Vieira André L G, Linares Edlaine, Augusto Ohara, Gomes Suely L
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Fungal Genet Biol. 2009 Aug;46(8):575-84. doi: 10.1016/j.fgb.2009.04.002. Epub 2009 Apr 23.
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 Nomega-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 Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis.
水生真菌艾美球囊霉的孢子形成阶段以大量游动孢子的形成并释放到培养基中达到顶点，这些游动孢子是负责真菌传播的运动细胞。1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮（ODQ）是一种有效且选择性的一氧化氮敏感型鸟苷酸环化酶抑制剂，其在孢子形成溶液中的存在完全阻止了游动孢子的生物合成。此外，该化合物能够显著降低环鸟苷酸（cGMP）水平，cGMP水平在孢子形成后期急剧增加，这表明存在一种依赖一氧化氮的cGMP合成机制。此外，在孢子形成过程中，通过使用DAF-2 DA的荧光测定法检测到一氧化氮衍生产物水平升高，在一氧化氮合酶抑制剂Nω-硝基-L-精氨酸甲酯（L-NAME）存在的情况下，其信号急剧降低。这些结果通过对一氧化氮衍生产物细胞内水平的定量化学发光测定得到证实。在整个孢子形成过程中检测到一种假定的一氧化氮合酶（NOS）活性，当将L-NAME和α-[2-（三氟甲基）苯基]咪唑（TRIM）添加到测定中时，这种酶活性显著降低。在乙二胺四乙酸（EGTA）存在下进行的NOS测定显示酶活性降低，表明钙离子参与酶的激活。此外，在艾美球囊霉EST数据库（http://blasto.iq.usp.br）中发现了编码假定鸟苷酸环化酶和一种cGMP磷酸二酯酶的表达序列标签（EST），并且观察到相应基因的mRNA水平在孢子形成过程中增加。总之，此处呈现的数据揭示了艾美球囊霉中鸟苷酸环化酶和cGMP磷酸二酯酶基因的存在和表达，并提供了一条Ca(2+)-(*)NO-cGMP信号通路在游动孢子生物合成中起作用的证据。
