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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Adenylate cyclase catalytic subunits from Neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with N-ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing MgATP. Maximal activation was observed at 550 urn. Transducin-depleted retinal membranes were not capable of reconstituting the heterologous light-stimulated adenylate cyclase system. Addition of a transducin preparation to depleted membranes restored the reconstitution capacity of these membranes. A similar heterologous adenylate cyclase system was reconstituted with Neurospora and mouse retinal whole membranes (pretreated with N-ethyhnaleimide). Membranes from mice suffering photoreceptor degeneration (rd homozygotes) did not reconstitute an heterologous adenylate cyclase system. [ABSTRACT FROM AUTHOR]

Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterotogous lutropin receptor-adrenal adenylate cyclase complexes were formed. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose)polymerase (PARP) is a nuclear enzyme present in most eukaryotes. Its activity depends on DNA strand breaks and has been involved in processes such as DNA replication, DNA repair and gene expression. Here we describe for the first time the cloning and characterization of a poly(ADP-ribose)polymerase from Trypanosoma cruzi (TcPARP). This enzyme absolutely required DNA for catalytic activity and nicked DNA strongly enhanced it. T. cruzi purified histones increased TcPARP activity and the covalent attachment of [32P]ADPribose to these proteins was demonstrated. The enzyme was active in the absence of divalent cations. It was inhibited by 3-aminobenzamide, nicotinamide, theophylline, thymidine and histamine, but not by menadione. We demonstrated that TcPARP automodification resulted inhibitory. TcPARP was localized into the cell nucleus. PAR synthesis was confirmed in vivo by indirect immunofluorescence with anti-PAR antibodies. When T. cruzi epimastigotes were exposed to DNA damaging agents, PAR formation drastically increased in the nucleus. ADPribose) glyeohydrolase, in T. cruzi genome databases. These results suggest an analogy with the known poly(ADP-ribose) metabolism in higher eukaryotes and point out a physiological role for PARP in trypanosomatid DNA repair. [ABSTRACT FROM AUTHOR]

Abstract: 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±6.5μM). 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. [Copyright &y& Elsevier]

Abstract: 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 10.46μM for rolipram and 1.3μM for etazolate. TcPDE4 is able to complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. The enzyme is specific for cAMP, Mg2+-dependent and its activity is not affected by cGMP or Ca2+. 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 compartimentalized. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 0.8mg/kg/day of EGCg for 45days, 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 311μM EGCg 120h after drug addition. No lysis, total culture growth inhibition or morphological changes were observed upon addition of 1–3mM EGCg at 24h. 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. [Copyright &y& Elsevier]

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)2Cys(X)4His(X)4Cys. A recombinant PZFP1 was generated in E. coli and the expected 21 kDa polypeptide co-purified with two other inducible products of about 42 and 63 kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41 kDa. 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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 U2AF65 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. [Copyright &y& Elsevier]

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.

The trypanocidal action of green tea catechins against two different developmental stages of Trypanosoma cruzi is reported for the first time. This activity was assayed with the nonproliferative bloodstream trypomastigote and with the intracellular replicative amastigote parasite forms. An ethyl acetate fraction from Camellia sinensis green tea leaves, which contains most of the polyphenolic compounds and the maximal trypanocidal activity, was obtained by fractionation of the aqueous extract with organic solvents. The active compounds present in this extract were further purified by LH-20 column chromatography and were identified by high-performance liquid chromatography analysis with a photo diode array detector and gas chromatography coupled to mass spectroscopy. The following flavan-3-ols derivatives, known as catechins, were identified: catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate. The purified compounds lysed more than 50% of the parasites present in the blood of infected BALB/c mice at concentrations as low as 0.12 to 85 pM. The most active compounds were gallocatechin gallate and epigallocatechin gallate, with minimal bactericidal concentrations that inhibited 50% of isolates tested of 0.12 and 0.53 pM, respectively. The number of amastigotes in infected Vero cells decreased by 50% in the presence of each of these compounds at 100 nM. The effects of the catechins on the recombinant T. cruzi arginine kinase, a key enzyme in the energy metabolism of the parasite, were assayed. The activity of this enzyme was inhibited by about 50% by nanomolar concentrations of catechin gallate or gallocatechin gallate, whereas the other members of the group were less effective. On the basis of these results, we suggest that these compounds could be used to sterilize blood and, eventually, as therapeutic agents for Chagas' disease.

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.