Antiporters that exchange alkali cations (Na+ or K+) for protons play an important role in the physiology of all known bacterial species. They are involved in regulating intracellular pH and maintaining cellular volume as well as the formation of a chemical Na+ gradient across the membrane, which is important to many bacteria as an energy source for processes such as accumulation of substrates, ATP synthesis, and flagellar rotation. Another important role of cation/proton antiporters is homeostasis of intracellular cation content. The situation of a thermodynamic equilibrium of Na+ or K+ on the membrane would result in toxic intracellular levels of these cations, so bacteria have cation/proton antiporters, which expel excess of alkali cations at the expense of the proton motive force. Of many antiporters described to date, the NhaP-type family is one of the most interesting groups. Its members collectively demonstrate a great diversity of their features. There are three antiporters of NhaP type encoded in the genome of the dangerous human pathogen, Vibrio cholerae. Phenotype analysis of engineered chromosomal VcnhaP1, VcnhaP2 and VcnhaP3 deletion mutants has proven the three NhaP paralogues to be essential for V. cholerae growth at low pH in the presence of high or low concentrations of K+. Genes encoding Vc-NhaP1-3 were cloned and antiporters expressed in their functional form in an antiporter-less strains of Escherichia coli. Although initially annotated as Na+/H+ antiporters, when assayed in everted membrane vesicles, all three isoforms of Vc-NhaP were shown to be K+/H+ antiporters with only limited ability to Na+/H+ antiport. None of three proteins was able to mediate Li+/H+ exchange. Overall, the three antiporters differed in their biochemical profiles, predicted topology, and their phenotypic manifestations. / February 2016

CABRAL, P. H. B. Efeitos cardiovasculares do composto [RU(BPY)2(Tioureia)(NO)](FOR 0812) em ratos normotensos e hipertensos. 2016. 124 f. Tese (Doutorado em Farmacologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-12-20T11:41:41Z No. of bitstreams: 1 2016_tese_phbcabral.pdf: 3593366 bytes, checksum: 9c3931febf1545f3558996f1c09a3c07 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-12-20T11:44:52Z (GMT) No. of bitstreams: 1 2016_tese_phbcabral.pdf: 3593366 bytes, checksum: 9c3931febf1545f3558996f1c09a3c07 (MD5) / Made available in DSpace on 2016-12-20T11:44:52Z (GMT). No. of bitstreams: 1 2016_tese_phbcabral.pdf: 3593366 bytes, checksum: 9c3931febf1545f3558996f1c09a3c07 (MD5) Previous issue date: 2016-08-17 / Ruthenium coordinated nitric oxide donors which do not release cyanide can be less toxic models for the development of drugs to treat angina and hypertensive crisis. The compound [Ru(bpy)2(tioureia)NO(PF6)3]– FOR0812, was synthesized and probed in vitro for its potential vasodilator activity and in vivo for its antihypertensive properties. First, we tested its effects on thoracic aortic rings harvested from male Wistar rats (200-250g). The rings were mounted in 5 ml organ baths in Krebs-Henseleit warmed to 37°C and gassed with 5%CO2 in 95%O2. The tissues were coupled to isometric force transducers under a 1g passive tension and coupled to a data acquisition system and recorded by using LabChart 7.0. FOR0812 was added cummulatively (10-12 a 10-4M) in aortic rings precontracted with phenylephrine or high potassium (60mM). On the other hand, in order to test the potential antihypertensive effects of FOR0812 the drug was administred at 1mg/kg by the oral route during 15 days to 2kidney-1clip hypertensive rats (2K-1C), Dahl salt-sentitive rats (DS) and to spontaneously hypertensive rats (SHR). In addition, the acute effects of intravenous bolus injections of 0.001- 1mg/Kg FOR0812 on systemic hemodynamics and cardiac eletromechanics were also studied by using microtip pressure-volume catheters inserted in the left ventricle and electrocardiogram analysis.FOR 0812 induced a concentration-dependent relaxation of 103±2.3 % with an IC50 of 0.24 µM[0.084 – 0.72] while the positive control sodium nitroprusside had a maximal relaxant effect of 108±5.3% with an IC50 of 0.05 µM[0.025 –0.12]. 100 µM L-NAME did not affect the maximal vasorelaxation induced by FOR0812 but reduced potency by 8-fold. Nevertheless, the vasorelaxant effect of FOR0812 was completely blunted by ODQ (10µM), (*p<0.05). The potency for the vasorelaxant of FOR0812 was reduced 5.8-fold by glibenclamide, 5.4-fold by apamin-carybdotoxin and by 11-fold by tetraethylammonium. The vasorelaxant response was greatly blunted by a nitric oxide scavenger, hydroxocobalamin (FOR0812=103±2.3 % vs. FOR0812+hydroxocobalamin=32.4%, *p<0.05). However, the removal of nitroxyl anions (NO-) with 3 mM L-cystein, did not affect the vasorelaxant effect of FOR0812. Similarly, the blockade of RhoA/Rho kinase (ROCK) with fasudil (HA-1077) did not affect the vasorelaxant effect of FOR0812. The blockade of prostaglandin synthesis with indomethacin did not affect the maximal vasorelaxation incuced by FOR0812 but reduced its potency by 2-fold. Strikingly, FOR0812 reduced mean arterial blood pressure (MAP) of 2K-1C rats from 181.5±3.6 mmHg to 149±3.1 mmHg after 4 days of treatment and continued to decrease MAP towards normal levels and at the end of the experimental period, i.e., 15th day the MAP of control rats was 171.1±3,2 mmHg while rats treated with FOR0812 had values of 111.4±6.9 mmHg. In the group treated only with vehicle (saline) the MAP continued high during the whole experimental period (>160mmHg). FOR also reduced MAP of DS rats from 158.8± 5.6 mmHg to 122.6±6.7mmHg after six days of treatment and this effect lasted throughout the experiment and at the end the control group treated with vehicle had MAP of 158.9±4.2 mmHg while DS rats treated with FOR0812 had MAP of 118.1±5.8mmHg. The intravenous injection of FOR at 0.001; 0.01; 0.1 and 1 mg/Kg decreased vascular resistance (expressed in mmHg x ml/min) of normotensive rats from control values of 6.5±0.8 to 5.6±0.7;4.9±0.6; 4.8±0.6 and 2.6±0.6, respectively. FOR0812 increases in a dose-dependent fashion the systolic work and cardiac output due to decreased afterload. At the higher doses, this compound decreases cardiac maximal dP/dt due to decreased left ventricular pressure. However, at the dose range used, the preload recruitable stroke work is not affected. In conclusion, FOR0812 has antihypertensive effect decreasing cardiac afterload and can decrease wall stress and myocardial oxygen demand in patients with hypertension and acute ischemic heart disease. / Complexos metálicos Ru-NO estáveis, não formadores de cianeto, são capazes de promover relaxamento do músculo liso vascular e são compostos potenciais para uso em distúrbio cardiovasculares como angina e crise hipertensiva. O composto [Ru(bpy)2(tioureia)NO(PF6)3] – FOR0812, foi estudado com objetivo de demonstrar possível efeito vasorelaxante in vitro e anti-hipertensivo in vivo. Ratos Wistar machos (200-250g) foram sacrificados, anéis de 4mm de aorta torácica foram montadas em banho de órgãos de 5 ml a 37°C e aerados com mistura carbogênica (5%CO2 em 95%O2). Os tecidos foram acoplados a um transdutor isométrico sob tensão passiva de 1g e os registros experimentais foram obtidos através de um sistema de aquisição de dados. A droga foi adicionada de maneira cumulativa (10-12 a 10-4M) no platô das contrações. Os estudos in vivo foram feitos em ratos com hipertensão renovascular (2 rins-1 clip), ratos Dahl sensíveis ao sal e ratos espontaneamente hipertensos (SHR) recebendo o composto FOR0812 (1mg/Kg) por via oral durante 15 dias. Os efeitos agudos na hemodinâmica sistêmica e eletromecânica cardíaca também foram estudados. Ademais, estudamos a razão da variação da pressão intracavernosa pela variação na pressão arterial em modelos de disfunção erétil por neuropatia diabética após tratamento agudo com FOR0812. Para os estudos hemodinâmicos um microcateter pressão-volume foi inserido na carótida direita e deslocado até o ventrículo esquerdo. Similarmente, a veia jugular esquerda foi canulada para infusão em bolus do composto nas doses 0,001 a 1mg/Kg. A adição de FOR 0812 induziu relaxamento na aorta da ordem de 103±2,3 % com IC50 de 0,24 µM[0,084 – 0,72] comparado com 108±5,3% e 0,05µM[0,025 –0,12] obtido com SNP. A potência do FOR 0812 foi reduzida em oito vezes por L-NAME(100µM). Por outro lado, a resposta relaxante do metalofármaco foi completamente abolida na presença de ODQ (10µM),(*p<0,05). A potência relaxante do composto FOR0812 também foi reduzida na presença de inibidores de canais de K+ na ordem de 5,8 vezes (glibenclamida), em 5,4 vezes (mistura apamina-iberotoxina) e em 11,2 vezes (tetraetilamônio). Em tecidos pré-tratados com hidroxicobalamina(100µM), um removedor de óxido nítrico (NO.) a resposta máxima relaxante para FOR0812 foi grandemente reduzida (Emáx=103±2,3 % vs. 32,4%, *p<0,05). No entanto a remoção do ânion nitroxil (NO-), em tecido pré-incubados com 3 mM de L-cisteína, não afetou a resposta relaxante para FOR0812. Similarmente, o bloqueio da enzima RhoA/Rho kinase (ROCK) com fasudil (HA-1077) não afetou a resposta relaxante para FOR0812. Em tecidos pretratados com indometacina, houve redução da potência em duas vezes quando comparado ao FOR0812. In vivo, o composto reduziu a pressão arterial de animais com hipertensão renovascular (2rins-1clipe) já no quarto de dia de tratamento (181,5±3,6 mmHg vs. 149±3,1 mmHg) e continuou diminuindo os níveis pressóricos até o fim do tratamento (171.1±3,2 mmHg –controle vs. 111.4±6,9mmHg - tratado). Animais tratados apenas com salina tiverem níveis pressóricos altos (>160mmHg) até o fim do período experimental. Nos animais com hipertensão sal-sensível, o composto reduz a pressão já no sexto dia de tratamento (158,8± 5,6 mmHg vs. 122,6±6,7mmHg-tratado) e o composto continua controlando a pressão até o final do tratamento (158,9±4,2 mmHg vs. 118,1±5,8mmHg-tratado). Animais tratados com salina (veículo) tiveram pressão elevada até o final do experimento.Este composto, nas doses de 0,001; 0,01; 0,1 e 1 mg/Kg , diminui a resistência periférica total (expressa em mmHg x ml/min) de ratos normotensos de valores controle de 6,5±0,8 para 5,6±0,7;4,9±0,6; 4,8±0,6 e 2,6±0,6. Aumenta gradativamente o trabalho sistólico e o débito cardíaco, por diminuição de pós-carga. Nas doses maiores (0,1 e 1 mg) tende a diminuir a dp/dt máxima por reduzir pressão do ventrículo esquerdo por diminuição de pós-carga. NO entanto, o trabalho sistólico recrutável não é prejudicado até a dose de 1mg/kg. O composto FOR0812 tem atividade antihipertensiva por reduzir resistência periférica total atuado como vasodilatador arterial como doador de NO de maneira estável, lenta e sem geração de cianeto.

Ion pumps establish homeostasis across the membranes of living cells. Hearing is a mechanotransduction event that takes place in a closed compartment containing a fluid high in K+ concentrations. In Drosophila melanogaster, this closed compartment is formed by a scolopale cell that wraps around the dendrite of sensory neurons. The receptor lymph is maintained by the scolopale cell. The lumenal membrane of the scolopale cell is the wall of the compartment containing the receptor lymph, the scolopale space. The ablumenal membrane of the scolopale cell creates the border of the scolopidium. The Na/K pump is located on the ablumenal membrane of the scolopale cell, bringing K+ into the scolopale cell cytoplasm and extruding K electrogenically (Roy et al, 2013). We explored other primary and secondary ion pumps that are involved in creating a K+-rich lumen in the Malpighian tubule (Day et al, 2008; Rodan et al, 2012). We used RNAi technology to knockdown one gene at a time and electrophysiology to measure a sound evoked potential (SEP) that reflects the fly’s ability to hear. We found that knocking down V-ATPase, a proton pump, subunits involved in proton extrusion significantly reduces the SEP of knockdown flies. The involvement of cation chloride cotransporters (CCCs) and cation proton antiporter (CPAs), both secondary ion pumps that use the gradients created by the Na/K pump and V-ATPase respectively to pump other ions up their gradient, is less clear. We found that knocking down Nhe3, a CPA, significantly reduced the SEP when knocked down in the scolopale cell, suggesting it as a partner to the V-ATPase. Knocking down CG31547, a CCC, statistically increased the SEP, possibly a type1 statistical error.

abstract: Na+/H+ antiporters are vital membrane proteins for cell homeostasis, transporting Na+ ions in exchange for H+ across the lipid bilayer. In humans, dysfunction of these transporters are implicated in hypertension, heart failure, epilepsy, and autism, making them well-established drug targets. Although experimental structures for bacterial homologs of the human Na+/H+ have been obtained, the detailed mechanism for ion transport is still not well-understood. The most well-studied of these transporters, Escherichia coli NhaA, known to transport 2 H+ for every Na+ extruded, was recently shown to bind H+ and Na+ at the same binding site, for which the two ion species compete. Using molecular dynamics simulations, the work presented in this dissertation shows that Na+ binding disrupts a previously-unidentified salt bridge between two conserved residues, suggesting that one of these residues, Lys300, may participate directly in transport of H+. This work also demonstrates that the conformational change required for ion translocation in a homolog of NhaA, Thermus thermophilus NapA, thought by some to involve only small helical movements at the ion binding site, is a large-scale, rigid-body movement of the core domain relative to the dimerization domain. This elevator-like transport mechanism translates a bound Na+ up to 10 Å across the membrane. These findings constitute a major shift in the prevailing thought on the mechanism of these transporters, and serve as an exciting launchpad for new developments toward understanding that mechanism in detail. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

Further in vivo functional studies were also performed. The SLC26A3 antibody was injected into the BALB/C mice seminiferous tubules using micropipette. The animals were sacrificed after three days, and CASA, daily sperm production (DSP) were used to evaluate sperm motility and spermatogenesis. The results showed that sperm motility was increased while there was no significant difference between DSP. Our results indicate that SLC26A3 on sperm does not play a dominant role in spermatogenesis, epididymal maturation and sperm motility. / In the first part of study, guinea pig sperm which were incubated in medium with various concentrations of Cl- resulted in varied percentages of capacitated sperm, in a concentration dependent manner. Depleting Cl-, even in the presence of HCO3 -, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO 3- dependent events, including cAMP production, protein tyrosine phosphorylation and pHi increase also depend on Cl - concentrations. Similar Cl- dependence was observed for sperm hyperactivated motility and sperm-egg fusion. The capacitation-associated events could also be significantly reduced by inhibitors or antibodies of CFTR and SLC26A3, with a more potent effect observed for niflumate, an inhibitor more selective for SLC26A3, over that of DIDS, an inhibitor more selective for SLC4 exchangers. The expression and localization of CFTR and SLC26A3 in guinea pig sperm were also demonstrated using immunostaining and Western blot analysis. Our results indicate that Cl- is required for the entry of HCO3- necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3 - with CFTR providing the recycling pathway for Cl- . / In the second part of study, GC-1 spg cell line that expresses SLC26A6 but not SLC26A3 was used as a negative control. The cells and sperm were pretreated with anion exchanger inhibitors and SLC26A3 antibody, and then membrane potential and intracellular calcium were measured. Our results showed that DIDS could inhibit the HCO3- deficiency induced depolarization of GC-1 spg cells as well as the depolarization induced by Cl- or HCO3- deficiency in sperm. Niflumate could inhibit the HCO3- induced [Ca 2+] i increase of the sperm but not GC-1 spg cells. SLC26A3 antibody had no effect on the GC-1 spg cells but it could block the depolarization caused by C--deficiency in sperm. / Our previous study has demonstrated the involvement of Cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate necessary for sperm capacitation. However, whether its involvement is direct or indirect remains unclear. The present study is design to investigate: (1) the possibility of a Cl-/HCO3- exchanger, solute carrier family 26, number 3 (SLC26A3), operating with CFTR during sperm capacitation, (2) the role and the underlying mechanisms of SLC26A3 in other sperm post-testicular processes and spermatogenesis. / Taken together, our results demonstrate the involvement of SLC26A3 in sperm function, particularly in transporting HCO3- necessary for sperm capacitation, which appears to be working with CFTR providing the recycling pathway for Cl- in parallel. The present results also provide an explanation to the observed subfertility in patients with SLC26A3 mutations. Further in vitro and in vivo studies also have shown that SLC26A3 does not play a predominant role in spermatogenesis but may affect other post-testicular maturation processes. / Chen, Wenying. / "November 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

De nombreux processus physiologiques tels que les mouvements stomatiques, l’absorption des nutriments, l’élongation cellulaire et la signalisation cellulaire impliquent des flux d’anions entre les membranes plasmique et vacuolaire des cellules végétales. Ces flux ioniques sont régulés par des canaux et transporteurs membranaires. Les canaux ioniques transportent passivement les ions au travers des membranes selon le gradient électrochimique. Les transporteurs actifs permettent le transport contre le gradient électrochimique de l’ion transporté induisant son accumulation dans un compartiment cellulaire. Dans les cellules végétales, le gradient de H+ entre différents compartiments constitue la principale source d’énergie couplée par les symports et les antiports au transport de NO₃⁻ et Cl⁻. Au cours de ma thèse, j’ai analysé ces flux ioniques avec deux approches. Une première approche a consisté en l’étude fonctionnelle par électrophysiologie de deux protéines membranaires, AtCLCc et AtCLCg impliquées dans le transport d’anions. Dans une deuxième approche, un biosenseur, clopHensor a été exprimé chez A. thaliana et a permis de mesurer simultanément la [NO₃⁻] et le pH cytosoliques in vivo. Les cellules de garde ont été choisies comme modèle cellulaire pour l’étude de la dynamique in vivo de la [NO₃⁻]cyt et du pH. Nous avons mis en évidence que la [NO₃⁻]cyt est influencée par les conditions extracellulaires dans ces cellules. Enfin l’expression de clopHensor en plantes KO pour un antiport NO₃⁻/H⁺ vacuolaire, AtCLCa, et d’un canal anionique de la membrane plasmique, SLAC1, nous a permis d’étudier la contribution de deux membranes dans la régulation de [NO₃⁻] et du pH cytosolique. Les travaux menés ont permis de visualiser l’activité de canaux et de transporteurs d’anions et H⁺ in vivo et de quantifier leur impact sur l’homéostasie du cytosol. / Many physiological processes like stomata aperture, nutrient up-take, cellular elongation and cell signalling involve anion fluxes at the two main membranes, the plasma and vacuolar membranes of plant cells. Specialized membrane proteins form active and passive anion transport systems mediating and regulating anion fluxes. Ion channels are passive transport systems mediating ion fluxes across membranes along the electrochemical gradient. Whereas active transporters work against the electrochemical gradient of the transported ion allowing its accumulation into a cellular compartment. In plant cells, the H⁺ gradient is the main energy source of antiporters and symporters that couple the transport of anions like NO₃⁻ and Cl⁻ to the transport of H⁺. In the presents work, we aimed at analysing anion and H⁺ fluxes at two levels. First, we used an electrophysiological approach to study the functional properties of two anion transport systems acting at the vacuolar membrane, AtCLCc and AtCLCg. We also expressed a biosensor, clopHensor in A. thaliana to dynamically measure in vivo the [NO₃⁻] and pH of the cytosol. We chose stomata guard cells as a cellular model to study these fluxes. Our results illustrate the in vivo dynamics of cytosolic [NO₃⁻] and pH variations in the cytosol of guard cells. Our data show that in guard cells the cytosolic [NO₃⁻] is highly influenced by the extracellular [NO₃⁻]. At last, clopHensor’s expression in plants KO for the vacuolar NO₃⁻/H⁺ antiporter AtCLCa and for the plasma membrane anion channel SLAC1 allowed us to dissect the role of the two membranes in controlling the variation of cytosolic [NO₃⁻] and pH. This work enabled to visualize the activity of an anion channel (SLAC1) and of a NO₃⁻/H⁺ antiporter (AtCLCa) in vivo and to quantify the impact of anion and proton fluxes on cytosolic homeostasis of guard cells.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O acÃmulo de Na+ no vacÃolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulaÃÃo dos volumes e conteÃdos dos vacÃolos de cÃlulas vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prÃtons (V-ATPase e V-PPase), aquoporinas e vÃrios sistemas de transportes ativos e/ou secundÃrios, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prÃtons transmembranares funcionam como sistemas de transporte primÃrio nas cÃlulas vegetais e ambas as enzimas geram uma diferenÃa de potencial eletroquÃmico de prÃtons atravÃs da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquÃmico de prÃtons gerado pelos transportadores primÃrios para transportar Na+ para dentro do vacÃolo. No presente trabalho inicialmente foram determinados os conteÃdos de Ãons Na+ e K+ em raÃzes, hipocÃtilos e folhas e em seguida a anÃlise da expressÃo dos genes das bombas de prÃtons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plÃntulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmÃtico. As plÃntulas foram crescidas em meio nutritivo na ausÃncia de NaCl e PEG (controle), na presenÃa de 100 mM de NaCl (estresse salino) ou na presenÃa de 200,67 g/L de PEG (estresse osmÃtico). O conteÃdo de Ãons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteÃdo de Ãons K+ diminuiu na mesma condiÃÃo. A expressÃo dos genes das bombas de prÃtons e dos contra-transportadores vacuolares de folhas e de raÃzes no estresse salino aumentou em todas as condiÃÃes estudadas, porÃm o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulaÃÃo paralela entre esses genes. JÃ no estresse osmÃtico, os resultados para as folhas mostraram que a expressÃo dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes nÃo sofreram mudanÃas significativas. Nossos resultados sugerem que o estresse salino e o estresse osmÃtico induziram uma regulaÃÃo diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmÃtico. / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress

Arginine decarboxylase is a key enzyme involved in the polyamine pathway of organisms. Pyruvoyl-dependent arginine decarboxylases are expressed in the form of proenzymes that self-cleave to form N-terminal [beta] and C-terminal [alpha] subunits generating an active pyruvoyl group at the [alpha] terminus. We have identified an archaeal homolog of a pyruvoyl-dependent arginine decarboxylase in Chlamydophila pneumoniae that could play a role in the persistence of the organism in the host. The recombinant enzyme showed highest activity at pH 3.4, which is the lowest optimum pH ever reported for a pyruvoyl dependent arginine decarboxylase. The proton-consuming decarboxylation raises intracellular pH, and thereby plays a role in acid-resistance. It could inhibit the pro-inflammatory nitric oxide synthase resulting in asymptomatic infection. A variant protein Thr⁵²Ser at the predicted cleavage site showed less pro-enzyme cleavage and activity compared to the wild-type. The homologs of arginine decarboxylase and flanking arginine-agmatine antiporter were also found in different biovariants of Chlamydia trachomatis. In the invasive L2 strain of C. trachomatis, the presence of a nonsense codon in the gene encoding arginine decarboxylase enzyme prevented the expression of an active enzyme. The variant protein with tryptophan replacing nonsense codon restored arginine decarboxylase activity. The non-invasive D strain of C. trachomatis had an intact arginine decarboxylase gene, but it was recombinantly expressed as a proenzyme that was uncleaved. The arginine-agmatine antiporters from both the strains were active and transported tritiated arginine into their cells. The polyamine pathway of the crenarchaeon Sulfolobus solfataricus uses arginine to make putrescine, but the organism lacks homologs of arginine decarboxylase. However, it has two paralogs of pyruvoyl dependent S-adenosylmethionine decarboxylase − SSO0536 and SSO0585. These enzymes were recombinantly expressed as pro-enzymes that self-cleaved into [beta] and [alpha] subunits. Even with a 47% amino acid sequence identity, the SSO0536 protein exhibited significant arginine decarboxylase activity whereas SSO0585 protein had significant S-adenosylmethionine decarboxylase activity. This is the first report of an S-adenosylmethionine decarboxylase enzyme showing alternative decarboxylase activity. The chimeric protein with the [alpha]-subunit of SSO0585 and [beta]-subunit of SSO0536 had arginine decarboxylase activity, suggesting that the residues responsible for substrate recognition are located in the amino terminus. / text

SOBREIRA, Alana Cecília de Menezes. Estudo da expressão dos genes das bombas de prótons (V-ATPase e V-PPase) e dos contra-transportadores vacuolares (NHX) de Vigna unguiculata (L.) Walp submetidos a estresses abióticos.2009 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, 2009. / Submitted by JOANA BEZERRA (joanabib@yahoo.com.br) on 2012-11-09T21:07:31Z No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Approved for entry into archive by Érica Barros(ericabarros@ufc.br) on 2012-11-12T22:49:16Z (GMT) No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Made available in DSpace on 2012-11-12T22:49:16Z (GMT). No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress. / O acúmulo de Na+ no vacúolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulação dos volumes e conteúdos dos vacúolos de células vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prótons (V-ATPase e V-PPase), aquoporinas e vários sistemas de transportes ativos e/ou secundários, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prótons transmembranares funcionam como sistemas de transporte primário nas células vegetais e ambas as enzimas geram uma diferença de potencial eletroquímico de prótons através da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquímico de prótons gerado pelos transportadores primários para transportar Na+ para dentro do vacúolo. No presente trabalho inicialmente foram determinados os conteúdos de íons Na+ e K+ em raízes, hipocótilos e folhas e em seguida a análise da expressão dos genes das bombas de prótons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plântulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmótico. As plântulas foram crescidas em meio nutritivo na ausência de NaCl e PEG (controle), na presença de 100 mM de NaCl (estresse salino) ou na presença de 200,67 g/L de PEG (estresse osmótico). O conteúdo de íons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteúdo de íons K+ diminuiu na mesma condição. A expressão dos genes das bombas de prótons e dos contra-transportadores vacuolares de folhas e de raízes no estresse salino aumentou em todas as condições estudadas, porém o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulação paralela entre esses genes. Já no estresse osmótico, os resultados para as folhas mostraram que a expressão dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes não sofreram mudanças significativas. Nossos resultados sugerem que o estresse salino e o estresse osmótico induziram uma regulação diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmótico.