A (Na+,K+)-ATPase é uma proteína integral da membrana plasmática que está sujeita a uma complexa regulação. Na fauna dos manguezais, dentre os crustáceos se destaca o caranguejo Cardisoma guanhumi (Latreille, 1825), um crustáceo decápode que desempenha um papel significativo na dinâmica deste ecossistema, considerado relevante recurso pesqueiro. Este estudo fornece o efeito das poliaminas, das enzimas do estresse oxidativo, da toxidade do amônio, e também investiga atividade K+-fosfatase e atividade (Na+,K+)-ATPase por estimulação sinérgica de K+/ NH4+ e NH4+/K+ na fração microsomal de brânquias do guaiamum. A atividade K+-fosfatase e a atividade (Na+,K+)-ATPase foram determinadas continuamente, a 25°C, em um espectrofotômetro Shimadzu U1800 equipado com células termostatizadas. Todos os experimentos foram feitos em duplicata utilizando-se pelo menos três preparações diferentes (N 3). A atividade PNFFase insensível à ouabaína representa 40% da atividade PNFFase total, e valor do KI foi de 370,0 18,5mol L-1. A atividade específica máxima estimada foi de 29,30 ± 1,46 nmol Pi min-1 mg-1 e o KM = 2,90 ± 0,14 mmol L-1. Por outro lado, a utilização do substrato fisiológico (ATP) permitiu a determinação de parâmetros cinéticos da atividade (Na+,K+)-ATPase em relação aos moduladores ATP, potássio, sódio, magnésio, amônio e, ouabaína. A atividade ATPase total na fração microsomal do tecido branquial de C. guanhumi recém-capturado (16 S) foi aproximadamente 166 nmol Pi min-1 mg-1 e uma atividade ATPase insensível à ouabaína de 26,55 nmol Pi min-1 mg-1, enquanto que aclimatado a 22 S a atividade ATPase total foi de 303,28 ± 15,16 nmol Pi min-1 mg-1 e a atividade insensível à ouabaína de 68,60 ± 3,43 nmol Pi min-1 mg-1. A (Na+,K+)-ATPase presente nessas duas preparações, não apresentam uma estimulação sinergística por K+ e NH4+. Houve alterações na afinidade da enzima para o ATP nas três diferentes concentrações de NH4Cl (120 mg/L; 240 mg/L; 360 mg/L) em comparação com o controle sem NH4Cl (KM= 0,1 ± 0,005 mmol L-1).Não foram observados efeitos significativos utilizando aminas biogênicas. Nossas análises mostraram também que as enzimas do estresse oxidativo estão atuando nestas diferentes preparações para combater os oxirradicais. Análise por Western blotting com anticorpo monoclonal revelou a presença de uma banda correspondente a subunidade da (Na+,K+)-ATPase com massa molecular 110 kDa. A imunolocalização mostrou que a subunidade da (Na+,K+)-ATPase encontra-se predominantemente distribuída por todo o citoplasma das células pilares branquiais, incluindo a região apical abaixo da cutícula. Identificamos o gene constitutivo da sequência parcial de nucleotídeos do cDNA da proteína ribosomal L10 (PRL10) das brânquias deCardisoma guanhumi. O estudo demonstrou que a (Na+,K+)-ATPase constitui um importante regulador da osmorregulação nesta espécie, contribuindo para um melhor entendimento dos papéis exercidos por essa enzima nos processos de osmorregulação e excreção de amônia nos crustáceos. / The (Na+,K+)-ATPase is an integral plasma membrane protein that is subject to complex regulation. In the mangrove fauna, the crustaceans include Cardisoma guanhumi crab (Latreille, 1825), a decapod crustacean that plays a significant role in the dynamics of this ecosystem, considered a relevant fishing resource. This study provides the effect of polyamines, oxidative stress enzymes, ammonium toxicity, and also investigates K+-phosphatase activity and (Na+,K+)-ATPase activity by synergistic K+/NH4+ and NH4+/ K+ stimulation in the microsomal fraction of guaiamum gills. The K+-phosphatase activity and (Na+,K+)-ATPase activity were determined continuously at 25°C on a Shimadzu U1800 spectrophotometer equipped with thermostated cells. All experiments were done in duplicate using at least three different preparations (N 3). The PNFFase activity insensitive to ouabain represents 40% of the total PNFFase activity, and KI value was 370,0 18,5mol L-1. The maximum specific activity estimated was 29.30 ± 1.46 nmol Pi min-1 mg-1 and KM = 2.90 ± 0.14 mmol L-1. On the other hand, the use of the physiological substrate (ATP) allowed the determination of kinetic parameters of the activity (Na+,K+)-ATPase in relation to the modulators ATP, potassium, sodium, magnesium, ammonium and ouabain.The total ATPase activity in the microsomal fraction of freshly caught C. guanhumi (16 S) gill tissue was approximately 166 nmol Pi min-1 mg-1 and a 26.55 nmol Pi min-1 mg-1 ouabain ATPase activity, while acclimated at 22 S the total ATPase activity was 303.28 ± 15.16 nmol Pi min-1 mg-1 and the ouabain insensitive activity of 68.60 ± 3.43 nmol Pi min-1 mg-1. The (Na+,K+)-ATPase present in these two preparations, do not present a synergistic stimulation by K+ and NH4+. There were changes in the enzyme affinity for ATP at the three different concentrations of NH4Cl (120 mg / L, 240 mg / L, 360 mg / L) compared to the control without NH4Cl (KM = 0.1 ± 0.005 mmol L-1). No significant effects were observed using biogenic amines. No significant effects were observed using biogenic amines. Our analyzes have also shown that oxidative stress enzymes are acting in these different preparations to combat oxirradicals. Analysis by Western blotting with monoclonal antibody revealed the presence of a band corresponding to sub subunit of (Na+,K+)-ATPase with molecular mass 110 kDa. Immunolocalization showed that the (Na+,K+)-ATPase sub subunit is predominantly distributed throughout the cytoplasm of the gill pillars, including the apical region below the cuticle. We identified the constitutive gene of the nucleotide partial sequence of the cDNA of ribosomal protein L10 (PRL10) of the gills of Cardisoma guanhumi. The study demonstrated that (Na+,K+)-ATPase is an important regulator of osmoregulation in this species, contributing to a better understanding of the roles played by this enzyme in the processes of osmoregulation and excretion of ammonia in crustaceans.

A digestão nos insetos ocorre no intestino médio de forma compartimentada. A digestão inicial dos polímeros ocorre no interior da membrana peritrófica. Os oligômeros resultantes difundem-se para o espaço luminal exterior à membrana peritrófica onde são atacados por outras enzimas. Na digestão final os dímeros resultantes são hidrolisados por enzimas imobilizadas na superfície do epitélio do intestino médio. Após o processo de digestão final os monômeros são absorvidos pelas células do epitélio intestinal. Os Díptera ditos superiores, incluindo a mosca doméstica, apresentam peculiaridades digestivas que aparentemente resultam de adaptações para digerir uma dieta que consiste principalmente de bactérias. No ventrículo anterior ocorre uma diminuição no conteúdo de amido do bolo alimentar. Na porção seguinte, o bolo alimentar passa para o ventrículo médio onde as bactérias são mortas pela ação combinada de baixo pH, uma lisozima digestiva e uma proteinase tipo catepsina D. O material liberado das bactérias é digerido no ventrículo posterior, como ocorre no ventrículo inteiro da maioria dos insetos de outros grupos taxonômicos. Com o objetivo de compreender a peculiar digestão em Musca domestica, foram utilizadas suas larvas para identificar funcionalmente as regiões absortivas de nutrientes, identificar as moléculas envolvidas na absorção de nutrientes, identificar as moléculas envolvidas com tamponamento e fluxos de fluidos intestinais, sequenciar as enzimas digestivas principais e identificar os seus sítios de secreção. Experimentos fisiológicos de absorção de glicose e análises de atividade enzimática permitiram acessar de maneira direta os aspectos da digestão. Contudo, experimentos de sequenciamento de bibliotecas de cDNA, análise de sequências transcritas e verificação de expressão de genes em diferentes tecidos foram abordagens fundamentais na identificação das moléculas subjacentes aos processos fisiológicos intestinas de Musca domestica. Os indícios de que absorção de glicose no intestino de Musca domestica se dê por transportadores do tipo SGLT, com a possível participação de facilitadores do tipo GLUT, permitem estabelecer um foco para futuros estudos. A descrição de sequências relacionadas ao tamponamento intestinal permitiu ampliar a discussão sobre tal processo. Ao detalhar os sítios de expressão da subunidade a da V-ATPase, do canal de cloreto e do transportador de amônia foi possível testar o modelo de tamponamento proposto anteriormente e propor a participação de outras moléculas no processo. Sequências correspondentes as atividades de carboxipeptidase, maltase e aminopeptidase descritas na literatura foram pesquisadas, gerando sequências candidatas a codificarem as referidas enzimas. Com isso, é possível descrever a digestão de oligômeros e dímeros com base nos genes transcritos e nas sequências de aminoácidos que formam as enzimas digestivas. A descoberta da sequência que transcreve uma metaloproteinase, por sua vez, abre caminhos para a descrição e caracterização de sua atividade proteolítica nos tecidos digestivos da larva de Musca domestica. Essa análise permitiu também elucidar a localização dos sítios de expressão e, portanto, as zonas de secreção de enzimas. De maneira geral, este estudo contribuiu para a compreensão de diversos aspectos da digestão de Musca domestica, elucidando questões da particular fisiologia digestiva desse inseto. / Digestion in insects occurs in the midgut in a compartmentalized way. Initial digestion takes place inside the peritrophic membrane. The resulting oligomers diffuse into the luminal space outside the peritrophic membrane where they are hydrolyzed by other enzymes. In the final digestion, the resulting dimers are hydrolyzed by enzymes immobilized on the midgut epithelium. After the final digestion, the monomers are absorbed by intestinal epithelial cells. The so-called higher Diptera, including the house fly, have digestive peculiarities apparently resulting of adaptations to digest a diet consisting mainly of bacteria. In the anterior midgut there is a decrease in the starch content of the food bolus. The bolus now passes into the middle midgut, where bacteria are killed by the combined action of low pH, a special lysozyme and a cathepsin D-like proteinase. Finally, the material released by bacteria is digested in the posterior midgut, as is observed in the whole midgut of insects of other taxa. In order to understand the peculiar digestion in Musca domestica, the larvae were used to identify (a) the functionally the nutrient absorptive regions, (b) the molecules involved in the absorption of nutrients, (c) the molecules involved in buffering and fluid flows, (d) the cDNA sequences corresponding to intestinal digestive enzymes, (e) the main sites of secretion. Physiological experiments of glucose absorption and enzyme activity analysis allowed a direct access to aspects of digestion. Otherwise, cDNA library sequencing followed by sequence annotation and tissue-specific expression analysis were fundamental approaches in the understanding of intestinal physiology of Musca domestica. Evidence that glucose absorption in the gut of Musca domestica occurs through SGLT-like transporters, with the possible participation of facilitators GLUT-like, allowed us to establish a focus for future studies. The description of cDNA sequences corresponding to proteins putatively responsible for intestinal buffering widened the discussion of this process. The finding of the expression sites of V-ATPase subunits, chloride channel, and ammonia transporter led to revising the present buffering model and the inclusion of other molecules in the process. The cDNA sequences corresponding to the activities of carboxypeptidase, aminopeptidase and maltase described in the literature were searched for as candidate sequences to encode those enzymes. This made it possible to describe the digestion of oligomers and dimers based on transcribed genes and enzyme amino acid sequences. The discovery of the metalloproteinase transcribing sequence opened a new research line: the description and characterization of its proteolytic activity in the midgut of the Musca domestica larvae. This study also allowed elucidating the location of digestive enzyme expression sites and, therefore, the putative zones of enzyme secretion. Overall, this study contributed to understanding many aspects of digestion of Musca domestica, clarifying aspects of the peculiar digestive physiology of this insect.

O Complexo de pré-replicação em T.brucei é composto por Orc1/Cdc6 e as helicases MCMs. Em um trabalho anterior mostramos que TbOrc1/Cdc6 pode ligar e hidrolisar ATP in vitro. Neste sentido, o objetivo deste trabalho é avaliar a importância da hidrólise e ligação de ATP para a formação e estabilidade do complexo pré-replicação de T.brucei. Para tanto, foram geradas proteínas recombinantes Orc1/Cdc6 de T. brucei mutadas nas regiões Walker A (TbOrc1/Cdc6K79T) ou sensor 2 (TbOrc1/Cdc6R251,252E) incapazes de ligar ou hidrolisar ATP, respectivamente. Finalmente, as células expressando TbOrc1/Cdc6K79T ou TbOrc1/Cdc6R251,252E foram avaliadas quanto a (i ) estabilidade da interação Orc1/Cdc6 -DNA, (ii) capacidade de estabilizar MCM no DNA, (iii) capacidade de replicar seu DNA. A mutação na região sensor 2 de T.brucei (TbOrc1/Cdc6R251,252E) reduziu drasticamente a atividade de ATPase em comparação com a proteína selvagem . TbOrc1/Cdc6 mutado no sitio de ligação ao ATP perdeu a capacidade de interagir com o ATP (TbOrc1/Cdc6K79T). A super expressão desses genes inibiu de forma significativa a proliferação celular, causou ineficiência no carregamento de MCM para o DNA e ocasionou falhas na progressão do ciclo celular, atrasando a fase S. / The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S.

O presente estudo indica que o pHi basal do segmento S3 do túbulo proximal é 7.10 ? 0.007 (n = 444/2117), sendo a extrusão celular de H+ feita pelo trocador Na+/H+ (marjoritariamante) e pela H+-ATPase. Nossos resultados sugerem um papel do cálcio citosólico na regulação do processo de recuperação do pHi após carga ácida, mediada pelo trocador Na+/H+ e pela H+-ATPase. O trocador é estimulado por Aldosterona (10-12, 10-10 e 10-8 M) e inibido por Aldosterona (10-6 M) via ação genômica e não-genômica. Esses resultados são compatíveis com a estimulação do trocador por moderado aumento da [Ca2+]i citosólico (com Aldosterona 10-12 M) e sua inibição por pronunciado aumento da [Ca2+]i (com Aldosterona 10-6 M). A H+-ATPase é estimulada por Aldosterona em todas as doses utilizadas via ação genômica e não-genômica e esses resultados são coincidentes com um aumento da [Ca2+]i, dose dependente. Esses nossos achados são também compatíveis com a demonstração de uma ação hormonal não-genômica (após 1 ou 15 min) e genômica (após 1 hora) na [Ca2+]i, no trocador e na H+-ATPase. Adicionalmente, nossos resultados indicando que os efeitos hormonais genômicos ocorrem via receptor MR são coincidentes com nossos dados demonstrando a expressão desses receptores no segmento S3. Esses efeitos da Aldosterona que acabamos de descrever podem representar uma regulação fisiológica relevante, em condições de depleção e expansão de volume no animal intacto. / The present study indicate that the basal pHi of proximal S3 segment is 7.10 ? 0.007 (n = 444/2117), being made the extrusion by of Na+/H+ exchanger (mainly) and H+-ATPase. Our results suggest a role for cell calcium in regulating the process of pHi recovery after the acid load induced by NH4Cl, mostly mediated by a basolateral Na+/H+ exchanger, and stimulated by Aldosterone (10-12, 10-10 e 10-8 M) and impaired by Aldosterone (10-6 M) via a genomic and nongenomic action. They are compatible with stimulation of the Na+/H+ exchanger by increases in cell calcium in the lower range (at Aldosterone 10-12 M) and inhibition at high cell calcium levels (at Aldosterone 10-6 M). The H+-ATPase is stimulated in all the used doses via a genomic and nongenomic action, this is coincident with the dose-dependent increase in [Ca2+]i. This finding is also compatible with the demonstration of a hormonal nongenomic (after 1 min or 15 min) and genomic (after 1 h) action on [Ca2+]i, on the Na+/H+ exchanger and on H+-ATPase. Our results indicating that the genomics effects are via MR receptor are in accordanc with our finding showing expression of the receptors in the proximal S3 segment of rat. These Aldosterone effects may represent physiologically relevant regulation in conditions of volume depletion or expansion in the intact animal.

Os conhecimentos sobre a regulação dos níveis de cálcio e manganês no Aspergillus fumigatus são bastante limitados, sendo que a homeostase destes íons pode ser diretamente controlada pela ação de ATPases específicas, dentre elas as cálcio ATPases da subfamília PMR1. Desta forma, o objetivo do presente estudo foi a expressão, caracterização e validação como alvo quimioterapêutico do gene Afpmr1 de A. fumigatus. Inicialmente, foi realizada a complementação funcional, de uma cepa de S. cerevisiae nocaute para a PMR1, em meios de cultura suplementados com EGTA ou manganês, revertendo o fenótipo da cepa nocute. Além disto, após expressão do gene Afpmr1, foi verificada uma reversão na intensa distribuição de quitina na parede celular da cepa nocaute. Paralelamente, para a RNAi, um fragmento do gene Afpmr1 apresentando baixa identidade com outros genes de cálcio ATPases de diferentes espécies foi clonado em vetor de expressão em A. fumigatus (pALB1). Após indução da expressão, a construção de RNA dupla fita para RNAi silenciou tanto o gene alb1 isoladamente (clone controle), quanto o duplo silenciamento com o gene de interesse Afpmr1, conferindo à ambas construções coloração branca às colônias. Uma vez confirmado o silenciamento gênico, por técnicas de RT-PCR quantitativo, os clones selecionados foram utilizados em ensaios de fagocitose e killing de macrófagos. O clone com o gene Afpmr1 silenciado apresentou diminuição na porcentagem de fagocitose, no número médio de conídios fagocitados e na eficiência de eliminação destes conídios quando comparados com seus controles. Estes resultados mostram que o gene Afpmr1 pode ser expresso funcionalmente em sistemas heterólogos e seu silenciamento, em A. fumigatus, influencia processos celulares que podem estar relacionados à manutenção da estrutura e composição da parede celular, além de desencadear alterações na fagocitose e killing de macrófagos. / The knowledge about the regulation of Aspergillus fumigatus calcium and manganese levels are very limited, while these ions homeostasis could be directly controlled by the function of specific ATPases, like the PMR1 calcium ATPase. In this way, the aim of the present work was the expression, characterization e validation, as chemotherapeutic target, of the A. fumigatus Afpmr1 gene. Initially, the functional complementation of a PMR1 knock-out strain phenotype was analyzed in EGTA or manganese supplemented culture media. Besides, after Afpmr1 expression, an intense distribution of chitin through the cell wall of the knock-out strain was reversed. At the same time, a fragment of the Afpmr1 gene, showing low identity values for another calcium ATPase genes, was cloned in an A. fumigatus expression vector (pALB1) for RNAi. After the induction of gene expression, a double strand RNA construct for RNAi has properly silenced either the alb1 gene alone (control clone), or the double silencing with the gene of interest Afpmr1, leading to both constructions white colored colonies. After confirmation of the gene silencing by quantitative RT-PCR techniques, the selected clones were used in macrophages killing and phagocytosis assays. The Afpmr1 silenced clone showed a decrease in the phagocytosis percentage, in the mean number of internalized conidia and in the killing percentage when compared with control groups. These results show that the Afpmr1 gene can be functionally expressed in eukaryotic heterologous systems and its silencing, in A. fumigatus, alters cellular processes that can be related with the maintenance of the cell wall structure and composition, as well as promote alterations in the macrophages phagocytosis and killing.

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial do sistema de regulação iônica e osmótica e de excreção ativa de NH4 +. Dessa forma, a caracterização cinética da (Na+,K+)-ATPase branquial de Clibanarius vittatus pode fornecer dados importantes para a compreensão dos mecanismos bioquímicos desses processos. Nesse sentido, foram realizados ensaios cinéticos com a fração microsomal obtida do tecido branquial de C. vittatus. Os dados obtidos revelaram a presença de um único pico com atividade ATPase. Os dados obtidos a partir da hidrólise do substrato ATP revelaram a presença de dois sítios, um de alta afinidade que apresenta interação sítio-sítio (nH=1,9; K0,5= 63,8 ± 2,9 nM e V= 19,1 ± 0,8 U/mg) e outro de baixa afinidade, com características Michaelianas (nH=1,0; KM= 44,1 ± 2,6 mM e VM= 123,5 ± 6,1 U/mg). Além disso, foi observada interação do tipo sítio-sítio (nH= 1,8) para estimulação dos íons magnésio (V= 132,0 ± 5,3 U/mg e K0,5= 0,36 ± 0,02 mM). Por outro lado, as interações dos íons sódio (K0,5= 7,4 ± 0,4 mM), potássio (K0,5= 1,51 ± 0,05 mM) e amônio (K0,5= 4,5 ± 0,2 mM) obedeceram a uma cinética Michaeliana. Os dados obtidos mostraram que em condições saturantes de íons potássio, a atividade enzimática aumenta de maneira concentração-dependente quando na presença de íons amônio (V= 290,8 ± 14,4 U/mg e KM= 2,9 ± 0,1 mM). A presença da ouabaína no meio reacional acarretou 66% de inibição (KI= 117,3 ± 3,5 mM), indicando a presença de outras ATPases. A atividade K+-fosfatase, medida usando o substrato sintético PNFF revelou a presença de um único sítio de hidrólise (V= 47,4 ± 1,9 U/mg e KM= 1,0 ± 0,04 mM) com características Michaelianas (nH= 1,1). Entretanto, para os íons Mg2+ (nH= 2,6 e K0,5= 0,6 ± 0,1 mM), K+ (nH= 1,4 e K0,5= 3,7 ± 0,1 mM) e NH4 + (nH= 1,9 e K0,5= 19,3 ± 0,7 mM) foram observadas interações sítio-sítio. Em conjunto, os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de C. vittatus, reforçam a possibilidade de que essa enzima contribui eficientemente com o mecanismo adaptativo desencadeado em resposta à exposição a altas concentrações externas de amônia. É importante ressaltar que o presente trabalho representa o primeiro estudo de caracterização cinética da enzima (Na+,K+)- ATPase no ermitão C. vittatus, um animal inserido em um microambiente constituído pela concha. Finalmente, este trabalho agrega informações relevantes relacionadas ao grupo dos Decapoda, em relação ao papel da (Na+,K+)-ATPase nos processos metabólicos e de osmorregulação nos crustáceos. / (Na+,K+)-ATPase is an essential component of osmotic and iono-regulatory system of crustacean gill and plays a relevant role in NH4 + excretion. Thus, the kinetic characterization of Clibanarius vittatus gill microsomal (Na+,K+)-ATPase, can provide crucial data to the understanding of osmoregulating and excretion processes of these animals in different environments, especially the shell microenvironment. In this way, kinetic assays were performed with gill microsomal fraction. The results showed a single protein peak with ATPase activity. The results from ATP hydrolysis revealed the presence of two different sites. One of them is related to the high-affinity with site-site interaction (nH=1.9; K0.5= 63.8 ± 2.9 nM and V= 19.1 ± 0.8 U/mg) and the other shows low-affinity, obeying Michaelis-Menten kinetics (nH=1.0; KM= 44.1 ± 2.6 mM and VM= 123.5 ± 6.1 U/mg). Moreover, site-site interaction (nH= 1.8) was observed to the magnesium ions stimulation (V= 132.0 ± 5.3 U/mg and K0.5= 0.36 ± 0.02 mM). On the other hand, the stimulations of Na+ (K0.5= 7.4 ± 0.4 mM), K+ (K0.5= 1.51 ± 0.05 mM) and NH4 + (K0.5= 4.5 ± 0.2 mM), followed Michaelis-Menten kinetics. At saturating concentrations of potassium ions, the enzyme activity was increased in a dependent-concentration manner with increasing ammonium ions concentration (V= 290.8 ± 14.4 U/mg and KM= 2.9 ± 0.1 mM). Ouabain inhibited 66% of (Na+,K+)-ATPase activity (KI= 117.3 ± 3.5 mM) of the gill microsomal enzyme, indicating the presence of other ATPase activities. The K+-fosfatase activity was also measured using PNPP, a synthetic substrate. PNPP hydrolysis resulted in single site hydrolysis site, obeying the Michaelis-Menten kinetics (nH= 1.1). However, it was observed site-site interactions for Mg2+ (nH= 2.6; K0.5= 0.6 ± 0.1 mM), K+ (nH= 1.4; K0.5= 3.7 ± 0.1 mM) and NH4 + (nH= 1.9; K0.5= 19.3 ± 0.7 mM) ion estimulations. Taken together, these results provide important informations on the kinetic properties of gill (Na+,K+)-ATPase of C. vittatus, emphasizing the synergiistic stimulation induced by K+ and NH4 +, which appear to contribute to the efficient adaptive mechanism triggered by exposure of high external ammonia concentrations. It is important to emphasize that this is the first study on the kinetic properties of (Na+,K+)-ATPase enzyme of the hermit crab C. vittatus. Finally, this study provide information concerning the (Na+,K+)-ATPase role in the metabolic and osmoregulating mechanisms of this Decapoda group

A caracterização bioquímica da (Na+,K+)-ATPase, uma importante enzima envolvida no controle osmo-iônico nos crustáceos osmorreguladores, foi realizada a partir de centrifugação diferencial de frações microsomais do tecido branquial do siri eurialino C. danae, coletado na Baía de Ubatuba e mantido a 33o/oo de salinidade (animais recém-capturados). A ultracentrifugação da fração microsomal em um gradiente contínuo de sacarose (10-50%) revelou a presença de um único pico de atividade (Na+, K+)-ATPase, coincidente com o pico de atividade K+-fosfatase. Ambas as atividades foram inibidas completamente pela ouabaína. O Western blotting da fração microsomal apresentou uma única banda imunoespecífica contra a subunidade alfa da (Na+, K+)-ATPase, sugerindo a presença de uma única isoforma para a cadeia alfa da enzima. A hidrólise do ATP ocorreu em sítios de alta afinidade que apresentaram interações sítio-sítio (nH=3,6) com uma atividade específica V= 35,4 ± 2,1 U/mg e K0,5= 54,0 ± 4,0 nM, bem como em sítios de baixa afinidade, que obedeceram uma cinética Michaeliana, com V= 271,5 ± 17,2 U/mg e KM = 55,0 ± 3,0 uM. A estimulação da atividade da enzima pelos íons Na+ (V= 302,1 ± 14,1 U/mg e K0,5= 5,80 ± 0,3 mM), Mg2+ (V= 309,7 ± 15,7 U/mg e K0,5= 0,48 ± 0,02 mM) e K+ (V= 294,0 ± 11,8 U/mg e K0,5= 1,61 ± 0,06 mM) ocorreu através de interações sítio-sítio, enquanto a estimulação pelos íons NH4+ obedeceu a uma cinética Michaeliana com V= 377,8 ± 22,7 U/mg e KM= 4,61 ± 0,27 mM). Interessantemente, os íons NH4+ estimularam sinergisticamente a atividade específica da enzima em cerca de 90% (V= 557,0 ± 28,3 U/mg), sugerindo que esses íons se ligam em diferentes sítios na molécula. A (Na+,K+)-ATPase do tecido branquial de C. danae hidrolisou o PNFF com V= 125,4 ± 7,5 U/mg, K0,5= 1,2 ± 0,1 mM, através de interações cooperativas (nH= 1,5). Além disso, essa atividade K+-fosfatase foi inibida competitivamente pelo ATP (KI= 57,2 ± 2,6 µM), sugerindo que os dois substratos foram hidrolisados no mesmo sítio da enzima. A estimulação da atividade K+-fosfatase da (Na+,K+)-ATPase pelos íons K+ (V= 121,0 ± 6,1 U/mg; K0,5= 2,1 ± 0,1 mM), Mg2+ (V= 125,3 ± 6,3 U/mg; K0,5= 1,0 ± 0,1 mM) e NH4+ (V= 126,1 ± 4,8 U/mg; K0,5= 13,7 ± 0,5 mM) ocorreram através de interações sítio-sítio, similarmente ao observado para o ATP. A ouabaína e o ortovanadato inibiram completamente a atividade (Na+,K+)-ATPase (KI= 147,2 ± 7,2 uM; KI= 11,2 ± 0,6 nM, respectivamente). Entretanto, para a atividade K+-fosfatase os valores determinados foram significativamente superiores (KI= 830,3 ± 42,5 uM; KI= 34,0 ± 1,4 nM, respectivamente). A inibição da atividade da (Na+,K+)-ATPase por essas duas substâncias foi afetada pela presença de íons NH4+. Entretanto, o mesmo não ocorreu com a atividade K+-fosfatase da enzima. A representação de Arrhenius revelou a ocorrência de uma transição de fase próximo a 19°C, com deltaH1= 15.939 cal/mol e outra a 38°C com deltaH2= 7.719 cal/mol. Temperaturas acima de 43°C provocaram uma rápida inativação da (Na+,K+)-ATPase. Esta é a primeira demonstração da presença de um sítio de alta afinidade para o ATP na (Na+,K+)-ATPase de crustáceo. Os resultados obtidos sugerem que as atividades (Na+,K+)-ATPase e K+-fosfatase pertencem à mesma enzima e que a preparação não apresenta contaminações por outras ATPases e/ou fosfatases. Do ponto de vista fisiológico, os resultados deste trabalho são relevantes em relação à excreção ativa dos íons NH4+ pelos crustáceos. / The modulation by Mg+2, Na+, K+, NH4+ ions and ATP of the (Na+, K+)-ATPase activity in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V= 35.4 ± 2.1 U/mg and K0.5= 54.0 ± 3.6 nM, obeying cooperative kinetics (nH= 3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with KM= 55.0 ± 3.0 uM and V= 271.5 ± 17.2 U/mg. This is the first demonstration of a crustacean (Na+, K+)-ATPase possessing two ATP hydrolyzing sites. Stimulation by sodium (K0.5= 5.80 ± 0.30 mM), magnesium (K0.5= 0.48 ± 0.02 mM) and potassium ions (K0.5= 1.61 ± 0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (KM= 4.61 ± 0.27 mM). Ouabain (KI= 147.2 ± 7.2 uM) and orthovanadate (KI= 11.2 ± 0.6 nM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule and that the presence of each ion modulates enzyme stimulation by the other. The kinetic properties of a microsomal gill (Na+,K+)-ATPase were also analyzed using p-nitrophenylphosphate as substrate. The (Na+,K+)-ATPase hydrolyzed the substrate obeying cooperative kinetics (n= 1.5) at rates of V= 125.4 ± 7.5 U/mg and K0.5= 1.2 ± 0.1 mM and ATP competitively inhibited K+-phosphatase activity (KI= 57.2 ± 2.6 µM). Enzyme stimulation by potassium (V= 121.0 ± 6.1 U/mg; K0.5= 2.1 ± 0.1 mM) and magnesium ions (V= 125.3 ± 6.3 U/mg; K0.5= 1.0 ± 0.1 mM) was cooperative. Ammonium ions stimulated the enzyme through site-site interactions to a rate of V= 126.1 ± 4.8 U/mg with K0.5= 13.7 ± 0.5 mM. However, the K+-phosphatase activity was not synergistically stimulated using potassium plus ammonium ions. Sodium ions (KI= 36.7 ± 1.7 mM), ouabain (KI= 830.3 ± 42.5 uM) and orthovanadate (KI= 34.0 ± 1.4 nM) completely inhibited K+-phosphatase activity. The data show that the K+-phosphatase activity corresponds strictly to the (Na+,K+)-ATPase. This is the first invertebrate (Na+,K+)-ATPase shown to exhibit both high- and low-affinity sites for ATP hydrolysis and synergistic stimulation by potassium and ammonium ions (Masui et al., 2002). Further characterization of the K+-phosphatase activity will reveal its specific kinetic characteristics and may become a useful tool in comparative osmoregulatory studies.

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O influxo de K+ na faixa de alta afinidade à controlado por transportadores dos tipos canal e carreador, os quais diferem na sua sensibilidade ao Ãon NH4 +. A atividade de ambos os componentes, nÃo-sensÃvel (canal) e sensÃvel (carreador) ao Ãon NH4 + dependem da enzima de membrana plasmÃtica, H+-ATPase. Objetivou-se a caracterizaÃÃo fisiolÃgica e molecular da absorÃÃo de K+ de alta afinidade em sorgo forrageiro [Sorghum bicolor (L.) Moench] sob a influÃncia do estado nutricional da planta e de diferentes fontes de nitrogÃnio inorgÃnico na soluÃÃo de crescimento. Sementes do genÃtipo CSF20 foram germinadas e cultivadas em soluÃÃes nutritivas contendo dois nÃveis de K+ (0,2 e 1,4 mM) e trÃs diferentes regimes de nitrogÃnio inorgÃnico (NO3 - e NH4 + a 4 mM, e a mistura NO3 -/NH4 +, ambos a 2 mM). ApÃs 15 dias de cultivo (t0) em soluÃÃes nutritivas completas, as plantas foram submetidas à deficiÃncia de K+ por 1 (t1), 2 (t2) e 3 (t3) dias. No t0, os teores de K+ da parte aÃrea foram reduzidos ignificativamente pela presenÃa do Ãon NH4 +, enquanto que, nas raÃzes a reduÃÃo foi significativa apenas nos cultivos com 0,2 mM de K+. Conforme o aumento do perÃodo de deficiÃncia, os teores de K+ nas raÃzes e na parte aÃrea tenderam a diminuir em razÃo da diluiÃÃo provocada pelo crescimento da planta. Em plantas cultivadas com 0,2 mM de K+ e com NO3 - e NO3 -/NH4 +, as eficiÃncias de absorÃÃo de K+ foram similares. Entretanto, a presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio, afetou severamente esse parÃmetro. O efeito do Ãon NH4 + na eficiÃncia de absorÃÃo foi mais ameno quando as plantas foram cultivadas a 1,4 mM de K+. A eficiÃncia de transporte de K+ nÃo diferiu em nenhum dos tratamentos testados. As curvas de depleÃÃo de K+ mostraram que as plantas cultivadas com NO3 -/NH4 + e NH4 + apresentaram maior capacidade para esgotar o K+ (100 μM) da soluÃÃo de depleÃÃo quando comparadas Ãquelas crescidas com NO3 -. No t2, maiores taxas de Imax foram observadas nas plantas cultivadas com NH4 + como Ãnica fonte de nitrogÃnio. O Km para o K+, das plantas provindas dos cultivos com 0,2 e 1,4 mM de K+, apresentou valores menores nas plantas tratadas com NH4 + e NO3 -/NH4 +. A induÃÃo da absorÃÃo de alta afinidade de K+ foi influenciada pelo conteÃdo de K+ dos tecidos aÃreos. Ensaios com inibidores mostraram que o influxo de K+ foi significativamente inibido pelo Ãon NH4 + a 1 mM em plantas cultivadas com NO3 - como Ãnica fonte de nitrogÃnio. Entretanto, a capacidade de absorÃÃo de K+ foi reduzida pela presenÃa de TEA nas plantas cultivadas em soluÃÃes contendo o Ãon NH4 + como Ãnica fonte de nitrogÃnio. Esses resultados sugerem que em plantas de sorgo sob deficiÃncia de K+ e na presenÃa do Ãon NH4 + no meio de crescimento, canais de K+ podem contribuir significativamente para o influxo de K+. Por outro lado, na ausÃncia do Ãon NH4 + durante o crescimento, um sistema de transporte mediado por carreadores de K+ seria a via principal para o influxo desse nutriente. A atividade de transporte de H+ da H+-ATPase de membrana plasmÃtica isolada de raÃzes mostrou que a deficiÃncia de K+ (t2) estimulou a capacidade de formaÃÃo do gradiente de H+ em presenÃa do Ãon NH4 + nas plantas provindas dos cultivos com 1,4 mM de K+, enquanto que naquelas provindas dos cultivos com 0,2 mM de K+, o Ãon NH4 + teve efeito na velocidade inicial do transporte de H+ e na hidrÃlise do ATP. A deficiÃncia de K+ na presenÃa do NO3 - nÃo estimulou as atividades da bomba de 11 H+. ImunodetecÃÃo com anticorpos especÃficos contra H+-ATPases de membrana plasmÃtica de plantas mostrou a induÃÃo de duas isoformas nas membranas plasmÃticas oriundas de plantas cultivadas com 0,2 mM de K+, independentemente da fonte de nitrogÃnio e dos perÃodos de deficiÃncia. SeqÃencias gÃnicas correspondentes a genes de H+-ATPases de membrana plasmÃtica (SBA1 e SBA2), canais (SbAKT1) e carreadores de K+ (SbHAK1) foram selecionadas no genoma do sorgo e seus nÃveis de expressÃo em raÃzes analisados por PCR em tempo real. Os genes SBA1 e SBA2 pertencem, respectivamente, aos grupos II e I da famÃlia das H+-ATPases. Em raÃzes provindas dos cultivos com 0,2 mM de K+ e na presenÃa do Ãon NH4 + os nÃveis dos transcritos de SBA1 e SBA2 foram significativamente expressos a partir do tempo t2 de deficiÃncia de K+, enquanto que na presenÃa do Ãon NO3 - eles foram reduzidos conforme o aumento do tempo de deficiÃncia. Na dose mais alta de K+, os transcritos de SBA1 tiveram sua expressÃo incrementada pela deficiÃncia de K+ em presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio. Ambos os genes tiveram um incremento transitÃrio dos nÃveis dos transcritos no t1 de deficiÃncia de K+ na presenÃa do Ãon NO3 -. Transcritos dos genes SbAKT1 e SbHAK1 nÃo foram detectados. AnÃlises filogenÃticas mostraram que SbAKT à um canal de K+ da famÃlia Shaker, compartilhando origem evolutiva comum com vÃrios canais de K+ de gramÃneas. Os resultados sugerem que a homeostase iÃnica do K+ à alterada pelo Ãon NH4 + em plantas de sorgo. No entanto, a adaptaÃÃo das plantas à presenÃa do Ãon NH4 + envolve a induÃÃo de um sistema altamente eficiente para a aquisiÃÃo de K+, com a participaÃÃo de canais de K+ e da H+-ATPase de membrana plasmÃtica / K+ influx in the range of high affinity is mediated by K+ carriers and channels, which can be distinguished by its differential sensibility to NH4+. The activity of the NH4+-sensitive component (carrier) and NH4+-insensitive component (channel) depend upon plasma membrane H+-ATPase. This work aimed the physiological and molecular characterization of system mediating K+ uptake in the high-affinity range of concentration in sorghum [(Sorghum bicolor (L.) Moench)]. The effect of K+ starvation and nitrogen inorganic source of the growth solution on high-affinity K+ uptake was also studied. Seeds of sorghum, genotype CSF20, were germinated and placed in modified one-fourth Hoagland solutions, which were formulated to contain 0,2 and 1,4 mM K+ and three nitrogen inorganic source, NO3- and NH4+ (4 mM) and NO3-/NH4+ in combination (2mM/2mM). Plants were grown for 15 days (t0) in complete nutritive solutions and then incubated in a K+-free solutions for one (t1), two (t2) and three (t3)days. At t0, K+ content of shoot was significantly decreased in plants grown in the presence of NH4+. In roots, the presence of NH4+ did altered the K+ content only of plants cultivated in solutions with lowest K+ concentration. The K+ content of the plant tissues was progressively reduced according to increasing of starvation period and increasing of dry matter (dilution). The highest reductions were observed in K+ content of the shoot. At the lowest level of K+ (0,2 mM), plants grown in solutions containing NO3- and NO3-/NH4+ showed similar uptake efficiency of K+. Whereas, the presence of NH4 as sole nitrogen source, reduced severely the absorption rates of K+. The effect of NH4+ on uptake efficiency of K+ was alleviated by increasing the external K+ concentration to 1,4 mM K+. At both levels of K+, the translocation rate of K+ was not altered by the presence of NH4 + and by K+ starvation. Assays of depletion in the external medium of K+ (100 μM) showed that plants growing in the presence of NO3-/NH4+ and NH4+ were more efficient to deplete external K+ than plants grown with NO3- , as sole nitrogen source. Kinetic parameters were significantly different at second day (t2) of deprivation. In the presence of NH4 +, as sole nitrogen source, Imax values were higher than those of plants grown in NO3-/NH4 + and NO3 -. On the other hand, Km values were lower in plants from solutions containing NH4+ and NO3 -/NH4 + than those cultivated in NO3-, as sole nitrogen source. High-affinity uptake of K+ responded to changes in plant K+ status, mainly to K+ content of aerial parts. K+ uptake by plants growing in NO3- was significantly inhibited by inclusion of NH4 at 1 mM in depletion solution, whereas it was not inhibited in plants grown with NH4 +, as sole nitrogen source. TEA (tetraethylammonium) inhibited the K+ influx of plants cultivated in solution containing NH4 +, as sole nitrogen source. The results suggest that factors such as presence of K+ or NH4 + during plant growth determine the relative contribution of each component to high-affinity uptake system of K+. In K+-starved plants grown in the presence of NH4 +, K+ channel could contribute to K+ uptake in the range of high-affinity concentration. On the other hand, in K+-starved plants grown without NH4 +, K+ carrier could constitute the principal route for K+ uptake. Active H+-transport driven by plasma membrane H+-ATPase of sorghum roots was stimulated by K+ deficiency and NH4 + in plants from solutions with high K+ level. However, these factors affected the initial rate 13 of H+-pumping and hydrolytic activity of ATP, but not H+-gradient formation, in plants from solutions containing 0,2 mM K+. These activities were not changed when plants were cultivated in growth solutions containing NO3 - and submitted to K+-starvation. Two specific isoforms of PM H+-ATPase by immuno-detection were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO3 --grown. cDNA sequences corresponding to plasma membrane H+-ATPase (SBA1 and SBA2), K+ channels (SbAKT) and K+ carrier (SbHAK1) from sorghum genome were identified. The expression level of these genes was analyzed by real-time PCR. SBA1 and SBA2 genes were included at subfamily II and I of plasma membrane H+-ATPase, respectively. In plants cultivated in solutions containing 0,2 mM K+ and with NH4+, the accumulation of SBA1 and SBA2 transcripts was observed after 48 h (t2) of K+ deficiency. Whereas, the expression level of these genes was reduced in plants cultivated with NO3 -. In roots came from solutions containing 1,4 mM K+ level, the accumulation of SBA1 transcripts was only observed in the presence of NH4 + and after 48 h (t2) of K+ deficiency. The transcript level of both genes increased only at t1 in roots cultivated in solution containing NO3 -, as sole nitrogen source. Transcipts of SbAKT and SbHAK1 were not detectable by real-time PCR. Phylogenetic analysis revealed that SbAKT belongs to Shaker channel family of plants and is also closely related to members of other gramineous species. The results suggest that K+ homeostasis in NH4+-grown sorghum plants may be regulated by a high capacity for K+ uptake, which is dependent upon the H+ -pumping activity of PM H+-ATPase and K+ channels

O caranguejo de mangue Ucides cordatus é um forte hiper-hipo-osmorregulador que se encontra iso-osmótico ao ambiente na salinidade próxima a 20‰. A osmorregulação é realizada por diversos órgãos, mas principalmente pelas brânquias posteriores, enquanto as brânquias anteriores relacionam-se com a respiração. A regulação iônica é feita por muitas enzimas, sendo a principal a Na+-K+-ATPase (NKA). Tal enzima pode variar sua atividade em diversas condições ambientais, como a salinidade e/ou temperatura; é alterada por características físicas das membranas, como fluidez; e também modulada por ácidos graxos de fosfolipídios de membrana encontrados nas brânquias. O objetivo deste trabalho foi elucidar a conformação dos fosfolipídios de membrana, no que tange aos ácidos graxos destes, em animais submetidos a diferentes salinidades. Os caranguejos foram divididos e submetidos a três situações (n=4 por grupo): controle /iso-osmótico (salinidade 20‰), hipo-osmótico (10‰) e hiper-osmótico (30‰). Um grupo de animais foi exposto a um período de curto prazo (6 horas) e outro grupo a um período de 120 horas. As brânquias foram removidas e as frações lipídicas extraídas para configurar o perfil de ácidos graxos. Após montado o perfil das classes dos ácidos graxos, os mesmos foram determinados por cromatografia gasosa. A atividade específica da NKA foi mensurada com base na diferença entre a taxa de liberação de fosfato a partir de ATP. Os resultados mostraram um aumento dos ácidos graxos poliinsaturados (PUFA) sob o estresse hipo-osmótico em brânquias posteriores em ambas as frações, fosfatidilcolina (FC) e fosfatidiletanolamina (FE), em animais submetidos ao tratamento de 120 horas. PUFA tende a aumentar a fluidez de membrana, portanto, nesta condição a membrana talvez estivesse sob tal estado, mais permeável. No estresse hiper-osmótico, por outro lado, o FE de brânquias posteriores mostrou um aumento dos ácidos graxos saturados em animais também expostos a 120 horas. Tais ácidos graxos fazem com que a conformação da membrana se torne menos fluida, e menos permeável a solutos. Não houve diferença no perfil de ácidos graxos para animais expostos a 6 horas em diferentes salinidades. A atividade da NKA variou entre as diferentes exposições. / The mangrove\'s crab Ucides cordatus is a good hyper-hyposmoregulator that is isosmotic in a 20‰ salinity environment. The osmoregulation is performed by many organs, however the most important structure is the posterior gills. On the other hand, anterior gills are related to breathing. Ionic regulation is performed by several pumps, primarily by Na+-K+-ATPase (NKA). This enzyme performs its role due to environmental conditions such as salinity and/or temperature, but also to membrane\'s physics characteristics such as fluidity and composition of fatty acids. The purpose of this study was to evaluate the membrane\'s conformation in relation to fatty acids of phospholipids, when acclimated to different salinities. The crabs were divided into three groups (n = 4 per group): isosmotic/control (20‰ salinity), hyposmotic (10‰ salinity) and hyperosmotic (30‰ salinity). A group of animals were exposed to a short-term period (6 hours) and another group to a long-term period (120 hours). The gills were removed and their total lipids was extracted to set up the fatty acids profile (phospholipid class). The fatty acids profile were determined as phospholipid class by gas chromatography. The specific activity of NKA was based upon measurement of the phosphate released through the breakdown of ATP. The results showed that phosphatidylethanolamine (PE) and phosphatidylcoline (PC) from posterior gills exposed to 10‰ salinity for long-term had significantly higher levels of polyunsaturated fatty acids (PUFA). PUFA tends to increase membrane\'s fluidity, thus in the hyposmotic stressful situation, the membrane\'s conformation could be more fluid. When exposed to 30‰ salinity for long term, the PE of posterior gills showed increased levels of saturated fatty acids that implies in a less fluid membrane\'s conformation. There were no significant differences among fatty acids of phospholipids in gills of crabs exposed to a short-term period (6 hours). The specific activity of NKA ranged among the different salinities.