The evolution of purinergic receptors involved in recognition of a blood meal by hematophagous insects

Many blood feeders use adenine nucleotides as cues for locating blood meal. Structure-activity relationship of adenine nucleotides as phagostimulants varies between closely-related species of blood feeders. It is suggested that a preexisting diverse pool of nucleotide-binding proteins present in all living cells, serves as a source of receptor proteins for the gustatory receptors involved in blood detection. It is proposed that the selection of any such nucleotide-binding protein is random.

Compositional Constraints in the Extremely GC-poor Genome of Plasmodium falciparum

We have analyzed the compositional properties of coding (protein encoding) and non-coding sequences of Plasmodium falciparum, a unicellular parasite characterized by an extremely AT-rich genome. GC% levels, base and dinucleotide frequencies were studied. We found that among the various factors that contribute to the properties of the sequences analyzed, the most relevant are the compositional constraints which operate on the whole genome

Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela

The mosquito Aedes aegypti is the main vector of dengue in Venezuela. The genetic structure of this vector was investigated in 24 samples collected from eight geographic regions separated by up to 1160 km. We examined the distribution of a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among 1144 Ae. aegypti from eight collections. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Seven haplotypes were detected throughout Venezuela and these were sorted into two clades. Significant differentiation was detected among collections and these were genetically isolated by distance.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Genetic variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of multiple introductions

To analyze the genetic relatedness and phylogeographic structure of Aedes aegypti, we collected samples from 36 localities throughout the Americas (Brazil, Peru, Venezuela, Guatemala, US), three from Africa (Guinea, Senegal, Uganda), and three from Asia (Singapore, Cambodia, Tahiti). Amplification and sequencing of a fragment of the mitochondrial NADH dehydrogenase subunit 4 gene identified 20 distinct haplotypes, of which 14 are exclusive to the Americas, four to African/Asian countries, one is common to the Americas and Africa, and one to the Americas and Asia. Nested clade analysis (NCA), pairwise distribution, statistical parsimony, and maximum parsimony analyses were used to infer evolutionary and historic processes, and to estimate phylogenetic relationships among haplotypes. Two clusters were found in all the analyses. Haplotypes clustered in the two clades were separated by eight mutational steps. Phylogeographic structure detected by the NCA was consistent with distant colonization within one clade and fragmentation followed by range expansion via long distance dispersal in the other. Three percent of nucleotide divergence between these two clades is suggestive of a gene pool division that may support the hypothesis of occurrence of two subspecies of Ae. aegypti in the Americas.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansiprecludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b.brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansiis alike to the BSF of T. b. bruceiin glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene. To analyze the genetic variability of populations of Aedes aegypti, 156 samples were collected from 10 municipalities in the state of Paraná, Brazil. A 311 base pairs (bp) region of the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene was examined. An analysis of this fragment identified eight distinct haplotypes. The mean genetic diversity was high (h = 0.702; p = 0.01556). AMOVA analysis indicated that most of the variation (67%) occurred within populations and the F ST value (0.32996) was highly significant. F ST values were significant in most comparisons among cities. The isolation by distance was not significant (r = -0.1216 and p = 0, 7550), indicating that genetic distance is not related to geographic distance. Neighbor-joining analysis showed two genetically distinct groups within Paraná. The DNA polymorphism and AMOVA data indicate a decreased gene flow in populations from Paraná, which can result in increased vectorial competence.

Potencial inseticida de plantas da família Annonaceae

A utilização de plantas para o controle de insetos tem aumentado em todo o mundo. Isto ocorre porque os inseticidas vegetais apresentam moléculas biodegradáveis, são menos tóxicos a mamíferos e potencialmente adequados para utilização no controle de pragas. As plantas da família Annonaceae estão ganhando destaque como biopesticidas por serem naturalmente bioativas, apresentando atividade citotóxica, antitumoral, vermicida, antimicrobiana, imunossupressora, antiemética, inibidora do apetite e crescimento, antimalárica e também inseticida. A atividade inseticida das anonáceas deve-se à presença de acetogeninas, substâncias que atuam nas mitocôndrias, inibindo a NADH - ubiquinona oxidorredutase, causando a morte dos insetos. Nesta revisão, relatamos a utilização de anonáceas no controle de insetos, mostrando que até o momento, apenas 42 espécies de anonáceas possuem informações de atividade inseticida contra pouco mais de 60 espécies de insetos-praga. Estas informações mostram que muita pesquisa ainda é necessária, principalmente para conhecer a atividade inseticida de outras espécies de anonáceas, além de seus efeitos sobre insetos-praga ainda não estudados. Assim, teremos como alternativa para o desenvolvimento sustentável, a utilização de novos inseticidas vegetais, como os obtidos das diferentes espécies de anonáceas, os quais podem atuar como mais uma ferramenta para equilibrar os exageros da agricultura química ou convencional.

Produtos naturais das esponjas marinhas Aaptos sp., Hymeniacidon aff. heliophila, e do nudibrânquio Doris aff. verrucosa

Herein we describe the isolation of homarine and piridiniumbetaine B from the sponge Aaptos sp. Although homarine has a common occurrence among animals, piridiniumbetaine B was only recently isolated from the marine sponge Agelas dispar. The isolation of piridiniumbetaine B from two taxonomically distant marine sponges corroborate previous assumptions that such betaines should be regarded rather as primary metabolites. We have also isolated (9-[5'-(methylthio)-beta-D-xylofuranosyl]adenine (xylosyl-MTA) from the mantle of a nudibranch identified as Doris aff. verrucosa. The occurrence of xylosyl-MTA in the mantle of this animal strongly suggests that it is the same nudibranch species described for the Mediterranean sea. We have been unable to detect any other compound in the mantle extract of D. aff. verrucosa other than xylosil-MTA and sterols. GC-MS analysis of the sterol fraction from the nudibranch and its prey, the sponge Hymeniacidon aff. heliophila, revealed the occurrence of the ubiquitous sterols, cholesterol, brassicasterol, cholestanol, 24-methylcholesterol and 24-ethylcholesterol, as the only common metabolites, therefore precluding any assumption concerning the sequestration of secondary metabolites by the nudibranch from H. aff. heliophila.

Biossensor enzimático para detecção de fungicidas ditiocarbamatos: estudo cinético da enzima aldeído desidrogenase e otimização do biossensor

Initially, all major factors that affect the rate of the AldH-catalyzed reaction (enzyme concentration, substrate concentration, temperature and pH) were investigated. Optimal activity was observed between pH values of 7.5 and 9.5 in the temperature range of 25 to 50 ºC. Kinetic parameters, such as Km (2.92 µmol L-1) and Vmax (1.33 10-2 µmol min-1) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola Blue-Reinecke salt (MBRS) combination. Operational conditions (NAD+ and substrate contents, enzyme loading and response time) were optimized. Also, two enzyme immobilization procedures were tested: entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) and crosslinking with glutaraldehyde. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde and also to construct inhibition curves with maneb and zineb fungicides. Best results were found with the following conditions: [NAD+] = 0.25 mmol L-1; [propionaldehyde] = 80 µmol L-1; enzyme loading = 0.8 U per electrode; response time = 10 min, and inhibition time = 10 min. Current intensities around 103 ± 13 nA with the sensors and good stability was obtained for both immobilization procedures. Detection limits, calculated using 10% inhibition were 31.5 µg L-1 and 35 µg L-1 for maneb and zineb, respectively. Results obtained with other MBRS-modified electrodes consisting of mono and bi-enzymic sensors were compared. The ability to catalyze NADH oxidation by MB was also highlighted.

Structural bioinformatics study of cyclin-dependent kinases complexed with inhibitors

The present work describes molecular models for the binary complexes CDK9, CDK5 and CDK1 complexed with Flavopiridol and Roscovitine. These structural models indicate that the inhibitors strongly bind to the ATP-binding pocket of CDKs and the structural comparison with the complexes CDK2:Flavopiridol and CDK2:Roscovitine correlates the structural differences with differences in inhibition of these CDKs by the inhibitors. These structures open the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures such as flavones and adenine derivatives.