Sodium chloride enhances the storage and conformational stability of BDNF and PEG-BDNF.

PURPOSE: BDNF, a noncovalent homodimer, was modified by covalently attaching polyethylene glycol (PEG) with an average molecular weight of 20kDa to the N-terminal methionine. Stability of modified BDNF (PEG-BDNF) in aqueous solution was compared to BDNF after storage at elevated temperature in the presence and absence of NaCl. METHODS: SDS-PAGE. Light Scattering and Size Exclusion Chromatography were used to assess conformational stability and chemical degradation. In addition, CD spectroscopy was used to follow changes in secondary and tertiary structures upon thermal stress of the protein. RESULTS: NaCl containing formulations are more stable than NaCl-free formulations. In NaCl-free formulations, the main degradation product of BDNF and PEG-BDNF had a molecular weight of monomer that was more chemically degraded than the dimer. Additionally, the degradation of PEG-BDNF occurred at an accelerated rate compared to BDNF in NaCl-free environments. CONCLUSIONS: The addition of NaCl to formulations enhances the shelf-life and conformational stability of both BDNF and PEG-BDNF.

Extending traditional query-based integration approaches for functional characterization of post-genomic data.

MOTIVATION: To identify and characterize regions of functional interest in genomic sequence requires full, flexible query access to an integrated, up-to-date view of all related information, irrespective of where it is stored (within an organization or across the Internet) and its format (traditional database, flat file, web site, results of runtime analysis). Wide-ranging multi-source queries often return unmanageably large result sets, requiring non-traditional approaches to exclude extraneous data. RESULTS: Target Informatics Net (TINet) is a readily extensible data integration system developed at GlaxoSmith- Kline (GSK), based on the Object-Protocol Model (OPM) multidatabase middleware system of Gene Logic Inc. Data sources currently integrated include: the Mouse Genome Database (MGD) and Gene Expression Database (GXD), GenBank, SwissProt, PubMed, GeneCards, the results of runtime BLAST and PROSITE searches, and GSK proprietary relational databases. Special-purpose class methods used to filter and augment query results include regular expression pattern-matching over BLAST HSP alignments and retrieving partial sequences derived from primary structure annotations. All data sources and methods are accessible through an SQL-like query language or a GUI, so that when new investigations arise no additional programming beyond query specification is required. The power and flexibility of this approach are illustrated in such integrated queries as: (1) 'find homologs in genomic sequence to all novel genes cloned and reported in the scientific literature within the past three months that are linked to the MeSH term 'neoplasms"; (2) 'using a neuropeptide precursor query sequence, return only HSPs where the target genomic sequences conserve the G[KR][KR] motif at the appropriate points in the HSP alignment'; and (3) 'of the human genomic sequences annotated with exon boundaries in GenBank, return only those with valid putative donor/acceptor sites and start/stop codons'.

An evaluation of ontology exchange languages for bioinformatics.

Ontologies are specifications of the concepts in a given field, and of the relationships among those concepts. The development of ontologies for molecular-biology information and the sharing of those ontologies within the bioinformatics community are central problems in bioinformatics. If the bioinformatics community is to share ontologies effectively, ontologies must be exchanged in a form that uses standardized syntax and semantics. This paper reports on an effort among the authors to evaluate alternative ontology-exchange languages, and to recommend one or more languages for use within the larger bioinformatics community. The study selected a set of candidate languages, and defined a set of capabilities that the ideal ontology-exchange language should satisfy. The study scored the languages according to the degree to which they satisfied each capability. In addition, the authors performed several ontology-exchange experiments with the two languages that received the highest scores: OML and Ontolingua. The result of those experiments, and the main conclusion of this study, was that the frame-based semantic model of Ontolingua is preferable to the conceptual graph model of OML, but that the XML-based syntax of OML is preferable to the Lisp-based syntax of Ontolingua.

The effects of alpha-helix on the stability of Asn residues: deamidation rates in peptides of varying helicity.

Asn deamidation was monitored in Ala-based octadecapeptides of varying alpha-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala --> Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of alpha-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.

Seamless integration of biological applications within a database framework.

There are more than two hundred biological data repositories available for public access, and a vast number of applications to process and interpret biological data. A major challenge for bioinformaticians is to extract and process data from multiple data sources using a variety of query interfaces and analytical tools. In this paper, we describe tools that respond to this challenge by providing support for cross-database queries and for integrating analytical tools in a query processing environment. In particular, we describe two alternative methods for integrating biological data processing within traditional database queries: (a) "light-weight" application integration based on Application Specific Data Types (ASDTs) and (b) "heavy-duty" integration of analytical tools based on mediators and wrappers. These methods are supported by the Object-Protocol Model (OPM) suite of tools for managing biological databases.

Advanced query mechanisms for biological databases.

Existing query interfaces for biological databases are either based on fixed forms or textual query languages. Users of a fixed form-based query interface are limited to performing some pre-defined queries providing a fixed view of the underlying database, while users of a free text query language-based interface have to understand the underlying data models, specific query languages and application schemas in order to formulate queries. Further, operations on application-specific complex data (e.g., DNA sequences, proteins), which are usually provided by a variety of software packages with their own format requirements and peculiarities, are not available as part of, nor integrated with biological query interfaces. In this paper, we describe generic tools that provide powerful and flexible support for interactively exploring biological databases in a uniform and consistent way, that is via common data models, formats, and notations, in the framework of the Object-Protocol Model (OPM). These tools include (i) a Java graphical query construction tool with support for automatic generation of Web query forms that can be either used for further specifying conditions, or can be saved and customized; (ii) query processors for interpreting and executing queries that may involve complex application-specific objects, and that could span multiple heterogeneous databases and file systems; and (iii) utilities for automatic generation of HTML pages containing query results, that can be browsed using a Web browser. These tools avoid the restrictions imposed by traditional fixed-form query interfaces, while providing users with simple and intuitive facilities for formulating ad-hoc queries across heterogeneous databases, without the need to understand the underlying data models and query languages.

PEGylation prevents the N-terminal degradation of megakaryocyte growth and development factor.

PURPOSE: Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. METHODS: Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. RESULTS: The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). CONCLUSIONS: PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.

Facilities for exploring molecular biology databases on the Web: a comparative study.

We discuss criteria for evaluating and comparing the main facilities provided by molecular biology databases (MBDs) for exploring (that is, retrieving and interpreting data) on the Web. We use these criteria for examining the facilities supported by typical MBDs such as Genbank, AtDB, GSDB, GDB, and MGD (as of September 5, 1996).