RESUMO
Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the contractile vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the contractile vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Equilíbrio Hidroeletrolítico , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Expressão Gênica , Microscopia Imunoeletrônica , Estrutura Terciária de Proteína , Vacúolos/químicaRESUMO
It is expected that the next generation of biotech crops displaying enhanced quality traits with benefits to both farmers and consumers will have a better acceptance than first generation biotech crops and will improve public perception of genetic engineering. This will only be true if they are proven to be as safe as traditionally bred crops. In contrast with the first generation of biotech crops where only a single trait is modified, the next generation of biotech crops will add a new level of complexity inherent to the mechanisms underlying their output traits. In this study, a comprehensive evaluation of the comparative safety approach on a quality-improved biotech crop with metabolic modifications is presented. Three genetically engineered potato lines with silenced polyphenol oxidase (Ppo) transcripts and reduced tuber browning were characterized at both physiological and molecular levels and showed to be equivalent to wild-type (WT) plants when yield-associated traits and photosynthesis were evaluated. Analysis of the primary metabolism revealed several unintended metabolic modifications in the engineered tubers, providing evidence for potential compositional inequivalence between transgenic lines and WT controls. The silencing construct sequence was in silico analysed for potential allergenic cross-reactivity, and no similarities to known allergenic proteins were identified. Moreover, in vivo intake safety evaluation showed no adverse effects in physiological parameters. Taken together, these results provide the first evidence supporting that the safety of next generation biotech crops can be properly assessed following the current evaluation criterion, even if the transgenic and WT crops are not substantially equivalent.
Assuntos
Inocuidade dos Alimentos , Engenharia Genética , Solanum tuberosum/genética , Alérgenos/análise , Animais , Catecol Oxidase/genética , Biologia Computacional , Feminino , Inativação Gênica , Camundongos , Camundongos Endogâmicos BALB C , FotossínteseRESUMO
Cytochrome P450 hemoproteins (CYPs) are involved in the synthesis of endogenous compounds such as steroids, fatty acids and prostaglandins as well as in the activation and detoxification of foreign compounds including therapeutic drugs. Cytochrome P450 reductase (CPR, E.C.1.6.2.4) transfers electrons from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. This work presents the complete sequences of three non-allelic CPR genes from Trypanosoma cruzi. The encoded proteins named TcCPR-A, TcCPR-B and TcCPR-C have calculated molecular masses of 68.6kDa, 78.4kDa and 71.3kDa, respectively. Deduced amino acid sequences share 11% amino acid identity, possess the conserved binding domains for FMN, FAD and NADPH and differ in the hydrophobic 27-amino acid residues of the N-terminal extension, which is absent in TcCPR-A. Every T. cruzi CPRs, TcCPR-A, TcCPR-B and TcCPR-C, were cloned and expressed in Escherichia coli. All of the recombinant enzymes reduced cytochrome c in a NADPH absolutely dependent manner with low K(m) values for this cofactor. They all were also strongly inhibited by diphenyleneiodonium, a classical flavoenzyme inhibitor. In addition, TcCPRs could support CYP activities when assayed in reconstituted systems containing rat liver microsomes. Polyclonal antiserum rose against the recombinant enzymes TcCPR-A and TcCPR-B demonstrated its presence in every T. cruzi developmental stages, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole. We suggest a participation of TcCPR-B in the detoxification metabolism of the parasite.
Assuntos
Resistência a Medicamentos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , DNA de Protozoário/isolamento & purificação , Escherichia coli/enzimologia , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme present in most eukaryotes and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catabolised by poly(ADP-ribose) glycohydrolase. Here, we describe the cloning and characterisation of a PARP from Trypanosoma cruzi (TcPARP). The recombinant enzyme (Mr=65) required DNA for catalytic activity and it was strongly enhanced by nicked DNA. Histones purified from T. cruzi increased TcPARP activity and the covalent attachment of [32P]ADP-ribose moieties to histones was demonstrated. TcPARP required no magnesium or any other metal ion cofactor for its activity. The enzyme was inhibited by 3-aminobenzamide, nicotinamide, theophylline and thymidine but not by menadione. We demonstrated an automodification reaction of TcPARP, and that the removal of attached PAR from this protein resulted in an increase of its activity. The enzyme was expressed in all parasite stages (amastigotes, epimastigotes and trypomastigotes). When T. cruzi epimastigotes were exposed to DNA-damaging agents such as hydrogen peroxide or beta-lapachone, PAR drastically increased in the nucleus, thus confirming PAR synthesis in vivo and suggesting a physiological role for PARP in trypanosomatid DNA repair signalling.
Assuntos
Dano ao DNA , Poli(ADP-Ribose) Polimerases/análise , Trypanosoma cruzi/enzimologia , Animais , Sequência de Bases , Clonagem Molecular , Reparo do DNA , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Escherichia coli/metabolismo , Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Parasitologia/métodos , Poli Adenosina Difosfato Ribose/biossíntese , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologiaRESUMO
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of cAMP and cGMP, and regulate a variety of cellular processes by controlling the levels of these second messengers. We have previously described the presence of both a calcium-stimulated adenylyl cyclase and two membrane-bound cAMP-specific PDEs (one of them strongly associated to the flagellum and the other one with a possible vesicular localization) in Trypanosoma cruzi. Here we report the identification and characterization of TcrPDEA1, a singular phosphodiesterase of T. cruzi which is resistant to the typical phosphodiesterase inhibitors, such as IBMX, papaverine and theofylline. TcrPDEA1 is a single copy gene that encodes a 620-amino acid protein, which is grouped with PDE1 family members, mainly with its kinetoplastid orthologs. TcrPDEA1 was able to complement a mutant yeast strain deficient in PDE genes, demonstrating that this enzyme is a functional phosphodiesterase. TcrPDEA1 is specific for cAMP with a high K(m) value (191.1+/-6.5 microM). Cyclic GMP neither activates the enzyme nor competes as a substrate. In addition, calcium-calmodulin did not affect the kinetic parameters and, as its counterpart in T. brucei, magnesium showed to be crucial for its activity and stability. Although TcrPDEA1 function remains unclear, its presence points out the high complexity of the cAMP signaling in trypanosomatids and the possible compartmentalization of the enzymes involved in the cAMP pathway.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Trypanosoma cruzi/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/química , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Calmodulina/farmacologia , Coenzimas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , DNA de Protozoário/química , DNA de Protozoário/genética , Ativadores de Enzimas/farmacologia , Estabilidade Enzimática , Dosagem de Genes , Teste de Complementação Genética , Guanosina Monofosfato/metabolismo , Magnésio/farmacologia , Dados de Sequência Molecular , Papaverina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Especificidade por Substrato , Teofilina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Cyclic nucleotide phosphodiesterases constitute the only known mechanism to inactivate regulatory signals involving cAMP or cGMP. In our laboratory a cAMP-specific phosphodiesterase associated to the flagellar apparatus, named TcPDE1, was identified in Trypanosoma cruzi. By using the catalytic domain sequence of TcPDE1 to screen a Trypanosoma cruzi genomic data base, a novel T. cruzi phosphodiesterase sequence was found and characterized. TcPDE4 encodes a 924-amino acid protein and shows homology with the PDE4 vertebrate subfamily. The sequence shows three conserved domains, FYVE, phosphohydrolase and PDEaseI. The FYVE zinc-finger domain is characteristic of proteins recruited to phosphatidylinosytol 3-phosphate-containing membranes, whereas the two others are characteristic of phosphohydrolases and members of the cyclic nucleotide phosphodiesterases. Sequence analysis shows all characteristic domains present at the type-4 phosphodiesterases specific for cAMP. Moreover, TcPDE4 shows the inhibition profile characteristic for PDE4 subfamily, with an IC50 of 10.46 microM for rolipram and 1.3 microM for etazolate. TcPDE4 is able to complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. The enzyme is specific for cAMP, Mg(2+)-dependent and its activity is not affected by cGMP or Ca(2+). The association of TcPDE4 with membranes was studied by subcellular fractionation of recombinant yeast and extraction in several conditions. Most of the enzyme remained associated to the membrane fraction after treatment with high salt concentration, detergent, or chaotropic agents. This support previous hypotheses that in this parasite cAMP phosphodiesterases, and consequently cAMP levels, are compartmentalized.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Membrana Celular/enzimologia , Trypanosoma cruzi/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA de Protozoário/análise , Etazolato/farmacologia , Cinética , Dados de Sequência Molecular , Inibidores de Fosfodiesterase/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rolipram/farmacologia , Alinhamento de Sequência , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Trypanosoma cruzi/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/análise , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Clonagem Molecular , Flagelos/enzimologia , Componentes do Gene , Teste de Complementação Genética , Microscopia Confocal , Dados de Sequência Molecular , Frações Subcelulares/química , Leveduras/enzimologia , Leveduras/genéticaRESUMO
L-Arginine plays an essential role in the energetic metabolism of Trypanosoma cruzi. In this work we propose a relationship between L-arginine uptake, arginine kinase activity and the parasite replication ability. In epimastigote cultures L-arginine uptake decreases continuously accompanying a cell replication rate reduction. The use of conditioned or fresh medium mimics uptake variations. Interestingly, in non-replicative trypomastigote cells, L-arginine uptake was undetectable. The association between L-arginine uptake and cell replication was demonstrated using the antimitotic agent hydroxyurea. Arginine kinase, the enzyme responsible for phosphoarginine and ATP synthesis, also shows a differential activity in epimastigote and trypomastigote parasite stages.
Assuntos
Arginina/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Arginina Quinase/metabolismo , Transporte Biológico , Estágios do Ciclo de Vida , Trypanosoma cruzi/metabolismoRESUMO
Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5-fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.
Assuntos
Arginina Quinase/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Arginina Quinase/biossíntese , Arginina Quinase/genética , Soluções Tampão , Meios de Cultura/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Transfecção , Trypanosoma cruzi/enzimologiaRESUMO
A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.
Assuntos
Genes de Protozoários , Proteínas Serina-Treonina Quinases/genética , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Clonagem Molecular , Globinas/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Trypanosoma cruzi/genéticaRESUMO
A poly-zinc finger protein, designated PZFP1 was identified in Trypanosoma cruzi for the first time. The protein has 191 amino acids, contains seven motifs Cys(X)(2)Cys(X)(4)His(X)(4)Cys. A recombinant PZFP1 was generated in E. coli and the expected 21kDa polypeptide co-purified with two other inducible products of about 42 and 63kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41kDa. Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins. The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis. PZFP1 interacted specifically with a T. cruzi serine-arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.
Assuntos
Proteínas de Protozoários , Trypanosoma cruzi/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA de Protozoário/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
A novel serine-arginine-rich protein designated TcSR was identified in Trypanosoma cruzi. The deduced amino acid sequence reveals that TcSR is a member of the SR protein family of splicing factors that contains two RNA-binding domains at the N-terminal side and several serine-arginine repeats at the COOH-terminus. Over expression of either TcSR or the human SR-protein associated splicing factor/splicing factor 2 (ASF/SF2) in wild-type Schizosaccharomyces pombe, provoked an elongated phenotype similar to that of fission yeast over expressing the SR-containing splicing factor Prp2, a U2AF(65) orthologue. When a double mutant strain lacking two SR protein-specific protein kinases was used, expression of TcSR or human SR ASF/SF2 splicing factor reverted the mutant to a wild-type phenotype. Transient expression of TcSR in HeLa cells stimulated the inclusion of the EDI exon of human fibronectin in an in vivo functional alternative cis-splicing assay. Inclusion was dependent on a splicing enhancer sequence present in the EDI exon. In addition, TcSR and peptides carrying TcSR-RS domain sequences were phosphorylated by a human SR protein kinase. These results indicate that TcSR is a member of the SR splicing network and that some components common to the trans- and cis-splicing machineries evolved from the early origins of the eukaryotic lineage.
Assuntos
Arginina/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Splicing de RNA , Serina/análise , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Células HeLa , Humanos , Proteínas de Protozoários/genética , Schizosaccharomyces/genética , Alinhamento de Sequência , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/fisiologiaRESUMO
Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H(2)O(2) or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways.
Assuntos
Morte Celular , Reparo do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Morte Celular/fisiologia , Núcleo Celular/metabolismo , Dano ao DNA , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologiaRESUMO
Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP(+)/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.
Assuntos
Vias Biossintéticas/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Esteróis/biossíntese , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animais , Membrana Celular/química , Expressão Gênica , NADP/análise , NADPH-Ferri-Hemoproteína Redutase/genética , Organelas/enzimologia , Fagocitose , Trypanosoma cruzi/químicaRESUMO
Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the "open field test" and the "nose-poking preference test", we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop.
Assuntos
Odorantes/análise , Tubérculos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/metabolismo , Análise de Variância , Animais , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Cor , Comportamento Exploratório/fisiologia , Feminino , Análise de Alimentos , Preferências Alimentares/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Percepção Olfatória/fisiologia , Oxirredução , Tubérculos/química , Tubérculos/genética , Plantas Geneticamente Modificadas/genética , Olfato/fisiologia , Solanum tuberosum/genéticaRESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.
Assuntos
Endocitose , Fosfatidilinositol 3-Quinases/metabolismo , Trypanosoma cruzi/enzimologia , Equilíbrio Hidroeletrolítico , Animais , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Pirofosfatase Inorgânica/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestruturaRESUMO
The trypanocidal activity of catechins on Trypanosoma cruzi bloodstream trypomastigotes has been previously reported. Herein, we present the effect of epigallocatechin gallate (EGCg) on parasitemia and survival in a murine model of acute Chagas' disease as well as on the epimastigote form of the parasite. Upon intraperitoneal administration of daily doses of 0.8 mg/kg/day of EGCg for 45 days, mice survival rates increased from 11% to 60%, while parasitemia diminished to 50%. No side effects were observed in EGCg-treated animals. Fifty percent inhibition of epimastigotes growth was achieved with 311 microM EGCg 120 h after drug addition. No lysis, total culture growth inhibition or morphological changes were observed upon addition of 1-3mM EGCg at 24 h. This treatment also produced oligosomal fragmentation of epimastigotes DNA, suggesting a programmed cell death (PCD)-like process. All these findings point out EGCg as a potential new lead compound for chemotherapy of Chagas' disease.
Assuntos
Catequina/análogos & derivados , Doença de Chagas/tratamento farmacológico , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Antioxidantes/toxicidade , Catequina/farmacologia , Catequina/uso terapêutico , Catequina/toxicidade , Doença de Chagas/parasitologia , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Distribuição Aleatória , Tripanossomicidas/uso terapêutico , Tripanossomicidas/toxicidade , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Because of the use of alternate exons 1, mammals express two distinct forms of Gsalpha-subunits: the canonical 394-aa Gsalpha present in all tissues and a 700+-aa extra-long alphas (XLalphas) expressed in a more restricted manner. Both subunits transduce receptor signals into stimulation of adenylyl cyclase. The XL exon encodes the XL domain of XLalphas and, in a parallel ORF, a protein called Alex. Alex interacts with the XL domain of XLalphas and inhibits its adenylyl cyclase-stimulating function. In mice, rats, and humans, the XL exon is thought to contribute 422.3, 367.3, and 551.3 codons and to encode Alex proteins of 390, 357, and 561 aa, respectively. We report here that the XL exon is longer than presumed and contributes in mice, rats, and humans, respectively, an additional 364, 430, and 139 codons to XLalphas. We called the N-terminally extended XLalphas extra-extra-long Gsalpha, or XXLalphas. Alex is likewise longer. Its ORF also remains open in the 5' direction for approximately 2,000 nt, giving rise to Alex-extended, or AlexX. RT-PCR of murine total brain RNA shows that the entire XXL domain is encoded in a single exon. Furthermore, we discovered two truncated forms of XXLalphas, XXLb1 and XXLb2, in which, because of alternative splicing, the Gsalpha domain is replaced by different sequences. XXLb proteins are likely to be found as stable dimers with AlexX. The N-terminally longer proteins may play regulatory roles.
Assuntos
Éxons , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades Proteicas/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Alinhamento de SequênciaRESUMO
This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.
Assuntos
Arginina Quinase/metabolismo , Trypanosoma brucei brucei/enzimologia , África , Animais , Arginina Quinase/genética , Clonagem Molecular , Metabolismo Energético , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , América do Sul , Especificidade da Espécie , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosomatina/enzimologia , Trypanosomatina/metabolismoRESUMO
Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.