Continuing professional development for public health: an andragogical approach.

The UK professional body for public health, the Faculty of Public Health (FPH), has a well-established approach towards continuing professional development (CPD) for its members and fellows. The FPH approach, initially designed for public health physicians, has evolved to meet CPD requirements of a workforce beyond that of public health medicine to what is now a multiprofessional public health workforce. Despite over 20 years of CPD activity in the field of public health, limited literature examines the underlying theoretical principles, or proposes the most effective approach to adopt. Public health CPD is a mandatory requirement for ongoing practice in many professional groups; it is therefore timely to examine theoretical principles and to propose the adoption of an andragogical approach to underpin CPD for all disciplines in public health. This paper concludes that adopting an andragogical approach to public health CPD, supported with critical reflective practice and undertaken in an ethical and moral framework, will equip practitioners to be ready to question practice, implement innovation and progress, and meet the challenges of a multiprofessional public health service.

Applying a causal framework to system modeling.

The emerging field of systems biology represents a revolution in our ability to understand biology. Perhaps for the first time in history we have the capacity to pursue biological understanding using a computer-aided integrative approach in conjunction with classical reductionist approaches. Technology has given us not only the ability to identify and measure the individual molecules of life and the way they change, but also the power to study these molecules and their changes in the context of a big picture. It is through the creation of a computer-aided framework for human understanding that we can begin to comprehend how these collections of molecules act as integrated biological systems, and to utilize this knowledge to rationally engineer the future of science and medicine.

Inferring gene regulatory networks from raw data--a molecular epistemics approach.

Biopathways play an important role in the functional understanding and interpretation of gene function. In this paper we present the results of an iterative algorithm for automatically generating gene regulatory networks from raw data. The algorithm is based on an epistemics approach of conjecture (hypothesis formation) and refutation (hypothesis testing). These operations are performed on a matrix representation of the gene network. Our approach also provides a way of incorporating external biological knowledge into the model. This is done by pre-assigning portions of the matrix--which represent previously known background knowledge. This background knowledge helps make the results closer to a human's rendition of such networks. We illustrate our approach by having the computer replicate a gene regulatory network generated by human scientists at an academic lab.

The Merck Gene Index browser: an extensible data integration system for gene finding, gene characterization and EST data mining.

MOTIVATION: To make effective use of the vast amounts of expressed sequence tag (EST) sequence data generated by the Merck-sponsored EST project and other similar efforts, sequences must be organized into gene classes, and scientists must be able to 'mine' the gene class data in the context of related genomic data. RESULTS: This paper presents the Merck Gene Index browser, an easily extensible, World Wide Web-based system for mining the Merck Gene Index (MGI) and related genomic data. The MGI is a non-redundant set of clones and sequences, each representing a distinct gene, constructed from all high-quality 3' EST sequences generated by the Merck-sponsored EST project. The MGI browser integrates data from a variety of sources and storage formats, both local and remote, using an eclectic integration strategy, including a federation of relational databases, a local data warehouse and simple hypertext links. Data currently integrated include: LENS cDNA clone and EST data, dbEST protein and non-EST nucleic acid similarity data, WashU sequence chromatograms. Entrez sequence and Medline entries, and UniGene gene clusters. Flatfile sequence data are accessed using the Bioapps server, an internally developed client-server system that supports generic sequence analysis applications. Browser data are retrieved and formatted by means of the Bioinformatics Data Integration Toolkit (B-DIT), a new suite of Perl routines.

Evolutionary relationship of the ligand-gated ion channels and the avermectin-sensitive, glutamate-gated chloride channels.

Two cDNAs, GluClalpha and GluClbeta, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707-711, 1994). Expression studies in Xenopus oocytes showed that GluClalpha and GluClbeta have pharmacological profiles distinct from the glutamate-gated cation channels as well as the gamma-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClalpha and GluClbeta genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClalpha and GluClbeta form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClalpha and GluClbeta, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClalpha and GluClbeta belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.

Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506.

The immunosuppressant FK506 is a 23-membered macrocyclic polyketide produced by several Streptomyces species. Sequencing of a 19.5-kb contiguous segment of DNA from the FK506 gene cluster of Streptomyces sp. MA6548 revealed the presence of a single 19.3-kb open reading frame designated fkbA. fkbA encodes a component of the FK506 polyketide synthase, a complex enzyme system which catalyzes synthesis of the polyketide portion of FK506. The predicted product of gene fkbA is a 630,660-Da protein (6420 amino acids) that contains 19 independent domains with a high degree of amino acid sequence similarity to the catalytic activities of known fatty acid synthases. The identified domains are arranged into four repeated modules with a linear organization precisely as that of animal fatty acid synthase and type I polyketide synthase. Each module participates in one round of chain extension and subsequent processing and thus FkbA polypeptide catalyzes four of the ten condensation steps required for synthesis of the FK506 macrolactone ring. Disruption of fkbA results in the generation of an FK506 non-producing mutant demonstrating direct involvement of fkbA in the biosynthesis of FK506.

Toward the development of a gene index to the human genome: an assessment of the nature of high-throughput EST sequence data.

A rigorous analysis of the Merck-sponsored EST data with respect to known gene sequences increases the utility of the data set and helps refine methods for building a gene index. A highly curated human transcript data base was used as a reference data set of known genes. A detailed analysis of EST sequences derived from known genes was performed to assess the accuracy of EST sequence annotation. The EST data was screened to remove low-quality and low-complexity sequences. A set of high-quality ESTs similar to the transcript data base was identified using BLAST; this subset of ESTs was compared with the set of known genes using the Smith-Waterman algorithm. Error rates of several types were assessed based on a flexible match criterion defining sequence identity. The rate of lane-tracking errors is very low, approximately 0.5%. Insert size data is accurate within approximately 20%. Reversed clone and internal priming error rates are approximately 5% and 2.5%, respectively, contributing to the incorrect identification of reads as 3' ends of genes. Follow-up investigation reveals that a significant number of clones, miscategorized as reversed, represent overlapping genes on the opposite strand of entries in the transcript data base. Relevance of these results to the creation of a high-quality index to the human genome capable of supporting diverse genomic investigations is discussed.

PROFILER: a tool for automatic searching of internally maintained databases.

A new application program, the BIOINFORMATICS PROFILER, is described which simplifies the analysis of new genome sequence information appearing on a daily basis. Control tasks are defined through an intuitive graphical user interface and are executed at user defined nightly intervals. Electronic mail is sent to indicate that search results have been found. All task output is presented to users in the form of hypertext (HTML), allowing easy browsing. Currently supported tasks include BLAST and FastA sequence searching, keyword based searching of network news articles and WAIS databases, examination of GenBank sequence entries using regular expressions and boolean operations and protein sequence motif searching.

The C-terminus of the B cell activator Oct-2 functions as an activation domain in yeast.

Oct-1 and Oct-2 are human transcriptional activators that bind to the same DNA element but activate distinct sets of genes. We expressed these factors in S. cerevisiae and observed greater than 5-fold stimulation of a lacZ reporter gene only with Oct-2. Transfer of the Oct-2 C-terminal domain onto either Oct-1 (Oct1.2) or a nonactivating DNA-binding domain from GAL4 created activators capable of greater than 15 and 10-fold stimulation of activity, respectively. Thus, the C-terminus of Oct-2 is sufficient to confer activation potential to nonactive DNA-binding fragments in yeast.

Carboxyl terminus of inducible nitric oxide synthase. Contribution to NADPH binding and enzymatic activity.

Cloning of a nitric oxide synthase (NOS) from RAW 264.7 mouse macrophages (Xie, Q.-w., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225-228) yielded two sets of cDNA: one with a longer coding region of 1144 amino acids, whose sequence matched that of the purified protein, and another with a shorter coding region of 1122 amino acids, in which the last 10 carboxyl-terminal amino acids differed completely from those of the long form. We have now found that the short form lacks NOS activity. To determine the basis of this defect, we prepared recombinant chimeric, deletional, and point mutants of the long and short NOS variants, monitored their expression by immunoblot, and tested their enzymatic activity. By itself, lack of the 22-carboxyl-terminal residues of the long form NOS was scarcely consequential. Mutation of Phe1122, the only aromatic residue within one of the longest conserved regions shared by all NOSs of reported sequence, reduced enzymatic activity by 41%. Deletion of 23 carboxyl-terminal amino acids (including Phe1122) reduced activity by 71%. Further loss of Ile1121, another completely conserved residue, reduced activity by 95%, and with the deletion of the rest of the conserved region, NOS activity was undetectable. Normal dimerization and binding of heme and calmodulin by the short variants militated against distortions of tertiary structure affecting the amino-terminal half or middle portion of the protein. In contrast, the short variants were deficient in binding to NADPH, as predicted by a model of tertiary structure based on that of spinach ferredoxin-NADP+ reductase. This is the first demonstration that the carboxyl terminus of NOS is a functionally critical region.

A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3.

Defects in the conversion of androstenedione to testosterone in the fetal testes by the enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) give rise to genetic males with female external genitalia. We have used expression cloning to isolate cDNAs encoding a microsomal 17 beta-HSD type 3 isozyme that shares 23% sequence identity with other 17 beta-HSD enzymes, uses NADPh as a cofactor, and is expressed predominantly in the testes. The 17 beta HSD3 gene on chromosome 9q22 contains 11 exons. Four substitution and two splice junction mutations were identified in the 17 beta HSD3 genes of five unrelated male pseudohermaphrodites. The substitution mutations severely compromised the activity of the 17 beta-HSD type 3 isozyme.

Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 alpha-hydroxysteroid dehydrogenase activity.

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is an enzyme crucial to the regulation of intracellular levels of biologically active steroid hormones in a variety of tissues. Here, we report the isolation, structure, and characterization of a cDNA encoding the human 17 beta-HSD type 2. A 1.4-kilobase cDNA was identified, and DNA sequence analysis indicated that 17 beta-HSD type 2 was a protein of 387 amino acids with a predicted molecular weight of 42,782. The protein contained an amino-terminal type II signal-anchor motif and a carboxyl-terminal endoplasmic reticulum retention motif, which suggested that 17 beta-HSD type 2 was associated with the membranes of the endoplasmic reticulum. 17 beta-HSD type 2 was capable of catalyzing the interconversion of testosterone and androstenedione as well as estradiol and estrone. The enzyme also demonstrated 20 alpha-HSD activity toward 20 alpha-dihydroprogesterone. The amount of 17 beta-HSD type 2 mRNA in placenta was found to be high. The data suggest that the 17 beta-HSD type 2 cDNA encodes the microsomal 17 beta-HSD of human placenta, described by several laboratories.

Optimal humanization of 1B4, an anti-CD18 murine monoclonal antibody, is achieved by correct choice of human V-region framework sequences.

The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.

Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.

FKB1 encodes a nonessential FK 506-binding protein in Saccharomyces cerevisiae and contains regions suggesting homology to the cyclophilins.

FK 506, a powerful immunosuppressant that blocks allograft rejection by preventing T-cell activation, binds to an 11-kDa protein called the FK 506-binding protein (FKBP). Like cyclophilin, a cytosolic protein that binds another immunosuppressant, cyclosporin A, FKBP possesses peptidylprolyl cis-trans isomerase activity. We have isolated a genomic clone encoding the yeast FKBP (FKB1). The gene encodes a protein of 114 amino acids having a calculated Mr of 12,158. Disruption of the gene shows that FKB1 is not essential for growth. A search of translated nucleic acid data bases revealed bacterial FKBP homologs in Neisseria meningiditis and Pseudomonas aeruginosa. Comparison of the conserved amino acids in FKBP homologs with the conserved amino acids in the cyclophilins has revealed a region of similarity that we speculate to be a homologous domain related to the functional similarities of the two proteins.