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PubMed2001

Evidence for paclitaxel from three new endophytic fungi of Himalayan yew of Nepal.

Taxus wallichiana, collected from the Himalayan region of Nepal, yielded, among others, three different endophytic fungi including Sporormia minima, Trichothecium sp., and an unidentified dimorphic fungus. Each was shown to produce paclitaxel in a culture medium. Each of these fungi represents a new report as a paclitaxel producer. The production of paclitaxel was confirmed by an immunoassay that utilized monoclonal antibodies, TLC chromatography as well as high performance liquid chromatography combined with on-line mass spectrometry.

Antibodies, Monoclonal↗
PubMed2003

[Incidence and detection of fungi and eosinophilic granulocytes in chronic rhinosinusitis].

BACKGROUND: Chronic Rhinosinusitis (CRS) is the most common chronic disease in the United States. Though for Europe no data are available, we have to assume that the situation is similar. Although the disease is defined very well by clinical symptoms, up to date the etiology and pathogenesis of chronic rhinosinusitis are unknown. CRS is considered to be multifactorial, with thickening of the mucosa and formation of polyps as an end stage of the disease. Treatment of choice includes corticosteroids and/or endoscopic or microscopic surgery. Antibiotics only help, if there is an acute bacterial exacerbation of the disease. They are not able to cure chronic rhinosinusitis per se. In 1999 Mayo Clinic researchers published data concerning the incidence of so-called "allergic" fungal sinusitis (AFS) in their patients suffering from chronic rhinosinusitis, demonstrating the majority of patients investigated presenting those criteria. Our own initial data from 2000 confirmed their findings. MATERIAL AND METHODS: In an open prospective study fungal cultures were obtained from nasal mucus of 238 consecutive patients suffering from CRS. As control group acted 23 members of our staff, who did not show any evidence for CRS. In addition, in 37 CRS patients surgical specimens (mucus and tissue) were investigated histologically for evidence of eosinophilic granulocytes and fungal elements. RESULTS: Using new techniques for fungal detection in culture and histology as proposed by Mayo Clinic researchers, positive detection of fungal cultures of the mucus of our CRS patients developed from 7 % in the past up to 87 % at present. 91.3 % of our control group yielded a positive result in fungal culture. Histologically, eosinophilic clusters were evident in 94.6 % and fungal elements were detected in 75.5 % within the mucus of 37 surgical CRS patients. Overall, 89.2 % of our surgical patients thus fulfilled the criteria of so-called AFS. Compared to our findings in the past, our latest results show an increase of 80 % in detection of fungal elements in our CRS patients. In all we were able to identify 654 positive fungal cultures in 238 CRS patients and 23 healthy controls respectively. 88 different genera grew, with 2.4 different species per patient and 3.1 different species per healthy control, on average. CONCLUSION: Utilizing new techniques of fungal culturing out of the mucus of CRS patients and healthy controls, the number of positive fungal cultures increased dramatically from 7 % to 87 % in our patients and 91.3 % in healthy controls respectively. To obtain these results it is crucial to perform special techniques of mucus sampling and pretreatment for culturing as well as for histological investigations. Our results show, that with suitable techniques fungi can be identified in almost everybody's nose, CRS patient or healthy. When inhaled, those airborne fungi are only "in transit" through the nose. Positive fungal cultures from nasal secretion therefore have to be considered normal findings. The reason for this delayed recognition has to be attributed to our inadequate methods in the past. In contrast to healthy controls, clusters of eosinophils and fungal elements are present simultaneously within the mucus of CRS patients and appear to be a marker of the disease.

Chronic Disease↗
PubMed2005

Evolution of a large ribosomal RNA multigene family in filamentous fungi: birth and death of a concerted evolution paradigm.

In eukaryotes, the primary components of the ribosome are encoded by multicopy nuclear ribosomal RNA (rRNA) genes: 28/26S, 18S, 5.8S, and 5S. Copies of these genes are typically localized within tandem arrays and homogenized within a genome. As a result, nuclear rRNA gene families have become a paradigm of concerted evolution. In filamentous fungi of the subphylum Pezizomycotina, 5S rRNA genes exist as a large and dispersed multigene family, with between 50 and 100 copies per genome. To determine whether these genes defy the concerted evolution paradigm, we examined the patterns of evolution of these genes by using sequences from the complete genomes of four species. Analyses of these sequences revealed (i) multiple 5S gene types within a genome, (ii) interspecies clustering of gene types, (iii) multiple identical gene types shared among species, (iv) multiple pseudogenes within a genome, and (v) presence/absence variation of individual 5S copies in comparisons of closely related species. These results demonstrate that the 5S family in these species is characterized by birth-and-death evolution under strong purifying selection. Furthermore, our results suggest that birth-and-death evolution occurs at different rates in the genera examined, and that the multiplication and movement of 5S genes across the genome are highly dynamic. As such, we hypothesize that a mechanism resembling retroposition controls 5S rRNA gene amplification, dispersal, and integration in the genomes of filamentous fungi.

Aspergillus nidulans↗
PubMed1989

Disparate evolution of yeasts and filamentous fungi indicated by phylogenetic analysis of glyceraldehyde-3-phosphate dehydrogenase genes.

Genes encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from several evolutionarily disparate organisms were used to construct a phylogenetic tree by evolutionary parsimony. The GAPDH tree indicates that, in contrast to the presently accepted taxonomy of fungi, the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii evolved separately from the filamentous ascomycetes (such as Aspergillus nidulans) with which these yeasts are classified. According to this tree, the Saccharomyces-like yeasts evolved very early in the course of eukaryotic evolution, whereas both ascomycete and basidiomycete filamentous fungi diverged much later through a common ancestor.

Biological Evolution↗
PubMed1997

Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.

Amino Acid Sequence↗
PubMed1999

Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins.

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.

Animals↗
PubMed2003

Molecular analysis of the pediatric ocular surface for fungi.

PURPOSE: To analyze the conjunctival flora of individuals 21 years of age or less for fungi using polymerase chain reaction (PCR) methodology. METHODS: Before povidone-iodine antisepsis, eye-swab specimens were collected from adolescent corneal donors preceding corneal excision and from children during preparation for strabismus surgery. Nucleic acid was extracted from the specimens and analyzed by PCR using primers designed for the detection of broad-spectrum fungal DNA and of Candida albicans-specific DNA. RESULTS: Twelve (38%) of 32 eye donor surfaces and 7 (23%) of 30 patient samples were positive for fungal DNA (P = 0.1). C. albicans DNA was detected in 6 (19%) of the decedents' eyes but from none of the surgical patients (P = 0.04). CONCLUSIONS: Fungi were present on the normal ocular surface of children and adolescents. C. albicans was more likely to be found postmortem than pre-surgically.

Adolescent↗
PubMed2002

Considerations on the use of the p-nitrophenyl phosphomonoesterase assay in the study of the phosphorus nutrition of soil borne fungi.

The p-nitrophenyl phosphomonoesterase assay (pNPPase) is commonly used to measure cell-wall-associated and extracellular phosphatase activity of soil fungi. pNPPases are usually assayed in the context of fungal nutrition, where inorganic P supply might be enhanced by the mineralisation of monoester organic P sources in the soil. The importance of the assay to the P nutrition of soil fungi is considered based on the evidence currently available including the consistency of methodological approach. The nature of organic P in the soil and the relevance of the assay to some specific soil substrates is discussed, particularly the chemistry and bioavailability of myo-inositol hexakisphosphate and the lower inositol phosphates. The evidence for the long-term stability of pNPPases in the soil is examined in the light of the persistence of pNPPase in soils. The role of persistent extracellular fungal pNPPases in the soil P cycle is discussed. Conclusions from pNPPase based studies must be based upon an appreciation of the constraints of the assay and the complex chemistry of organic P and pNPPase in the soil.

Fungi↗
PubMed2004

Quality test of the MicroSeq D2 LSU Fungal Sequencing Kit for the identification of fungi.

Fungal contaminants may be health risk factors in general indoor and occupational environments. The identification of microfungi by macro- and micro-morphological characters is known to be very difficult and time consuming. An alternative method for identification of fungi using a molecular biological method based on a commercially available kit is described. The method is based on the DNA-extraction and subsequent amplification of the D2 expansion region of the large subunit of the fungal rDNA. DNA extraction, amplification, sequencing and database screening can be performed within 24 hours. A total of 28 different fungal species were identified on a morphological basis and compared with identification by the molecular biological method. The database comparison revealed identification down to the species for 12 samples and to the genus level of 11 samples. Five samples could not be identified. It is concluded that the identification of fungi by molecular biological method needs to be improved. This can partly be reached by expanding the fungal DNA-sequence datasets.

Air Pollution, Indoor↗
PubMed1975

Chromomycosis in the toad (Bufo marinus) and a comparison of the etiologic agent with fungi causing human chromomycosis.

Of 75 toads (Bufo marinus) 2 were found infected by black molds. The internal organs of these animals had granulomatous lesions containing brown fungi identical to those found in human chromomycosis. Cultures gave rise to slow-growing black molds but all attempts to induce sporulation failed. The fungi did not grow at 36 degrees C or above and failed to hydrolyse gelatin or casein. Immunodiffusion and immunoelectrophoresis revealed that both isolates were identical and shared common antigens with the recognized human pathogens P. pedrosoi, P. verrucosa and C. carrioni. The findings are compared with other reports of black mold infections in amphibians.

Animals↗
PubMed1979

The preservation of medically important fungi.

Details are given of a method, first described in 1962, for preservation of fungi in anhydrous silica gel. It is shown that the procedure is suitable for use with dermatophytes, yeasts and other fungi of medical importance and that they may be preserved for periods of 5 years or more.

Fungi↗
PubMed1981

The effect of 5-fluorocytosine on the synthesis of 80S ribosomes by pathogenic fungi.

We studied the influence of 5-fluorocytosine(5FC) on the monomeric 80S ribosomes isolated by gradient ultracentrifugation from Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and Wangiella dermatitidis. Labelling of the cultures with 32PO4 and [3H]-leucine showed marked inhibition of both ribosomal RNA and ribosomal protein synthesized during the contact of the fungi with the drug. The degree of inhibition was very similar for the synthesis of both ribosomal constituents, (mostly 60-80% each during the initial hours of the contact). This indicates that some ribosomes were still synthesized in the presence of 5FC and that the drug inhibits the synthesis of complete ribosomes to the same extent as the synthesis of ribosomal RNA and protein. By labelling fungi with [14C]-5FC it was shown that in those 80S ribosomes that were still synthesized, large quantities of the drug were incorporated--doubtless as 5-fluorouracil (5FU) in RNA. It can be concluded that RNA containing 5FU is still capable of combining with protein to form complete ribosomes of normal conformation. This is, however, no proof that these ribosomes would have a normal function.

Cell-Free System↗
PubMed1986

Occurrence of nitroso compounds in fungi-contaminated foods: a review.

Dimethylnitrosamine (NDMA), diethylnitrosamine (NDEA), methylbenzylnitrosamine, and a new N-nitroso compound, N-3-methylbutyl-N-1-methylacetonylnitrosamine (NMAMBA), were found in cornbread that was inoculated with Fusarium moniliforme and underwent an incubation and the addition of a small amount of NaNO2. The carcinogenicity of NMAMBA was shown by the induction of forestomach carcinomas and liver tumors in mice and rats. Another new nitrosamine, N-2-methylpropyl-N-methylacetonyl nitrosamine, was isolated in millet and wheat flour after similar treatment. Some species of fungi not only can reduce nitrates to nitrites but also can increase the amount of secondary amines in the moldy foods and provide favorable conditions for the synthesis of nitrosamines. Some fungi may utilize primary amines to synthesize nitrosamines in appropriate culture mediums. Furthermore, the cornmeal may naturally contain trace amounts of nitrosamines, as detected by a gas chromatography/thermoenergy analyzer. Prolonged feeding of F. moniliforme-inoculated cornbread alone also induced the development of forestomach carcinoma in rats. This indicates the occurrence of potential carcinogens, perhaps certain Fusarium mycotoxins, in the moldy cornbread in addition to the carcinogenic nitrosamines. Another nitroso compound, Roussin red methylester (di-mu-methanethiolatotetranitrosodiiron) was found in pickled vegetables that are eaten daily in the Linxian (China) area. Roussin Red may provide NO-2 ions, which would react readily with secondary amines to form nitrosamines. Possibly this compound is a natural tumor promoter and is able to induce epithelial hyperplasia of the upper digestive tract and forestomach papilloma in treated mice. Also, trace amounts of NDMA, NDEA, and some other nitrosamines were found in the extract of pickled vegetables.

Animals↗
PubMed1994

Phylogeny of dermatophytes and dimorphic fungi based on large subunit ribosomal RNA sequence comparisons.

The phylogeny of dermatophytes and dimorphic fungi was considered using the large-subunit of ribosomal RNA (25S rRNA). Aligned sequences of 595 nucleotides covering the two most divergent domains D1 and D2, permitted a comparison of phylogenetic relationships at different levels. The dimorphic species (Onygenaceae) were significantly separated from dermatophytes (Arthrodermataceae) and from a third group including geophilic or very weakly pathogenic species (Onygenaceae and Gymnoascaceae). On a species level, the varietal status of Histoplasma duboisii and Histoplasma farciminosum, as close relations of Histoplasma capsulatum, was confirmed. The dimorphic fungus Emmonsia parva, in spite of a completely different parasitic form (adiaspores instead of yeast-like cells), clustered with Blastomyces dermatitidis which has a perfect form resembling that of H. capsulatum. From our data, teleomorphs of E. parva, Paracoccidioides brasiliensis and H. farciminosum, three dimorphic fungi known only under their anamorphic states, should belong to the family of Onygenaceae and the genus Ajellomyces. Among Arthrodermataceae, and family containing the most keratinophilic species, it was not possible to establish a clear hierarchy of species. Only Ctenomyces serratus, the species adapted to degrade keratin of feathers, Trichophyton ajelloi and Trichophyton terrestre were significantly separated. The speciation of true dermatophytes resulted most likely from a very recent evolution by adaptation to parasitism. Among species used as outgroups, the two emerging pathogens Pseudallescheria boydii (Scedosporium apiospermum) and Scedosporium prolificans (Scedosporium inflatum) were shown to be closely related to each other.

Arthrodermataceae↗
PubMedDate not supplied

Rapid detection and identification of pathogenic fungi by polymerase chain reaction amplification of large subunit ribosomal DNA.

We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens.

Base Sequence↗
PubMedDate not supplied

Expression of PZ-peptidases by cultures of several pathogenic fungi. Purification and characterization of a collagenase from Trichophyton schoenleinii.

Peptidolytic activity was studied in the broken-cell extracts of 17 isolates of pathogenic fungi tested with phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (PZ-PLGPA) as a substrate. All the fungi studied except Candida spp., Cryptococcus neoformans and two actinomycetes hydrolyzed the substrate and therefore contained a so-called PZ-peptidase activity. Of all the positive strains, Trichophyton schoenleinii, a pathogenic fungus showed the highest activity and was therefore chosen as a source for PZ-peptidase purification. The four chromatographic steps, a 'negative' dye column, a 'positive' dye column, hydroxyapatite Ultrogel, and modified TSK (HW 55), gave a highly purified peptidase with a 12% overall yield. Inhibitor studies suggested that the 82 000 M(r) PZ-peptidase is a metalloproteinase. Moreover it cleaved native rat type I collagen. Partial peptide sequencing showed a strong sequence homology to regions of two metalloproteinases previously identified in the yeast Saccharomyces cerevisiae and in rat.

Amino Acid Sequence↗
PubMed1999

Molecular mechanisms of chromosomal rearrangement in fungi.

Both sexual and asexual fungi undergo chromosomal rearrangements, which are the main cause of karyotype variability among the populations. Different recombination processes can produce chromosomal reorganizations, both during mitosis and meiosis, but other mechanisms operate to limit the extent of the rearrangements; some of these mechanisms, such as the RIP (repeat-induced point mutations) of Neurospora crassa, have been well established for sexual fungi. In laboratory strains, treatments such as mutation and transformation enhance the appearance of chromosomal rearrangements. Different DNA sequences present in fungal genomes are able to promote these reorganizations; some of these sequences are involved in well-regulated processes (e.g., site-specific recombination) but most of them act simply as substrates for recombination events leading to DNA rearrangements. In Penicillium chrysogenum we have found that short specific DNA sequences are involved in tandem reiterations leading to amplification of the cluster of the penicillin biosynthesis genes. In some cases, specific chromosomal rearrangements have been associated with particular phenotypes (as occurs in adaptive-like mutants of Candida albicans and Candida stellatoidea), and they may play a role in genetic variability for environmental adaptation.

Chromosomes, Fungal↗
PubMed1996

Review of quantitative standards and guidelines for fungi in indoor air.

Existing quantitative standards/guidelines for fungi in indoor air issued by governmental agencies are based primarily on baseline data (rather than health effects data), and are either absolute (numerical) or relative (indoor/outdoor comparisons) or a combination of the two. The Russian Federation is the only governmental agency that has binding quantitative regulations for bioaerosols. Recommended guidelines have been proposed or sponsored by North American and European governmental agencies and private professional organizations. A considerable number of frequently cited guidelines have been proposed by individuals based either on baseline data or on personal experience. Quantitative standards/guidelines range from less than 100 CFU/m3 to greater than 1000 CFU/m3 (total fungi) as the upper limit for non-contaminated indoor environments. Major issues with existing quantitative standards and guidelines are the lack of connection to human dose/response data, reliance on short term grab samples analyzed only by culture, and the absence of standardized protocols for data collection, analysis, and interpretation. Urgent research needs include the study of human responses to specific fungal agents, development and widespread use of standard protocols using currently available sampling methodologies, and the development of long term, time-discriminating personal samplers that are inexpensive, easy to use, and amenable to straightforward, relevant analysis.

Air Microbiology↗