D-Aspartate (D-Asp) is an endogenous compound found in the central nervous system (CNS) of a variety of organisms. Despite its prevalence, however, relatively little understood of its physiological role. The prevailing theory is that D-Asp is an alternate agonist of N-methyl-D-aspartate receptor (NMDAR) channels. The goal of this work was to characterize the currents activated by D-Asp in neurons Aplysia californica, focusing on cells of the buccal S cluster (BSC). First, a general electrophysiological characterization was carried out, examining ion permeability, agonist dose-response, and the kinetics of activation, inactivation, and desensitization. D-Asp activated non-specific cation currents characterized by permeability to Na+ and K+. D-Asp-induced currents shared similar current-voltage relationships and time courses of activation and inactivation with L-glutamate (L-Glu)-induced currents. D-Asp currents, however, were subject to prolonged desensitization. Additionally, D-Asp activated currents independently of L-Glu, the known agonist of NMDAR channels, suggesting a non-NMDAR-dependent role of D-Asp. Next, select antagonists were used in an effort to pharmacologically characterize D-Asp receptor channels. These experiments suggested that D-Asp whole cell currents may be characterized by activation of multiple receptor sites, including NMDARS, excitatory amino acid transporters (EAATs), and a putative non-L-Glu D-Asp receptor. Furthermore, bath-applied D-Asp attenuated L-Glu-activated currents. Finally, D-Asp currents were compared to those evoked by acetylcholine (ACh) and serotonin (5-HT) in BSC cells. Results suggested that D-Asp activated receptor channels independently of ACh and 5-HT. Ten minute bath application of 5-HT was found to potentiate D-Asp current responses, likely through activation of a protein kinase C (PKC)-dependent mechanism, suggesting that D-Asp induced currents may be subject to synaptic plasticity associated with learning. While the identity of the putative D-Asp receptor remains elusive, the current work has advanced our understanding of the role D-Asp may play in the nervous system. These results should provide the groundwork for future studies aimed at identifying this unknown receptor channel, as well as investigation of the potential relationship of D-Asp receptor modulation to learning and memory in Aplysia, which may have relevance in higher organisms.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 2000. / Includes bibliographical references. Also available on the Internet.

NOGUEIRA, Vanessa Lúcia Rodrigues. Papel de proteínas na tinta liberada pela lesma-do-mar Aplysia dactylomela Rang 1828 os mecanismos de defesa do animal. 2005. 90 f. Dissertação (Mestrado em Ciências Marinhas Tropicais) - Instituto de Ciências do Mar, Universidade Federal do Ceará, Fortaleza, 2005. / Submitted by Debora Oliveira (deby_borboletinha@hotmail.com) on 2011-12-22T15:10:49Z No. of bitstreams: 1 2005_dis_vlrnogueira.pdf: 12654022 bytes, checksum: 35936f35caac0bc23dbf25e1b2ffcafc (MD5) / Approved for entry into archive by Nadsa Cid(nadsa@ufc.br) on 2012-01-23T17:07:24Z (GMT) No. of bitstreams: 1 2005_dis_vlrnogueira.pdf: 12654022 bytes, checksum: 35936f35caac0bc23dbf25e1b2ffcafc (MD5) / Made available in DSpace on 2012-01-23T17:07:24Z (GMT). No. of bitstreams: 1 2005_dis_vlrnogueira.pdf: 12654022 bytes, checksum: 35936f35caac0bc23dbf25e1b2ffcafc (MD5) Previous issue date: 2005 / The sea hare Aplysia dactylomela is known by discharging a purple ink when disturbed. As it doesn't show any external structure of protection, it is believed that this secretion, rich in bioactive substances, participates in the chemical defense against pathogens and/or predators. The ink is constituted mainly by pigments, proteins and low molecular mass substances. The pigments are originated from red algae, nevertheless, there is lack of information about the origin, processing, storage and function of the ink proteins. This work describes the protein composition of the ink and present some physiochemical and biological characteristics of the dactylomelin-P, an antibacterial protein from the ink besides information concerning the location of this protein in the purple gland. The ink was obtained from specimens collected at Fleixeiras beach, Ce. The protein composition of the ink was studied by bi-dimensional electrophoresis. The purification of dactylomelin-P consisted basically in ion exchange and hydrophobic interaction chromatography. Dactylomelina-P was analyzed as to molecular mass, isoeletric point, amino acid composition, carbohydrates, heat stability, pH and proteinase resistance. Several biological activities were tested with the ink and with dactylomelina-P, including antimicrobial, enzymatic, haemagglutinating, anticoagulant, hemolytic, cytotoxic and toxic activities. The immunolocalization assays of dactylomelina-P were carried out in different sea hare tissues by western blot and immunohistochemistry and the interaction with Staphylococcus aureus was investigated by immunocytochemistry. The ink of A. dactylomela contains more than 40 proteins/peptides, with molecular masses below 70 kDa and acid pIs. The most abundant protein in the ink is the dactylomelin-P, a monomeric protein with 59,8 kDa, pI 5,0, high methionin content, and less than 1% carbohydrates. It is denatured at 60 ºC for 10 minutes and it resists to pH range of 3-12. Dactylomelin-P shows a wide antibacterial action spectrum, but no antifungal, unlike the ink that possesses some factor with this activity. It is particularly efficient against sea bacteria, can be bactericidal (4,0μg/ml) or bacteriostatic (0,2μg/ml), depending on the concentration. Dactylomelin-P agglutinated rabbit, mice and rat erythrocytes, but it did not show anticoagulating, hemolytic and cytotoxic activities. The LD50 for mice was 60-100 mg/Kg, being considered moderately toxic. Dactylomelin-P was only found in the ink gland, being located preferentially in the cells of the producing vesicles. The electronic transmission microscopy revealed that the dactylomelina-P crosses the cell wall of S. aureus and interacts mainly with the plasmatic membrane, probably interfering in the metabolism of the bacterium, instead of causing mechanic damage to the cell. / O gastrópode marinho Aplysia dactylomela é conhecido por liberar uma tinta púrpura sempre que é importunado. Como não possui nenhuma estrutura externa de proteção, acredita-se que essa secreção, rica em substâncias biologicamente ativas, participe da defesa química do animal. A tinta é composta de pigmentos, proteínas e substâncias de baixa massa molecular. Os pigmentos da tinta são originados de algas vermelhas, mas quanto às proteínas, pouco é conhecido sobre a sua origem, processamento, local de armazenamento e função no animal. Este trabalho descreve pela primeira vez a composição protéica da tinta e apresenta algumas características físico-químicas e biológicas da dactylomelina-P, uma proteína antibacteriana presente na tinta dessa lesma, além de trazer informações acerca da localização desta proteína na glândula de tinta. A tinta foi obtida a partir de espécimes encontrados na praia de Fleixeiras, Ce. A composição de proteínas da tinta foi determinada por eletroforese bidimensional e a purificação da dactylomelina-P foi feita através de cromatografias de troca iônica e interação hidrofóbica. Dactylomelina-P foi analisada quanto à massa molecular, ponto isoelétrico, composição de aminoácidos, presença de carboidratos, estabilidade térmica, resistência ao pH e a proteases. Várias atividades biológicas foram testadas com a tinta e com a dactylomelina-P, incluindo atividades antimicrobianas, enzimáticas, hemaglutinante, anticoagulante, hemolítica, citotóxica e tóxica. Os ensaios de localização da proteína foram realizados em diferentes tecidos da lesma por western blot, na glândula de tinta por imunohistoquímica e a interação com a bactéria Staphylococcus aureus foi feita por imunocitoquímica. A tinta de A. dactylomela contém mais de 40 proteínas/ peptídeos, com massas abaixo de 70 kDa e pIs na faixa ácida. A proteína mais abundante na tinta é a dactylomelina-P, que é uma molécula monomérica, de 59,8 kDa, pI 5,0, que possui alto teor de metionina e menos de 1% de carboidratos, é desnaturada a 60 ºC por 10 minutos e resiste a pHs entre 3-12. Dactylomelina-P mostra um amplo espectro de ação antibacteriano, mas não antifúngico, ao contrário da tinta que possui um fator com esta atividade. É particularmente eficiente contra bactérias marinhas, podendo ser bactericida (4,0μg/ml) ou bacteriostática (0,2μg/ml), dependendo da concentração. Dactylomelina-P aglutinou eritrócitos de coelho, ratos e camundongos, não apresentou atividade anticoagulante, hemolítica e nem citotóxica. A DL50 para camundongos ficou entre 60-100 mg/Kg, sendo considerada moderadamente tóxica. Dactylomelina-P só foi encontrada na glândula de tinta, localizando-se preferencialmente nas células das vesículas produtoras. Ensaios de microscopia eletrônica de transmissão revelaram que a dactylomelina atravessa a parede celular da bactéria S. aureus e, interage principalmente com a membrana citoplasmática, provavelmente interferindo no metabolismo, ao invés de causar danos à célula.

No description available.

O gastrÃpode marinho Aplysia dactylomela à conhecido por liberar uma tinta pÃrpura sempre que à importunado. Como nÃo possui nenhuma estrutura externa de proteÃÃo, acredita-se que essa secreÃÃo, rica em substÃncias biologicamente ativas, participe da defesa quÃmica do animal. A tinta à composta de pigmentos, proteÃnas e substÃncias de baixa massa molecular. Os pigmentos da tinta sÃo originados de algas vermelhas, mas quanto Ãs proteÃnas, pouco à conhecido sobre a sua origem, processamento, local de armazenamento e funÃÃo no animal. Este trabalho descreve pela primeira vez a composiÃÃo protÃica da tinta e apresenta algumas caracterÃsticas fÃsico-quÃmicas e biolÃgicas da dactylomelina-P, uma proteÃna antibacteriana presente na tinta dessa lesma, alÃm de trazer informaÃÃes acerca da localizaÃÃo desta proteÃna na glÃndula de tinta. A tinta foi obtida a partir de espÃcimes encontrados na praia de Fleixeiras, Ce. A composiÃÃo de proteÃnas da tinta foi determinada por eletroforese bidimensional e a purificaÃÃo da dactylomelina-P foi feita atravÃs de cromatografias de troca iÃnica e interaÃÃo hidrofÃbica. Dactylomelina-P foi analisada quanto à massa molecular, ponto isoelÃtrico, composiÃÃo de aminoÃcidos, presenÃa de carboidratos, estabilidade tÃrmica, resistÃncia ao pH e a proteases. VÃrias atividades biolÃgicas foram testadas com a tinta e com a dactylomelina-P, incluindo atividades antimicrobianas, enzimÃticas, hemaglutinante, anticoagulante, hemolÃtica, citotÃxica e tÃxica. Os ensaios de localizaÃÃo da proteÃna foram realizados em diferentes tecidos da lesma por western blot na glÃndula de tinta por imunohistoquÃmica e a interaÃÃo com a bactÃria Staphylococcus foi feita por imunocitoquÃmica. A tinta de A. dactylomela contÃm mais de 40 proteÃnas/ peptÃdeos, com massas abaixo de 70 kDa e pIs na faixa Ãcida. A proteÃna mais abundante na tinta à a dactylomelina-P, que à uma molÃcula monomÃrica, de 59,8 kDa, pI 5,0, que possui alto teor de metionina e menos de 1% de carboidratos, à desnaturada a 60 ÂC por 10 minutos e resiste a pHs entre 3-12. Dactylomelina-P mostra um amplo espectro de aÃÃo antibacteriano, mas nÃo antifÃngico, ao contrÃrio da tinta que possui um fator com esta atividade. à particularmente eficiente contra bactÃrias marinhas, podendo ser bactericida (4,0μg/ml) ou bacteriostÃtica (0,2μg/ml), dependendo da concentraÃÃo. Dactylomelina-P aglutinou eritrÃcitos de coelho, ratos e camundongos, nÃo apresentou atividade anticoagulante, hemolÃtica e nem citotÃxica. A DL50 para camundongos ficou entre 60-100 mg/Kg, sendo considerada moderadamente tÃxica. Dactylomelina-P sà foi encontrada na glÃndula de tinta, localizando-se preferencialmente nas cÃlulas das vesÃculas produtoras. Ensaios de microscopia eletrÃnica de transmissÃo revelaram que a dactylomelina atravessa a parede celular da bactÃria S. aureus e, interage principalmente com a membrana citoplasmÃtica, provavelmente interferindo no metabolismo, ao invÃs de causar danos à cÃlula. / The marine gastropod Aplysia dactylomela is known to release a purple ink when it is plagued. How has no external structure of protection, it is believed that secretion, rich in biologically active substances, participate in the chemical defense of the animal. The ink is composed of pigments, proteins and low molecular weight substances. The pigments of ink are originated from red algae, but as the protein, little is known about its origin, processing, storage location and function in animals. This paper describes for the first time the protein composition of the ink and presents some physico-chemical and dactylomelina of organic-P, an antibacterial protein present in the ink that slug, and get information about the location of this protein in the ink gland. The paint was obtained from specimens found on the beach in Fleixeiras, Ce. The protein composition of the ink was determined by two-dimensional electrophoresis and purification of dactylomelina-P was done by ion exchange chromatography and hydrophobic interaction. Dactylomelina-P was analyzed on the molecular mass, isoelectric point, composition of amino acids, the presence of carbohydrates, thermal stability, resistance to proteases and pH. Several biological activities were tested with the ink and the dactylomelina-P, including antimicrobial activity, enzyme, haemagglutinating, anticoagulant, hemolytic, cytotoxic and toxic. Tests for localization of the protein were performed in different tissues of the slug by western blot in the ink gland by immunohistochemistry and the interaction with the bacterium Staphylococcus was performed by immunocytochemistry. The ink of A. dactylomela contains more than 40 proteins / peptides, with masses below 70 kDa and PIs in the acidic range. The most abundant protein in the ink is dactylomelina-P, which is a monomeric molecule of 59.8 kDa, pI 5.0, which has high content of methionine and less than 1% of carbohydrates, is denatured at 60  C for 10 minutes and resist the pH between 3-12. Dactylomelina-P shows a broad spectrum of antibacterial action, but not antifungal, unlike paint that has a factor with this activity. It is particularly effective against marine bacteria and may be bactericidal (4.0 g / ml) or bacteriostatic (0.2 g / ml), depending on the concentration. P-Dactylomelina agglutinate rabbit erythrocytes, rats and mice, showed no anticoagulant activity, and cytotoxic and hemolytic. The LD50 for mice was between 60-100 mg / kg, is considered moderately toxic. Dactylomelina-P was found in the ink gland, is located preferentially in cells producing the vesicles. Tests for transmission electron microscopy revealed that the dactylomelina through the cell wall of the bacterium S. aureus and mainly interacts with the cytoplasmic membrane, probably interfering with metabolism, rather than damage the cell.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

In predator-prey interactions, prey species have complex defensive behaviors to protect themselves from predators. Chemical defenses are one tool that is employed to protect against predators, especially for slow-moving or otherwise susceptible prey. Many of these chemical defenses have been studied and the effective compounds identified, but few studies were performed on their mechanisms of detection. In my research, I used the sea hare, Aplysia californica, as chemically defended prey. This slow moving mollusk is soft-bodied with no external shell, but it has adapted a number of defenses including chemical defenses. Ink is a sticky mixture of the products of the ink gland and the opaline gland which are mixed in the mantle cavity and released toward an attacker. I show that this ink secretion protects the sea hare from predation by a fish predator. Because many deterrent compounds taste bitter, bitter taste receptors are thought to protect predators from ingesting harmful compounds in prey. Studies of deterrent taste detection have commonly utilized bitter compounds from human hedonics to study the responses in animals, such as fruit flies, fishes, rats, and monkeys. In my dissertation, I argue that the study of chemical defenses allows us to ask more questions about detection of relevant deterrents and interactions between predators and prey at the individual and population levels. My results show that diet-derived pigments in Aplysia ink, aplysioviolin and phycoerythrobilin, are strongly deterrent to fish predators. Electrophysiological analyses of the gustatory system show that these compounds are equipotent and cross-adapt each others’ responses completely. Aplysioviolin and phycoerythrobilin produced incomplete reciprocal cross-adaptation with amino acids and adapted bile salt responses but were not significantly adapted by these latter stimuli. These results showed multiple pathways that are sensitive to aplysioviolin and phycoerythrobilin, which may have different effects on the physiology and behavior of the predatory fish. My findings demonstrate the value to the fields of chemical ecology and chemosensory biology of studying sensory processing of relevant deterrent compounds. This work lays the foundation for how a diet-derived photopigment is adapted by a species to protect itself from predators by stimulating their chemosensory systems.

Made available in DSpace on 2014-06-12T15:53:07Z (GMT). No. of bitstreams: 2 arquivo5100_1.pdf: 1414667 bytes, checksum: adaa042e8a901926ad1151535e3a2641 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Uma sulfatase (EC 3.1.6.1.), heparina específica, foi identificada no fígado do molusco Aplysia cervina, largamente encontrado na costa nordestina do Brasil. Esta enzima foi purificada por precipitações sucessivas com sulfato de amônio e acetona e por cromatografia de afinidade em Heparina-Sepharose CL-6B. Algumas das propriedades físico-químicas e cinéticas desta preparação purificada 89,7 vezes (rendimento de 5,37%) foram investigadas usando o p-nitrofenil sulfato (pNFS) como substrato. Seus valores ótimos de pH e temperatura foram 5,0 e 45oC, respectivamente. Ela reteve mais de 90% de sua atividade quando incubada por 15 minutos a 45ºC enquanto que perdeu 60% a 55ºC. Seu Km foi igual a 3,71 ± 0,41 mM. Sua atividade foi estimulada por MgCl2, CaCl2 e FeCl2 e inibida por Na2S2O3, Na2SO4, KCl, C6H5Na3O7 (citrato de sódio), HgCl2, Na2HPO4 e NaH2PO4. Heparina de baixa massa molecular competiu com o pNFS pelo centro ativo da enzima mais do que a de massa molecular elevada. Esta enzima foi covalentemente imobilizada ao Dacron e a uma rede semi-interpenetrada de polisiloxano e álcool polivinílico (POS/PVA), ambos magnetizados, resultando em derivados com atividade específica e retenção de 3,17 unidades/mg proteína, 1,85 unidades/mg proteína, 36,5% e 21,23%, respectivamente. Estas preparações foram removidas facilmente da mistura de reação por um campo magnético e foram reutilizadas diversas vezes sem perda de suas atividades. Elas foram mais termoresistentes do que a enzima solúvel e apresentaram o mesmo pH ótimo, temperatura ótima e Km aparente. O derivado sintetizado com o Dacron ferromagnético apresentou uma vida útil mais elevada do que aquele em POS/PVA. O MgCl2, CaCl2 e EDTA ativaram ambos os derivados de sulfatase insolúveis em água enquanto que Na2HPO4 e NaH2PO4 e a heparina inibiram. A ação pelos outros íons variou de acordo com o suporte

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No description available.