bluuebunny/crossref_metadata_2025_split huggingface dataset:
System peaks in capillary zone electrophoresis. 3. Practical rules for predicting the existence of system peaks in capillary zone electrophoresis of anions using indirect spectrophotometric detection
Authors: Macka Miroslav , Haddad Paul R. , Gebauer Petr , Boček Petr 
Published: May 2005

AbstractA theoretical and experimental study of the existence and evolution of system peaks in capillary zone electrophoresis (CZE) with indirect spectrophotometric detection is presented with respect to the effect of the number of coions present in the background electrolyte (BGE). It is shown that in BGEs having only one coion (i.e., the UV‐absorbing probe anion), the sample produces only negative peaks due to each analyte anion and no system peaks, with the number of sample peaks corresponding to the number of analytes present in the sample injected. In BGEs containing two coions, a sample with one analyte anion produces one negative indirect detection peak and one system peak. The transition between BGEs having one coion and those with two coions has also been studied and it has been shown that an addition of ca. 5% of the second coion to a single coion BGE causes the resulting BGE to behave macroscopically as a regular two‐coion BGE. A descriptive model is proposed, based on transient isotachophoresis (transient stacking) of the sample species and of the coion from the BGE which has the closest mobility to the sample ion. This model explains qualitatively the formation and evolution of the sample peak (containing the sample species and being detected by indirect detection due to displacement of the UV‐absorbing probe in its zone) and the system peak (containing no sample species and being a vacancy in the continuum of coions of the BGE). It is shown that the system peak may be positive or negative as it corresponds to the situation where the vacancy of one component of the BGE results in an enhanced concentration of the other component. It has been demonstrated that the system peak is created by a vacancy of that component of the BGE which has the greatest difference in mobility relative to that of the sample species. On indirect detection in BGEs containing two coions the sample displaces predominantly the BGE coion which has a mobility closest to that of the analyte anion. In systems with BGEs containing two coions, a sample having n analytes produces n sample peaks and one system peak, the sign and magnitude of which are dependent on the sum of the UV absorbances of the analytes involved. The effect of bicarbonate from atmospheric CO2 has also been studied and it has been shown that weakly alkaline BGEs with a single anionic UV‐absorbing coion, such as those currently used for anionic analyses with indirect detection, may suffer from the presence of system peaks due to bicarbonate.
Elution concentration of proteins cut from two‐dimensional polyacrylamide gels using Pasteur pipettes
Authors: Kristensen Dan Bach , Inamatsu Mutsumi , Yoshizato Katsutoshi 
Published: May 2005

AbstractThe present study devised a new procedure for concentrating proteins cut from primary two‐dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two‐dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene diffluoride membrane, where the protein was subjected to tryptic on‐membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two‐dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on‐membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on‐membrane area occupied by the concentrated proteins.
Interleukin‐1β induced changes in the protein expression of rat islets: A computerized database
Authors: Andersen Henrik U. , Fey Stephen J. , Larsen Peter Mose , Nawrocki Arkadiusz , Hejnæs Kim R. , Mandrup‐Poulsen Thomas , Nerup Jørn 
Published: May 2005

AbstractInsulin‐dependent diabetes mellitus is caused by an autoimmune destruction of the β‐cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to β‐cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1‐mediated β‐cell cytotoxicity are unknown. However, previous studies have found an association of β‐cell destruction with alterations in protein synthesis. Thus, two‐dimensional (2‐D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin‐dependent diabetes mellitus. 2‐D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the β‐cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2‐D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%–36.1%. When the same sample was analyzed repeatedly in one set of gels (intra‐assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interlenkin‐1β (IL‐1β) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2‐D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL‐1βPart of this work was carried out at the Institute of Medical Microbiology, Århus University, Århus, Denmark
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Capillary electrophoresis of biotechnology‐derived proteins
Authors: Strege Mark A. , Lagu Avinash L. 
Published: May 2005

AbstractApplications of capillary electrophoretic techniques for the analysis of biotechnology‐derived proteins continue at a steady pace. This review summarizes the analyses of these proteins achieved using capillary isoelectric focusing, micellar electrokinetic capillary chromatography, capillary electrophoresis/mass spectrometry and capillary gel electrophoresis. A very brief summary of each technique is included. Application of capillary isoelectric focusing to the analysis of recombinant tissue plasminogen activator in quality control type applications is examined in some detail. Analyses of dosage forms of biotechnology derived proteins of pharmaceutical importance as well as the identification and examination of the peptides, obtained after enzymatic cleavage of proteins, by capillary electrophoresis have been included. Examples of capillary gel electrophoresis as a substitute sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of biotechnology‐derived proteins are also reviewed.
Capillary zone electrophoresis of proteins
Authors: Dolník Vladislav 
Published: May 2005

AbstractThis review article with 237 references is focused on capillary zone electrophoresis (CZE) of proteins. It includes discussion of modeling electrophoretic migration of proteins, sample pretreatment before the analysis, methods reducing the sorptions of proteins on the capillary wall, and techniques for increasing selectivity by using electrolyte additives including the sieving matrices. Significant progress in detection techniques, namely in laser‐induced fluorescence and mass spectrometry, is emphasized. Modifications of CZE using specific interactions, such as affinity capillary electrophoresis or capillary immunoelectrophoresis are debated as well as combination of CZE with other separation methods such as high performance liquid chromatography (HPLC). A number of practical applications of CZE of proteins are described.
Developments in amino acid analysis using capillary electrophoresis
Authors: Smith Joel T. 
Published: May 2005

AbstractThe current status in the analysis of amino acids using capillary electrophoresis is addressed. This area of biological analysis has received increased attention with more than 200 articles being published in the last five years. This review discusses pre‐, post‐, and on‐column derivatization techniques used to tag amino acids providing a detectable moiety. Several separation methodologies which provided resolution for large sets of amino acids are presented. An overview of advances in the enantiomeric resolution methodologies for amino acids is given. Both direct and indirect enantiomeric separation schemes are summarized. Recent advances in detection strategies for both derivatized and underivatized amino acids are presented. Applications utilizing amino acid analysis by capillary electrophoresis are described. This review covers articles published between 1991 and 1996.
Use of free‐flow electrophoresis for the analysis of cellular uptake of picornaviruses
Authors: Kronenberger Peter , Schober Daniela , Prchla Elisabeth , Blaas Dieter , Fuchs Renate 
Published: May 2005

AbstractFree‐flow electrophoresis is a powerful tool to separate subcellular vesicles such as early and late endosomes from plasma membranes. Using this technique, the intracellular distribution of poliovirus type 2 Sabin (PV2) and its derived subviral particles was analyzed upon infection of HeLa cells. Comparison of various infection conditions showed that maximally 30% of total cell associated PV2 was found in endosomal compartments with the remainder being associated with plasma membrane fractions; 2% of viral label was recovered from the cytoplasm in form of free virions. Sucrose gradient centrifugation analysis of the viral material recovered from the respective fractions revealed that intracellular virus was exclusively in its native conformation. This is in sharp contrast to human rhinovirus serotype 2 (HRV2), which is rapidly modified to RNA‐free subviral particles upon accumulation in endosomes. The data suggest that productive poliovirus uncoating can occur at the plasma membrane whereas internalized virus is most probably aborted.
Mycobacterial phagosome maturation, rab proteins, and intracellular trafficking
Authors: Deretic V. , Via Laura E. , Fratti Rutilio A. , Deretic Dusanka 
Published: May 2005

AbstractOne of the most prominent features of pathogenic mycobacteria, which include the potent human pathogensMycobacterium tuberculosisandMycobacterium lepraeand their opportunistic relativesMycobacterium aviumandMycobacterium marinum, is their ability to survive and multiply in phagosomes of mononuclear phagocytic cells. The phagocytosed mycobacteria reside in a vacuolar compartment which is exempted from maturation into the phagolysosome. Recently, the arrest of the maturation of phagosomes containingM. tuberculosiscomplex organisms (Mycobacterium bovisBCG) has been linked to the accumulation on the phagosomal membrane of the small GTP binding protein rab5, specific for the control of fusion within the early endosomal compartment. Furthermore,M. bovisBCG phagosome is devoid of rab7, a rab protein associated with the late endosome. The selective accumulation of rab5 and exclusion of rab7 defines the check point that has been compromised in mycobacterial phagosome maturation. Here we summarize these observations and relates them to other phenomena in the area of membrane and protein trafficking with the emphasis on phagosomes containing intracellular pathogens.
The interaction between Mycobacterium and the macrophage analyzed by two‐dimensional polyacrylamide gel electrophoresis
Authors: Sturgill‐Koszycki Sheila , Haddix Pryce L. , Russell David G. 
Published: May 2005

AbstractThe intramacrophage pathogen Mycobacterium avium resides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG‐opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle‐containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two‐dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between the M. avium and IgG‐bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse‐chase labeling experiments revealed greater differences in the accessibility of Mycobacterium avium and IgG‐bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellular M. avium for comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellular M. avium express several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly after infection.
Subcellular fractionation of polarized epithelial cells and identification of organelle‐specific proteins by two‐dimensional gel electrophoresis
Authors: Fialka Irene , Pasquali Christian , Lottspeich Friedrich , Ahorn Horst , Huber Lukas A. 
Published: May 2005

AbstractProtein targeting and sorting is accomplished by complex vesicular transport processes that are tightly regulated within a cell. This is especially important for epithelial cells because correct delivery of newly synthesized proteins as well as recycling and sorting of internalized membrane proteins is essential for the establishment and preservation of cellular polarity. Many transport events, linking various subcellular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify proteins specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by continuous sucrose gradient centrifugation in order to further analyze their protein composition by high‐resolution two‐dimensional gel electrophoresis (2‐DE). The successful separation of late endosomes (LE), early endosomes (EE) and most of the rough endoplasmic reticulum (RER) was confirmed by subsequent analysis of gradient fractions for compartment‐specific enzymes and marker proteins. Both Golgi and plasma membrane (PM) were found to partially co‐purify with EE in such gradients. Characteristic polypeptide patterns were revealed on 2‐DE gels for fractions enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue, C. Pasqualiet al., Electrophoresis, 1997,18, 2573–2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassiestained spots. This will be the basis for a further characterization of organellespecific molecules in epithelial cells as well as for the establishment of a 2‐DE reference map of membrane proteins from murine mammary epithelium.
Identification of components of trans‐Golgi network‐derived transport vesicles and detergent‐insoluble complexes by nanoelectrospray tandem mass spectrometry
Authors: Shevchenko Andrej , Keller Patrick , Scheiffele Peter , Mann Matthias , Simons Kai 
Published: May 2005

AbstractEpithelial cells have to deliver newly synthesized proteins to the apical and the basolateral plasma membrane domains of the polarized cell surface. Sorting takes place in the trans‐Golgi network and at least two vesicular carriers exist for apical and basolateral delivery. After immuno‐isolation, the composition of these vesicle preparations was analyzed by two‐dimensional (2‐D) gel electrophoresis and detergent extraction. In this paper we compare the constituents of detergent‐insoluble complexes in different cell lines of polarized or nonpolarized origin and present the identification of five previously uncharacterized proteins. We show that our protein identification strategy can be successfully applied to the problem of small hydrophobic proteins from organisms that have not been substantially sequenced. The high sensitivity of nanoelectrospray tandem mass spectrometry allowed us to identify two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking. Furthermore we have mapped CD9 and CD81, two members of a large family of proteins consisting of highly hydrophobic four transmembrane proteins. In addition we have identified caveolin‐2 as a constituent of basolateral transport vesicles. We have also extended our analysis of immunoisolated vesicles to a more basic pI range and show that this region on 2‐D gels is devoid of proteins. With these approaches and with the previously published data we have now identified most of the major low molecular weight proteins recovered in detergent‐insoluble glycolipid‐enriched complexes.
Mapping the protein composition of trans‐Golgi network (TGN)‐derived carrier vesicles from polarized MDCK cells
Authors: Fiedler Klaus , Kellner Roland , Simons Kai 
Published: May 2005

AbstractIn polarized MDCK cells, proteins and lipids are sorted in the trans‐Golgi network /TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN‐derived carrier vesicles from perforated MDCK cells. The composition of immuno‐isolated apical and basolateral carriers was mapped by two‐dimensional (2‐D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2‐D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epidermal keratinocyte lysates. This allowed us to assign eleven known components by a comparison with the comprehensive keratinocyte 2‐D gel database. These comprised two members of the 14‐3‐3 family of proteins that have been implicated in vesicular trafficking. Five proteins were purified from preparative 2‐D gels and identified by peptide microsequencing, including the β1 and β2 subunit of trimeric G proteins and an annexin II variant. A member of the SNARE family of proteins was identified by immunoblotting. The combination of 2‐D gel electrophoresis and 2‐D gel databases allows the rapid assessment of the purity of subcellular fractions and to characterize components involved in vesicular transport.
Two‐dimensional electrophoresis reveals a nuclear matrix‐associated nucleolin complex of basic isoelectric point
Authors: Gotzmann Josef , Eger Andreas , Meissner Michael , Grimm Rudolf , Gerner Christopher , Sauermann Georg , Foisner Roland 
Published: May 2005

AbstractA monoclonal antibody was raised against a salt‐extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an ∼ 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two‐dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5–6.1, characteristic for nucleolin, and at pI 8.5–9. Whereas the protein focusing at acidic pI was found in Triton X‐100‐soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two‐dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse‐released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix‐derived antigen was positioned at pI 5–6. The present data indicate that the nuclear matrix‐bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.
Separation of early steps in endocytic membrane transport
Authors: van der Goot F. Gisou 
Published: May 2005

AbstractWe describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane‐bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl‐6‐(biotinamido)hexanoate (NHS‐LC) biotin at 4°C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol‐glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore‐forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.