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At least 955 records · Page 53Linked to original sources
PubMed1999

Prostate cancer expression profiling by cDNA sequencing analysis.

Prostate cancer is a frequently diagnosed solid tumor that is originated mostly from prostate epithelium. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. Automated, single-pass cDNA sequencing was utilized to rapidly identify expressed genes in a number of cDNA libraries constructed from various normal and tumor prostatic tissues. These included cell lines as well as short-term epithelial culture. A total of 6604 expressed sequence tags (ESTs) were generated and searched against on-line nucleotide and protein databases. A relational database centric software system was constructed to process, store, and analyze EST data rapidly. cDNA contigs were also obtained by assembly of multiple EST sequences. Protein structural signatures were annotated using motif analysis tools including BLOCKS and an in-house-designed neural network. Cross-library comparisons revealed their unique gene expression profiles. Several differentially expressed cDNA clones were identified, and their expression patterns were confirmed by RNA dot blot and RT-PCR analyses.

Cell Line↗
PubMed1998

Optimizing energy potentials for success in protein tertiary structure prediction.

BACKGROUND: Success in solving the protein structure prediction problem relies on the choice of an accurate potential energy function. for a single protein sequence, it has been shown that the potential energy function can be optimized for predictive success by maximizing the energy gap between the correct structure and the ensemble of random structures relative to the distribution of the energies of these random structures (the Z-score). Different methods have been described for implementing this procedure for an ensemble of database proteins. Here, we demonstrate a new approach. RESULTS: For a single protein sequence, the probability of success (i.e the probability that the folded state is the lowest energy state) is derived. We then maximize the average probability of success for a set of proteins to obtain the optimal potential energy function. This results in maximum attention being focused on the proteins whose structures are difficult but not impossible to predict. CONCLUSIONS: Using a lattice model of proteins, we show that the optimal interaction potentials obtained by our method are both more accurate and more likely to produce successful predictions than those obtained by other averaging procedures.

Computer Simulation↗
PubMed1999

Proteomics for genetic and physiological studies in plants.

Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology, for deciphering the function and role of the genes that are or will be sequenced. In this review we focus on the various, mainly genetic, applications of the proteomic tools that have been developed in recent years: characterization of individuals or lines, estimation of genetic variability within and between populations, establishment of genetic distances that can be used in phylogenetic studies, characterization of mutants and localization of the genes encoding the revealed proteins. Improvements in specifically devoted software have permitted precise quantification of the variation in amounts of proteins, leading to the concept of "protein quantity loci" which, combined with the "quantitative trait loci" approach, results in testable hypotheses regarding the role of "candidate proteins" in the metabolism or phenotype under study. This new development is exemplified by the reaction of plants to drought, a trait of major agronomic interest. The accumulation of data regarding genomic and cDNA sequencing will be connected to the protein databases currently developed in plants.

Chromosome Mapping↗
PubMed1997

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.

Kinetics↗
PubMed1992

Isolation of a putative retrovirus pol gene fragment from trout.

1. A 220 bp DNA fragment was obtained from three different species of salmonids during PCR analysis using a primer sequence based on human beta-2 microglobulin. All of the 220 bp fragments showed strong homology to each other. 2. Several of the DNA sequences also contained protein reading frames. Searching DNA and protein databases revealed significant homology to a segment of the pol gene (reverse transcriptase) from various retroviruses. Phylogenetic analysis at both the DNA and the protein levels showed clustering of the fish sequences and the closest viral sequence was the Moloney murine leukaemia virus (MoMuLV). Southern analysis indicated that there are several copies of the gene dispersed throughout the salmonid genome. 3. Preliminary results suggest that these sequences may be unique to the family Salmonidae. This would suggest that this retrovirus was incorporated in the DNA of an ancestral salmonid prior to the evolution and divergence of this family of fish.

Amino Acid Sequence↗
PubMed1998

The cadmium-stress stimulon of Escherichia coli K-12.

The influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 microM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argI, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.

Amino Acid Sequence↗
PubMed2006

Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein.

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.

Amino Acid Sequence↗
PubMed1997

[Application of subtractive hybridization in screening for colorectal cancer negatively related genes].

OBJECTIVE: To search previously uncertained factors that corelated with the occurence and development of human colorectal cancer. METHOD: Subtractive hybridization technique was employed to screen down-regulated genes of colorectal cancer. RESULTS: We obtained 46 clones which highly were expressed in normal intestinal mucosa but much lower or none in colorectal cancer. Two of the 46 clones, SCN6 and SCN19, were accepted by the Genbank Database as novel genes with the accession numbers U17714 and U20248. Full length cDNA (2932bp) of SNC6 was sequenced, and supposed to encode a protein comprised 271 amino acids, MW30kd, which shared less than 30% homology with the protein database. The gene was localized to human chromosome 22q13. RNA dot blot analysis showed that the expression level of SNC6 was lower in colorectal cancer than in normal intestinal mucosa, and similar results were found in breast cancer. Northern blot showed higher expression levels of SNC6 in liver, lung, prostate, testis, ovary and K562 cell line than in other tissues. CONCLUSION: SNC6 can be further investigated as a novel colorectal cancer negatively related gene.

Colonic Neoplasms↗
PubMed2004

Integrative analysis of cancer-related data using CAP.

The development of human cancer is a highly complex process and can be considered the result of several combined events, such as genetic alterations, disturbance of signal transduction, or failure of immunological surveillance. Cancer-related databases usually focus on specific fields of research, e.g., cancer genetics or cancer immunology, whereas the complexity of cancer genesis requires an integrated analysis of heterogeneous data from several sources. Here we present the cancer-associated protein database (CAP), a novel analysis system for cancer-related data. CAP integrates data from multiple external databases, augments these data with functional annotations, and offers tools for statistical analysis of these data. We have employed CAP to analyze genes that have been found to cause an autoimmune response in cancer. In particular, we explored the connection between the autoimmune response, mutations, and overexpression of these genes. Our preliminary results suggest that mutations are not significant contributors to raising an antibody response against tumor antigens, whereas overexpression seems to play a more important role. We hereby demonstrate how different types of data can be integrated and analyzed successfully, providing interesting results. As the amount of available data is growing rapidly, a combined analysis will play an important role in exploring the genetic and immunological basis of cancer. CAP is freely available at the following web site: http://www.bioinf.uni-sb.de/CAP/.

Autoimmunity↗
PubMed1993

Backbone-dependent rotamer library for proteins. Application to side-chain prediction.

A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20 degrees by 20 degrees blocks for the main-chain angles phi, psi is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone phi, psi values. These probabilities are used to place the side-chains on the known backbone in test applications for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both chi 1 and chi 2 values in the correct position (to within 40 degrees) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme. The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.

Amino Acid Sequence↗
PubMed2003

The relationship between sequence and interaction divergence in proteins.

There is currently a gap in knowledge between complexes of known three-dimensional structure and those known from other experimental methods such as affinity purifications or the two-hybrid system. This gap can sometimes be bridged by methods that extrapolate interaction information from one complex structure to homologues of the interacting proteins. To do this, it is important to know if and when proteins of the same type (e.g. family, superfamily or fold) interact in the same way. Here, we study interactions of known structure to address this question. We found all instances within the structural classification of proteins database of the same domain pairs interacting in different complexes, and then compared them with a simple measure (interaction RMSD). When plotted against sequence similarity we find that close homologues (30-40% or higher sequence identity) almost invariably interact the same way. Conversely, similarity only in fold (i.e. without additional evidence for a common ancestor) is only rarely associated with a similarity in interaction. The results suggest that there is a twilight zone of sequence similarity where it is not possible to say whether or not domains will interact similarly. We also discuss the rare instances of fold similarities interacting the same way, and those where obviously homologous proteins interact differently.

Amino Acid Sequence↗
PubMed2003

SDAP: database and computational tools for allergenic proteins.

SDAP (Structural Database of Allergenic Proteins) is a web server that provides rapid, cross-referenced access to the sequences, structures and IgE epitopes of allergenic proteins. The SDAP core is a series of CGI scripts that process the user queries, interrogate the database, perform various computations related to protein allergenic determinants and prepare the output HTML pages. The database component of SDAP contains information about the allergen name, source, sequence, structure, IgE epitopes and literature references and easy links to the major protein (PDB, SWISS-PROT/TrEMBL, PIR-ALN, NCBI Taxonomy Browser) and literature (PubMed, MEDLINE) on-line servers. The computational component in SDAP uses an original algorithm based on conserved properties of amino acid side chains to identify regions of known allergens similar to user-supplied peptides or selected from the SDAP database of IgE epitopes. This and other bioinformatics tools can be used to rapidly determine potential cross-reactivities between allergens and to screen novel proteins for the presence of IgE epitopes they may share with known allergens. SDAP is available via the World Wide Web at http://fermi.utmb.edu/SDAP/.

Allergens↗
PubMed2004

Identification of trace element-containing proteins in genomic databases.

Development of bioinformatics tools provided researchers with the ability to identify full sets of trace element-containing proteins in organisms for which complete genomic sequences are available. Recently, independent bioinformatics methods were used to identify all, or almost all, genes encoding selenocysteine-containing proteins in human, mouse, and Drosophila genomes, characterizing entire selenoproteomes in these organisms. It also should be possible to search for entire sets of other trace element-associated proteins, such as metal-containing proteins, although methods for their identification are still in development.

Animals↗
PubMed2005

CysMap and CysJoin: database and tools for protein disulphides localisation.

We have developed a computer program able to make user-customised databases derived from the public PIR non-redundant reference protein database. When the database of interest has been created, the user will generate the map of all the possible linear peptides containing one and two cysteines for each protein and combine them to calculate the mass of all the possible clusters of linear peptides linked by a disulphide bridge with a cysteine pair. It is also possible to create selected maps corresponding to peptides formed by the action of specific proteases. In this way, mass spectrometric data obtained from the hydrolysis of proteins of unknown sequence can be related to that contained in the database for quick disulphide assignment and protein identification. To confirm signal attribution, the program will also furnish the expected mass of cluster peptides after performing a cycle of Edman degradation. The utility of the program is discussed and examples of application are given.

Amino Acid Sequence↗
PubMed2004

The identification of an adenovirus receptor by using affinity capture and mass spectrometry.

A tandem mass spectrometry--based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors.

Antigens, CD↗
PubMedDate not supplied

Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.

The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.

Amino Acid Sequence↗
PubMed2003

Mimicry between the hepatitis C virus polyprotein and antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis C virus infection.

Autoantibodies to smooth muscle (SMA) and nuclear components (ANA) arise in the natural course of chronic infection with hepatitis C virus. In view of the growing evidence for 'molecular mimicry' as a mechanism of autoimmunity we investigated whether cross-reactive immune reactions between host smooth muscle/nuclear components and HCV antigens may contribute to the formation of SMA and ANA in chronic HCV infection. Computer-assisted protein database search methods were used to identify three smooth muscle (smoothelin698-717, myosin1035-1054, vimentin69-88) and three nuclear (matrin722-741, histone H2A11-30, replication protein A133-152) host antigens with the highest local sequence similarity to the HCV polyprotein and 20-mer peptides corresponding to these regions were constructed. Sera from 51 children with chronic HCV infection [median age: 8 (2-16); 27 boys], 26 SMA positive and five ANA positive, were tested for reactivity to the synthesized HCV peptides and their human homologues by enzyme linked immunosorbent assay (ELISA). Sera from patients with HBV infection and chronic liver disease of different aetiologies were used as controls. 'Double reactivity' to HCV peptides and smooth muscle/nuclear homologues was associated strongly with HCV infection (P < 0.001 for both). Humoral cross-reactivity was established as the basis for double recognition by competition ELISA. Double-reactivity to smooth muscle and HCV peptide antigens correlated with SMA positivity by indirect immunofluouresence (P = 0.05). Of 15 patients double-reactive to myosin1035-1054 and its HCV homologue, 13 recognized whole myosin by immunoblot. These results suggest that ANA and SMA in chronic HCV infection may arise, at least in part, as a consequence of cross-reactive immune responses to HCV and host smooth muscle/nuclear antigens.

Adolescent↗