PubMed Health⌕ Search

Biomedical subjects

J E Bennett

Publications and source records attributed to J E Bennett.

At least 19 recordsLinked to original sources
PubMed1992

Small subunit ribosomal RNA of Blastomyces dermatitidis: sequence and phylogenetic analysis.

We determined the small subunit (18S) ribosomal RNA sequence of the dimorphic fungus Blastomyces dermatitidis. The sequence was compared to that of fourteen other eukaryotic organisms, ten of which were higher fungi, and an evolutionary tree was constructed based on these sequences. B. dermatitidis aligned most closely with the Ascomycetes Neurospora crassa and Podospora anserina, in agreement with previous phylogenetic analysis based on morphological criteria. Phase-specific cDNA clones derived by reverse transcription of RNA isolated from the yeast and mycelial phases of B. dermatitidis were also sequenced. The 18S ribosome sequence was found to be the same in both phases. Heterogeneity was found at both the genomic and RNA level at position 1352.

Base Sequence↗
PubMed1992

Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization.

In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast.

Amino Acid Sequence↗
PubMed1992

Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium.

Spontaneous mutants requiring uracil were isolated from both varieties of Cryptococcus neoformans by plating on 5-fluoroorotic acid (5-FOA) medium. Of the 36 strains tested (18 var. neoformans and 18 var. gattii), 24 (12 of each variety) generated 5-FOA-resistant cells requiring uracil for growth. Six of the 12 C. neoformans var. gattii strains produced ura3 cells while the remaining six strains produced ura5 cells. None of the 12 strains produced both ura3 cells and ura5 cells. All 12 isolates of var. neoformans, however, produced ura5 cells and one of them produced ura3 as well as ura5 cells. A genetic lesion in the URA5 gene of an isolate of C. neoformans var. gattii was confirmed by complement with the cognate URA5 gene of C. neoformans var. neoformans. The ura3 isolates were tentatively identified by their ability to grow on a medium containing uridine but not on a medium with orotic acid or orotidine. Enzymatic assays for orotidine-5'-phosphate decarboxylase activity confirmed the isolates to be ura3 mutants. Hybridization analysis of total DNA, digested with EcoRI or StuI and probed with pURA5g2, revealed the presence of only one copy of URA5 in the strains of either variety, regardless of the prevalence of ura5 mutants. Extensive polymorphism was observed in the restriction patterns of the fragments containing the URA5 locus. The prevalence of spontaneously arising ura3 mutants among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans, is one more biological difference that distinguishes the two varieties.

Animals↗
PubMed1991

In vivo interferon-gamma therapy augments the in vitro ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae.

During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-gamma (rIFN-gamma) therapy in chronic granulomatous disease (CGD), a metabolic assay of neutrophil damage to Aspergillus fumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 10(4) conidia that had germinated into hyphae were exposed to 5 x 10(5), 15 x 10(5), or 50 x 10(5) CGD neutrophils. By analysis of variance, neutrophils from patients on rIFN-gamma were found to produce significantly more damage to hyphae than those from the placebo group (P less than .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x 10(5) CGD neutrophils, where neutrophils from patients receiving rIFN-gamma produced significantly more damage to the hyphae than those from the placebo group (P less than .05). In vivo rIFN-gamma therapy improves the ability of CGD neutrophils to damage Aspergillus fumigatus hyphae in an in vitro assay.

Analysis of Variance↗
PubMed1991

Beta 1,4-oligoglucosides inhibit the binding of Aspergillus fumigatus conidia to human monocytes.

The binding of Aspergillus fumigatus conidia to human monocytes is mediated by a barley beta-glucan-inhibitable receptor. The simplest linkages in this glucan are present in the disaccharides laminaribiose (beta 1,3) and cellobiose (beta 1,4). Although laminaribiose gave strong inhibition of conidial binding to monocytes, cellobiose and oligosaccharides with beta 1,4-linked glucose residues were more potent as specific inhibitors of this binding over similar concentrations. Increasing the number of beta 1,4-linked glucose residues led to greater inhibition of conidial binding by human monocytes.

Aspergillus fumigatus↗
PubMed1991

Comparative safety, tolerance, and pharmacokinetics of amphotericin B lipid complex and amphotericin B desoxycholate in healthy male volunteers.

Amphotericin B lipid complex (ABLC), a lipid complex formulation of amphotericin B, and amphotericin B desoxycholate (AB) were compared for safety, tolerance, and pharmacokinetics in two groups of eight healthy male volunteers. After a 1-mg test dose, study drug was infused at 0.1, 0.25, and 0.5 mg/kg; the 0.5-mg/kg dose was not given to subjects receiving AB. ABLC caused few acute adverse effects except for mild somnolence (drowsiness) in six volunteers. In addition, three of eight ABLC recipient had asymptomatic, transient serum transaminase elevations that resolved spontaneously. The AB recipients experienced more acute side effects, but only one had a mild shaking chill: three of eight also experienced sleepiness. No significant changes in vital signs, electrocardiogram, oximetry, pulmonary function, or clinical status were observed in either group. Due to its increased estimate volume of distribution and estimated clearance. ABLC yielded decreased amphotericin B levels and area under the serum concentration versus time curve relative to AB.

Adult↗
PubMed1991

Standardization of a fluconazole bioassay and correlation of results with those obtained by high-pressure liquid chromatography.

An improved bioassay for fluconazole was developed. This assay is sensitive in the clinically relevant range (2 to 40 micrograms/ml) and analyzes plasma, serum, and cerebrospinal fluid specimens; bioassay results correlate with results obtained by high-pressure liquid chromatography (HPLC). Bioassay and HPLC analyses of spiked plasma, serum, and cerebrospinal fluid samples (run as unknowns) gave good agreement with expected values. Analysis of specimens from patients gave equivalent results by both HPLC and bioassay. HPLC had a lower within-run coefficient of variation (less than 2.5% for HPLC versus less than 11% for bioassay) and a lower between-run coefficient of variation (less than 5% versus less than 12% for bioassay) and was more sensitive (lower limit of detection, 0.1 micrograms/ml [versus 2 micrograms/ml for bioassay]). The bioassay is, however, sufficiently accurate and sensitive for clinical specimens, and its relative simplicity, low sample volume requirement, and low equipment cost should make it the technique of choice for analysis of routine clinical specimens.

Biological Assay↗
PubMed1991

Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity.

We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de-O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation.

Animals↗
PubMed1990

Administration of potassium iodide to normal volunteers does not increase killing of Sporothrix schenckii by their neutrophils or monocytes.

The mechanism by which iodide cures sporotrichosis was postulated to be an improvement in phagocyte-mediated killing of the causal fungus, Sporothrix schenckii. We found that both neutrophils and monocytes could readily kill the fungus and that killing could be blocked by adding inhibitors of oxidative metabolism. Iodide, administered to four volunteers for 1 week, raised their serum iodide level 213-fold but failed to improve killing of S. schenckii by their neutrophils or monocytes in a killing assay in vitro. Furthermore addition of iodide directly to neutrophils at concentrations as high as 100 microM did not augment killing. The mechanism by which iodide acts remains obscure.

Azides↗
PubMed1990

Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae.

Using a metabolic test of hyphal viability, the interaction between neutrophils and Aspergillus hyphae was investigated over a broad range of hyphae-to-neutrophil ratios. Normal neutrophils were found to damage hyphae whereas neutrophils from patients with both chronic granulomatous disease (CGD) and myeloperoxidase (MPO) deficiency did not. Further, both azide and catalase + superoxide dismutase inhibited the ability of normal neutrophils to damage hyphae, suggesting that this damage is mediated by products of the respiratory burst and by the MPO-halide system. Also, mixtures of small numbers of normal neutrophils with larger numbers of CGD neutrophils (range, 1:5 to 1:15) damaged hyphae more efficiently than either population of cells alone. Further, mixtures of CGD and MPO-deficient neutrophils, neither of which alone could efficiently damage hyphae, were able to damage the hyphae almost as well as a comparable number of normal neutrophils. These data demonstrate that intact neutrophils can cooperate to synergistically damage Aspergillus hyphae, possibly by extracellular mixing of hydrogen peroxide and MPO.

Aspergillus fumigatus↗
PubMed1990

Potential for laboratory exposures to biohazardous agents found in blood.

The magnitude of risk for occupational exposures to biohazardous agents found in blood was assessed by 800 environmental samples taken from a total of 10 clinical and research laboratories at the National Institutes of Health (NIH). Thirty-one samples from 11 work stations in three laboratories contained hepatitis B virus surface antigen (HBsAg). Observations of workers indicated that environmental contamination arose from several sources. Among the 11 work stations with HBsAg environmental samples, eight had high work loads, seven had inappropriate behaviors, and nine had flawed laboratory techniques. This information suggests that a multifactorial approach is needed to minimize the risk of laboratory-associated infections.

Blood↗