Magister Scientiae - MSc (Biotechnology) / It has been estimated that the world population will reach about 10 billion by the year 2050 and in order to accommodate the increased demand of food, the world agricultural production needs to rise by 70 % in the year 2030. However, the realisation of the goal in food production is hindered by limited arable land caused by urbanisation, salinisation, desertification and environmental degradation. Furthermore, abiotic and biotic stresses affect plant growth and development, which lead to major crop losses. The long term goal of this study is to improve food security by producing genetically engineered agricultural crops that will be tolerant to diverse stresses. This research aims at developing stress tolerant crops through the determination of important signalling molecules and second messengers, such as nitric oxide (NO), carbon monoxide (CO) and calcium ion (Ca2+), which can bind to plant proteins such as AtNOGC1 in order to induce stress tolerance in plants.

Cyclic AMP and cyclic GMP (cAMP and cGMP) are important second messengers in key signal-transduction pathways that mediate various physiological functions in bacteria and eukaryotes. Adenylyl Cyclases (ACs) and Guanylyl Cyclases (GCs) cyclize ATP and GTP to produce cAMP and cGMP, respectively. Though most nucleotide cyclises show exquisite specificity for their substrates, there are instances where ACs were observed to have low GC activity as well, and vice versa. To understand structural basis of substrate (ATP or GTP) recognition, discrimination and binding by an adenylyl cyclase, we have taken up Ma1120, an AC from Mycobacterium avium, for our studies. Work presented in the thesis includes crystal structures of Ma1120 in the presence of substrate (ATP or GTP), by-product pyrophosphate and ATP analogue 2′,5′-dideoxyd-3′-adenosine triphosphate (2′,5′-dd-3′-ATP). A triple mutant of Ma1120 (K101→E, D157→G, A167→Y) was generated to increase specificity of Ma1120 towards GTP by mutation in the substrate specifying residues, but the enzyme showed equal specificity for ATP as well as for GTP. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. ATP bound to Ma-Cat has two different conformations, one with C2′-endo and the other with C3′-endo puckering for the ribose. C3′-endo conformation is favourable for catalysis as it brings 3′-OH group of ribose and free oxygen of α-phosphate closer to each other. The crystal structure of GTP bound to Ma-Cat showed a novel mode of GTP binding to AC. This is the first report of GTP bound to AC. ATP bound to Ma-Cat-KDA→EGY forms non-cognate substrate complex and ATP is stabilized by stacking of adenines over each other with Tyr167 flanking on both sides of adenines. Ma-Cat-KDA→EGY+GTP complex is the first report of GTP bound to a guanylyl cyclase. GTP is bound in reverse orientation when compared to ATP bound to AC. Reverse orientation of GTP is attained to stabilize the guanine in highly electronegative guanine binding pocket. Also, O3' of GTP is placed in opposite orientation as compared to ATP bound to Ma-Cat. Therefore, during cyclization reaction guanine and ribose changes their orientation to bring O3' atom of ribose closer to α-phosphate, after cleavage of the bond between α- and β-phosphates. Thus, this study has revealed novel modes of binding of ATP and GTP to catalytic domains of Ma1120 and its triple mutant, mechanism of substrate discrimination and residual activity for the non-cognate substrate.

Recentemente, uma molécula baseada em nucleotídeos, a adenosina monofosfato dimérica cíclica (c-di-AMP), foi identificada em várias bactérias patogênicas, ganhando rapidamente o status de um sinalizador central no controle de diversos processos bacterianos essenciais, tais como transporte iônico e vigilância a danos no DNA. Em Staphylococcus aureus, por exemplo, o c-di-AMP é essencial para homeostase da parede celular e resistência aos estresses ambientais. A biossíntese de c-di-AMP envolve a condensação de duas moléculas de ATP, catalisadas por enzimas que contenham o domínio DAC (diadenilato ciclase). Especialmente em S. aureus, somente uma ciclase (DacA) é responsável pela síntese desse segundo-mensageiro, através da enzima DacA. Recentemente, nosso grupo determinou a estrutura da DacA de S. aureus, demonstrando a importância do seu estado oligomérico para o mecanismo catalítico. Diferentemente da primeira diadenilato caracterizada, a DisA, uma enzima octamérica, a atividade enzimática da DacA ocorre através do encontro de dois dímeros distintos, formando uma interface cataliticamente competente. Dadas essas características, o foco desse trabalho foi a busca de novos inibidores para a DacA, através de triagens em larga escala (HTS), triagem cristalográfica por fragmentos e docagem molecular. Foram encontrados 30 compostos com atividade inibitória nos ensaios de HTS, 15 compostos derivados de fragmentos da triagem cristalográfica e construída uma biblioteca de 480 compostos docados virtualmente no sítio ativo. Os compostos encontrados na triagem cristalográfica foram caracterizados com respeito ao seu valor de IC50 frente à atividade enzimática. O composto AN-584/43409544 se demonstrou bastante promissor, com valor de IC50 de 54 µM. Ainda, um análogo desse composto foi encontrado na interface dimérica da DacA nos estudos cristalográficos, o que mostra um possível sítio alostérico para o desenvolvimento e busca de inibidores. Os resultados gerados nesse projeto podem servir de base para estudos futuros e desenvolvimento de novos antibióticos, que em combinação com os tratamentos atualmente disponíveis pode levar ao controle das infecções até mesmo das formas mais resistentes de S. aureus. / Recently, a nucleotide-based molecule, the cyclic dimeric adenosine monophosphate (c-di-AMP), has been identified in several pathogenic bacteria, rapidly gaining the status of a central signaling controlling several essential bacterial processes, such as ion transport and DNA damage surveillance. In Staphylococcus aureus, for example, c-di-AMP is essential for homeostasis of the cell wall and resistance to environmental stresses. The biosynthesis of c-di-AMP involves the condensation of two molecules of ATP, catalyzed by enzymes that contain the DAC (diadenylate cyclase) domain. Especially in S. aureus, only one cyclase (DacA) is responsible for the synthesis of this second-messenger. Recently, our group determined the structure of the S. aureus diadenylate cyclase, demonstrating the importance of their oligomeric state for the catalytic mechanism. Unlike DisA, a octameric diadenylate cyclase, the enzymatic activity of DacA occurs through the meeting of two dimers forming a catalytically competent interface. Given these characteristics, the focus of this work was the search for new inhibitors through high-throughput screening (HTS), crystallographic screening of fragments and molecular docking. It was found 30 compounds with inhibitory activity in HTS assays, 15 compounds derived from fragments of crystallographic screening and built a 480 compound library virtually docked in the active site. The compounds found in the crystallographic screening were characterized with respect to its IC50 value. The compound AN-584/43409544 has been shown verry promising, with IC50 of 54 µM. Also, an analog of this compound was found in the dimeric interface of DacA in crystallographic assays, which shows a possible allosteric site for inhibitors search and development. The results generated in this project can serve as a basis for future studies and development of new antibiotics, which in combination with the currently available treatments can lead to infection control even of the most resistant strains of S. aureus.

Recentemente, uma nova molécula de sinalização bacteriana, o AMP dimérico cíclico (c-di-AMP) emergiu como um regulador central dos processos fisiológicos essenciais, tais como a homeostase celular, verificação da integridade do DNA e virulência bacteriana, entre outros. O c-di-AMP é produzido a partir da condensação de duas moléculas de adenosina trifosfato (ATP) por proteínas denominadas diadenilato ciclases, que contém o domínio DisA_N, também denominado DAC. Existem 2842 sequências de proteínas que contém o domínio DAC, provenientes de 2386 organismos encontradas no banco de dados Protein Families Database (Pfam). Essas proteínas são divididas em subfamílias sendo as três subfamílias mais abundantes: DacA (69,1%), proteínas de membrana associadas a sinalização intracelular de alterações decorrentes do meio externo; DisA (24,1%), primeira diadenilato ciclase a ser amplamente estudada, é uma proteína intracelular encontrada na forma de octâmeros ativos em solução, a qual, indiretamente, controla a divisão celular através da verificação da integridade do DNA e DacB (5,5%), proteínas citoplasmáticas expressa, particularmente, durante a formação de esporos bacterianos. Uma característica interessante é que a maioria dos organismos contém uma única e essencial proteína com domínio DAC. Os organismos que contém duas ou mais proteínas-DAC, tais como Clostridium e Bacillus spp., são uma exceção. Em Staphylococcus aureus (S. aureus), um patógeno humano oportunista e responsável por inúmeras doenças infecciosas, uma única diadenilato ciclase é encontrada pendurada na porção interna da membrana celular (Sau_DacA). A atividade desta proteína é potencialmente regulada através da interação direta com uma proteína YbbR-like, que contém um domínio sensor extracelular. Sau_DacA conserva todos os elementos-chave de uma diadenilato ciclase bacteriana, e por ser a única presente em S. aureus, revela-se um excelente alvo de estudo para o desenvolvimento de novos fins terapêuticos. No entanto, até o presente momento, existem poucas informações em relação a estrutura proteica, ao mecanismo de síntese de c-di-AMP e regulação do mecanismo de síntese de nucleotídeo destas proteínas, sendo, portanto, neste aspectos que o presente trabalho pretendeu contribuir. Através de uma série de ensaios, estruturais, calorimétricos, espectroscópicos e bioquímicos, aliados a mutações sítio-dirigidas, identificou-se a relevância de uma conformação dimérica para a estabilidade conformacional e térmica para a proteína ser funcionalmente ativa, assim como a importância dos motivos conservados DGA (Aspartato-Glicina-Alanina) e RHR (Arginina-Histidina-Arginina) para a atividade da Sau_DacA. O loop L5 localizado entre o sítio ativo e a interface dimérica mostrou-se relevante, uma vez que nele é encontrado o motivo DGA - de ligação ao ATP - e o mesmo encontra-se estabilizado em uma posição favorável para ligação do ATP, apenas na conformação dimérica da proteína. Nossos resultados aliados a dados da literatura possibilitaram a proposição de um mecanismo de síntese de c-di-AMP que deve ocorrer via encontro face-a-face de dois sítios de ligação de ATP presentes em dímeros proteicos distintos, podendo a taxa de síntese de o nucleotídeo sofrer interferência via interação proteína-proteína com a proteína receptora de sinal Sau_YbbR. Desta forma, contribuímos para uma melhor compreensão da estrutura e função da Sau_DacA, possibilitando o uso desta como alvo para o desenvolvimento de novos fármacos, uma vez que é sabido que a biossíntese de c-di-AMP é essencial para a maioria dos patógenos que o sintetizam. / Recently, a new bacterial signaling molecule, the dimeric cyclic AMP (c-di-AMP) has emerged as a central regulator of essential physiological processes, such as cell wall homeostasis, DNA integrity and bacterial virulence, among others. C-di-AMP is synthesized from two molecules of adenosine triphosphate (ATP) by proteins containing DisA_N domain, also called diadenilato cyclases (DACs). A survey in the Protein Families Database database (Pfam) found 2842 protein sequences containing the DAC domain, from 2386 different organisms. These proteins are divided into subfamilies and the three most abundant are: DacA (69,1%), a membrane protein associated with intracellular signaling resulting from an external environment change; DisA (24,1%), the first and most widely studied diadenilate cyclase, an intracellular protein found as active octamers in solution which indirectly controls cell division by DNA integrity verification; and DacB (5,5%), a cytoplasmic proteins, particularly expressed during bacterial spores formation. An interesting feature is that most organisms contain just a single and essential DAC-protein. Organisms containing two or more DAC-containing proteins, such as Clostridium and Bacillus spp., are exceptions. In Staphylococcus aureus (S. aureus), an opportunistic human pathogen responsible for some life-threating diseases, there is a single membrane attached diadenilate cyclase, hanging in the inner portion of the cell membrane (Sau_DacA). The activity of this protein is potentially regulated through direct interaction with YbbR, which contains an extracellular sensor domain. Sau_DacA conserves all key elements of bacterial di-adenylate cyclase, and for being the only di-adenylate cyclase from S. aureus, proves to be an excellent study target for new therapeutic purposes. However, to date, there is a lack of information about structure, c-di-AMP synthesis mechanism and regulation of nucleotide synthesis by Sau_DacA. Therefore, in this context the present work aims to contribute. Through a series of structural, calorimetric, spectroscopic and biochemical assays combined with site-directed mutations, we solved the structure of a soluble construct of Sau_DacA and identified a dimeric interface relevance for the conformational and thermal stability to the protein. This dimer is functionally active and highlights the importance of conserved motifs DGA (Aspartate-Glycine-Alanine) and RHR (Arginine-Histidine-Arginine) for the activity of Sau_DacA. The L5 loop, located between the active site and the dimer interface where is allocated the ATP binding motif (DGA), is stabilized in a favorable position for ATP binding, just in protein dimeric conformation. Our results combined with literary allowed us infer the synthesis of c-di-AMP occurs by face-to-face encounter of two distinct ATP binding site and its rate of synthesis could be regulated through direct protein interaction with. In this way, we contribute to a better understanding of Sau_DacA structure and function, assisting in its use as a target for new drugs development since it is known the biosynthesis of c-di-AMP is essential for most pathogens that synthesize.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Previamente, foi demonstrado que o tratamento via intravenosa com 1-nitro-2-feniletano (NFE) induziu hipotensÃo que resulta principalmente de sua aÃÃo vasodilatadora diretamente sobre o mÃsculo liso. Nesse estudo, estudamos o mecanismo subjacente ao efeito miorrelaxante do NFE em tecidos vasculares isolados de ratos. Registros isomÃtricos foram obtidos a partir de anÃis isolados de artÃria aorta e do segundo ramo da mesentÃrica de ratos atravÃs de sistema de aquisiÃÃo de dados. Experimentos in silico (docking) de simulaÃÃo da formaÃÃo de complexos entre o NFE e a enzima guanilato ciclase foram realizados. Em preparaÃÃes de aorta isolada com endotÃlio intacto, NFE (1-300 &#61549;g/mL) relaxou a contraÃÃo induzida por fenilefrina ou K+, com valores de CI50 de 35,0 [23,3â52,6] e 73,2 [39,5â134,3] &#61549;g/mL, respectivamente. Os efeitos vasorrelaxantes do NFE foram significativamente (P < 0,05, teste de Mann-Whitney) reduzidos pelo tratamento prÃvio com ODQ (10 ÂM), azul de metileno (10 ÂM), TEA (5 mM), glibenclamida (10 ÂM) ou 4-aminopiridina (1 mM), mas nÃo pela remoÃÃo do endotÃlio vascular ou pelo prÃ-tratamento com L-NAME (100 &#956;M), indometacina (10 &#956;M), MDL-12.330A (3 &#956;M), KT5823 (0.5 ÂM) ou KT5720 (1 &#956;M). A potÃncia do NFE para induzir efeito vasorrelaxante foi significantemente maior (p < 0,01, teste de Mann-Whitney) nas preparaÃÃes de artÃria mesentÃrica quando comparado à aorta, tanto nas contraÃÃes induzidas por K+ como por noradrenalina com valores de CI50 de 5,3 [2,6â10,5] e 6,5 [2,8â14,9] Âg/mL, respectivamente. Em um meio sem cÃlcio, na presenÃa de K+ 60 mM ou fenilefrina 1 ÂM, as contraÃÃes induzidas CaCl2 foram significativamente reduzidas e atà mesmo abolidas pelo NFE na concentraÃÃo de 100 Âg/mL, respectivamente. Em meio sem cÃlcio, contendo EGTA, a resposta contrÃtil da fenilefrina foi significantemente reduzida pelo NFE (100 Âg/mL ), efeito impedido pelo tratamento com ODQ (10 ÂM), enquanto nÃo teve efeito significativo sobre as contraÃÃes induzidas por cafeÃna. Resultados semelhantes foram obtidos com nitroprussiato de sÃdio. NFE tambÃm foi capaz de inibir a contraÃÃo induzida pela entrada capacitativa de cÃlcio e pelo Ãster de forbol. AlÃm disso, os resultados de docking revelam que existem clusters de provÃveis interaÃÃes do NFE com a enzima guanilato ciclase. O presente estudo sugere que a atividade vasorrelaxante do NFE em aorta de rato à causada por provÃvel estimulaÃÃo da guanilato ciclase e consequente ativaÃÃo da cascata enzimÃtica guanilato ciclase/GMPc/canais de K+. / It was early shown that intravenous treatment with 1-nitro-2-phenylethane (NPE) induced hypotension resulting mainly from its direct vasodilatory action on vascular smooth muscle. Here, it was sudied the underlying mechanism involved in the vasorelaxant effect of NPE in isolated rings of rat arteries. Isometric recordings were obtained from rings made from aorta or mesenteric artery using a digital acquisition system. Experiments in silico (docking) for simulation of molecular interactions between NPE and the enzyme guanylate cyclase were performed. In endothelium-intact aortic preparations, NPE (1-300 &#61549;g/mL) relaxed the phenylephrine or K+-induced contractions with IC50 values of 35.0 [23.3- 52.6] and 73.2 [39.5-134.3] &#61549;g/mL, respectively. Vasorelaxant effects of NPE were significantly (P <0.05, Mann-Whitney test) decreased by pretreatment with ODQ (10 ÂM), methylene blue (10 ÂM), TEA (5 mM), glibenclamide (10 ÂM) or 4-aminopyridine (1 mM) but not by vascular endothelium removal or by pretreatment with L-NAME (100 ÂM), indomethacin (10 ÂM), MDL-12.330A (3 ÂM), KT5823 (0.5 ÂM ) or KT5720 (1 ÂM). Pharmacological potency of NPE was significantly greater (p <0.01, Mann-Whitney test) in preparations of mesenteric artery compared to that of aorta, as in K+- and norepinephrine-induced contraction with IC50 values of 5.3 [2.6-10.5] and 6.5 [2.8-14.9] &#61549;g/mL, respectively. In calcium-free medium, in presence of K+ 60 mM or phenylephrine 1 &#956;M, the CaCl2-induced contractions were significantly reduced and almost abolished by NPE at 100 &#956;g/mL, respectively. In calcium-free medium, containing EGTA, the contractile response of phenylephrine was significantly reduced by NPE (100 &#61549;g/mL), an effect prevented by treatment with ODQ (10 ÂM), whereas NPE was deprived of any significant effect on caffeine-induced contractions. Similar results were obtained with sodium nitroprusside. NPE also inhibited the contractions induced by the capacitive calcium entry or by phorbol ester. In addition, docking results revealed clusters of interactions of NPE with the guanylate cyclase molecule. The present study suggests that vasorelaxant activity of NPE on rat aorta is due to its stimulatory properties on guanylate cyclase, which activates the guanylate cyclase/cGMP/K+ channels pathway.

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:44:09Z (GMT). No. of bitstreams: 1 Silverio_CamilaBitencourtMendes_M.pdf: 1655028 bytes, checksum: e42d500e98127c5551d4f94269778bcd (MD5) Previous issue date: 2013 / Resumo: O BAY 60-2770 constitui-se um novo ativador da guanilil ciclase solúvel (GCs) independente de óxido nítrico (NO). Há relatos de que ativadores da GCs independente de NO podem reativar esta enzima, mesmo em seu estado heme-oxidada, condição encontrada em doenças vasculares. Neste trabalho, temos por hipótese que a oxidação do grupamento heme da GCs pelo ODQ leva à potencialização dos efeitos antiplaquetários in vitro do BAY 60-2770. Foram utilizadas plaquetas humanas purificadas de voluntários sadios. Realizamos ensaios de agregação e de adesão plaquetária, bem como análise da ativação da integrina plaquetária, ?IIb?3, por citometria de fluxo. Realizamos Também ensaios de Western blotting para a quantificação protéica das subunidades ?1 e ?1 da GCs. Os níveis intracelulares de Ca2+ foram quantificados por fluorimetria, utilizando-se o fluoróforo FluoFort. O BAY 60-2770 (0,001 - 10 ?M) produziu inibição significativa da agregação plaquetária induzida pelo colágeno (2 ?g/mL) ou trombina (0,1 U/mL), sendo este efeito marcantemente potencializado pelo inibidor da GCs, ODQ (10 ?M). Em placas recobertas pelo fibrinogênio, o BAY 60-2770 (0,1 - 10 ?M) inibiu significativamente a adesão plaquetária, sendo esta inibição também potencializada pelo ODQ (10 ?M). O BAY 60-2770 (0,01 - 10 ?M) aumentou os níveis de GMPc e reduziu a mobilização de Ca2+, e ambos os efeitos foram potencializados pelo ODQ. O análogo permeável do GMPc, 8-Bromo-GMPc (8-Br-GMPc; 100 ?M), inibiu a agregação plaquetária e os níveis de Ca2+ de maneira ODQ-resistente. Os níveis de AMPc não foram alterados pelo BAY 60-2770. O colágeno (2 ?g/mL) e trombina (0,1 U/mL) ativaram a integrina ?IIb?3. Este efeito foi inibido pelo BAY 60-2770 (0,01 - 10 ?M), e potencializado pelo ODQ. Os efeitos antiplaquetários do nitroprussiato de sódio (SNP) foram totalmente revertidos pelo ODQ. O tratamento com ODQ (10 ?M) reduziu significativamente os níveis protéicos das subunidades ?1 e ?1 da GCs, os quais foram prevenidos pelo tratamento com BAY 60-2770. Os efeitos inibitórios do BAY 60-2770 sobre a agregação e adesão plaquetária, mobilização intracelular de Ca2+ e ativação da integrina ?IIb?3 foram todos potencializados em condições de heme-oxidação. O BAY 60-2770 impediu a diminuição dos níveis protéicos da GCs produzida por ODQ. Assim, o BAY 60-2770 pode ser de grande interesse terapêutico em doenças cardiovasculares associadas às complicações tromboembólicas / Abstract: Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested. Human washed platelet aggregation and adhesion assays, as well as flow cytometry for ?IIb?3 integrin activation and Western blot for ?1 and ?1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte). BAY 60-2770 (0.001-10 ?M) produced significant inhibition of collagen (2 ?g/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 ?M). In fibrinogen-coated plates, BAY 60-2770 (0.1 - 10 ?M) significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 (0.01 - 10 ?M) increased the cGMP levels and reduced the intracellular Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 ?M) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced ?IIb?3 activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 ?M) were all prevented by ODQ. Incubation with ODQ (10 ?M) significantly reduced the protein levels of ?1 and ?1 sGC subunits, which were prevented by BAY 60-2770. The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and ?IIb?3 activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications / Mestrado / Farmacologia / Mestra em Farmacologia

No description available.

PURPOSE. Na, K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na, K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na, K-ATPase response. METHODS. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na, K-ATPase activity. RESULTS. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na, K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na, K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. CONCLUSIONS. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na, K-ATPase activity.

African horse sickness virus (AHSV) affects equine populations around the world. It is the cause of a high rate of morbidity and associated large economic losses in affected regions. The virus is a segmented double stranded RNA virus and a member of Orbivirus genus in the Reoviridae family. The prototype member of the orbiviruses is bluetongue virus (STY) and other members include Chuzan virus and St. Croix River virus. These viruses are all characterized by a genome of ten dsRNA segments that encode at least ten different proteins. Three of the minor core proteins are found within the core of BTV. These are all associated with the RNA transcription complex and the enzymatic activities with which they are associated include an RNA polymerase (VP1), an RNA capping enzyme (VP4) and an RNA helicase (VP6). Genes homologous to the BTV genes that encode these proteins are found in all members of the Orbivirus genus. The aim of this thesis is to characterize VP4 of AHSV, the capping enzyme candidate, and to compare it to other orbivirus capping enzymes. Possible functional motifs and regions of importance within the orbivirus capping enzymes will be identified. The gene will also be expressed and used to perform assays to characterize the different enzymatic activities of VP4. The VP4 cDNA of AHSV serotype 3 was cloned and sequenced. From the full-length verified nucleotide sequence an open reading frame was identified and used to predict the amino acid sequence. These were compared to other orbivirus species including STY, Chuzan virus and St. Croix River virus. These alignments identified a number of highly conserved regions, consisting of four or more amino acids conserved between all the sequences analyzed. A fibronectin type 3-like motif, containing 12 conserved amino acids, was identified which could be responsible for protein binding. This motif contains 12 conserved amino acids making it a good candidate for a functional motif. Conservation does not, however, always predict regions of importance. In BTV a lysine-containing motif was identified to be responsible for GMP binding. This region is not conserved between the different viruses. AHSV has a motif containing a lysine residue similar to the motif identified in rotavirus and reovirus. Two other motifs described in BTV were also not conserved in the other viruses. One of them, a leucine zipper, was shown to dimerize BTV VP4. Phylogenetically, AHSV and Chuzan virus are the most closely related while BTV is more distant and St. Croix River virus forms a distinct out-group when the different VP4 sequences are compared. AHSV-3 VP4 was expressed as a histidine-tagged protein in the baculovirus expression system. Not unexpectedly, the protein was found to be insoluble, similar to BTV VP4 produced by means of the same system. However, whereas BTV VP4 could be solubilized by the addition of salt the AHSV VP4 remained insoluble at high salt concentrations. Several adjustments were made. Cells were lysed in a high salt buffer, the pH of the buffers was adjusted and sucrose cushions were used but none of the methods was found to improve the yield of soluble VP4 significantly. However, the pellet containing VP4 was relatively empty of contaminating protein and, therefore, a number of enzymatic assays were performed with the pellet. Assays for inorganic phosphatase and nucleotide phosphatase were performed. Strikingly, both assays indicated the presence of active phosphatases in the WT and VP4 pellets. Also, an assay was performed for guanylyltransferase activity but no activity was observed for this assay. The sequence data therefore points to VP4 as the probable capping enzyme although it may have a different structural complex. The failure to produce a reliable source of soluble purified AHSV VP4 made it impossible to provide evidence to confirm the associated enzymatic activities. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

No description available.