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At least 991 records · Page 55Linked to original sources
PubMed2006

Comparative analysis of two-dimensional electrophoresis maps (2-DE) of Helicobacter pylori from Brazilian patients with chronic gastritis and duodenal ulcer: a preliminary report.

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Bacterial Proteins↗
PubMed2004

Differential proteomic analysis of Aeromonas salmonicida outer membrane proteins in response to low iron and in vivo growth conditions.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids. Bacterial phenotypes are known to change in vivo compared to the in vitro state. Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass. Using an in vivo growth chamber model, the pathogenic fish bacterium A. salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis. Growth of A. salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions. Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor. When cultured in vivo, A. salmonicida up-regulated the identical 73, 76 and 85 kDa proteins. The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A. salmonicida within the peritoneal cavity of salmon.

Aeromonas↗
PubMed2007

Proteomic discovery of 21 proteins expressed in human plasma-derived but not platelet-derived microparticles.

Microparticles (MPs) are small membrane vesicles generated by essentially all cell types. In the plasma, most MPs are derived from platelets, but those from other sources, particularly leukocytes (macrophages, lymphocytes, and neutrophils), endothelial cells, and even smooth muscle cells can be detected and appear to play an important role in normal physiology and various diseases. In previous work we analyzed the proteome of MPs generated from isolated platelets (platelet MPs). Here, we report on a comparative analysis of microparticles isolated from plasma (plasma MPs) versus platelet MP using two complementary methods of comparative analysis. The first method, spectral count analysis, yielded 21 proteins detected in plasma MPs (with a total spectral count of 10 or greater) that were essentially absent in platelet MPs (with a total spectral count of 1 or 0). An additional two proteins (von Willebrand Factor, albumin) were present in both types of MPs but enriched in the plasma MPs. The second method, isotope-coded affinity tag (ICAT) labeling of proteins, supported the spectral count results for the more abundant proteins and provided better relative quantitation of differentially expressed proteins. Proteins present only in the plasma MPs include several associated with apoptosis (CD5-like antigen, galectin 3 binding protein, several complement components), iron transport (transferrin, transferrin receptor, haptoglobin), immune response (complement components, immunoglobulin J and kappa chains), and the coagulation process (protein S, coagulation factor VIII).

Apoptosis↗
PubMed2001

The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.

Bacterial Adhesion↗
PubMed2001

Identification and characterization of a bovine myosin light chain-1 fast polymorphism.

This study aimed to identify genes or gene products associated with high lean muscle mass in bovines that may serve as potential markers for selection. An animal with a genetic predisposition to high lean muscle mass, the Belgium Blue, was chosen as a model to compare with the Holstein Friesian, a model that does not. Two-dimensional polyacrylamide gel electrophoresis analysis was utilized to compare the exhibited skeletal muscle proteome between the two animal types at two stages of foetal development. A previously uncharacterized polymorphism of a high expression myofibrillar protein, myosin light chain 1 fast (MLC-1f), was observed. The characterization of this polymorphism revealed a two amino acid insertion in a part of the protein that has been implicated in modulating myosin S1 ATPase activity. Furthermore, this polymorphism was shown to be the product of two alleles that are codominant. Screening studies were carried out on selected herds and showed a very high frequency of one allele. Both isoforms of MLC-1f were produced by recombinant means and purified. The recombinant proteins were exchanged into purified myosin hexamers that were then subject to assays measuring ATP consumption. The sensitivity of the assay utilized could not reveal any significant difference in ATPase activity between hexamers containing one or the other isoform.

Actins↗
PubMed2005

Protein abnormality in denervated skeletal muscles from patients with brachial injury.

A proteomic analysis was performed to compare protein expression between normal sternocleidomastoid muscle and denervated muscle. Two-dimensional electrophoresis (2-DE) of muscle proteins showed that 26 proteins among about 800 spots in 2-DE gel displayed a decrease and 6 proteins an increase in expression in muscles with denervation atrophy compared to normal controls; the identified proteins that were abnormally expressed could be generally grouped together as metabolic proteins, chaperone proteins, and contractile-apparatus proteins. The significance of these altered proteins is discussed. In particular, the decrease in hD54 may reduce the activity of transmembrane signaling in atrophied muscle, while the disregulation of DnaJC 1 showed a possible role of molecular chaperones in the functional recovery of atrophied muscles.

Adult↗
PubMed2005

The role of informatics in glycobiology research with special emphasis on automatic interpretation of MS spectra.

This paper reviews the current status of bioinformatics applications and databases in glycobiology, which are based on bioinformatics approaches as well as informatics for glycobiology where an explicit encoding of glycan structures is required. The availability of the complete sequence of the human genome has accelerated the systematic identification of so far unidentified glycogenes considerably in many areas of glycobiology using well-established bioinfomatics tools. Although there has been an immense development of new glyco-related data collections as well as informatics tools and several efforts have been started to cross-link and reference the various data deposited in distributed databases, informatics for glycobiology and glycomics is still poorly developed compared to the genomics and proteomics area. The development of algorithms for the automatic interpretation of MS spectra - currently, a severe bottleneck, which hampers the rapid and reliable interpretation of MS data in high-throughput glycomics projects - is reviewed. A comprehensive list of web resources is given. Several lines of progression are discussed. There is an urgent need for the development of decentralised input facilities of experimentally determined glycan structures. Simultaneously, agreements of standards for the structural description of glycans as well as formats for the related data have to be established. The integration of glycomics with genomics/proteomics has to increase.

Computational Biology↗
PubMed2006

Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle.

The cytosolic Ca2+ -binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+ -leak channels and luminal Ca2+ -binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+ -handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+ -handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+ -handling proteins may result in an insufficient maintenance of Ca2+ -homeostasis and an abnormal regulation of Ca2+ -dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.

Aging↗
PubMed2006

A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis.

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. Fifteen percent of generated peptides are predicted to bind to HLA with halftime of dissociation T(1/2) >or=100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of no-namers by different HLA molecules was studied employing structural modeling of HLA class I-peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-à-vis the limitations of the use of subunit vaccine and DNA vaccine.

Amino Acid Sequence↗
PubMed2007

Oxidized transthyretin in amniotic fluid as an early marker of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls. The analysis was carried out by two-dimensional electrophoresis followed by peptide mapping and tandem mass spectrometric analysis. Besides the implementation of the previously published AF proteomic maps, our results show that transthyretin (TTR), the protein responsible for transporting both the thyroid hormone tyroxine and the retinol binding protein, is present in the AF of both preeclamptic and control women as a mixture of dimeric and post-translationally modified monomeric forms. Although the nature of these forms is similar in both groups, the preeclamptic women showed a significant increase in the amount of monomeric proteins with respect to the control group. Since the TTR monomeric forms are the results of different oxidizing reactions, we hypothesize that the higher oxidative stress in preeclampsia is the major destabilizing factor of the TTR functional dimeric form in the preeclamptic women.

Amniocentesis↗
PubMed2006

Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.

Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70-75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had approximately 75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.

Animals↗
PubMed2006

Identification of proteins cleaved downstream of caspase activation in monocytes undergoing macrophage differentiation.

We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.

Carcinogens↗
PubMed2006

Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins.

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.

Amino Acids↗
PubMed2004

Neoantigens in osteoarthritic cartilage.

PURPOSE OF REVIEW: Osteoarthritis has been considered a degenerative disease. However, recent evidence supports involvement of immunologic mechanisms in this pathophysiology: for example, inflammation of synovial tissue is observed in osteoarthritis. In osteoarthritis, the proinflammatory cytokine interleukin-1, which is produced by activated synoviocytes and mononuclear cells and has catabolic effects on chondrocytes, is one of the most involved. The immune reaction would require driving antigens. This review describes autoantigens in osteoarthritis and discusses their roles in triggering and/or perpetuating synovitis and joint cartilage destruction in osteoarthritis. RECENT FINDINGS: Several autoantigens/autoantibodies have been reported in osteoarthritis, such as the cartilage intermediate layer protein. Furthermore, recent comprehensive proteomic surveillance has revealed that comparable numbers of autoantigens were detected in osteoarthritis and rheumatoid arthritis, and that some of them were recognized predominantly in osteoarthritis rather than in rheumatoid arthritis. In addition, it was revealed that the cartilage intermediate layer protein immunization of mice developed calcification of tendons, thus indicating that autoimmunity modulates functions of target molecules. SUMMARY: Osteoarthritis-specific autoantigens may drive chronic synovitis and may thereby contribute to production of cytokines to upregulate proteases, which lead to chondrocyte and cartilage damage. In addition, autoimmunity may damage joint components by modulating functions of the target molecules.

Autoantibodies↗
PubMedDate not supplied

The use of microarrays for studying the pathogenesis of Helicobacter pylori.

At present, the genomes of various microorganisms have been completely sequenced, and many others are in progress. The availability of this level of information and the computational analysis of the described sequences have led to the development of new genomic areas such as: analysis in silico, comparative genomics, functional genomics, transcriptomics, proteomics, and pharmacogenomics. Microarray technology is a powerful tool for analyzing the expression profile of thousands of genes in a global way and can be applied to the study of various biological systems. Using the complete sequences for both the H. pylori and human genome that are available in the data bases, a number of researchers have revealed important information. Some of these data offer a glimpse into the great genetic diversity of H. pylori, the differential genetic expression between the strains that shows the complexity of the response of microorganisms to different conditions of development, and into the association of gene cluster expression with clinical outcome. Other groups have examined the global transcriptional response of gastric epithelial cells to H. pylori. The majority of these studies report an alteration in gene expression related to transcription functions, transduction signals, cell cycle regulation and differentiation, development factors, proliferation/apoptosis balance, expression of membrane proteins, and inflammatory response.

Animals↗
PubMed2006

Proteome analysis of yeast response to various nutrient limitations.

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids beta-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.

Ammonia↗
PubMed2000

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation.

Human epidermal stem cells express higher levels of beta1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of beta1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2-fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA-FABP, also known as E-FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA-FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA-FABP is a novel marker of epidermal transit amplifying cells.

3T3 Cells↗
PubMed2006

Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study.

The Human Proteome Organization (HUPO) recently completed the first large-scale collaborative study to characterize the human serum and plasma proteomes. The study was carried out in different locations and used diverse methods and instruments to compare and integrate tandem mass spectrometry (MS/MS) data on aliquots of pooled serum and plasma from healthy subjects. Liquid chromatography (LC)-MS/MS data sets from 18 laboratories were matched to the International Protein Index database, and an initial integration exercise resulted in 9,504 proteins identified with one or more peptides, and 3,020 proteins identified with two or more peptides. This article uses a rigorous statistical approach to take into account the length of coding regions in genes, and multiple hypothesis-testing techniques. On this basis, we now present a reduced set of 889 proteins identified with a confidence level of at least 95%. We also discuss the importance of such an integrated analysis in providing an accurate representation of a proteome as well as the value such data sets contain for the high-confidence identification of protein matches to novel exons, some of which may be localized in alternatively spliced forms of known plasma proteins and some in previously nonannotated gene sequences.

Blood Proteins↗