共查询到20条相似文献,搜索用时 31 毫秒
1.
《Fungal genetics and biology : FG & B》2009,46(8):575-584
The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nω-Nitro-l-arginine methyl ester (l-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when l-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca2+-NO-cGMP signaling pathway playing a role in zoospore biogenesis. 相似文献
2.
3.
Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane
Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [3H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles. 相似文献
4.
Luis Alberto Luévano-Martínez Camille C. Caldeira da Silva Gianlucca G. Nicastro Robert I. Schumacher Alicia J. Kowaltowski Suely L. Gomes 《Fungal biology》2019,123(1):59-65
Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii. 相似文献
5.
Yujing Zhang Pascal Benz Daniel Stehle Shang Yang Hendrikje Kurz Susanne Feil Georg Nagel Robert Feil Shiqiang Gao Markus Bender 《Open biology》2022,12(8)
Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology. 相似文献
6.
《Cell differentiation》1980,9(3):169-179
The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the control of Blastocladiella emersonii germination was studied. This differentiative transition may be induced by replacing K+, a classical inducer, by cyclic AMP or by competitive inhibitors of cyclic AMP phosphodiesterase activity. When zoospores are treated simultaneously with two inducers at non-effective concentrations, a synergistic effect is observed between cyclic AMP and either KCl or adenine. The calcium ionophore A23187 per se is not able to elicit germination, but the association of A23187 and sub-optimal concentrations of cyclic AMP is effective. These results suggest that germination may depend on a correlation between the intracellular mobilization of calcium and cyclic AMP levels. 相似文献
7.
The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (TL and TH) were found to be independent of the external K+, Ca2+, or Mg2+ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (TM) was markedly influenced by the external Ca2+ concentration. Ca2+ increased TM from 12°C (no Ca2+ added) to 22°C (10 mM Ca2+), i.e., growth temperature. K+ reversed this Ca2+ effect, downshifting TM from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in TM, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment. 相似文献
8.
《Experimental mycology》1984,8(1):13-24
Ribosomes fromBlastocladiella emersonii zoospores stimulatein vitro protein synthesis in a system using soluble factors extracted from wheat germ. Aurintricar☐ylic acid inhibits less than 40% of thein vitro protein synthetic activity of the zoospore ribosomes, indicating that messenger RNA is already complexed to the ribosomes. In addition to the mRNA complexed to the ribosomes, zoospores contain an mRNA fraction which is not bound to the ribosomes. Extraction of RNA from zoospore ribosomes and deacylation followed by reacylation with labeled amino acids demonstrated the presence of tRNA molecules specific for methionine and other amino acids on zoospore ribosomes. Transfer RNA from zoospore ribosomes contained 9.8% methionyl-tRNA compared to 2.4% methionyl-tRNA bound to ribosomes isolated from growth-phase plants. The fourfold enrichment of methionyl-tRNA on zoospore ribosomes suggests that between 12 and 25% of the zoospore ribosomes exist in arrested 80 S initiation complexes. Collectively, the data indicate that zoospore ribosomes complexed to mRNA have completed initiation and are somehow blocked at one or more of the elongation steps of protein synthesis. The data are compatible with the idea that an inhibitor is associated with the zoospore ribosomesin vivo. 相似文献
9.
The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T1, T2, and A, which can be labeled with 32PO4 and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs. 相似文献
10.
Joseph Donald Smith Philip M. Silverman 《Biochemical and biophysical research communications》1973,54(3):1191-1197
The lipid content of zoospores is 5 pg/cell or about 13% of dry weight. Within the first few minutes of germination 60–70% of total zoospore lipid is lost, with neutral lipid, glycolipid and phospholipid fractions decreasing to about the same extent. These changes in lipid content precede the breakdown during germination of the complex and extensive membrane system of zoospores. During growth, which immediately follows germination, net phospholipid synthesis resumes so that total lipid is maintained at 6% of dry weight, but net synthesis of neutral and glycolipid does not begin until induction of sporulation. During sporulation the phospholipid level decreases so that the distribution of lipid among the three fractions approaches that found in zoospores. These changes in lipid content suggest that zoospore membranes containing neutral and glycolipids are synthesized during spore formation. 相似文献
11.
C.P. Selitrennikoff D.R. Sonneborn 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,451(2):408-416
Forward direction assays have been developed for the last two pathway-specific enzymes of hexosamine biosynthesis using crude extracts from Blastocladiella emersonii zoospores. The specific enzyme activities measured are substantially higher than those reported with enzyme preparations from other organisms. During the development of one of the assays, another enzyme activity was observed which converts one of the intermediates of the pathway, N-acetylglucosamine-6-phosphate, to N-acetylglucosamine. The finding of these three enzyme activities in zoospore extracts completes the demonstration that all the enzyme activities necessary to synthesize some 2% by weight as chitin early during zoospore germination (de novo cell wall formation) pre-exist in the zoospore. This demonstration is consistent with the conclusion that the hexosamine pathway is regulated at the post-translational level during zoospore germination. 相似文献
12.
13.
《Experimental mycology》1984,8(2):132-145
To evaluate gene expression during sporulation and early development of the aquatic fungus Blastocladiella emersonii, the nucleotide sequence complexity of the polysomal RNA has been measured at different stages. To assess the effect of medium composition on gene expression, similar experiments were completed during early development in a range of simple to complex media. The polysomal RNA sequence complexity was measured by hybridization with single-copy tracer DNA and with a complex class-enriched cDNA fraction copied from the stored zoospore poly(A+)RNA. Forty-four to eighty-six percent (8.2 × 106 to 16 × 106 nucleotides) of the single-copy DNA sequence complexity was found on polysomes, depending upon the stage examined or the medium used, compared to 42.5% (8 × 106 nucleotides) in the stored RNA pool of zoospores. The highest levels of complexity occurred during the two periods of active differentiation, sporulation and germination. During starvation-induced sporulation, and average of 82% of the total asymmetrically transcribed complexity was expressed; half of this complexity was lost prior to the completion of zoospore differentiation and was missing from the zoospore-stored RNA pool. During the first 30 min of zoospore germination the level of sequence complexity increased by 46 to 66% over the zoospore level, depending upon the medium used. The polysomal RNA complexity then decreased by a nearly equal amount between 30 and 60 min when the cells entered the growth phase. An inverse relationship was found between the richness of the medium and the level of sequence complexity found on polysomes. The data indicate that sequences representative of most of the zoospore-stored poly(A+)RNA were expressed at all other stages and maintained by turnover and resynthesis. In addition, significant numbers of new sequences were also expressed, particularly during stages of active differentiation. Cells that germinated and completed early development in an inorganic starvation medium showed a marked loss of the middle and high abundance classes of poly(A+)RNA and slight enrichment for the low abundance class. 相似文献
14.
《Experimental mycology》1981,5(1):1-14
A quantitative bioassay forBlastocladiella emersonii zoospore maintenance factor (ZMF) is documented analyzed. The basis of the assay is an antagonistic interaction between NaCl and ZMF; the former elicits rapid encystment in zoospore populations whereas the latter counteracts this effect. Dilution techniques are used to quantitate the effects of each variable in terms of percentage rapid encystment. Each conditional bioassay variable examined (NaCl concentration, assay pH, cell density, zoospore age, time of assay) appears to affect the sensitivity of zoospore populations to ZMF rather than the level of ZMF activity per se. In particular, zoospore populations “age,” in terms of lowered sensitivity to ZMF, in the following ways: (a) “zoospore aging” without NaCl added and either with or without ZMF added; and (b) “delayed encystment” with NaCl and different fixed levels of ZMF added. ZMF activity in the medium (buffered CaCl2) accumulates gradually during sporulation; under bioassay conditions and even at subsaturating levels of ZMF activity, activity recoverable from the medium remains stable over long time intervals (4.5–24 h) whether populations remain zoospores or germinate. Populations incubated without added ZMF, whether they remain zoospores or germinate, do not detectably release ZMF activity into the medium. We discuss the following proposals concerning the functions of ZMF: (a) ZMF acts as a negative regulator of themechanics of zoospore encystment; and (b) ZMF acts as a self-generated, natural dispersal signal for the organism. 相似文献
15.
Ana Lucia T.O. do Nascimento Giuseppe Cilento 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,843(3)
Exposure of the zoospore of Blastocladiella emersonii to the peroxidase-catalyzed aerobic oxidation of phenylacetaldehyde results in: (i) an increased rate of O2 uptake; (ii) malondialdehyde formation; (iii) light emission with a biphasic kinetics arising from two emitters, one of which is probably a flavin. The process in the zoospore which is responsible for these observations is induced by energy transfer from, or reaction with, the excited species generated in the phenylacetaldehyde/peroxidase/O2 system prior to lysis. In the case of the germling stage, emission is observed, but no malondialdehyde is formed. Phenylacetaldehyde alone induces a weak emission from non-lysed zoospores or germlings, suggesting in situ excited-state generation. 相似文献
16.
Concanavalin A (ConA) induces lysis of zoospores of the aquatic fungus Blastocladiella emersonii. The rapid lysis can be completely blocked by α-methyl-
-mannoside. At lower temperatures the cells are more tolerant towards lysis compared with cells at room temperature. Dimeric ConA derivatives and Lens culinaris hemagglutinin A are less effective, whilst other lectins tested do not induce lysis at all. We suggest that cluster formation of adjacent ConA receptors precedes lysis. 相似文献
17.
The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (TL and TH) were found to be independent of the external K+, Ca2+, or Mg2+ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (TM) was markedly influenced by the external Ca2+ concentration. Ca2+ increased TM from 12°C (no Ca2+ added) to 22°C (10 mM Ca2+), i.e., growth temperature. K+ reversed this Ca2+ effect, downshifting TM from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in TM, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment. 相似文献
18.
Microbodies isolated from sporangia of the aquatic fungus have a mean buoyant density of 1.222 g/cm3 after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm3 while the spore's single mitochondrion has a buoyant density of 1.219 g/cm3. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers. 相似文献
19.
Thomas E. Hutchinson Marie E. Cantino Edward C. Cantino 《Biochemical and biophysical research communications》1977,74(2):336-342
Energy selective X-ray elemental microanalysis was used to demonstrate the presence of Ca, K and P (relative amounts about 1 : 1 : 4, respectively) in the virus-like Gamma Particles found in the zoospores of the aquatic fungus . Some Gamma Particles, however, lack detectable amounts of K. The possible significance of these observations for understanding the triggering of encystment and the initiation of wall synthesis is underscored. 相似文献
20.
《Experimental mycology》1987,11(3):176-186
The poly(A) RNA in zoospores ofBlastocladiella emersonii contains RNA synthesized during the growth phase (GP poly(A) RNA) and late sporulation (LS poly(A) RNA). LS poly(A) RNA synthesized during the final 30 minutes of sporulation is bound exclusively to polyribosomes which comprise approximately 50% of the total zoospore ribosome population. In contrast, GP poly(A) RNA is bound to zoospore monoribosomes. During the final 30 minutes of sporulation, GP poly(A) RNA which is bound to polyribosomes makes a transition to monoribosomes. Zoospore monoribosomes and RNA extracted from zoospore monoribosomes are inactivein vitro while both zoospore polyribosomes and RNA extracted from zoospore polyribosomes stimulate protein synthesis in the wheat germin vitro system. Sedimentation of poly(A) RNA from zoospore monoribosomes on dimethyl sulfoxide gradients revealed that the GP poly(A) RNA was of sufficiently high molecular weight to code for average-sized proteins. These denaturing gradients failed to activate the zoospore monoribosome RNA. The results suggest that the inability to translate zoospore monoribosomesin vitro is due to some property or modification of the zoospore monoribosome poly(A) RNA. Zoospore monoribosomes bound to poly(A) RNA contain an average of two tRNA molecules while zoospore polyribosomes have an average of less than one tRNA bound. This suggests the two classes of ribosomes are blocked at different steps in the elongation process. 相似文献