Biomedical subjects

J C Sanchez

Publications and source records attributed to J C Sanchez.

At least 55 records · Page 3Linked to original sources

PubMed1998

Analyzing glycoproteins separated by two-dimensional gel electrophoresis.

Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha2-HS glycoprotein (human fetuin) and alpha1-antitrypsin (alpha1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE.

Amidohydrolases↗

PubMed1998

Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number.

Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.

Bacillus subtilis↗

PubMed1998

Towards an automated approach for protein identification in proteome projects.

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.

Automation↗

PubMed1998

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Bacterial Proteins↗

PubMed1998

Multiple parameter cross-species protein identification using MultiIdent--a world-wide web accessible tool.

Recent increases in the number of genome sequencing projects means that the amount of protein sequence in databases is increasing at an astonishing pace. In proteome studies, this is facilitating the identification of proteins from molecularly well-defined organisms. However, in studies of proteins from the majority of organisms, proteins must be identified by comparing analytical data to sequences in databases from other species. This process is known as cross-species protein identification. Here we present a new program, MultiIdent, which uses multiple protein parameters such as amino acid composition, peptide masses, sequence tags, estimated protein pI and mass, to achieve cross-species protein identification. The program is structured so that protein amino acid composition, which is highly conserved across species boundaries, first generates a set of candidate proteins. These proteins are then queried with other protein parameters such as sequence tags and peptide masses. A final list of database entries which considers all analytical parameters is presented, ranked by an integrated score. We illustrate the power of the approach with the identification of a set of standard proteins, and the identification of proteins from dog heart separated by two-dimensional gel electrophoresis. The MultiIdent program is available on the world-wide web at: http://www.expasy.ch/sprot/multiident.h tml.

Amino Acid Sequence↗

PubMed1998

A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multigene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes. There are four genetic complementation groups (A, B, C and D), reflecting the existence of four MHC-II regulators. The factors defective in groups A (CIITA), C (RFX5) and D (RFXAP) have been identified. CIITA is a non-DNA-binding co-activator that controls the cell-type specificity and inducibility of MHC-II expression. RFX5 and RFXAP are two subunits of RFX, a multi-protein complex that binds the X box motif of MHC-II promoters. Mutations in the genes encoding RFX5 (RFX5) or RFXAP (RFXAP) abolish binding of RFX (refs 7,8,12). Similar to groups C and D, group B is characterized by a defect in RFX binding, and although it accounts for the majority of patients, the factor defective in group B has remained unknown. We report here the isolation of RFX by a novel single-step DNA-affinity purification approach and the identification of RFXANK, the gene encoding a third subunit of RFX. RFXANK restores MHC-II expression in cell lines from patients in group B and is mutated in these patients. RFXANK contains a protein-protein interaction region consisting of three ankyrin repeats. Its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC-II promoters.

Amino Acid Sequence↗

PubMed1998

Identification of a basophil leukocyte interleukin-3-regulated protein that is identical to IgE-dependent histamine-releasing factor.

This study aimed to identify basophil leukocyte proteins associated with interleukin (IL)-3 and/or anti-IgE activation by two-dimensional (2-D) gel electrophoresis. We noticed one particular protein showing increased synthesis after recombinant human (rh)IL-3 and, to a lesser extent, anti-IgE stimulation. The protein was also present in the culture medium in increased amounts after rhIL-3 stimulation. On the basis of comigration with proteins in published 2-D gel electrophoresis databases and immunoblotting with a specific monoclonal antibody, we identified this protein as translationally controlled tumor protein (TCTP), also known as p23 or IgE-dependent histamine-releasing factor. The antibody was shown to be specific for TCTP/IgE-dependent histamine-releasing factor by blotting on 2-D gels of proteins from human lymphocytes and the human basophilic cell line KU812, followed by N-terminal amino-acid sequencing of the bound protein. Densitometric analysis of the gels showed that the synthesis of IgE-dependent histamine-releasing factor in human basophil leukocytes was dose dependent upon rhIL-3 stimulation with an optimum of 100 ng/ml. The level of the protein in the medium was also highest at an optimal rhIL-3 concentration of 100 ng/ml. Supernatants from cultured basophils were able to stimulate histamine release from other basophils. This histamine release was decreased by precipitation of TCTP/IgE-dependent histamine-releasing factor from these supernatants.

Amino Acid Sequence↗

PubMed1997

Elevation of apolipoprotein E in the CSF of cattle affected by BSE.

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt-Jakob disease (CJD) display two unique polypeptide chains by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In the absence of a well-defined ante-mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post-mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10-fold increase). These elevated polypeptide chains were identified as apolipoprotein E.

Amino Acid Sequence↗

PubMed1997

Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting.

A novel approach is reported for the analysis and identification of proteins separated by 2D-PAGE with scanning infrared matrix-assisted laser desorption/ionization mass spectrometry (scanning IR-MALDI-MS). The proteins of human blood plasma were separated by 2D-PAGE, electroblotted onto PVDF membranes, incubated in matrix solution, and then scanned by IR-MALDI-MS. Mass contour plots of selected spots were obtained. Protein separation is shown to be conserved by comparison with silver-stained gels. The sensitivity for the protein detection is comparable if not better than that of silver-stained gels. Posttranslational modifications were identified by comparing the measured mass to the one calculated from the known DNA sequence. Adduct formation to unprotected cysteine residues during gel separation is demonstrated for selected proteins.

Blood Proteins↗

PubMed1997

Translationally controlled tumor protein: a protein identified in several nontumoral cells including erythrocytes.

The translationally controlled tumor protein (TCTP) is a growth-related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two-dimensional gel electrophoresis, monoclonal antibodies, and 45Ca-gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post-translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.

Amino Acid Sequence↗

PubMed1997

A role for Edman degradation in proteome studies.

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.

Amino Acid Sequence↗

PubMed1997

Melanie II--a third-generation software package for analysis of two-dimensional electrophoresis images: I. Features and user interface.

Although two-dimensional electrophoresis (2-DE) computer analysis software packages have existed ever since 2-DE technology was developed, it is only now that the hardware and software technology allows large-scale studies to be performed on low-cost personal computers or workstations, and that setting up a 2-DE computer analysis system in a small laboratory is no longer considered a luxury. After a first attempt in the seventies and early eighties to develop 2-DE analysis software systems on hardware that had poor or even no graphical capabilities, followed in the late eighties by a wave of innovative software developments that were possible thanks to new graphical interface standards such as XWindows, a third generation of 2-DE analysis software packages has now come to maturity. It can be run on a variety of low-cost, general-purpose personal computers, thus making the purchase of a 2-DE analysis system easily attainable for even the smallest laboratory that is involved in proteome research. Melanie II 2-D PAGE, developed at the University Hospital of Geneva, is such a third-generation software system for 2-DE analysis. Based on unique image processing algorithms, this user-friendly object-oriented software package runs on multiple platforms, including Unix, MS-Windows 95 and NT, and Power Macintosh. It provides efficient spot detection and quantitation, state-of-the-art image comparison, statistical data analysis facilities, and is Internet-ready. Linked to proteome databases such as those available on the World Wide Web, it represents a valuable tool for the "Virtual Lab" of the post-genome area.

Algorithms↗

PubMed1997

Make2ddb: a simple package to set up a two-dimensional electrophoresis database for the World Wide Web.

Two-dimensional electrophoresis (2-DE) has become a highly reproducible protein separation technique that currently serves as the main basis for proteome research and in particular for protein identification. Also, the Internet provides large utilities for exchanging data, and we can observe increased interest among scientists to build remote 2-DE databases, since many members of the concerned community are now able to access the data. By preparing the data and programs that are required to create a federated 2-DE database, the Make2ddb package, described here, helps to build such a database on the user own World Wide Web site.

Computer Communication Networks↗

PubMed1997

A two-dimensional protein map of human amniotic fluid at 17 weeks' gestation.

Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2-D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.

Adult↗

PubMed1997

Protein expression profiles in human breast ductal carcinoma and histologically normal tissue.

Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin beta isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.

Aged↗

PubMed1997

Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis.

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.

Colorectal Neoplasms↗

PubMed1997

Serum "B" esterases as a nondestructive biomarker for monitoring the exposure of reptiles to organophosphorus insecticides.

A field study was conducted to validate serum B esterases as nondestructive biomarkers (NDBs) in lizards. Serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities were measured in lizards and four species of birds collected in an area of 0.5 ha sprayed with 0.36 kg a.i./ha of Folidol SE5 (5% parathion). Serum B esterase activities were determined in a total of 213 lizards (Gallotia galloti) and 81 birds of four species (Sylvia melanocephala, Serinus canaria, Parus caeruleus, and Erithacus rubecula) collected for 23 days after the spraying. A control group of 39 lizards and 58 birds was sampled before the spraying. No relationship was found between serum B esterases and sex or biometric parameters in all species. Inhibition of BChE (> 40%) and CbE (> 50%) activities was recorded in lizards 23 days after spraying. BChE activity was found to be more sensitive than CbE to inhibition by parathion. Inhibition of serum B esterase activities was recorded in only two bird species (S. melanocephala and S. canaria), but the number of individuals collected was much less than the lizards. The advantages and disadvantages of G. galloti as bioindicator of exposure to organophosphorus insecticides in the Canary Islands (Spain) are discussed in relation to birds commonly used for this purpose.

Animals↗

PubMed1997

The clinical, neuroradiographic, and endocrinologic profile of patients with bilateral optic nerve hypoplasia.

PURPOSE: The purpose of the study was to expand on ophthalmologic and endocrinologic data and report the neuroimaging findings of 35 patients with bilateral optic nerve hypoplasia (BONH). METHODS: A retrospective chart review of 35 children with BONH was conducted. Data on visual acuity, refractive error, and presence of nystagmus and strabismus were collected. Twenty-six children underwent full-endocrinologic evaluation and magnetic resonance imaging or computed tomography scanning. RESULTS: The male:female ratio was 2:1. Ten percent of eyes had visual acuity of 20/60 or better, whereas 34% had no light perception. Eighty-six percent of eyes had acuity less than 20/200, and 80% of patients were legally blind. Most patients (86%) had nystagmus or strabismus or both. Forty-six percent had absence of the septum pellucidum and corpus callosum on neuroimaging. Twenty-seven percent of patients had endocrinologic abnormalities, with growth hormone deficiency being the most common. Panhypopituitarism occurred in 11.5% of children. CONCLUSIONS: Although the visual prognosis of children with BONH generally is poor, 10% of such patients have excellent acuity. In contrast to previous reports, endocrine abnormalities were seen in only one quarter of patients, and the full-blown deMorsier syndrome (septo-optic dysplasia with panhypopituitarism) was seen in only 11.5% of patients with BONH.

Abnormalities, Multiple↗