臺灣博碩士論文加值系統

水溶性鳥苷酸環化酶 (soluble guanylyl cyclase; sGC) 在NO/cGMP訊息傳遞中受到一氧化氮 (nitric oxide, NO) 的活化，進而將GTP轉變成cGMP。本研究從蜜蜂Apis mellifera L.工蜂的腦部選殖得一β次單元的基因，並將之命名為Amgcβ1。Amgcβ1全長為2010 bp，具有大小為1809 bp之開放讀架 (open reading frame)，序列末端具有23個腺嘌呤 (A) 所組成之poly (A) tail，5’-及3’-端分別具有94及106 bp之未轉譯序列 (untranslation sequence)。Amgcβ1由603個胺基酸所組成，預估分子量為66.33 kDa，經序列比對後發現Amgcβ1與已發表之昆蟲sGC β次單元基因最為相近，與菸草天蛾 (Manduca sexta) 的MsGC-β1有76.7%的一致性，與甘比亞瘧蚊 (Anopheles gambiae) 的GCSβ一致性有57.2%，而與黃果蠅 (Drosophila melanogaster) 的Dgcβ1有55.7%的一致性。RT- PCR的分析結果顯示，Amgcβ1在頭、胸及腹均有表現，但頭部表現量較高。Amgcβ1在羽化後第0天即有表現，但在羽化後第7天及第14天之工蜂表現量則降低，然後在羽化後第21天及第28天之工蜂表現量則再度上升。對初羽化工蜂處理100 μg青春激素異構物(juvenile hormone analogue, JHA)-百利普芬 (pyriproxyhen)，在處理後的24及48小時內 Amgcβ1的表現沒有明顯變化。原位雜合結果顯示Amgcβ1表現在視葉 (optic lobes) 的lamina monopolar cells、lamina、outer chiasma及medulla，嗅葉的glomeruli、soma，以及蕈狀體 (mushroom bodies) 的Kenyon cells等組織，推測Amgcβ1在視覺及嗅覺的形成，以及經視覺產生的行為可能扮演重要的角色。

Soluble guanylyl cyclases (sGCs) are an important target for endogenous nitric oxide (NO). sGC are heterodimeric enzymes consisting of α and β subunits, and have been considered as a key factor in the regulation of synthesis of cyclic GMP, a second messenger of signal transduction. The aim of this study is to discover the possible function of sGC in honeybee brain, and to investigate the gene at different ages. The first β1 subunit gene (Amgcβ1) was cloned from the honeybee brain. The Amgcβ1 consisting of 2010 bp contains an open reading frame and encodes a protein of 603 amino acids with a predicted molecular weight of 66.33 kDa. The sequence exhibited 76.7 % amino acid identity with the tabacco hornworm, Manduca sexta β1 amino acid sequence, 57.2% with malaria mosquito, Anophele gambiae, 55.7% with fruit fly, Drosophila melanogaster. RT-PCR analysis demonstrated that the transcripts was mainly present in head and indicated that Amgcβ1 transcript was present at a variable levels in any stages of development of adult workers. Thus, RT-PCR analysis that treated with 100 μg of JHA show no significantly different from the normal worker brain after 24 and 48 hours of the treatment. In situ hybridization analyses show that Amgcβ1 was expressed at high level in the laminar monopolar cells, lamina, outer chiasma and medulla of optic lobe, the soma of Kenyon cells in the mushroom body and the glomeruli of antennal lobe. sGC was shown in the visual and olfactory systems using in situ hybridization for the Amgcβ1, suggesting that cGMP-dependent signaling pathway may play an important role in the visual and olfactory systems of honeybee.

中文摘要 -------------------------------------------- 1
英文摘要 -------------------------------------------- 2
前言 -------------------------------------------- 3
前人研究 -------------------------------------------- 5
材料方法 -------------------------------------------- 12
結果 -------------------------------------------- 20
討論 -------------------------------------------- 23
參考文獻 -------------------------------------------- 27
圖表 -------------------------------------------- 37
附錄 -------------------------------------------- 53

Andreopoulos, S. and A. Papapetropoulos. 2000. Molecular aspects of soluble guanylyl cyclase regulation. Gen. Pharmacol. 34: 147-157.
Ashman, D. F., R. Lipton, M. M. Melicow and T. D. Price. 1963. Isolation of adenosine 3’, 5’- monophosphate and guanosine 3’, 5’- monophosphate from rat urine. Biochem. Biophys. Res. Commun. 11: 330-334.
Behrends, S. C., A. Steenpass, H. Porst and H. Scholz. 2000. Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum. Biochem. Pharmacol. 59: 713-717.
Bellamy, T. C., J. Wood and J. Garthwaite. 2002. On the activation of soluble guanylyl cyclase by nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 99: 507-510.
Ben-Shahar, Y. and G. E. Robinson. 2001. Satiation differentially affects performance in a learning assay by nurse and forager honey bees. J. Comp. Physiol. A. 187: 891-899.
Ben-Shahar, Y., A. Robichon, M. B. Sokolowski and G. E. Robinson. 2002. Influence of gene action across different time scales on behavior. Science 296: 741-744.
Ben-Shahar, Y., C. K. Thompson, S. M. Hartz, B. H. Smith and G. E. Robinson. 2000. Differences in performance on a reversal learning test and division of labor in honey bees colonies. Anim. Congit. 3: 119-125.
Bicker, G. 1998. NO news from insect brains. Trends Neurosci. 21: 349-355.
Bicker, G. 2001a. Sources and targets of nitric oxide signalling in insect nervous systems. Cell Tissue Res. 303: 137-146.
Bicker, G. 2001b. Nitric oxide: an unconventional messenger in the nervous system of an Orthopteroid insect. Arch. Insect Biochem. Physiol. 48: 100-110.
Bloch, G., D. P. Toma and G. E. Robinson. 2001. Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain. J. Biol. Rhythms 16: 444-456.
Bloch, G., D. E. Wheeler and G. E. Robinson. 2002. Endocrine influences on the organization of insect societies. Pp. 195-236. In: Pfaff D. W. ed. “Hormones, Brain and Behavior Vol. 3”. Elsevier Science, USA.
Bullerjahn, A. and H. J. Pflüger. 2003. The distribution of putative nitric oxide releasing neurons in the locust abdominal nervous system: a comparison of NADPHd histochemistry and NOS- immunocytochemistry. Zoology 106: 3-17.
Dizhoor, A. M. 2000. Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina. Cell. Signal. 12: 711-719.
Elekonich, M. M., D. J. Schulz, G. Bloch and G. E. Robinson. 2001. Juvenile hormone levels in honeybee (Apis mellifera L.) foragers: foraging experience and diurnal variation. J. Insect Physiol. 47: 1119-1125.
Elphick, M. R. and I. W. Jones. 1998. Localization of soluble guanylyl cyclase α-subunit in identified insect neurons. Brain Res. 800: 174-179.
Elphick, M. R., L. Williams and M. Shea. 1996. New features of the locust optic lobe: evidence of a role for nitric oxide in insect vision. J. Exp. Biol. 199: 2395-2407.
Fahrbach, S. E., D. Moore, E. A. Capaldi, S. M. Farris and G. E. Robinson. 1998. Experience-expectant plasticity in the mushroom bodies of the honeybee. Learn. Mem. 5: 115-123.
Friebe, A. and D. Koesling. 2003. Regulation of nitric oxide-sensitive guanylyl cyclase. Circ. Res. 93: 96-105.
Friebe A., B. Wedel, C. Harteneck, F. Foerster, G. Schultz and D. Koesling. 1997. Functions of conserved cysteines of soluble guanylyl cycalse. Biochemistry 36: 1194-1198.
Garbers, D. L. 1999. The guanylyl cyclase receptors. Methods 19: 477-484.
Gibb B. J. and J. Garthwaite. 2001. Subunits of the nitric oxide receptor, soluble guanylyl cyclase, express in rat brain. Eur. J. Neurosci. 13: 539-544.
Gibbs, S. M. and J. W. Truman. 1998. Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila. Neuron 20: 83-93.
Gibbs, S. M., A. Becker, R. W. Hardy and J. W. Truman. 2001. Soluble guanylate cyclase is required during development for visual system function in Drosophila. J. Neurosci. 21: 7705-7714.
Gibson, N. J. and A. Nighorn. 2000. Expression of nitric oxide synthase and soluble guanylyl cyclase in the developing olfactory system of Manduca sexta. J. Comp. Neurol. 422: 191-205.
Gibson, N. J., W. Rössler, A. J. Nighorn, L. A. Oland, J. G. Hildebrand and L. P. Tolbert. 2001. Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta. Dev. Biol. 240: 326-339.
Giray T., Z. Y. Huang, E. Guzmán-Novoa and G. E. Robinson. 1999. Physiological correlates of genetic variation for rate of behavioral development in the honeybee, Apis mellifera. Behav. Ecol. Sociobiol. 47: 17-28.
Giuili G., U. Scholl, F. Bulle and G. Guellaen. 1992. Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain. FEBS Lett. 304: 83-88.
Grünbaum, L. and U. Müller. 1998. Induction of a specific olfactory memory leads to a long-lasting activation of protein kinase C in the antennal lobe of the honeybee. J. Neurosci. 18: 4384-4392.
Gupta G., J. Kim, L. Yang, S. L. Sturley and R. S. Danziger. 1997. Expression and purification of soluble, active heterodimeric guanylyl cyclase from baculovirus. Protein Expr. Purif. 10: 325-330.
Hammer, M. and R. Menzel. 1995. Learning and memory in honeybee. J. Neurosci. 15: 1617-1630.
Harteneck, C., B. Wedel, D. Koesling, J. Malkewitz, E. Böhme and G. Schultz. 1991. Molecular cloning and expression of a new α-subunit of soluble guanylyl cyclase. FEBS Lett. 292: 217-222.
Hiledbrandt, H. and U. Müller. 1995. PKA activity in the antennal lobe of honeybees is regulated by chemosensory stimulation in vivo. Brain Res. 679: 281-288.
Hofmann F., A. Ammendola and J. Schlossmann. 2000. Rising behind NO: cGMP-dependent protein kinases. J. Cell Sci. 113: 1671-1676.
Huang, Z. Y. and G. E. Robinson. 1996. Regulation of honeybee division of labor by colony age demography. Behav. Ecol. Sociobiol. 39: 147-158.
Huang, Z. Y., G. E. Robinson and D. W. Borst. 1994. Physiological correlates of division of labor among similarly aged honeybees. J. Comp. Physiol. A. 174: 731-739.
Huang, Z. Y., G. E. Robinson, S. S. Tobe, K. J. Yagi , C. Strambi, A. Strambi and B. Stay. 1991. Hormone regulation of behavioral development in the honey bee is based on changes in the rate of juvenile hormone biosynthesis. J. Insect Physiol. 37: 733-741.
Jassim, O., Z. Y. Huang and G. E. Robinson. 2000. Juvenile hormone profiles of worker honeybees, Apis mellifera, during normal and accelerated behavioral development. J. Insect Physiol. 46: 243-249.
Koesling, D. 1999. Studying the structure and regulation of soluble guanylyl cyclase. Methods 19: 485-493.
Koesling D., J. Herz, H. Guasepohl, F. Niroomand, K. D. Hinsch, A. Mulsch, E. Bohme, G. Schultz and R. Frank. 1988. The primary structure of the 70 kDa subunit of bovine soluble guanylyl cyclase. FEBS Lett. 239: 29-34.
Koglin, M. and S. Behrends. 2000. Cloning and functional expression of the rat α2 subunit of soluble guanylyl cyclase. Biochim. Biophys. Acta 1494: 286-289.
Koglin, M. and S. Behrends. 2003. A functional domain of the α1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding. J. Biol. Chem. 278: 12590-12597.
Kojima, M., K. Hisaki, H. Matsuo and K. Kangawa. 1995. A new type soluble guanylyl cyclase, which contains a kinase-like domain: its structure and expression. Biochem. Biophys. Res. Commun. 217: 993-1000.
Laber, U., T. Kober, V. Schmitz, A. Schrammel, W. Mayer, M. Weber and G. Kojda. 2002. Effect of hypercholesterolemia on expression and function of vascular soluble guanylyl cyclase. Circulation 105: 855-860.
Laura, R. P., A. M. Dizhoor and J. B. Hurley. 1996. The membrane guanylyl cyclase, retinal guanylyl cyclase-1, is activated through its intracellular domain. J. Biol. Chem. 271: 11646-11651.
Leboulle G. and U. Müller. 2004. Synergistic activation of insect cAMP-dependent protein kinase A (type II) by cyclicAMP and cyclicGMP. FEBS. 576: 216-220.
Lucas, K. A., G. M. Pitari, S. Kazerounian, I. Ruiz-Stewart, J. Park, S. Schulz, K. P. Chepenik and S. A. Waldman. 2000. Guanylyl cyclase and signaling by cyclic GMP. Pharmacol. Rev. 52: 375-414.
Meller, V. H. and R. L. Davis. 1996. Biochemistry of insect learning: lessons from bees and flies. Insect Biochem. Mol. Biol. 26: 327-335.
Menzel, R. and M. Giurfa. 2001. Cognitive architecture of a mini-brain: the honeybee. Trends Cogn. Sci. 5: 62-66.
Menzel, R., M. Hammer, U. Müller and H. Rosenboom. 1996. Behavioral, neural and cellular components underlying olfactory learning in the honeybee. J. Physiol. 90: 395-398.
Menzel, R., K. Geiger, J. Joerges, U. Müller and L. Chittka. 1998. Bees travel novel homeward routes by integrating separately acquired vector memories. Anim. Behav. 55: 139-152.
Mergia, E., M. Russwurm, G. Zoidl and D. Koesling. 2003. Major occurrence of the new α2β1 isoform of NO-sensitive guanylyl cyclase in brain. Cell. Signal. 15: 189-195.
Mikami, T., T. Kusakabe and N. Suzuki. 1998. Molecular cloning of cDNA and expression of mRNAs encoding α and β subunits of soluble guanylyl cyclase from medaka fish Oryzias latipes. Eur. J. Biochem. 253: 42-48.
Morton D. B. and P. J. Simpson. 2002. Cellular signaling in eclosion hormone action. J. Insect Physiol. 48: 1-13.
Müller, U. 1997. Neuronal cAMP-dependent protein kinase type II is concentrated in mushroom bodies of Drosophila melanogaster and the honeybee Apis mellifera. J. Neurobiol. 33: 33-44.
Müller, U. 1999. Second messenger pathway in the honeybee brain: immunohistochimistry of protein kinase A and protein kinase C. Microsc. Res. Tech. 45: 165-173.
Müller, U. 2000. Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees. Neuron 27: 159-168.
Müller, U. and H. Hiledbrandt. 1995. The nitric oxide/cGMP system in the antennal lobe of Apis mellifera is implicated in integrative processing of chemosensory stimuli. Eur. J. Neurosci. 7: 2240-2248.
Müller, U. and H. Hiledbrandt. 2002. Nitric oxide/cGMP-mediated protein kinase A activation in the antennal lobes plays an important role in appetitive reflex habituation in the honeybee. J. Neurosci. 22: 8739-8747.
Nakane, M., A. Arai, S. Saheki, T. Kuno, W. Buechler and F. Murad. 1990. Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung. J. Biol. Chem. 265: 16841-16845.
Nighorn, A., K. A. Byrnes and D. B. Morton. 1999. Identification and characterization of a novel β subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide. J. Biol. Chem. 274: 2525-2531.
Nighorn, A., P. J. Simpson and D. B. Morton. 2001. The novel guanylyl cyclase MsGC-I is strongly expressed in higher-order neuropils in the brain of Manduca sexta. J. Exp. Biol. 204: 305-314.
Nighorn, A., N. J. Gibson, D. M. Rivers, J. G. Hildebrand and D. B. Morton. 1998. The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta. J. Neurosci. 18: 7244-7255.
Osborne, K. A., J. S. de Belle and M. B. Sokolowdki. 2001. Foraging behaviour in Drosophila larvae: mushroom body ablation. Chem. Senses 26: 223-230.
Osborne, K. A., A. Robichon, E. Burgess, S. Butland, R. A. Shaw, A. Coulthard, H. S. Pereira, R. J. Greenspan and M. B. Sokolowski. 1997. Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science 277: 834-836.
Pankiw, T., R. E. Page and M. K. Fondrk. 1998. Brood pheromone stimulates pollen foraging in honey bees (Apis mellifera). Behav. Ecol. Sociobiol. 44: 193-198.
Robinson, G. E. 1985. Effects of a juvenile hormone analogue on honey bee foraging behaviour and alarm pheromone production. J. Insect Physiol. 31: 277-282.
Robinson, G. E. 1987. Regulation of honey bee age polyethism by juvenile hormone. Behav. Ecol. Sociobiol. 20: 329-338.
Robinson, G. E. and E. L. Vargo. 1997. Juvenile hormone in adult eusocial hymenoptera: gonadotropin and behavioral pacemaker. Arch. Insect Biochem. Physiol. 35: 559-583.
Russwurm M., N. Wittau and D. Koesling. 1998. Guanylyl cyclase/ PSD-95 interaction: targeting of the nitric oxide-sensitive alpha2 beta1 guanylyl cyclase to synaptic membranes. J. Biol. Chem. 276: 44647-44652.
Russwurm M., S. Behrends, C. Harteneck and D. Koesling. Functional properties of a naturally occurring isoform of soluble guanylyl cyclase. Biochem. J. 335: 125-130.
Schmachtenberg, O. and G. Bicker. 1999. Nitric oxide and cyclic GMP modulate photoreceptor cell responses in the visual system of the locust. J. Exp. Biol. 202: 13-20.
Schulz, D. J. and G. E. Robinson. 1999. Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies. J. Comp. Physiol. A. 184: 481-488.
Schulz, D. J., J. P. Sullivan and G. E. Robinson. 2002. Juvenile hormone and octopamine in the regulation of division of labor in honey bee colonies. Horm. Behav. 42: 222-231.
Schwarz, U. R., U. Walter and M. Eigenthaler. 2001. Taming platelets with cyclic nucleotides. Biochem. Pharmacol. 62: 1153-1161.
Seeley T. D. 1985. Honeybee ecology. A Study of Adaptation in Social Life. Princeton. Princeton UP.
Shah, S. and D. R. Hyde. 1995. Two Drosophila genes that encode the α and β subunits of the brain soluble guanylyl cyclase. J. Biol. Chem. 270: 15368-15376.
Simpson, P. J., A. Nighorn and D. B. Morton. 1999. Identification of a novel guanylyl cyclase that is related to receptor guanylyl cyclases, but lacks extracellular and transmembrane domains. J. Biol. Chem. 274 : 4440-4446.
Sokolowski, M. B. 1998. Genes for normal behavioral variation: recent clues from flies and worms. Neuron 21: 463-466.
Stasch, J.-P., E. M. Becker, C. Alonso-Alija, H. Apeler, K. Dembowsky, A. Feurer, R. Gerzer, T. Minuth, E. Perzborn, U. Pleiss, H. Schroeder, W. Schroeder, E. Stahl, W. Steinke, A. Straub and M. Schramm. 2001. NO-independent regulatory site on soluble guanylyl cyclase. Nature 410: 212-215.
Stone J. R. and M. A. Marletta. 1994. Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33: 5636-5640.
Stone J. R. and M. A. Marletta. 1995a. Heme stoichiometry of heterodimeric soluble guanylate cyclase. Biochemistry 34: 14668-14674.
Stone J. R. and M. A. Marletta. 1995b. The ferrous heme of soluble guanylate cyclase: formation of haxacoodinate complexes with carbon monoxide and nitrosomethane. Biochemistry 34: 16397-16403.
Sullivan, J. P., O. Jassim and S. E. Fahrbach. 2000. Juvenile hormone paces behavioral development in the adult worker honey bee. Horm. Behav. 37: 1-14.
Thorpe, D. S. and E. Morkin. 1990. The carboxyl region contains the catalytic domain of the membrane from of guanylyl cyclase. J. Biol. Chem. 265: 14717-14720.
Toma, D. P., G. Bloch., D. Moore and G. E. Robinson. 2000. Changes in period mRNA levels in the brain and division of labor in honey bee colonies. Proc. Natl. Acad. Sci. U.S.A. 97; 6914-6919.
Tremblay, J., R. Gerzer and P. Hamet, 1988. Cyclic GMP in cell function. Adv. Second Mess. Phosphoprotein Res. 22: 319-383.
Wedel, B., C. Harteneck, J. Foerster, A. Friebe, G. Schultz and D. Koesling. 1995. Function domains of soluble guanylyl cyclase. J. Biol. Chem. 270: 24871-24875.
Wedel, B. J. and D. L. Garbers. 1998. Guanylyl cyclases: approaching year thirty. Trends Endocrinol. Metab. 9: 213-219.
Whitfield, C. W., A. M. Cziko and G. E. Robinson. 2003. Gene expression profiles in the brain predict behavior in individual honey bees. Science 302: 296-299.
Whitfield, C. W., M. R. Band, M. F. Bonaldo, C. G. Kumar, L. Liu, J. R. Pardinas, H. M. Robertson, M. B. Soares and G. E. Robinson. 2002. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee. Genome Res. 12: 555-566.
Zhuo, M. and R. D. Hawkins. 1995. Long-term depression: a learning-related type of synaptic plasticity in the mammalian central nervous system. Rev. Neurosci. 6: 259-277.