spectra was consistent with the presence of both primary and secondary metabolites. In addition, the intensity of the metabolite peaks corresponded reasonably well with the respective dose and in vivo time interval associated with the urine collection. This study shows that MEKC with DAD has potential for further exploration as a clinical assay for detecting surreptitious abuse of sulfonylurea drugs.