SEARCH · PubMed Health

Results for “Proteomics techniques”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources

PubMed2006

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin alpha-1 chain, beta-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-alpha, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, alpha-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE.

Adult↗

PubMed2006

Mass spectrometrical analysis of galectin proteins in primary rat cerebellar astrocytes.

Galectins are a family of animal lectins with specificity for beta-galactosides and are involved in a host of cellular activities, ranging from development to cancer. The molecules are expressed by neural and non-neural cells intracellularly as well as extracellularly. Using two-dimensional gel electrophoresis coupled to tandem mass spectrometry, the present work aimed to identify and characterize galectins in primary rat cerebellar astrocytes. The protein-chemical method identified nine spots representing two members of the galectin family, namely galectin-1 and galectin-3. These findings suggest that high abundant expression of galectin in astrocytes is limited to the two abundant galectin family members. As these family members are linked to human astrocytic tumors, their reliable detection in astrocytes by proteomic techniques would enable us to further understand their role in neural development, injury, and regeneration in general and astrocytoma in particular.

Amino Acid Sequence↗

PubMed2006

Tumour banks in pediatric oncology.

INTRODUCTION: The information offered by the new genomic and proteomic techniques will play a central role in our knowledge of cancer; but it is limited by the lack of available tissue samples. Cancer in children is a sum of infrequent diseases, so tumor banks are support tools for translational research, providing access to a sufficiently large series of samples, which would minimize the asymmetric effect of the diverse origin. MATERIAL AND METHODS: From 2003 a Molecular Pathology Network in Pediatric Solid Tumors Netwoks exists in Spain, and we are a part of it. Our aim was to create a pediatric tumor bank program and consensus documents about its use. RESULTS: Standard Operating Procedures for collection and transport of samples have been created. CONCLUSIONS: Thinking about the fast progress in Molecular Biology and the low frequency in pediatric tumors, it is vital to consider the importance of a bio bank.

Adolescent↗

PubMed2006

Addition of electrophilic lipids to actin alters filament structure.

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

Actins↗

PubMed2006

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Dual kinase-mediated regulation of PITK by CaMKII and GSK3.

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIdelta), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.

Amino Acid Sequence↗

PubMed2006

Histologic grading of noninvasive papillary urothelial neoplasms.

OBJECTIVES: In 1998, a revised system of classifying noninvasive papillary urothelial neoplasms of the urinary bladder was proposed and subsequently formally adopted by the World Health Organization (WHO). The introduction of this new system was justified as being potentially superior on a number of levels to the 1973 WHO classification system that it replaced. Specifically, a new category of neoplasms, designated papillary urothelial neoplasm of low malignant potential (PUNLMP), was considered advantageous for several reasons. The new system was expected to gain widespread acceptance, improve reproducibility of diagnoses among pathologists, and enhance the correlation between urine cytology and tumor histology. We examine the history of the changes in terminology for these lesions, the relative merits of PUNLMP terminology, the extent to which the expectations accompanying the new grading system have been met, and the extent to which the new system has enhanced the management of patients with noninvasive papillary urothelial neoplasms of the bladder. METHODS: A PubMed literature search after the introduction of this new classification was performed and relevant papers reviewed. RESULTS AND CONCLUSIONS: The 2004 WHO classification is a positive initiative in attempting to standardize urothelial tumor grading by expanding and clearly defining the morphologic characteristics of noninvasive papillary urothelial neoplasms. The new terminology used in this system is of questionable validity and utility. Full-genome searches for prognostic and predictive molecular gene expression signatures, GeneChip technology and proteomics techniques, and several new biomarkers and molecular tests may be useful in future grading schemes after their clinical utility is better established.

Carcinoma, Transitional Cell↗

PubMed2005

Evaluation of a sensitive detection method for peptide arrays prepared by SPOT synthesis.

The growing range of applications for peptide arrays prepared by SPOT synthesis confirms that they are a powerful proteomics technique to study numerous aspects of molecular interaction events. The most frequent application for peptide arrays prepared by SPOT synthesis is the identification of linear epitopes that are recognized by antibodies. In the conventional format using secondary antibodies for detection unspecific binding and high background have been observed. This leads to difficulties in evaluation of developed membranes. Especially for application with combinatorial libraries false positive results are to be avoided. To circumvent this issue, we directly labeled compounds of interest with biotin and detected binding by incubation with streptavidin-horseradish peroxidase via chemiluminescence. Optimization of method conditions led to a very sensitive detection technique with no or low number of unspecific spots, which is superior to conventional detection with secondary antibodies. As one consequence, evaluation of competitive assays got more reliable.

Epitope Mapping↗

PubMed2006

Recent surprises in protein targeting to mitochondria and plastids.

The functions of mitochondria and chloroplasts rely on thousands of proteins, mostly imported from the cytosol through specialized import channels. Neither the detailed import mechanisms nor the identities of all targeted proteins are known. Recent surprises include unexpected results concerning import receptors, unexpectedly frequent dual-targeting of proteins, and the discovery of novel routes of protein trafficking. Such findings make it more difficult to predict which proteins really are targeted to organelles. By combining experimental and bioinformatics data, we estimate the size of the mitochondrial and plastid proteomes to be approximately 2000 and 2700 proteins, respectively. Advances in cell and organelle fractionation coupled with modern proteomics techniques are probably the best route to understanding organellar protein composition.

Mitochondria↗

PubMed2005

SELDI-MS-based expression profiling of ductal invasive and lobular invasive human breast carcinomas.

Expression profiling using proteomic techniques has a great potential to identify new biomarkers that might help to better diagnose and treat diseases such as breast cancer, which is one of the leading causes of cancer death in women. Surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) combines chromatographic separation of peptides and proteins with mass spectrometry and is a fast, user-friendly tool to analyze protein and peptide profiles. SELDI-MS was employed for a comparative analysis of lobular invasive versus ductal invasive breast tumors to find differentially expressed proteins and peptides, and to validate this technique for biomarker identification using complex samples such as tissue. After optimization of sample preparation using HMEC and MCF-7 cell lines, 20 breast tumors were analyzed, and about 550 mass signals corresponding to an estimated 140 native peptides and proteins were detected in each tumor. Only 14% of the mass signals were present in more than six tumors of one subgroup or in more than 12 tumors of both groups showing a great overall heterogeneity of the peptide and protein profiles obtained. Peptide mass signals specific for each of the analyzed groups were identified. In addition, we detected peptides from laser-microdissected ductal invasive and intraductal tumor parts corresponding to peptides present in whole tumors. The low amount of identified peptides and proteins and the observed heterogeneity suggest that SELDI-MS is not well suited for biomarker identification of and profiling experiments on complex samples such as tumor tissue.

Breast Neoplasms↗

PubMed2004

Impact of genomics on microbial food safety.

Genome sequences are now available for many of the microbes that cause food-borne diseases. The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress. The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host. New concepts are emerging about how pathogens function, both within foods and in interactions with the host. The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.

Biofilms↗

PubMed2002

Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat.

Chronic rejection is the leading cause of late renal transplant failure. Various structural lesions are observed in grafts undergoing chronic rejection including glomerular basement membrane (GBM) duplications. The well-established Fisher (F344) to Lewis (LEW) rat renal transplant model for chronic rejection was used to assess the presence and role of the humoral immune response against graft antigens during chronic rejection. LEW recipients of F344 allografts develop transplant glomerulopathy and produce IgG1 antibodies directed against F344 GBM preparations that are detectable 3 weeks after transplantation. Glomerular IgG1 deposition was observed that in vitro co-localized with a rabbit anti-rat GBM antiserum in rejecting F344 grafts; elution experiments of isolated glomeruli yielded IgG1 antibodies reactive in vitro with F344 GBM, but not LEW GBM. Prevention of acute rejection by transient treatment of the recipients with cyclosporin A completely abrogated the production of anti-GBM antibodies. Using proteomic techniques we identified the antigens recognized by the LEW posttransplant sera as being the heparan sulfate proteoglycan perlecan and the alpha1 chain of collagen type VI in association with the alpha5 chain of collagen type IV. In conclusion, LEW recipients of F344 kidney grafts produce IgG1 antibodies against donor type perlecan and alpha1(VI)/alpha5(IV) collagen and develop transplant glomerulopathy. These data implicate an important role for the humoral immune response in the development of glomerulopathy during chronic rejection.

Albuminuria↗

PubMed2005

Structure of UDP-glucuronosyltransferases in membranes.

This chapter presents the most recent experimental approaches to the investigation of UDP-glucuronosyltransferase (UGTs) in membranes. The first topic described is the subcellular localization of UGTs with special emphasis on the association of these proteins with the endoplasmic reticulum (ER). Experimental methods include subfractionation of tissue for microsome preparation, evaluation of the purity of the membrane fraction obtained, and measurement of UGT activity in the presence of detergents. Next, the recently demonstrated formation of UGT homo- and heterodimer formation and its functional relevance is discussed and the appropriate methods used to characterize such interactions are given (radiation inactivation, size exclusion chromatography, immunopurification, cross-linking, two-hybrid system). The structural determinants of UGTs in relation to membrane association, residency, and enzymatic activity are the next topic, supplemented by a description of the appropriate methods, including the design and expression of chimeric proteins, membrane insertion, and subcellular localization by immunofluorescence. Also presented is new information on the structure and function of UGTs obtained by molecular modeling, bioinformatics (sequence alignment), and comparison with selected crystallized glycosyltransferases. Finally, we discuss the important, and still not fully developed, issue of UGT active site architecture and organization within the ER. This is addressed from two perspectives: (1) chemical modification of UGT active sites by amino acid-specific probes and (2) photoaffinity labeling of UGTs. The detailed synthesis of a photoaffinity probe for an aglycon-binding site is provided and the use of this probe and direct photoaffinity labeling with retinoids is discussed. The application of proteomics techniques, including proteolytic digestion and protein sequencing by liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption ionization/time of flight, to the identification of crucial amino acids of the active sites, and subsequent site-directed mutagenesis of identified amino acids, is discussed in detail.

Animals↗

PubMed2005

Protein nitration in biological aging: proteomic and tandem mass spectrometric characterization of nitrated sites.

Proteomic techniques for the identification of 3-nitrotyrosine-containing proteins in various biological systems are described with emphasis on the direct mass spectrometric detection and sequencing of 3-nitrotyrosine-containing peptides. Strengths and weaknesses of various separation and mass spectrometric techniques are discussed. Some examples for the MS/MS analysis of nitrated peptides obtained from aging rat heart and skeletal muscle are provided, such as nitration of Tyr105 of the mitochondrial electron-transfer flavoprotein and Tyr14 of creatine kinase.

Aging↗

PubMed2002

Human brain cytosolic histamine-N-methyltransferase is decreased in Down syndrome and increased in Pick's disease.

Histamine-N-methyltransferase (HMT) inactivates the neurotransmitter histamine. Central histaminergic deficits may contribute to the cognitive impairment of neurodegenerative disorders including Alzheimer's disease (AD) and Down syndrome (DS). However, there is no evidence for histaminergic deficits in Pick's disease (PiD). HMT levels were measured in the frontal cortex and cerebellum of brains of patients with AD, DS, and PiD, and normal aged subjects using proteomics techniques. In frontal cortex, HMT was significantly decreased in DS, but significantly increased in PiD compared with controls. HMT levels were comparable in cerebellum of all groups. Elevated HMT in PiD could lead to increased histamine degradation that in turn would be in agreement with impaired cognitive functions of PiD. Decreased HMT in DS would be compatible with findings of decreased histamine synthesis, thus reflecting a compensation mechanism to antagonize reduced synthesis by decreased degradation.

Aged↗

PubMed2002

In vitro toxicology methods: impact on regulation from technical and scientific advancements.

The impressive advancement of technologies in biomedical research, and particularly in the area of in vitro experimental models, has opened up new possibilities related to co-cultures, micromass or stem cell cultures. Engineered cells to study specific targets and/or mechanisms are also available. Moreover, a very subtle approach in the study of toxicological effects is represented by the very recent genomics and proteomics techniques. New mechanistically based methods could be established from all these approaches, which, once validated, could enter the regulatory procedure. So far, in toxicology, only a few in vitro tests are accepted for regulatory purposes, such as those related to corrosion, phototoxicity and absorption. Many others are in the pre-validation or validation phase. An area where in vitro tests play a key role is the genetic toxicology. In this context, the most recent testing strategies and test methods will be presented, with particular attention to the recently updated guidelines for food additives by the EU Scientific Committee on Food. An improvement in the implementation of validated methods could arise from a better coordination on the matter at national and international levels, the harmonisation of different legislations, and a strict control of the national rules in order to make them up-to-date with respect to validated methods.

Animals↗

PubMed2001

Applications of peptide arrays prepared by the SPOT-technology.

The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.

B-Lymphocytes↗

PubMed2003

Pre-mRNA splicing: awash in a sea of proteins.

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.

Animals↗