Biomedical subjects
J W Taylor
Publications and source records attributed to J W Taylor.
Dimeric structure and conformational stability of brain-derived neurotrophic factor and neurotrophin-3.
We have examined the molecular structure of the related neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) by physical methods, including gel filtration, velocity sedimentation, sedimentation equilibrium, urea gel electrophoresis, fluorescence spectroscopy, and far-ultraviolet circular dichroism. The results of these studies indicate that at physiologically relevant concentrations both recombinant proteins exist as tightly associated dimers. The dimers are stable even in 8 M solutions of urea. In solutions of guanidine hydrochloride, BDNF and NT-3 undergo slow unfolding between 3 and 5 M concentration of denaturant. Circular dichroism spectroscopy revealed approximately 70% beta-sheet and 20% beta-turn content in the native structure of both neurotrophic factors. In this respect, BDNF and NT-3 resemble other polypeptide growth factors whose receptors are also integral protein-tyrosine kinases.
Probing the functional conformation of neuropeptide Y through the design and study of cyclic analogues.
The functional importance of the PP-fold conformation in neuropeptide Y (NPY) was investigated. NPY and N alpha-Ac-NPY(10-36), and corresponding cyclic analogues cyclo18,22-[Lys18,Asp22]-NPY and N alpha-Ac-cyclo18,22-[Lys18,Asp22]-NPY(10-36), were synthesized. Strategies for synthesis of the cyclic analogues included the use of the Kaiser oxime resin and a segment condensation approach. Circular dichroism studies in phosphate buffer, pH 5.0, indicated self-association of all four peptides at low micromolar concentrations. Monomeric N alpha-Ac-NPY(10-36) showed only 13% alpha-helix, compared to 32% alpha-helix for monomeric NPY, demonstrating a helix-stabilizing effect of residues 1-9 that is consistent with the PP fold. The [Lys18,Lys22] lactam bridge stabilized the helical conformation in N alpha-Ac-NPY(10-36)(51% alpha-helix), but was helix destabilizing in NPY (21% alpha-helix). In rat brain receptor binding assays, the cyclic and linear N alpha-Ac-NPY(10-36) analogues were equipotent (IC50 = 13 nM for 125I-BH-NPY displacement), although the cyclic analogue was twice as potent in rat vas deferens assays. NPY was more potent than its cyclic analogue in the brain receptor binding assays (IC50 = 0.07 and 0.25 nM, respectively), but these peptides were equipotent in the vas deferens assays. These results support a functional role for the PP fold in NPY and correlate with the solution conformations of the monomeric peptides.
Alkaline phosphatase-somatostatin hybrid proteins as probes for somatostatin-14 receptors.
By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand-binding sites on their membrane-bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18-residue peptide containing the receptor-binding segment of somatostatin-14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand-binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild-type enzyme and the hormone-enzyme hybrid displaced 125I-labeled somatostatin from rat brain membrane receptors only at high concentrations. However, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor-binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild-type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand-binding site of somatostatin receptors.
Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.
For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.
Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence.
Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release. Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms. The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes. The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently. Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge. In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates. A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test. Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge. Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA. The transition-to-transversion ratio was 1.9. Of all 12 possible substitutions, 29% were from U to C. At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4). In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).
Two ascomycete classes based on fruiting-body characters and ribosomal DNA sequence.
Traditional fruiting body-based classification of ascomycetes has been under attack for 2 decades. Fruiting-body types can converge, and few researchers now assume that either the closed fruiting bodies (cleistothecia) characterizing the class Plectomycetes or the flask-shaped fruiting bodies (perithecia) characterizing the class Pyrenomycetes are stable, unifying characters. Unless we identify characters uniting major ascomycete groups, orders of ascomycetes remain narrowly defined, and supraordinal classification is impossible. We sequenced both strands of 18s rDNA from nine ascomycete fungi, adding three sequences from GenBank into our analysis. The phylogeny, inferred from 162 informative sites in 1,700 bp of DNA sequence data and using yeast as an outgroup, divided the fungi into two groups correlating well both with fruiting-body type and with the traditional classes Plectomycetes and Pyrenomycetes. Each group received strong statistical support. Genera producing cleistothecia, such as Talaromyces (with a Penicillium asexual state) and the human pathogen Ajellomyces capsulatus (causing histoplasmosis), fall within the plectomycete group. Plectomycetes also includes Eremascus albus and the bee pathogen Ascosphaera apis, although both lack typical fruiting bodies. The Dutch elm disease fungus groups with pyrenomycetes such as Neurospora, in spite of its confusing mixture of class-level characters.
Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes.
Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.
Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA.
Ribosomal DNA variation was used to study evolutionary relationships among five fungal-like protoctistan Phytophthora species. On the basis of morphological and ecological characteristics, four of these species--P. palmivora, P. megakarya, P. capsici, and P. citrophthora--were once thought to be related. Variation within a species was extensively studied in a fifth, outgroup species--P. cinnamomi--known, on the basis of ecological, isozyme, and mitochondrial DNA studies, to be variable. Internal transcribed spacer regions (ITS I, between the 18S and 5.8S rDNAs; and ITS II, between the 5.8S and 25S rDNAs) from 27 isolates of these five species were analyzed by polymerase chain reaction amplification and direct sequencing. Intraspecific variability was undetected or low. Interspecific nucleotide difference was 0.3%-14.6%, and comparisons of variable regions permitted the evaluation of phylogenetic relationships among species. Both neighbor-joining and parsimony analysis of ITS variability support a close relationship between cacao isolates of P. capsici and P. citrophthora and a common lineage for P. palmivora and P. megakarya. Large distance values were estimated between P. cinnamomi and the other species. Inferred relationships based on ITS variability were compared with those based on other characters. The catalog of sequences provides the information necessary to design taxon-specific probes potentially useful in taxonomic, ecological, and population-level studies.
Molecular evolution of the fungi: human pathogens.
The morphological, ecological, and clinical diversity among ascomycete fungi that are pathogenic to humans suggest that the potential for pathogenicity may have arisen multiple times within these higher fungi. We have obtained 18S ribosomal DNA sequences from a diverse group of human pathogenic fungi in order to determine their evolutionary origins. The fungi studied include a skin pathogen that is confined to humans (Trichophyton rubrum) and three systemic, facultative parasites that cause histoplasmosis (Histoplasma capsulatum), blastomycosis (Blastomyces dermatitidis) and coccidioidomycosis (Coccidioides immitis) in humans and other higher animals. Also included in our analysis are representatives of non-pathogenic fungi, as well as two opportunistic pathogens, Pneumocystis carinii and Candida albicans, that cause severe disease in immunocompromised individuals, especially those with AIDS. Two of the fungi we sequenced, T. rubrum and C. immitis, are limited to asexual modes of reproduction and therefore lack the sexual structures that are most useful for evolutionary comparison as well as being essential for classification among the higher fungi. Coccidioides immitis is particularly problematic owing to its contradictory and confusing asexual morphologies, which have caused it to be placed in three fungal classes and the protista. Our analysis shows that the specialized, superficial parasite and the systemic, facultative parasites, including C. immitis, are closely related ascomycetes, which clearly demonstrates the power of molecular characters to compensate for missing or confusing reproductive morphology. Analysis also shows that the opportunistic pathogens are more distantly related, with the likely explanation that pathogenicity has arisen more than once within the Ascomycetes.
Itraconazole in antifungal therapy.
OBJECTIVE: This overview compares and contrasts the pharmacotherapy of itraconazole with that of other antifungal agents. DATA SOURCES: Primary literature on itraconazole was identified through a medical literature search from 1976 through 1991. This search included journal articles, abstracts, conference proceedings, and reports of animal and human research published in the English language. STUDY SELECTION: All primary literature was reviewed regardless of the study design or outcome. Literature evaluations of efficacy were ranked using a literature rating scale (Dalen JE, Hirsh J. Arch Intern Med 1986;146:462-72), which was slightly modified to include case reports and observations. DATA EXTRACTION: All data were collected and represented with a primary focus on itraconazole's mechanism of action, pharmacokinetics, clinical efficacy in systemic mycotic infections, drug interactions, and adverse reactions. All articles were referenced in the final data presentation unless grouped data had been accurately reviewed and published. DATA SYNTHESIS: Despite the paucity of controlled comparative trials with itraconazole in patients with deep mycoses, results on efficacy are encouraging. It is still unclear what role itraconazole will have in the prophylaxis of fungal infections in immunocompromised hosts. The favorable pharmacokinetic profile permits once- or twice-daily administration and itraconazole appears to be safe and well tolerated. CONCLUSIONS: Itraconazole should prove to be a useful replacement for ketoconazole on hospital formularies. This recommendation is based on itraconazole's greater apparent safety and efficacy. Reevaluation of this agent will be necessary upon the release of newer imidazoles and triazoles.
Comparison of 5.8S ribosomal DNA sequences among the basidiomycetous yeast genera Cystofilobasidium, Filobasidium and Filobasidiella.
Nucleotide sequences obtained from regions of the ribosomal DNA repeat were compared by phylogenetic methods and combined with a statistical evaluation to clarify the relationships among the genera Cystofilobasidium, Filobasidium (F.) and Filobasidiella (Fl.), to assess the affinity of Filobasidiella neoformans and Filobasidiella depauperata, and to compare the varieties of Fl. neoformans. With appropriate primers, the nuclear 18S, 5.8S and internal transcribed spacer (ITS) regions of the ribosomal RNA genes (rDNA) of 10 strains were amplified with the polymerase chain reaction. The resulting DNA products were compared by digestion with endonucleases and analysis of restriction fragments. Single strands of the 5.8S rDNA and ITS regions were subsequently sequenced by the dideoxy method. Statistical support for the phylogeny inferred from parsimony analysis of aligned 5.8S rDNA sequences was determined by bootstrapping. There were no nucleotide substitutions in this region, nor in the ITS, among strains of Fl. neoformans that differ in variety and serotype. There was strong support for retaining Fl. depauperata and Fl. neoformans in the same genus, but nucleotide substitutions can be used to distinguish the two species. There was no support for combining the genera Filobasidium, Filobasidiella or Cystofilobasidium.
Development of clinical practice standards for nurses.
Nursing, with leadership from the American Nurses Association (ANA), has a long-standing commitment to the development of standards. The first nursing practice standards were published in 1973. Since that time, both the ANA and specialty nursing organizations have developed standards. However, the proliferation of standards, reflecting a wide divergence of intent, format, and scope, has limited their usefulness. In 1989, the ANA appointed a Task Force (now a Committee) to address this issue. The Committee, working with representatives of the specialty nursing organizations, has developed a framework for development of both nursing practice standards and guidelines. Standards and guidelines are defined and differentiated. The relationship of standard and guidelines development to the federal government's effectiveness initiative is discussed.
Automatic stop-order procedure for antibiotics needs evaluation.
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The segmental rectus abdominis free flap for ankle and foot reconstruction.
The reconstruction of soft-tissue defects of the ankle and foot usually requires free-tissue transfer. Although certain local flaps have been described for the reconstruction of these injuries, their utility may be compromised by significant crush injury or the size and location of the defect. Part of the rectus abdominis muscle, the segmental rectus abdominis free flap, is ideally suited for this use because of the muscle's versatility, reliability, and negligible donor deformity when harvested through a low transverse abdominal incision. Seven patients reconstructed with this flap are presented, and the technique is discussed. All patients have been successfully reconstructed with preservation of the ankle and foot. At present, all patients are fully or partially weight-bearing. The segmental rectus abdominis free flap is recommended for the reconstruction of such wounds.
Peptide models of dynorphin A(1-17) incorporating minimally homologous substitutes for the potential amphiphilic beta strand in residues 7-15.
Two peptide models of dynorphin A(1-17) have been synthesized. These peptides incorporate a minimally homologous substitute sequence for residues 6-17, including alternating lysine and valine residues substituting for the potential amphiphilic beta-strand structure in positions 7-15. Model 1 retains Pro10 from the native sequence, but model 2 does not. Compression isotherms of peptide monolayers at the air-water interface and CD spectra of peptide films adsorbed from aqueous solution onto siliconized quartz slides were evaluated by comparison to those of idealized amphiphilic alpha-helical, beta-sheet, and disordered peptides. Dynorphin A(1-17) was mostly disordered, whereas beta-endorphin was alpha helical. Dynorphin model 1 had properties similar to those of dynorphin A(1-17) at these interfaces, but model 2 formed strongly amphiphilic beta sheets. In binding assays to mu-, delta-, and kappa-opioid receptors in guinea pig brain membranes, model 1 reproduced the high potency and selectivity of dynorphin A(1-17) for kappa receptors, and model 2 was only 3 times less potent and less selective for these receptors. Both peptide models retained the high, kappa-selective agonist activity of dynorphin A(1-17) in guinea pig ileum assays, and like dynorphin A(1-17), model 1 had little activity in the rat vas deferens assay. In view of the minimal homology of the modeled dynorphin structures, these studies support current models of membrane-catalyzed opioid ligand-receptor interactions and suggest a role for the amphiphilic alpha-helical and beta-strand structures in beta-endorphin and dynorphin A(1-17), respectively, in this process.
Introduction of guest peptides into Escherichia coli alkaline phosphatase. Excision and purification of a dynorphin analogue from an active chimeric protein.
High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution not only to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichia coli alkaline phosphatase polypeptide by linker insertion mutagenesis of the phoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues (Sowadski, J. M., Foster, B. A., and Wycoff, H. W. (1981) J. Mol. Biol. 150, 245-272). Mutant protein enzymatic function was generally tolerant of insertions in exterior loops, but was inactivated by insertion within alpha-helical or beta-strand structural elements. We further demonstrate that these tolerant surface loops can serve as vehicles for high level expression and stabilization of larger foreign peptide sequences, using a 15-residue analogue of dynorphin as an example. Insertion of the dynorphin "guest" peptide probably caused only a local structural perturbation of the alkaline phosphatase carrier since the hybrid protein retained enzymatic activity, was exported efficiently to the periplasmic space, and could be purified by anion-exchange chromatography using a protocol developed for alkaline phosphatase itself. The gust peptide was recovered from one of these fusion proteins intact and in high yield by protease digestion in vitro and was then purified by cation-exchange chromatography to near homogeneity in a single step.
Mosaic trisomy 16 in a live newborn infant.
Trisomy 16 is thought to be incompatible with fetal survival. A boy with mosaic trisomy 16 who lived for 11 weeks is reported. Chromosome analysis was carried out on skin fibroblasts grown during life and confirmed on samples taken at necropsy. We believe that this is the first report of mosaic trisomy 16 that has been confirmed by cytogenetic banding.