beta-Bungarotoxin (beta-BuTx), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide subunits. A phospholipase A2 subunit named A chain, and a non-phospholipase A2 subunits named B chain. The A chain and B chain are covalently linked by one disulfide bridge. Although it has been widely accepted that the toxic effect of beta-BuTx is attributed to the disturbance of presynaptic transmitter release, however the inhibition of transmitter release by beta-BuTx is still obscure. Here we investigate the mechanism that mediates facilitation of transmitter release at the neuromuscular junction induced by beta-BuTx, using Xenopus nerve-muscle coculture. Application of beta-BuTx and isotoxins BM12, BM13 led to a marked increase in the frequency of spontaneous synaptic currents (SSCs) after a short period (12~18 min) of latency. The synaptic potentiation induced by these toxins was abolished when Ca2+ in the medium is substituted by Ba2+ (a potent phospholipase A2 inhibitor). Application of PLP-BM12 and PLP-BM13, which have been chemical-modification to lose their PLA2 activity from BM12 and BM13, failed to potentiate the transmitter release.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19142号 / 生博第325号 / 新制||生||43 / 32093 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 豊島 文子, 教授 千坂 修 / 学位規則第4条第1項該当

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

No description available.

Für die Bildung eines funktionalen Nervensystems in Vertebraten ist ein Gleichgewicht zwischen inhibitorischen und exzitatorischen Neuronen essentiell. Ein Schlüsselfaktor in der Regulation dieses Gleichgewichts ist der bHLH Transkriptionsfaktor Ptf1a, welcher GABAerge inhibitorische Neurone in der Retina, dem Hinterhirn und im Rückenmark von Vertebraten spezifiziert, zugleich jedoch glutamaterge exzitatorische Neurone unterdrückt. In diesem Zusammenhang benötigt die Aktivität von Ptf1a die Bildung eines trimeren Komplexes, in welchem Ptf1a an ein allgemein exprimiertes E-Protein und an ein Mitglied der Su(H)-Familie bindet. Ptf1a fördert ebenfalls generelle neuronale Differenzierung in X. laevis Embryonen und Explantaten, was darauf hinweist, dass Ptf1a proneurale Aktivität besitzt. In dieser Doktorarbeit wurde die Rolle von Ptf1a im Zusammenhang mit genereller Neurogenese (frühe Funktion) und neuronaler Subtypen-Spezifizierung (späte Funktion) untersucht. Durch eine zeitliche Expressionsanalyse bekannter Gene konnte gezeigt werden, dass Ptf1a durch die Aktivierung von nachgeschalteten Genen, ähnlich dem proneuralen Transkriptionsfaktor Ngn2, in animalen Kappen (naives Ektoderm) zu frühen Zeitpunkten Neurogenese induziert. In späteren Stadien hingegen aktivierte Ptf1a die Expression von Markergenen, die GABAerge Neurone kennzeichnen, während neuronale glutamaterge Markergene von Ngn2 induziert wurden. Eine mutierte Version von Ptf1a (Ptf1aW224A/W242A), welche nicht in der Lage ist, mit dem Kofaktor Su(H) zu interagieren, behielt die Fähigkeit, generelle Neurogenese zu induzieren, nicht aber GABAerge Markergene zu aktivieren. Diese Ergebnisse lassen darauf schließen, dass Ptf1a in der Entwicklung des Nervensystems kontext-spezifische Transkriptionskomplexe bildet: einen Su(H)-unabhängigen Komplex zur Aktivierung genereller Neuorgenese und einen Su(H)-abhängigen Komplex zur Spezifizierung GABAerger Neurone. Da die Zielgene von Ptf1a in der Entwicklung des Nervensystems nicht genau bestimmt sind, wurden zwei unabhängige Transkriptom-Analysen durchgeführt, um das Ptf1a nachgeschaltete genetische Netzwerk aufzuzeigen. In diesen Untersuchungen wurde eine zeitliche Analyse von Genen durchgeführt, die durch wildtyp Ptf1a, Ptf1aW224A/W242A und Ngn2 in X. laevis animalen Kappen aktiviert werden; direkte Zielgene für Ptf1a und Ptf1a/Su(H) wurden bestimmt durch die Aktivierung dieser Transkriptionsfaktoren unter Vorhandensein eines Proteinsyntheseinhibitors (CHX). Durch dieses Vorgehen konnten viele mutmaßlich neue frühe und späte Zielgene von Ptf1a identifiziert werden. Eine weitere Analyse dieser nachgeschalteten Zielgene dürfte darüber Aufschluss geben, wie Ptf1a generelle Neurogenese und neuronale Subtypen-Spezifizierung reguliert.

In eukaryotes, the ubiquitin-proteasome system (UPS) degrades most cellular protein. Inhibition of the UPS has been associated with different disease states and can affect various intracellular processes including the activation of heat shock protein (hsp) gene expression. During cellular stress, HSPs act as molecular chaperones by inhibiting protein aggregation and assisting in their refolding once normal conditions are re-established. In the present study, Withaferin A (WA), a steroidal lactone with possible anti-inflammatory and antitumor properties, was found to inhibit proteasome activity and induce the expression of hsp genes in the amphibian model system, Xenopus laevis. Treatment of Xenopus kidney epithelial A6 cells with WA produced an increase in the accumulation of ubiquitinated protein and a significant decrease in chymotrypsin-like activity. Furthermore, immunoblot analysis revealed that WA induced HSP30 and HSP70 accumulation. For example, cells treated with 5 μM WA for 18 h resulted in the optimal accumulation of HSP30 and HSP70. Northern blot analysis revealed that exposure of cells to 5 μM WA induced hsp30 and hsp70 mRNA accumulation in a time-dependent manner up to 12 h. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in WA-induced hsp gene expression in A6 cells, since pretreatment with the HSF1 inhibitor, KNK437, reduced the accumulation of HSP30 and HSP70. Also, WA acted synergistically with mild heat shock to enhance HSP accumulation to a greater extent than the sum of both stressors individually. In cells recovering from WA, the relative levels of HSP30 and HSP70 accumulation remained elevated from 6 to 12 h after removal of WA. Immuocytochemical analysis and laser scanning confocal microscopy revealed that WA-induced HSP30 accumulation occurred primarily in the cytoplasm with some staining in the nucleus in a granular or punctate pattern. Prolonged exposure to WA resulted in some disorganization of the actin cytoskeleton as well as large cytoplasmic HSP30 staining structures in some cells. Prior exposure of cells to WA treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 °C. In conclusion, this study has shown that WA can induce an inhibition of proteasome activity and an increase hsp gene expression. Activating the heat shock response is a potential avenue for novel drug therapies, which can confer cytoprotection in disease states involving cytotoxic protein aggregation.

Progestagenic compounds are emerging contaminants found in surface and ground water around the world. Information on the effects and potency of progestagens is needed in order to understand the environmental risks posed by these compounds. Using the Xenopus (Silurana) tropicalis test system, developmental and reproductive toxicity after exposure to selected progestagens were determined. Larval exposure to levonorgestrel (LNG) severely impaired oviduct and ovary development causing sterility. No effects on testicular development, spermcount or male fertility were observed. Hepatic mRNA expression of the androgen receptor was increased in the females indicating that the receptor is involved in LNG-induced developmental reproductive toxicity. Exposure of adult females to LNG, norethindrone (NET) or progesterone (P) increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes compared with the controls, indicating an inhibited vitellogenesis. The effects on oocyte development were ascertained at environmentally relevant concentrations of LNG, NET and P (1.3, 1 and 10 ng/L respectively). Since unintentional co-exposure of progestagens and ethinylestradiol (EE2) occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development are needed. Co-exposure during development showed antagonistic effects of EE2 and LNG. EE2 caused a female biased sex ratio which showed a tendency to be antagonized by LNG. Moreover, the hepatic AR induction by LNG was counteracted by co-exposure to EE2. In conclusion, the results show that female amphibians are susceptible to reproductive toxicity of progestagens after developmental exposure as well as after adult exposure during the breeding period. The differentiating Müllerianduct and ovary, and the egg development are sensitive targets for progestagens. Finally, the findings reported in this thesis show that environmental progestagens impairs reproductive function in amphibians and may present a threat to reproduction in wild populations.

In frogs, part of the important immune defence system of their skin is the secretion of antimicrobial peptides from granular glands. This study investigated the immune function of skin peptides in protection against bacterial pathogens associated with infectious bacterial dermatosepticemia under a number of environmental conditions and at certain stages of the life cycle of frogs. The natural peptide mixture of skin peptides was collected from skin secretions of three semi-aquatic Litoria frog species L. aurea, L. raniformis and L. ewingii and aquatic Xenopus laevis and assayed for activity against the bacterial pathogens: Aeromonas hydrophila, Chryseobacterium meningosepticum, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Serratia liquefaciens. The peptide mixtures of three frog species Xenopus laevis, Litoria aurea and Litoria raniformis showed activity against C. freundii, C. meningosepticum, K. pneumoniae and P. aeruginosa in vitro indicating a likely protective function. One Litoria species, L. ewingii, had a peptide mixture that did not have activity against any pathogen. Subsequently, in experimental exposure of animals to the pathogen K. pneumoniae, this species was found to be susceptible to disease while the other sympatric species L. raniformis was found to be resistant. A strong correlation was shown between composition of skin peptides and resistance to disease. A comparison of the production and activity of skin peptides from four frog species showed the aquatic X. laevis to have more effective immune defence against bacterial pathogens than three tested Litoria species. X. laevis produced significantly greater amount of bioactive peptide mixture than three tested Litoria species. Three pathogens A. hydrophila, P. mirabilis and S. liquefaciens are abundant components of the skin microbiota of healthy frogs and were found to be resistant to the peptide mixtures of all four frog species tested. It was shown that one pathogen, A. hydrophila, had the ability to secrete proteases which could inactivate skin peptides. Thus while skin peptides could function against several pathogens, some pathogens might have co-evolved to resist skin peptides. A comparison of the peptide mixtures from skin secretions of adults, metamorphs and larvae of L. ewingii using liquid chromatography-mass spectrometry analyses showed that peptide mixtures of post metamorphic animals, adults and metamorphs, had a species-specific profile that included the antimicrobial peptide uperin 7.1, while the larval peptide mixture did not contain uperin 7.1 or any other known species-specific peptide. This finding indicates the absence of a secretory mechanism that could compensate for the absence of granular glands in larvae. Analyses of the production and activity of skin peptides of L. raniformis after exposure to two different environmental stressors, low environmental temperature and pesticide carbaryl, showed that the total amount of bioactive peptide was significantly reduced which could consequently increase susceptibility to disease. Thus suppression of skin peptides could be a possible mechanism for synergism between the important stressors and pathogens in disease development.