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At least 19 recordsLinked to original sources

PubMed1990

Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.

Blotting, Southern↗

PubMed1991

Molecular cloning and characterization of two distinct hsp85 sequences from the steroid responsive fungus Achlya ambisexualis.

In Achlya ambisexualis, hsp85 is one of the characteristic mycelial heat shock proteins induced in response to a rapid elevation in temperature (Silver et al. 1983). This heat shock protein has the same electrophoretic mobility on two-dimensional gels and is antigenically related to an 85 kDa steroid hormone-regulated protein which constitutes a component of the putative Achlya steroid hormone-receptor complex. We report here the isolation of two distinct, yet highly related, hsp85 gene sequences from Achlya genomic libraries. Northern analyses, using these two Achlya genomic sequences as probes, suggest that there are two hsp85 message population in Achlya and that at least one of these is regulated by the steroid hormone antheridiol.

Animals↗

PubMed1987

Heat-shock-induced changes in the phosphorylation of ribosomal and ribosome-associated proteins in the filamentous fungus Achlya ambisexualis.

In the filamentous fungus Achlya ambisexualis, heat shock resulted in a rapid reduction in the rate of protein synthesis. This was accompanied by dephosphorylation of a prominent basic 30 kD protein associated with the small subunit of Achlya ribosomes and which may be analogous to ribosomal protein S6 of vertebrates. A large ribosomal subunit protein with a relative molecular weight (MW) of 24,500 exhibited increased phosphorylation during heat shock, while a second large subunit protein having a relative MW of 22,000 was dephosphorylated. Several proteins which could be dissociated from Achlya ribosomes by 0.5 M KCl also exhibited altered patterns of phosphorylation during heat shock. These KCl-soluble proteins included proteins at 50, 21, 20 and 19 kD, which exhibited decreased phosphorylation with heat shock and proteins at 32 and 23.5 kD, which exhibited increased phosphorylation with heat shock. Such alterations in the phosphorylation of components of the Achlya translational apparatus may be involved in the qualitative and quantitative changes in protein synthesis which are observed with heat shock in Achlya.

Fungi↗

PubMed1979

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.

Animals↗

PubMed1990

Characterization and nucleosomal core localization of Achlya histones involved in stress-induced chromatin condensation.

To better understand the basis for heat shock-induced chromatin condensation in Achlya, a further characterization of the histones of this organism was carried out. The nucleosomal location (i.e., core vs linker), partial peptide map, and electrophoretic behavior of each Achlya histone was determined and compared to the well-characterized histones of rabbit kidney. The results of this and previous studies suggest that in Achlya, no nucleosome linker-associated histone analogous to histone H1 of higher eucaryotes is observed and that the Achlya histone designated alpha is a novel nucleosomal core histone. These observations may reflect the existence of a mechanism of stress-induced chromatin condensation which does not involve histone H1.

Animals↗

PubMed1977

DNA sequence organization in the water mold Achlya.

Experiments are described that characterize the organization of DNA sequences in the water mold Achlya bisexualis. These experiments demonstrate that repetitive and single copy sequences in the Achlya genome are arranged in a long-period interspersion pattern. Estimates of the spacing intervals between repetitive and single copy DNA indicate, however, that the interspersion pattern in Achlya is longer than has been previously reported in other eukaryotes. These data and measurements of structural gene expression in Achlya [Timberlake, W.E., Shumard, D. S. & Goldberg, R. B. (1977) Cell 10, 623-632] make it difficult to propose a regulatory function for repeated DNA in this eukaryote.

Journal Article↗

PubMed1984

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.

Cell Nucleus↗

PubMed1975

Differential uptake and metabolism of sitosterol and cholesterol by Achlya, Pythium, and Phytophthora species.

The relative ability of isolates of Achlya bisexualis and A. ambisexualis and isolates of Pythium and Phytophthora to take up and metabolize sitosterol and cholesterol was studied. Species of Pythium and Phytophthora took up cholesterol and sitosterol efficiently, whereas Achlya species took up booth sterols inefficiently. Species of Pythium and Phytophthora produced a polar metabolite and esters from sitosterol as they did from cholesterol. Achlya species did not produce the polar metabolite from either sterol. In these experiments Achlya species produced esters only from cholesterol; however, their failure to produce esters from sitosterol may have been due to the higher sitosterol than cholesterol concentration.

Cholesterol↗

PubMed1998

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya.

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.

Animals↗

PubMed1979

Chromatin organization in the oomycete Achlya ambisexualis.

Nuclei from the Oömycete Achlya ambisexualis and rabbit kidney nuclei were digested with micrococcal nuclease and the resultant DNA fragments analyzed on slab gels. The average DNA repeat size was found to be 159 +/- 1.2 base pairs for Achlya and 199.8 +/- 3.7 base pairs for rabbit kidney. The presence of a DNA repeat size of 159 base pairs for Achlya extends the characterization of eukaryotic chromatins to this most primitive and perhaps unique microbe.

Biological Evolution↗

PubMed1986

Cellular localization of steroid hormone-regulated proteins during sexual development in Achlya.

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.

Cell Compartmentation↗

PubMed1983

Effect of heat shock on synthesis and phosphorylation of nuclear and cytoplasmic proteins in the fungus Achlya.

Heat shock induced by an increase in temperature from 28 to 37 degrees C led to changes in synthesis and phosphorylation of cytoplasmic and nuclear proteins in the aquatic fungus Achlya. In the cytoplasmic fraction a marked increase in [35S]methionine labelling of proteins in the molecular weight range of 96 000, 85 000, 74 000, and 70 000 was observed. Two-dimensional electrophoresis resolved each of these classes of proteins into several components. Major changes in the nuclear fraction included the increased [35S]methionine labelling of 43 000 and 28 000-23 000 proteins. A marked decline in the synthesis of many other proteins was also evident. The heat-shock-induced changes in labelling patterns became evident as early as 20 to 60 min after treatment, but they were transient. With continued incubation at the heat-shock temperature, the cells appeared to adapt to the new temperature conditions. Both cytoplasmic and nuclear proteins returned to nearly normal labelling patterns within 100 to 140 min at 37 degrees C. Changes in phosphorylation of histone and nonhistone nuclear proteins were also noted. Achlya histone H3 and the putative oomycete-specific histone "alpha" appeared highly phosphorylated after heat shock. Since phosphorylation of histone H3 is primarily associated with chromatin condensation, it is possible that rapid chromatin condensation is an initial response to heat shock in Achlya.

Cells, Cultured↗

PubMed2007

Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and Saprolegnia.

The Saprolegniales are responsible for various fish mycoses worldwide and considered the most important fungi afflicting fresh water fish. Saprolegniosis leads to massive epidermal destruction and macrophage recruitment, yet little is known regarding the cytological response of their piscine hosts. The objective of this study was to explore the response of fish macrophage to members of the Saprolegniales using the rainbow trout monocyte/macrophage cell line, RTS11. After 48 h in co-culture, RTS11 demonstrated chemotaxis, adherence and homotypic aggregation to both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11 and Achlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viable throughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses. Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderately up-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1beta and TNFalpha were strongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor and associated molecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increased dependence of macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH II receptor expression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic pathogens.

Achlya↗

PubMed1974

Differential effects of analogs of cycloheximide on protein and RNA synthesis in Achlya.

Analogs of the glutarimide antibiotic cycloheximide were tested for their effect on growth and incorporation of proline and uridine into acid-insoluble material in Achlya bisexualis. Each of the compounds tested had reduced antibiotic activity as compared to cycloheximide. The effects of the antibiotics on protein and RNA synthesis were varied. While cycloheximide inhibited both protein and RNA synthesis immediately, two of the analogs inhibited proline incorporation without effect on uridine incorporation, while three, each representing a modification of the hydroxyl of cycloheximide, stimulated uridine incorporation and either had no effect on or inhibited protein synthesis. These results indicate that the control of RNA synthesis by protein synthesis in Achlya can be released by glutarimide antibiotics.

Antifungal Agents↗

PubMed1985

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis.

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Binding Sites↗

PubMed1985

Calmodulin from the water mold Achlya ambisexualis: isolation and characterization.

A protein-activator of bovine cyclic nucleotide phosphodiesterase from the water mold Achlya ambisexualis has been affinity-purified to apparent electrophoretic homogeneity. The heat-stable protein is similar in amino acid content and electrophoretic mobility on SDS acrylamide gels, to bovine brain calmodulin. It also cross-reacts with antibodies raised to the bovine protein. Achlya calmodulin activates PDE increasing its activity up to 9-fold in a Ca2+-dependent manner. The mold protein appears unusual in that its tyrosine fluorescence is unaltered by Ca2+ or by EGTA.

Amino Acids↗

PubMed1986

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording.

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.

Amino Acids↗

PubMed1984

Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium.

Growing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8.5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.

Adaptation, Physiological↗