Biomedical subjects

J W Taylor

Publications and source records attributed to J W Taylor.

At least 19 recordsLinked to original sources

PubMed1998

Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus.

Aspergillus flavus, like approximately one-third of ascomycete fungi, is thought to be cosmopolitan and clonal because it has uniform asexual morphology. A. flavus produces aflatoxin on nuts, grains, and cotton, and assumptions about its life history are being used to develop strategies for its biological control. We tested the assumptions of clonality and conspecificity in a sample of 31 Australian isolates by assaying restriction site polymorphisms from 11 protein encoding genes and DNA sequences from five of those genes. A. flavus isolates fell into two reproductively isolated clades (groups I and II). The lack of concordance among gene genealogies among isolates in one of the clades (group I) was consistent with a history of recombination. Our analysis included five strains of the closely related industrial fungus A. oryzae, all of which proved to be clonally related to group I.

Aflatoxins↗

PubMed1998

Insulin increases fatty acid synthase gene transcription in human adipocytes.

The purpose of this study was to investigate the molecular mechanism whereby insulin increases expression of a key de novo lipogenic gene, fatty acid synthase (FAS), in cultured human adipocytes and hepatoma cells. RNA isolated from cultured adipocytes or from Hep G2 cells treated with or without insulin (20 nM) was analyzed. In addition, run-on transcription assays and measurements of RNA half-life were performed to determine the controlled step in FAS gene regulation by insulin. We demonstrated that FAS mRNA was expressed in both Hep G2 cells and human adipocytes. Insulin induced an approximately five- and three-fold increase in FAS mRNA content in adipocytes and hepatoma cells, respectively. Similar regulation of FAS was observed in adipocytes from lean and obese human subjects. Furthermore, we demonstrated that the induction of human FAS expression by insulin was due to increased transcription rate of the FAS gene in human adipocytes, whereas mRNA stabilization accounted for increased FAS mRNA content in hepatoma cells. In conclusion, we report here for the first time expression of human FAS mRNA and its specific transcriptional induction by insulin in cultured human adipocytes.

Adipocytes↗

PubMed1998

Suction-assisted lipectomy for the correction of stomal dysfunction.

Poorly fitting appliances for urinary or fecal stream stomas can lead to frequent leaking and subsequent social embarrassment, stomal stenosis, hyperkeratosis, skin breakdown, and increased cost secondary to frequent changing. Obese patients with abnormal skin folds frequently are more prone to these problems. Herein is described the use of suction-assisted lipectomy for abdominal wall contouring in an effort to improve and simplify appliance application as well as lengthen appliance life. Between 1989 and 1996, five patients (three females, two males) ages 13 to 47 years, were treated with abdominal wall suction-assisted lipectomy in an attempt to improve stomal function. Preoperatively, an average of 1.25 appliance changes a day were required for leaking. Three patients had one procedure removing 400 to 600 cc of fat. Two patients had staged suction, removing a total of 900 to 1600 cc of fat. At 6-month follow-up, the average appliance life was 4 days, and the incidence of skin problems and leaking was markedly reduced. The technique of suction-assisted lipectomy is a simple outpatient procedure with low morbidity that can successfully be used to improve stomal function in obese patients with poorly fitting appliances secondary to skin folds, poor body habitus, or abdominal scarring.

Abdominal Muscles↗

PubMed1997

Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis.

Simple cladogenetic theory suggests that gene genealogies can be used to detect mixis in a population and delineate reproductively isolated groups within sexual taxa. We have taken this approach in a study of Coccidioides immitis, an ascomycete fungus responsible for a recent epidemic of coccidioidomycosis (Valley fever) in California. To test whether this fungus represents a single sexual species throughout its entire geographic range, we have compared genealogies from fragments of five nuclear genes. The five genealogies show multiple incompatibilities indicative of sex, but also share a branch that partitions the isolates into two reproductively isolated taxa, one centered in California and the other outside California. We conclude that coccidioidomycosis can be caused by two distinct noninterbreeding taxa. This result should aid the future study of the disease and illustrates the utility of the genealogical approach in population genetics.

Amino Acid Sequence↗

PubMed1997

A set of electrophoretic molecular markers for strain typing and population genetic studies of Histoplasma capsulatum.

A set of eleven biallelic and three multiallelic molecular markers have been developed to analyze populations of Histoplasma capsulatum. All markers are amplified by polymerase chain reaction (PCR) and can be readily scored using minimal amounts of template DNA. The 11 biallelic loci have polymorphic restriction endonuclease sites or small insertions or deletions which may be assessed by agarose gel electrophoresis. These markers are inherited in an unambiguous manner and are ideal for assessing structure and gene flow within US populations of H. capsulatum, but are monomorphic in non-US populations. Both length and sequence variation are present in the multiallelic loci, which can be scored by direct sequencing, polyacrylamide gel electrophoresis, or single-strand conformation polymorphism (SSCP): As they are hypervariable, the multiallelic loci can be used to type isolates and to assess the level of genetic variation within populations. Preliminary results indicate that the three multiallelic markers presented are sufficient to distinguish isolates at the individual level and are polymorphic in both US and non-US populations. This collection of molecular markers will be a useful tool in population and epidemiology studies of H. capsulatum.

Alleles↗

PubMed1997

The ultrastructure of skin from a patient with mucopolysaccharidosis IIID.

Mucopolysaccharidosis IIID (MPS-IIID) is the rarest of the MPS-III syndromes. It is caused by deficient activity of lysosomal N-acetylglucosamine-6-sulfatase (G6S). To date, the clinical and biochemical features of seven patients with MPS-IIID have been reported, but no biopsy or autopsy findings have been described. The purpose of this report is to define the ultrastructure of affected cells seen in a skin biopsy from a 14-year-old boy. The child presented with progressive mental deterioration, hyperactivity and mild to moderate dysmorphism. The diagnosis of a mucopolysaccharidosis was suggested, but the initial urine analyses were negative for elevated mucopolysaccharides, and only the third analysis showed abnormal excretion of heparan sulfate. Because of the diagnostic difficulties posed by this case, a skin biopsy was performed for morphological and biochemical studies. Numerous vacuoles were noted in Schwann cells, fibroblasts, smooth muscle cells, eccrine gland and ductal epithelium in resin-embedded sections stained with toluidine blue. Ultrastructurally, many lysosomes were distended with abundant, fibrillar material. Occasionally, lamellated membranous structures were present within the same lysosomes. These findings are consistent with those seen in other forms of MPS, in which the lysosomal storage occurs predominantly, but not exclusively, in mesenchymal cells. Furthermore, deficient activity of G6S was confirmed in cultured skin fibroblasts. This study demonstrates that electron microscopy of skin biopsies is a useful method for identification of patients with clinical features of MPS-IIID whether or not heparan sulfaturia is present.

Adolescent↗

PubMed1997

Molecular markers reveal differentiation among isolates of Coccidioides immitis from California, Arizona and Texas.

Coccidioides immitis causes coccidioidomycosis, a fungal disease of both immuno-compromised and otherwise healthy people; it is capable of causing large epidemics and the disease is often refractory to chemotherapy. To quantify the magnitude of population differentiation and estimate levels of gene flow in C. immitis, multilocus genotypes were scored for 20-25 clinical isolates from each of Bakersfield (California), Tucson (Arizona), and San Antonio (Texas). The molecular markers used were PCR products with polymorphic restriction endonuclease sites, found and characterized in a previous study of the Tucson population. The data show very highly significant differences in allele frequencies between all three populations, and suggest very low levels of migration between populations. One isolate in the San Antonio sample was an outlier, showing the California-specific allele at all four of the loci distinguishing the two populations, and subsequent inquiries indicated that the infection had indeed been acquired in California. Thus, genetic information can be used to infer the geographical origin of a fungal infection.

Arizona↗

PubMed1997

Angiotensinogen gene expression in adipose tissue: analysis of obese models and hormonal and nutritional control.

Synthesis of angiotensin II (ANG II) has recently been described in adipose cells and has been linked to regulation of adiposity. Angiotensinogen (AGT), the substrate from which ANG II is formed, was previously shown to be elevated in adipose tissue of obese (ob/ob and db/db) mice and regulated by nutritional manipulation. It is unknown, however, whether overexpression of adipose AGT can be extended to other models of obesity and whether hormonal and/or nutritional factors directly regulate AGT expression in adipocytes. We investigated these possibilities by analyzing AGT mRNA levels in adipose tissue of obese Zucker rats, viable yellow (Avy) mice, and humans and by treating 3T3-L1 adipocytes with insulin, glucose, and a beta-adrenergic agonist. We demonstrate that AGT mRNA is decreased by approximately 50 and 80%, respectively, in adipose tissue of obese vs. lean Zucker rats and Avy mice. We also report that AGT is expressed at variable levels in human adipose tissue. Finally, we show that AGT mRNA is upregulated by insulin and downregulated by beta-adrenergic stimulation in adipocytes.

3T3 Cells↗

PubMed1997

Human mucopolysaccharidosis IIID: clinical, biochemical, morphological and immunohistochemical characteristics.

Mucopolysaccharidosis IIID (MPS IIID) is one of the rarest of the MPS-III syndromes. To date, the clinical manifestations of 10 patients have been reported, the deficient N-acetylglucosamine 6-sulfatase (G6S) enzyme has been purified, and the G6S gene has been cloned, sequenced and localized. However, morphological manifestations of this condition have not been reported and the pathogenesis of the severe neurological deficits remains an enigma. In this paper we describe and correlate the clinical, biochemical and pathological observations for 2 cases of MPS IIID. We used monoclonal antibodies against heparan sulfate (HS) and GM2-ganglioside, thin layer chromatography, mass spectrometry, and morphological techniques to demonstrate the nature and the distribution of the uncatabolized substrates. The majority of the cells in various tissues showed morphological changes expected with lysosomal storage of HS. The central nervous system (CNS) was most severely affected because of the secondary storage of GM2 and GM3 gangliosides in addition to the primary accumulation of HS. The extent as well as the distribution of the diverse storage materials varied within and among different neurons as observed in MPS-III A, B, and C syndromes. This study supports the hypothesis that the neurological dysfunction and neurodegeneration common to the Sanfilippo syndromes is, in part, due to the secondary metabolic perturbations induced by HS accumulation.

Adolescent↗

PubMed1996

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis.

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.

Base Sequence↗

PubMed1996

Human pathogeneic fungi and their close nonpathogenic relatives.

In order to understand the relationships between human pathogenic fungi and their close, nonpathogenic relatives, we compared small-subunit ribosomal DNA sequences among four closely related pathogens, Histoplasma capsulatum, Blastomyces dermatitidis, Trichophyton rubrum, and Coccidioides immitis, and seven nonpathogenic fungi expected on morphological grounds to be their nearest relatives. We sequenced small-subunit RNA genes from these fungi and used both genetic distance and parsimony algorithms to evaluate their evolutionary relationships to the pathogens. We show that the pathogens are not a monophyletic group, but rather are interspersed among nonpathogenic fungi; thus it is likely that pathogenicity has arisen multiple times within this group. The saprobic fungi Chrysosporium parvum and Uncinocarpus reesii are the closest known relatives of the highly pathogenic Blastomyces dermatitidis and Coccidioides immitis, respectively, and thus may be considered primary candidates for model systems for researchers studying these fungi. The branching order suggests that the conidium (asexual spore) types aleurioconidia and arthroconidia do not define monophyletic groups and may be less distinct than their names suggest. Fungi with a complete life cycle are shown to have closest relatives that lack a known sexual cycle. Such analyses offer a means by which the sexual and asexual fungi could be integrated into a single classification system.

Base Sequence↗

PubMed1996

Phylogenetic origins of the asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes.

The phylogenetic relationship of the asexual mycorrhizal fungus Cenococcum geophilam Fr. among sexual ascomycetes was examined by phylogenetic analysis of nucleotide sequence data from the nuclear small subunit (18S) ribosomal RNA genie region. A specific focus of this study was to test the hypothesis that the genus Elaphomyces is the closest sexual relative of C. geophilum. Thus nucleotide sequence data of five C. geophilum isolates, three Elaphomyces species, and 44 additional genera of ascomycetes were included in the phylogenetic analyses. The percentage of similarity among the 18S rDNA sequences of the C. geophilum isolates examined was 99.8 to 100%, indicating that C. geophilum is monophyletic. Percent similarity of nucleotide sequence among the three Elaphomyces species was also high and ranged from 99.4 to 99.5%. DNA parsimony and distance analysis of the sequence data separated these 2 genera on distant clades when sequence from 44 additional genera of ascomycetes was included. Parsimony and distance analyses positioned C. geophilum as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothidiales, and strongly supported Elaphomyces to be of Plectomycete origin. Among the sexual Ascomycetes examined, which included representative taxa from four classes of filamentous Ascomycetes (Plectomycetes, Pyrenomycetes, Discomycetes, and Loculoascomycetes), no close sexual relative to C. geophilum was identified. At least four independent lineages of mycorrhizal fungi were identified among the ascomycetes examined.

Ascomycota↗

PubMed1996

Insulin increases lipogenic enzyme activity in human adipocytes in primary culture.

Studies with human adipose tissue have demonstrated the presence of key enzymes of fat synthesis. However, long-term regulation of these enzymes has not been reported. To address this issue, we used human adipocytes in primary culture. Human adipose tissue was obtained from abdominal fat of patients undergoing abdominal surgery. Adipocytes were isolated by collagenase digestion and cultured in media supplemented with 1% fetal bovine serum. To evaluate metabolic activity of cultured cells, we assessed the following during the culture: DNA pattern, cell size, glucose consumption and activities for two lipogenic enzymes, fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH). Analysis of DNA pattern showed that human adipocytes cultured under the above condition did not undergo cell apoptosis. In addition, no significant change in the cell size occurred during 22 d of culture. Glucose consumption by cultured cells was also constant during the culture and was 60% greater in the presence of 10 nmol/L of insulin. Treatment of cultured human adipocytes with insulin for 3-22 d increased GPDH and FAS activity by 60% and 2.8-fold, respectively, compared to cells cultured without insulin. Furthermore, the increase in FAS activity due to insulin treatment was dose dependent and maximal at 10 nmol/L. Our studies show for the first time that human adipocytes can be maintained viable and metabolically active for 2-3 wk in culture. Interestingly, cultured cells remain responsive to insulin. Therefore, this system will allow further characterization of long-term regulation of lipogenesis in human adipocytes and will be useful for developing pharmacological treatments of obesity.

Adipocytes↗

PubMed1996

Clinical isolates of Histoplasma capsulatum from Indianapolis, Indiana, have a recombining population structure.

A sample of 30 clinical isolates of Histoplasma capsulatum was analyzed to determine (i) whether genetic exchange is important in the life cycle of this fungus and (ii) whether distinct subpopulations which correlate with disease severity or host immune status exist. Eleven biallelic molecular markers were developed, with the frequency of the least common allele at each molecular locus ranging from 10 to 50%. Every isolate had a different, unique multilocus genotype. Data analysis indicated that frequent recombination occurs within the Indianapolis, Ind., population. There were no associations between isolates from the immunocompromised population or from those with different clinical manifestations of histoplasmosis.

Genes, Fungal↗

PubMed1995

Structural and conformational requirements for human calcitonin activity: design, synthesis, and study of lactam-bridged analogues.

The conformational and pharmacological effects of the introduction of conformational constraints in the form of i-(i + 4) lactam-bridges in the potential amphiphilic alpha-helical region (8-21) of human calcitonin (hCT) were studied. The following three cyclic hCT analogues were synthesized: cyclo17,21-[Lys17,Asp21]hCT (1), cyclo17,21- [Asp17,Lys21]hCT (2) and cyclo10,14-[Lys10,-Asp14]hCT (3). For their syntheses, solid-phase methodology was used in combination with either direct side chain to side chain cyclization on the solid support or a segment-condensation strategy. Circular dichroism studies in aqueous buffer, pH 7.0, indicated that the conformational effects were different for each lactam bridge introduced. Significant induction of alpha-helical structure was observed only for peptide 3. In contrast, peptide 1 and hCT had similar CD spectra, indicative of mixed disordered and beta-sheet conformations, and peptide 2 had a weaker spectrum consistent with the formation of a more ordered but nonhelical structure. In rat brain receptor binding assays, peptide 2 showed a nearly 80-fold higher potency than hCT or peptides 1 and 3. All three analogues stimulated adenylyl cyclase in the rat kidney membrane at 5-fold lower concentrations than hCT and with similar maximal effects. In vivo hypocalcemic assays, performed in mice by analysis of serum calcium levels 1 h after sc injection, indicated that peptide 2 had similar maximal effects to hCT and was 10-20 times more potent than hCT at doses giving half-maximal effects. In contrast, peptides 1 and 3 were not significantly more potent than hCT. Our findings indicate compatibility of all three lactam bridges and, most probably, also the amphiphilic alpha-helix, with the pharmacological activities of hCT. However, the properties of peptide 2 also suggest that another conformation, possibly a type I beta-turn involving residues 17-20, may play an important role. A multistep mechanism of receptor recognition by hCT that might account for these results is discussed.

Adenylyl Cyclases↗

PubMed1995

Phylogeny of Discomycetes and early radiations of the apothecial Ascomycotina inferred from SSU rDNA sequence data.

We used nucleotide sequences of the small subunit ribosomal genes (SSU rDNA) to examine evolutionary relationships of apothecial ascomycetes (division Ascomycota; class Discomycetes sensu), commonly known as the cup fungi. The apothecial ascomycetes include both lichen-forming and free-living fungi. We sequenced the SSU rDNA from representatives of 10 fungal genera from four orders: Pezizales (Ascobolus lineolatus, Morchella elata agg., Peziza badia); Leotiales (Leotia lubrica, Sclerotinia sclerotiorum); Caliciales (Calicium tricolor, Mycocalicium albonigrum, Sphaerophorus globosus); and Lecanorales (Lecanora dispersa, Porpidia crustulata). Of these, C. tricolor, S. globosus, L. dispersa, and P. crustulata are lichen-forming fungi. Based on parsimony analyses of approximately 1750 aligned nucleotides of their SSU rDNA, we determined a most parsimonious tree (MPT). This hypothesis suggests that the apothecial ascomycetes are a paraphyletic assemblage, basal to other groups of filamentous ascomycetes including representatives of the perithecial fungi and cleistothecial fungi. The most parsimonious tree produced using this dataset supported the monophyly of the orders Pezizales, Leotiales, and Lecanorales. However, there was no support for monophyly of the representative Caliciales; S. globosus had affinities with members of the Lecanorales. This phylogenetic hypothesis recognizes Pezizales as basal and supports Nannfeldt's hypothesis (1932) of a primitive apothecial ascomata with subsequent evolution of perithecial and cleistothecial forms. This MPT provides a foundation for understanding evolution of the ascomycetous fungi.

Ascomycota↗

PubMed1995

Pretreatment regimens for adverse events related to infusion of amphotericin B.

Infusion-related adverse events (IRAEs) such as nausea, vomiting, fever, chills, and thrombophlebitis that are associated with amphotericin B therapy often lead clinicians to prescribe a number of adjunctive pretreatment medications in an attempt to reduce the incidence and severity of these events. The purpose of this study was to determine the incidence of IRAEs during the first week of systemic amphotericin B therapy and to identify pretreatment regimens that are effective in preventing these IRAEs. Three hundred ninety-seven adult inpatients receiving amphotericin B therapy were prospectively monitored, and data regarding IRAEs and pretreatment regimens were collected. Of these patients, 282 (71%) developed at least one IRAE during the first 7 days of therapy. The IRAEs most commonly reported were fever (51% of patients) and chills (28%), followed by nausea (18%), headache (9%), and thrombophlebitis (5%). The most common regimens included diphenhydramine, a corticosteroid, acetaminophen, and heparin, administered alone or in combination with these or other drugs. Overall, common pretreatment regimens were similar in efficacy to no pretreatment in the prevention of IRAEs. Thus empirical premedication for IRAEs associated with amphotericin B cannot be routinely advocated; instead, patients should be treated when symptoms first arise and then premedicated for subsequent amphotericin B infusions.

Acetaminophen↗

PubMed1995

Positions of multiple insertions in SSU rDNA of lichen-forming fungi.

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.

Base Sequence↗