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PubMed1986

The molybdate-stabilized glucocorticoid binding complex of L-cells contains a 98-100 kdalton steroid binding phosphoprotein and a 90 kdalton nonsteroid-binding phosphoprotein that is part of the murine heat-shock complex.

This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from 32P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98-100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98-100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis. This antibody recognizes an epitope that is conserved in 90 kdalton phosphoproteins from rodent and human cells, and it reacts with the 90 kdalton phosphoprotein that copurifies with the molybdate-stabilized, untransformed chick oviduct progesterone receptor. The 90 kdalton nonsteroid-binding phosphoprotein is an abundant cytosolic protein that dissociates from the glucocorticoid receptor when it is transformed, and unlike the steroid-binding protein, it does not bind to DNA. The 90 kdalton phosphoprotein determines the acidic behavior of the untransformed glucocorticoid receptor on DEAE-cellulose. This abundant cytosolic 90 kdalton phosphoprotein reacts with rabbit antiserum raised against the gel purified 89 kdalton chicken heat-shock protein (hsp89). This antiserum recognizes 90 kdalton heat-shock proteins in human, rodent, frog and Drosophila cells. Immunoadsorption of molybdate-stabilized cytosol with antibody directed against the 98-100 kdalton steroid receptor results in the immune-specific adsorption of a 90 kdalton phosphoprotein that reacts with anti-hsp89 antibody on Western blots. These observations suggest that, like the transforming proteins from several avian sarcoma viruses, the untransformed glucocorticoid receptor exists in a complex with the 90 kdalton heat-shock protein.

Animals↗

PubMed1985

Prostaglandins or prostaglandin like substances are implicated in normal growth and development in oomycetes.

The prostaglandin synthesis inhibitors aspirin and indomethacin inhibit the growth of Achlya caroliniana, A. ambisexualis and Saprolegnia parasitica in a dose-related manner. In addition, the inhibitors cause the formation of a characteristic asterisk-shaped colony. This abnormal colony morphology does not appear to be dependent on medium composition, since three different nitrogen and five differentcarbon sources all support the abnormal growth in the presence of 0.1 mM indomethacin. The abnormal colony morphology is the result of abnormal branching. Inhibitor grown colonies are more densely branched than controls, with shorter distances between branches. Inhibited colonies allowed to grow for greater than ten days escape the inhibition and assume a normal gross colony morphology and size, however, they do not reproduce sexually. The addition of 2 micrograms/ml PGF1 alpha to the growth medium partially overcomes the growth inhibition caused by indomethacin. The data suggest a role for prostaglandin or prostaglandin-like compounds in oomycete development.

Alprostadil↗

PubMed2004

Image analysis of hyphal morphogenesis in Saprolegniaceae (Oomycetes).

Because of their wide range of apical morphology, several members of saprolegniaceous fungi (Oomycetes) were chosen to examine concordance with the vesicle supply center (VSC) model of hyphal morphogenesis. Two computer routines were devised to measure diameter changes over long stretches of hyphae and to test compatibility with the theoretical hyphoid shape, y = xcot(xV/N). In all four genera examined, the apex followed closely the contour described by the hyphoid equation; divergences became evident in the subapex. The hyphae of Saprolegnia parasitica showed maximum concordance with the VSC model, i.e., their profile matched a hyphoid curve from the apex to the entire length of the mature hyphal tube. In Aphanomyces and Leptolegnia, growth in the subapical region subsided becoming less than that specified by the hyphoid equation. In Achlya bisexualis, the reverse was true, the subapical region expanded beyond that specified by the hyphoid equation. The two divergent subapical tendencies gave the hyphal tips a cylindroid or conoid appearance, respectively. Since the hyphal apex of all four species conformed to the curvature dictated by the hyphoid equation, we concluded that a basic VSC mechanism operates in all of these oomycetous fungi. Accordingly, we suggest that the shape of an oomycetous hypha is generated by a VSC-driven gradient of wall formation, which is subject to additional modification in the subapex to produce a range of hyphal tip morphologies. The mathematical basis for generating a conoid hyphal tip by elongating the VSC is described in Appendix A.

Hyphae↗

PubMed1986

The effect of papulacandin B on (1----3)-beta-D-glucan synthetases. A possible relationship between inhibition and enzyme conformation.

The antibiotic, papulacandin B, inhibited growth or (1----3)-beta-D-glucan synthetase (or both) in the fungi Saccharomyces cerevisiae, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune and Wangiella dermatitidis. No effect was observed on Achlya ambisexualis. There was no apparent correlation between the inhibition of growth and that of the synthetase. With most of the fungal extracts, the inhibition of glucan synthetase by papulacandin B became less pronounced as the substrate (UDP-glucose) concentration was decreased. At very low levels of UDP-glucose, with the enzymes from S. cerevisiae and W. dermatitidis, the antibiotic stimulated the activity of glucan synthetase. As further studied with the W. dermatitidis enzyme, those low concentrations of UDP-glucose corresponded to a sigmoidal portion of the rate vs. substrate curve. The sigmoid segment of the curve extended to higher concentrations of UDP-glucose as the temperature was increased. Concomitantly, the range of substrate concentrations at which papulacandin B stimulated the reaction or was noninhibitory was broadened. It is tentatively concluded that glucan synthetase may exist in more than one interconvertible form. The stimulatory effect of papulacandin B is possibly due to preferential binding to the active form of the enzyme. The equilibrium between these forms could be shifted by structural changes in the membrane in which the enzyme is embedded. The lack of correlation between the effects of papulacandin B in whole cells and in extracts is discussed in terms of the variations in membrane structure in the two situations.

Aminoglycosides↗

PubMed1998

Molecular analysis of a glucose-regulated gene (grp78) of Neurospora crassa.

The nucleotide sequence of the gene encoding the glucose-regulated protein 78 (GRP78) of Neurospora crassa was determined. The ORF codes for a protein of 662 amino acids (72 kDa) and belongs to the heat shock protein 70 (hsp70) gene family, which is characterized by three HSP70 'signature sequences'. The grp78 gene contains 5 introns. The protein carries the ER retention signal HDEL at its carboxy terminus and is most homologous to the KAR2/GRP78 protein of Saccharomyces cerevisiae (78%) and to KAR2/BiP of Yarrowia lipolytica (76%). The expression of grp78 is constitutive and can be enhanced by starvation, treatment with tunicamycin, the calcium ionophore A23187 or elevated temperatures (40 degrees C). An uninterrupted ORF was found on the reverse cDNA strand of grp78. The putative peptide shows 47% homology to the NAD-specific glutamate dehydrogenase of Achlya klebsiana.

Amino Acid Sequence↗

PubMed2001

Characterization of Aphanomyces invadans isolates using pyrolysis mass spectrometry (PyMS).

Twenty-one isolates of Aphanomyces invadans, the fungal pathogen associated with the Asian fish disease epizootic ulcerative syndrome (EUS), were compared with other Oomycete fungi in terms of their pyrolysis mass spectrometry (PvMS) profiles. Canonical variate analysis (CVA) of the pyrolysis mass spectra distinguished the Aphanomyces species from a wide scatter of Achlya and Saprolegnia isolates. Further CVA and hierarchal cluster analysis (HCA) separated the Aphanomyces species into two main groups. The first group clustered A. invadans isolates from EUS outbreaks in Thailand, Bangladesh, Indonesia, Philippines, Australia and Japan together. However, HCA also included the crayfish plague fungus Aphanomyces astaci within this group. Non-pathogenic Aphanomyces strains isolated from ulcerative mycosis (UM)-affected fish were shown to be distinct from A. invadans, and instead clustered with saprophytic Aphanomyces strains to form the second group. Recently, an invasive Aphanomyces pathogen has been isolated from UNI-affected fish, but that was not tested here. This is the first report using PyMS in the study of Oomycete systematics. The technique was not sensitive enough to show any intraspecific differences, but it was considered a useful technique for the discrimination of species where taxonomic relationships are uncertain.

Animals↗

PubMed1983

Evidence for metabolic and functional discrimination of sterols by Phytophthora cactorum.

When fed 10 ppm of one of the following sterols: cholesterol (cholest-5-en-3beta-ol), wingsterol (21-isopentylcholesterol), desmosterol [cholesta-5,24(25)-dien-3beta-ol], 24-methylenecholesterol [ergosta-5,24(28)-dien-3beta-ol], or fucosterol [stigmasta-5,24(28)-dien-3beta-ol], the pathogenic fungus Phytophthora cactorum, which is naturally unable to epoxidize squalene, accumulated each of the test compounds to similar levels. Fucosterol, the only sterol metabolized, was reduced to yield 24-ethylcholesterol. All the sterols tested induced the formation of sex structures. Fertilization and subsequent maturation of oospores capable of germination occurred only with the naturally occurring sterols. Wingsterol treatments resulted in aborted oospores. None of the sterols tested was inhibitory to growth, measured as changes in the 21-day mycelial dry weight. The results are consistent with the view that the accumulated sterol functions to regulate the life cycle of P. cactorum. However, the metabolism and kinds of recognition of the sterol molecule, in terms of uptake and effects on growth and induction of the various sexual events, contrast sharply with what is known for other oomycetous fungi such as Achlya and Saprolegnia. This implies that the evolutionary histories of the Oomycetes may be different.

Journal Article↗

PubMedDate not supplied

Five Serbian reservoirs contain different fungal propagules.

This paper presents results of a mycological survey conducted in five large reservoirs in Serbia (Sjenica, Barje, Garasi, Meduvrsje and Ovcar-Kablar) that have different hydrobiological and production characteristics. The sampling was conducted in March, June, August and October 2002. Quantitative analysis of fungal communities showed that the average number of colony-forming units in these reservoirs was 1966-5682/L of water. The highest number of CFUs was found in the alpha-mesosaprobic (Class III) Ovcar-Kablar Reservoir and the lowest was in the oligosaprobic (Class I) Sjenica Reservoir. CFUs were higher in samples taken near lake bottoms and in littoral zones rich with macrovegetation than in the middle depths. In these reservoirs 48 species were identified from 720 isolates. The dominant genera were Penicillium, Aspergillus, Cladosporium, Fusarium, Rhizopus, Mucor, Phoma and Verticillium. The autochthonous aquatic fungal community comprised these species: Achlya americana, A. diffusa, A. racemosa, Dictyuchus sterile, Isoachlya toruloides, Leptomitus lacteus, Pythium ultimum, Saprolegnia ferax, S. hypogyna and S. monica. My results indicate that aquatic fungi on the whole are better represented in lakes with a higher trophic status.

Colony Count, Microbial↗

PubMed1984

Transhyphal electrical currents in fungi.

Representative mycelial fungi from the phycomycete, ascomycete and basidiomycete groups (Achlya bisexualis, Neurospora crassa, Aspergillus nidulans, Schizophyllum commune and Coprinus cinereus) all generated steady electrical currents around their hyphal tips; the generation of a transhyphal ion current may therefore be a universal characteristic of hyphal growth. As with all other tip growing organisms, positive current always entered apically and left distally; non-growing hyphae did not drive transcellular currents. The current density, measured approximately 30 micron from the membrane surface at the hyphal tips, varied between 0.05 and 0.06 microA cm-2 in different fungi and tended to be larger in wider, rapidly extending hyphae than in thinner, slow growing hyphae. The possibility that these currents serve to localize growth at the apex is discussed.

Electricity↗

PubMed1997

CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica.

PCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsici and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.

Amino Acid Sequence↗

PubMed2006

Fungal heat-shock proteins in human disease.

Heat-shock proteins (hsps) have been identified as molecular chaperones conserved between microbes and man and grouped by their molecular mass and high degree of amino acid homology. This article reviews the major hsps of Saccharomyces cerevisiae, their interactions with trehalose, the effect of fermentation and the role of the heat-shock factor. Information derived from this model, as well as from Neurospora crassa and Achlya ambisexualis, helps in understanding the importance of hsps in the pathogenic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Trichophyton rubrum, Phycomyces blakesleeanus, Fusarium oxysporum, Coccidioides immitis and Pneumocystis jiroveci. This has been matched with proteomic and genomic information examining hsp expression in response to noxious stimuli. Fungal hsp90 has been identified as a target for immunotherapy by a genetically recombinant antibody. The concept of combining this antibody fragment with an antifungal drug for treating life-threatening fungal infection and the potential interactions with human and microbial hsp90 and nitric oxide is discussed.

Fungal Proteins↗

PubMed1965

Heterothallism in biflagellate aquatic fungi: preliminary genetic analysis.

Genetic analysis of several hundred progeny from crosses of two heterothallic species of Achlya and Dictyuchus provides preliminary information about the life cycle and pattern of sexuality in the biflagellate Phycomycetes. Extensive testing of mycelial progeny indicates a diploid life cycle. Control of sexual expression and mating competence appears to be based on a complex genetic system.

Female↗

PubMed1985

Stimulation of beta(1----3)glucan synthetase of various fungi by nucleoside triphosphates: generalized regulatory mechanism for cell wall biosynthesis.

Particulate fractions from the taxonomically diverse fungi Achlya ambisexualis, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune, and Wangiella dermatitidis were found to catalyze the time-dependent incorporation of glucose from UDP-[14C]glucose into a water-insoluble material. The reaction was stimulated by bovine serum albumin. The product was characterized as beta(1----3)glucan on the basis of its resistance to alpha- and beta-amylase and susceptibility to beta(1----3)glucanase. With the exception of the preparation from A. ambisexualis, all others were stimulated by nucleoside triphosphates and their analogs. The best activators were GTP and guanosine 5'-(gamma-thio)triphosphate. It is concluded that the stimulation by nucleotides, previously found with the glucan synthetase of Saccharomyces cerevisiae, is a regulatory mechanism that was well conserved during fungal evolution, presumably because of its importance in controlling cell wall biosynthesis and cell growth.

Cell Wall↗

PubMed1997

The tigA gene is a transcriptional fusion of glycolytic genes encoding triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in oomycota.

Genes encoding triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are fused and form a single transcriptional unit (tigA) in Phytophthora species, members of the order Pythiales in the phylum Oomycota. This is the first demonstration of glycolytic gene fusion in eukaryotes and the first case of a TPI-GAPDH fusion in any organism. The tigA gene from Phytophthora infestans has a typical Oomycota transcriptional start point consensus sequence and, in common with most Phytophthora genes, has no introns. Furthermore, Southern and PCR analyses suggest that the same organization exists in other closely related genera, such as Pythium, from the same order (Oomycota), as well as more distantly related genera, Saprolegnia and Achlya, in the order Saprolegniales. Evidence is provided that in P. infestans, there is at least one other discrete copy of a GAPDH-encoding gene but not of a TPI-encoding gene. Finally, a phylogenetic analysis of TPI does not place Phytophthora within the assemblage of crown eukaryotes and suggests TPI may not be particularly useful for resolving relationships among major eukaryotic groups.

Amino Acid Sequence↗

PubMed1991

Heat shock protein 88 and Aspergillus infection.

Immunoblotting was used to dissect the antibody responses in the sera of 50 patients with proven invasive aspergillosis, 28 patients with suspected invasive aspergillosis, 35 patients with allergic bronchopulmonary aspergillosis, and 10 patients with an aspergilloma. This demonstrated the immunodominance of antigenic bands at 88, 84, 51, and 40 kDa. Monoclonal antibodies against the heat shock protein 90 complexes of Candida albicans and the water mold Achlya ambisexualis identified these four antigenic bands as homologous proteins. Similar antigens have been described in humans, mice, Saccharomyces cerevisiae, chickens, and Drosophila species. The antibody against A. ambisexualis has previously been shown to cross-react with antigens belonging to the human heat shock protein 90 complex. Aspergillus heat shock protein 90 was extracted from the sera of patients with invasive aspergillosis by affinity chromatography. This was done with both a rabbit hyperimmune antiserum raised against an extract of Aspergillus fumigatus NCPF 2109 and a monoclonal antibody against the heat shock protein 90 of C. albicans. In vivo expression of the antigen was demonstrated in an aspergilloma surgically removed from a patient. The role of the antigen as an allergen in allergic bronchopulmonary aspergillosis is also discussed.

Allergens↗

PubMed1975

A rapid and sensitive auxin binding system for detecting N6-substituted adenines, and some urea and thiourea derivatives, that show cytokinin activity in cell division tests.

A selective, sensitive and rapid (2 min or less) method for detecting compounds with potential for cytokinin activity is described. The method does not measure cytokinesis; instead, it determines the ability of cytokinin-active agents to (i) activate the intake of either L-tryptophan or indoleacetic acid by germinated spores of the water-mould Achlya, while inhibiting the energy-dependent transport of all L-amino acids usually found in proteins; (ii) inhibit the energy-dependent transport of nucleosides and sugars by the same organism. The compounds with cytokinin activity generally activate auxin (tryptophan) intake at 10(-8) M or greater and inhibit at 10(-6) M or greater. The most effective activating compounds were N6-(delta2-isopentenyl)adenine, N6-benzyladenine. N6-furfuryladenine, and N6-(trans-hydroxy-3-methyl-but-2-enyl)adenine. These compounds are classed generally as cytokinins in plant growth studies. A cell membrane - localized glycopeptide of molecular weight 6000 was isolated from this organism and shown to be the site at which cytokinins, auxin, and tryptophan bind. An earlier study had also established that calcium ions bind to this entity as well. Tryptophan binding to the glycopeptide was enhanced by cytokinins, suggesting that this may be the way in which whole cells display enhanced tryptophan binding in the bioassay. On the other hand, calcium binding was antagonized by cytokinin. The results suggest that this may be an important experimental system for use in studying one possible way in which cytokinin may regulate plant growth.

Adenine↗

PubMed1975

Amino acid transport in a water-mould: the possible regulatory roles of calcium and N6-(delta2-isopentenyl)adenine.

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different systems elucidated are: (i) methionine, (ii) cysteine. (iii) proline, (iv) serine-threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine-tyrosine-tryptophan and leucine-isoleucine-valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.

Adenine↗

PubMed1977

The diguanosine nucleotides: do they exist in aquatic fungi?

The aquatic fungi Achlya ambisexualis and Blastocladiella emersonii were grown in the presence of 32Pi and the 32P-labeled acid-soluble nucleotide fractions were analyzed by ion-exchange chromatography on DEAE-cellulose. Selected column fractions containing diguanosine tri- and tetra-phosphates (Gp3G and Gp4G) added as chromatographic markers were analyzed further for 32P by chromatography and (or) enzyme hydrolysis. The results of these experiments clearly indicate that neither Gp4G nor Gp3G is synthesized during vegetative growth of these organisms and cast doubt on the hypothesis that diguanosine nucleotides are important metabolic regulators in fungi.

Blastocladiella↗