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Showing papers on "Gel electrophoresis published in 2002"


Journal ArticleDOI
TL;DR: Global quantification of protein expression between laser capture microdissection-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.

378 citations


Journal ArticleDOI
TL;DR: SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy.

298 citations


Journal ArticleDOI
TL;DR: In this paper, the detection of proteins in SDS-PAGE is an important first step for protein analysis, and various experimentalefforts have been directed to develop and improve theprotein detection methods.
Abstract: Department of Chemistry, Sungkyunkwan University, Suwon 440-746, KoreaReceived August 12, 2002Key Words : Coomasie brilliant blue, 2-Dimensional gel electrophoresis, ProteomicsDetection of proteins in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) is an important firststep for protein analysis. For years, various experimentalefforts have been directed to develop and improve theprotein detection methods

278 citations


Journal ArticleDOI
TL;DR: It is found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses.
Abstract: Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

270 citations


Journal ArticleDOI
TL;DR: A comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification is reported on.
Abstract: Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.

215 citations


Journal ArticleDOI
TL;DR: The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line‐derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.
Abstract: The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of approximately 33-45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is approximately 16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at approximately 40 pg/ml, or 1 pM, and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.

210 citations


Journal ArticleDOI
TL;DR: Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.
Abstract: Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.

198 citations


Journal ArticleDOI
TL;DR: It was shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose/polyacrylamide composite gels.
Abstract: A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly(vinylidene fluoride) membrane, reductive β-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human α-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecul...

197 citations


Journal ArticleDOI
TL;DR: The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.
Abstract: Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.

197 citations


Journal ArticleDOI
TL;DR: The combination of RT‐PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.
Abstract: Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.

196 citations


Journal ArticleDOI
TL;DR: A rapid and convenient extraction procedure of human hair proteins to examine their biochemical properties in detail was developed and found to be effective when wool, chicken feathers, rat hair and human nails were used as starting materials.
Abstract: We developed a rapid and convenient extraction procedure of human hair proteins to examine their biochemical properties in detail. This procedure is based upon the fact that the combination of thiourea and urea in the presence of a reductant can effectively remove proteins from the cortex part of human hair. The extracted fraction mainly consisted of hard alpha-keratins with molecular masses of 40-60 kDa, matrix proteins with 12-18kDa, and minor components with 110-115kDa and 125-135kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein phosphorylation in human hair was investigated by immunoblotting with antibodies against phosphoserine, phosphothreonine and phosphotyrosine. We found serine phosphorylation in alpha-keratins and matrix proteins and threonine phosphorylation in alpha-keratins. The extraction was also found to be effective when wool, chicken feathers, rat hair and human nails were used as starting materials.

Journal ArticleDOI
TL;DR: The N terminus of the MUC2 mucin is assembled into trimers that contain proteolytically stable parts, suggesting that M UC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.

Journal ArticleDOI
TL;DR: Oxidized proteins observed in blood plasma from AD subjects and age-matched controls were isoforms of fibrinogen gamma-chain precursor protein and of alpha-1-antitrypsin precursor, both of which have been previously implicated in the pathology of Alzheimer's disease.

Journal ArticleDOI
TL;DR: The results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease.
Abstract: The degradation of different isoforms of human recombinant tau (R-tau; T39, T40, and T44) and fetal tau (F-tau) by cathepsin D (CD) was investigated. Gel electrophoresis and Coomassie Blue staining of different R-tau species digested at pH 3.5 showed very little differences in CD susceptibility. Immunoblotting analyses revealed that amino and carboxy termini of tau were cleaved before other regions. F-tau was most vulnerable to proteolysis at both termini. Digestion of R-tau with 0.01 unit of CD/ml at pH 3.5 resulted in cleavage between Phe8-Glu9, Met419-Val420, Thr427-Leu428-Ala429, and Leu436-Ala437 as determined by amino acid sequencing and mass spectroscopy (numbering of amino acids was based on T40). With higher concentrations of CD (1 unit/ml), additional sites of digestion were detected between amino acids 34-161, 200-257, and 267-358. The cleavage sites at amino acids 34-161 and 267-358 were observed at pH 3.5, whereas that at amino acids 200-257 was detected at pH 7.0. Our results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease. Such proteolysis might also contribute to the changes of PHF phenotype observed in intracellular and extracellular tangles.

Journal ArticleDOI
TL;DR: To find a target of PAD V, granulocyte-differentiated HL-60 granulocytes were incubated with the calcium ionophore A23187 and deiminated proteins were studied by immunocytochemistry and immunoblotting using a monospecific antibody to modified citrulline residues.

Journal ArticleDOI
TL;DR: Present results reveal for the first time the potency of CT in initiating multiple events linked with defense/stress response(s) in the leaves of whole rice plants.

Journal ArticleDOI
TL;DR: The ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.
Abstract: For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5−20 components (all within ∼5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of ∼400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of ∼19 000 protein forms. This A...

Journal ArticleDOI
TL;DR: It is shown that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations and in combination with specific hydrolases, it can be used to analyze the structure of polySaccharides.

Journal ArticleDOI
TL;DR: Internal sequencing with a quadrupole time‐of‐flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins.
Abstract: Time-course analysis of root protein profiles was studied by two-dimensional gel electrophoresis and silver staining in the model plant Medicago truncatula, inoculated either with the arbuscular mycorrhizal fungus Glomus mosseae or with the nitrogen fixing bacterium Sinorhizobium meliloti. Protein modifications in relation to the development of both symbioses included down- and upregulations, as well as newly induced polypeptides. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry after trypsin digestion clearly identified one polypeptide induced in nodulated roots as a M. truncatula leghemoglobin. Internal sequencing with a quadrupole time-of-flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins. In nodulated roots, one polypeptide was identified as an elongation factor Tu from S. meliloti, while another one could not be assigned a function. In mycorrhizal roots, analyzed proteins also included a protein of unknown function, as well as a glutathione-S-transferase, a fucosidase, a myosin-like protein, a serine hydroxymethyltransferase and a cytochrome-c-oxidase. These results emphasize the usefulness of proteome analysis in identifying molecular events occurring in plant root symbioses.

Journal ArticleDOI
TL;DR: 2‐D blue‐native gel electrophoresis was applied to the mitochondrial proteome and by coupling to tryptic peptide fingerprinting using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, a partial mitochondrial proteomes map has been assembled.
Abstract: The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.

Journal ArticleDOI
TL;DR: VCaB45 is an “in vitro” substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity suggesting potential functions.
Abstract: A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 ± 0.142 mm and 0.64 ± 0.08 mm, and has an estimated 24.7 ± 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an “in vitro” substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.

Journal ArticleDOI
TL;DR: These results show that P1 and P2 do not interact with each other or DNA to form a discrete complex or subunit structure that is dependent upon particular P1/P2 stoichiometries.
Abstract: We have compared the relative proportion of protamine 1 (P1) and protamine 2 (P2) bound to DNA in the sperm of a variety of eutherian mammals to obtain insight into how these two proteins interact in sperm chromatin. Gel electrophoresis (combined with microdensitometry) and high performance liquid chromatography (HPLC) were used to determine the content of the two protamines, and the identity of each protein was confirmed by amino-terminal sequencing or amino acid analysis. The sperm of all species examined contained P1, but P2 was found to be present only in certain species. Unlike the fixed ratio of core histones that package DNA into nucleosomes in all somatic cells, the proportion of P2 present in mature sperm was found to be continuously variable from 0 to nearly 80%. These results show that P1 and P2 do not interact with each other or DNA to form a discrete complex or subunit structure that is dependent upon particular P1/P2 stoichiometries. Data obtained from a number of closely and distantly related species also indicate that while the P2 content of sperm chromatin is allowed to vary over a wide range during the course of evolution, the relative proportion of P1 and P2 are tightly regulated within a genus.

Journal ArticleDOI
TL;DR: In this paper, the characteristics and the microbial diversity of denitrifying phosphate-accumulating organisms (DNPAOs) that are capable of conducting enhanced biological phosphorus removal (EBPR) using nitrate as electron acceptor, three sequencing batch reactors were operated under three different acceptor conditions, i.e., only oxygen, oxygen together with nitrate and only nitrate.

Journal ArticleDOI
TL;DR: Data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni, and identify two such glycoproteins, the first non‐flagellin campylobacter gly coproteins to be identified, and demonstrated that their glycan components contain α‐linked N‐acetylgalactosamine residues.
Abstract: It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To characterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), Coomassie-stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild-type strains were analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA-reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA-reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid-glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of alpha-linked N-acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non-flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain alpha-linked N-acetylgalactosamine residues.

Journal ArticleDOI
TL;DR: The findings suggest that animal monoclonal antibodies specific for cow's milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins with mares and donkeys.
Abstract: Background Cross-reactivity between food allergens occurs when they share part of their amino acid sequence, or when their three-dimensional molecular structure causes them to have a similar capacity to bind specific antibodies. Objectives To review data from our laboratory on cross-reactivity between mammalian proteins (milk and meat allergens). Methods Studies used immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/polyacrylamide gel electrophoresis and immunoblotting), and animal monoclonal antibodies. Results The findings suggest that animal monoclonal antibodies specific for cow's milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins from mares and donkeys. None of the antibodies used in our studies reacted with proteins from an exotic mammalian species: the camel. Similar cross-reactions were found with human circulating immunoglobulin E from children allergic to milk. With regard to beef allergy, monoclonal antibodies specific for bovine serum albumin cross-reacted only with ovine serum albumin, whereas the number of sera from allergic children able to recognize other mammalian serum albumins depended directly on the closeness of phylogenetic relationship between animal species and inversely on the percent identity with human serum albumin in the main epitopic sequence. Conclusion An area of heterogeneity between animal and human species in a critical amino acid sequence (epitope) of an allergen can determine the degree of immunogenic activity.

Journal ArticleDOI
TL;DR: The identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo and the hypothesis that E-fABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120 are indicated.

Journal ArticleDOI
TL;DR: The usability of nucleic acids extracted from banked human tissues for further molecular analyses was determined and gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues.
Abstract: The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, stored for various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RTPCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision. Clinical and molecular pathology are undergoing a revolution based on the accelerated advances in biotechnology such as DNA microarrays and proteomics. Answers to fundamental questions such as “How does the DNA sequence differ between individuals?” and “What makes one individual more prone for a certain disease?” are eagerly sought in this postgenomic era. Several novel genes and their products have been identified in human cancers by screening archival tissue samples using molecular methods. 1,2 More important, molec

Journal ArticleDOI
TL;DR: The results suggest that the balance of protein oxidation and degradation is altered in AD, and this method can be applied to study oxidative changes of individual proteins in brain.
Abstract: There is a growing body of evidence that oxidative stress plays a major role in Alzheimer's disease (AD) pathogenesis. Identification of oxidatively altered proteins in AD is important for understanding the relationship between protein oxidation, protein aggregation and neurodegeneration. In this communication, we report a method that can be applied to study oxidative changes of individual proteins in brain. In order to analyze protein oxidation by detection of protein-bound carbonyls, cytosolic protein extracts were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then separated by two-dimensional (2-D) gel electrophoresis. After electrotransfer to polyvinylidene difluoride (PVDF) membranes, proteins were first stained with Sypro Ruby protein stain, and then the oxidized proteins were detected with anti-dinitrophenyl (DNP) antibody. About 150 proteins and more than 100 oxidized proteins were detected and quantified in both AD and control cases by 2-D image analysis. The amount of protein-bound carbonyls was decreased for six and increased for one protein in AD. The amount of protein was increased for three proteins in AD. Furthermore, the degree of oxidation was calculated as the ratio of protein-bound carbonyls to the total amount of an individual protein. Two proteins showed a significant decrease in the degree of oxidation in AD. Our results suggest that the balance of protein oxidation and degradation is altered in AD.

Journal ArticleDOI
TL;DR: A subdivision of the mitochondrial proteome into defined sets of proteins is reported, which is based on the combination of three different gel electrophoresis procedures, which allows the separation of isoforms of subunits forming part of protein complexes.
Abstract: We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.

Journal ArticleDOI
TL;DR: The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.
Abstract: Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.