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.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

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.     

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.     

Hormone-dependent lipoxygenase activity in Achlya ambisexualis

Homogenates of male Achyla ambisexualis oxidize exogenously added [¹⁴C]arachidonic acid to an unidentified lipoxygenase product. Synthesis occurs at a rate of 10.6 ± 1.3 μg mg⁻¹ protein 30 min⁻¹. Activity in homogenates of female mycelium is only 2.1 ± 1.2. Conversion is eliminated by the lipoxygenase inhibitor nordihydroguaiaretic acid (10⁻⁴ M). Homogenates prepared from the male grown in chemical but not physical contact with female mycelium had decreased lipoxygenase activity (3.1 ± 1.5), suggesting that antheridiol produced by the female decreases lipoxygenase activity in the male. To confirm this, actively growing male cultures were exposed to 10⁻⁹ M 7-deoxy-7-dihydroantheridiol, a stable analog of antheridiol, for 24 h. Homogenates from these cultures also had diminished lipoxygenase activity (2.7 ± 1.0). 7-Deoxy-7-dihydroantheridiol added to the incubation mixture at 10⁻⁹ M had no effect on lipoxygenase activity (9.0 ± 1.8), excluding a direct action of 7-deoxy-7-dihydroantheridiol on the enzyme. These data support the hypothesis that lipoxygenase products are associated with vegetative growth and suggest the antheridiol initiation of reproductive growth suppresses lipoxygenase activity.     

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.     

Analysis of the steroid receptor of Achlya ambisexualis

We have previously reported the discovery of a specific high-affinity binding protein for the fungal sex steroid pheromone antheridiol in the cytosol of Achlya ambisexualis male cells. In this report, we describe the fractionation of the binding protein from the cytosol by ammonium sulfate precipitation, the optimization of in vitro conditions for radioligand binding assays, and some of the biochemical properties of the binding protein. In the presence of sodium molybdate, the macromolecule has a sedimentation coefficient of 8.3 S in sucrose gradients of low ionic strength, a Stokes radius of 56.6 A (Sephacryl S-300 columns), a molecular weight of approximately 192,000, a frictional ratio of 1.5, and an axial ratio of 8.9. The binding protein can be eluted with 0.24 M KCl as a single peak from DEAE-Sephadex A-25 columns. These results indicate that this steroid-binding protein from a primitive eukaryotic microbe has in vitro biochemical properties that are similar to those of other known steroid receptors in higher organisms.     

Chromatin organization in the oomycete Achlya ambisexualis.

Nuclei from the O?mycete 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.     

Cellulase Localization in Hyphae of Achlya ambisexualis

Cellulase (EC 3.2.1.4; beta-1, 4-glucan glucanohydrolase) was localized at the ultrastructural level and found to occur in dictyosomes and vesicles, around the periphery of unidentified storage bodies, between the plasmalemma and the cell wall, and on the outer surface of the cell wall in the male strain (E87) of Achlya ambisexualis after treatment with the sex hormone antheridiol.     

Structural and physiological changes during sporangial development inAchlya intricata beneke

Summary Sporangial development in the zoosporic fungusAchlya intricata has been studied using light microscopy, a plasmolytic technique, KCl-filled microelectrodes and ion-selective microelectrodes. The completion of cleavage (spore delimitation) is accompanied by a change in appearance of the sporangium, loss of turgor and membrane potential, decrease in volume and release of K⁺. The measured loss of K⁺ is in agreement with previous measurements of extracellular ionic currents around developing sporangia ofAchlya using a vibrating probe. The relationship between these changes and the mechanism of spore liberation is discussed.     

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.     

Steroid effects on the gene expression of peripheral myelin proteins

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA(A) receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).     

Biological significance of a thyroid hormone-regulated secretome

The thyroid hormone, 3,3,5-triiodo-L-thyronine (T₃), modulates several physiological processes, including cellular growth, differentiation, metabolism and proliferation, via interactions with thyroid hormone response elements (TREs) in the regulatory regions of target genes. Several intracellular and extracellular protein candidates are regulated by T₃. Moreover, T₃-regulated secreted proteins participate in physiological processes or cellular transformation. T₃ has been employed as a marker in several disorders, such as cardiovascular disorder in chronic kidney disease, as well as diseases of the liver, immune system, endocrine hormone metabolism and coronary artery. Our group subsequently showed that T₃ regulates several tumor-related secretory proteins, leading to cancer progression via alterations in extracellular matrix proteases and tumor-associated signaling pathways in hepatocellular carcinomas. Therefore, elucidation of T₃/thyroid hormone receptor-regulated secretory proteins and their underlying mechanisms in cancers should facilitate the identification of novel therapeutic targets. This review provides a detailed summary on the known secretory proteins regulated by T₃ and their physiological significance. This article is part of a Special Issue entitled: An Updated Secretome.     

Sex hormone-regulated renal transport of perfluorooctanoic acid

The biological half-life (t1/2) of perfluorooctanoic acid (PFOA) in male rats is 70 times longer than that in female rats. The difference is mainly due to the difference in renal clearance (CL(R)), which was significantly reduced by probenecid, suggesting that PFOA is excreted by organic anion transporter(s). Castration of male rats caused a 14-fold increase in the CL(R) of PFOA, which made it comparable with that of female rats. The elevated PFOA CL(R) in castrated males was reduced by treating them with testosterone. Treatment of male rats with estradiol increased the CL(R) of PFOA. In female rats, ovariectomy caused a significant increase in CL(R) of PFOA, which was reduced by estradiol treatment. Treatments of female rats with testosterone reduced the CL(R) of PFOA as observed in castrated male rats. To identify the transporter molecules that are responsible for PFOA transport in rat kidney, renal mRNA levels of organic anion transporter 1 (OAT1), OAT2, OAT3, organic anion transporting polypeptide 1 (oatp1), oatp2 and kidney specific organic anion transporter (OAT-K) were determined in male and female rats under various hormonal states and compared with the CL(R) of PFOA. The level of OAT2 mRNA in male rats was only 13% that in female rats. Castration or estradiol treatment increased the level of OAT2 mRNA whereas treatment of castrated male rats with testosterone reduced it. In contrast to OAT2, mRNA levels of both oatp1 and OAT-K were significantly higher in male rats compared with female rats. Castration or estradiol treatment caused a reduction in the levels of mRNA of oatp1 and OAT-K in male rats. Ovariectomy of female rats significantly increased the level of OAT3 mRNA. Multiple regression analysis suggests that the change in the CL(R) of PFOA is, at least in part, due to altered expression of OAT2 and OAT3.     

Ultrastructural localization of basic proteins ofBlastocystis hominis

Summary Basic proteins ofBlastocystis hominis were detected by the ammoniacal silver and ethanolic phosphotungstic acid techniques using electron microscopy. The central vacuole showed many silver grains when treated with ammoniacal silver and an increased electron density when treated with phosphotungstic acid. The intensity of positive reactions correlated with the electron density of the central vacuole, because cells having an electron-lucent central vacuole showed no silver grain deposits. Since it is known that the concentration of electron-dense materials in the central vacuole increases during log phase of growth, and then decreases in stationary phase, this organelle must accumulate basic proteins during cell growth.     

The basic proteins of bovine allantoic fluid

This paper reports the separation of highly cationic proteins (i.e. pI greater than 9.0) of bovine allantoic fluid and their possible pathogenic properties. Experimentally, polycations and cationic proteins of pI greater than 10 induced intravascular coagulation and hemolysis, as well as precipitation of fibrinogen and proteinuria. Bovine allantoic fluid collected at the time of calving contains from 0.6 to 1.3 g of proteins per liter (11 samples). Ion-exchange chromatography, followed by either chromatofocusing or heparin-sepharose-6 beta binding, and, finally, gel filtration separated several fractions and subfractions. These were examined later using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and at least ten constituents were visualized. Two components, Mr 34,000, pI 9.7, and Mr 38,000, pI 9.6-9.0, accounted for 33% of the basic proteins present in allantoic fluid and 0.7% of its total protein content. Electrophoretic mobility was unaltered by beta-mercaptoethanol, and periodic acid-Schiff staining was negative. These proteins were not found in ox plasma. The major basic proteins were bound to red cells and platelets. Cell electrophoretic mobility decreased linearly with the logarithm of protein concentration. At concentrations between 10(-6) and 10(-5) M, red cell clumping was rare; hemolysis and platelet agglutination were not observed.