Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR.
(41/1936)
The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus species. A number of primers with the least homology to equivalent human or Candida gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of Aspergillus DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients. (+info)
Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species.
(42/1936)
We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical Aspergillus (n = 82) and Fusarium (n = 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for Aspergillus isolates, with minor variations among species. MICs for Fusarium isolates were higher than those for Aspergillus isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant Aspergillus fumigatus isolates and Fusarium solani. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for F. solani were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B. (+info)
Territrem B, a tremorgenic mycotoxin that inhibits acetylcholinesterase with a noncovalent yet irreversible binding mechanism.
(43/1936)
Territrem B (TRB) is a fungal metabolite isolated from Aspergillus terreus shown previously to be a potent and irreversible inhibitor of acetylcholinesterase (AChE). In the present study, a number of binding and inhibition assays were carried out to further characterize the inhibitory effect of TRB. The results indicate that the binding of TRB (a) is much more selective than a well characterized selective inhibitor of AChE, BW284C51, (b) adopts a one-to-one stoichiometry with the enzyme, (c) cannot be undone by an AChE-regenerating oxime agent, which contrasts the ability of 8 M urea to release AChE-bound TRB, (d) is enhanced by high concentration NaCl but prevented, unless preincubated, by Triton X-100, and (e) exhibits quasi-first order kinetics with an overall inhibition constant of 0.01 nM(-1) min(-1). Together these results suggest a very different irreversible binding (a noncovalent type) from that of the covalent type, which involves typical irreversible AChE inhibitors such as diisopropylfluorophosphate and neostigmine. According to the prediction of a molecular modeling study, the distinct AChE inhibitory characteristics of TRB may arise from the inhibitor being noncovalently trapped within a unique active-site gorge structure of the enzyme. It was predicted that an optimal TRB. AChE binding would position a narrowing connection of the TRB structure at a constricted area near the entrance of the gorge, thereby providing a structural basis for the observed irreversible binding. (+info)
Aspergillus tracheobronchitis after allogeneic bone marrow transplantation.
(44/1936)
We describe a patient who developed Aspergillus tracheobronchitis after BMT. She complained of progressive dyspnea on day +165 and her respiratory function deteriorated rapidly. Although neither early chest X-rays nor CT scans were negative, bronchoscopy revealed formation of a pseudomembrane around the bronchial walls. Based upon pathological and microbiological examinations, she was diagnosed as having invasive Aspergillus tracheobronchitis. Retrospectively analyzed, the Aspergillus circulating antigen detection tests became positive before clinical symptoms developed, and may be beneficial for early diagnosis of Aspergillus tracheobronchitis. This form of aspergillosis should be regarded as one of the serious complications after BMT. (+info)
Proton-pumping-ATPase-targeted antifungal activity of a novel conjugated styryl ketone.
(45/1936)
NC1175 (3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinyle ne]-1- ethyl-4-piperidinol hydrochloride) is a novel thiol-blocking conjugated styryl ketone that exhibits activity against a wide spectrum of pathogenic fungi. Incubation of NC1175 with various concentrations of cysteine and glutathione eliminated its antifungal activity in a concentration-dependent fashion. Since NC1175 is a lipophilic compound that has the potential to interact with cytoplasmic membrane components, we examined its effect on the membrane-located proton-translocating ATPase (H(+)-ATPase) of yeast (Candida albicans, Candida krusei, Candida guilliermondii, Candida glabrata, and Saccharomyces cerevisiae) and Aspergillus (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus nidulans) species. The glucose-induced acidification of external medium due to H(+)-ATPase-mediated expulsion of intracellular protons by these fungi was measured in the presence of several concentrations of the drug. NC1175 (12.5 to 50 microM) inhibited acidification of external medium by Candida, Saccharomyces, and Aspergillus species in a concentration-dependent manner. Vanadate-inhibited hydrolysis of ATP by membrane fractions of C. albicans was completely inhibited by 50 microM NC1175, suggesting that the target of action of NC1175 in these fungi may include H(+)-ATPase. (+info)
The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases.
(46/1936)
We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii. (+info)
Enzymatic function of the nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus.
(47/1936)
The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1' keto group of norsolorinic acid to the 1' hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro. (+info)
The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids.
(48/1936)
A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit gamma of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box. A single nucleotide exchange in this region (AAAATTCAAT --> AAGATCAAT) uncouples the expression of an AtpC promoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene. By screening an Arabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C. Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex. At least eight genes with homologies to CBF-C are present in the Arabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1. In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels, i.e. extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e. extracts from light-grown seedlings and etiolated seedlings treated with cytokinin. Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence. The data are consistent with the idea that the assembly of CBF at the AtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect. The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function in AtpC gene expression. (+info)