Alterações estruturais in vivo dos isômeros todo-trans, 9-cis e 13-cis do beta-caroteno

Com o objetivo de verificar alterações estruturais nos isômeros todo-trans, 9- e 13-cis do beta-caroteno foi realizado um ensaio biológico baseado no modelo de esgotamento das reservas hepáticas de carotenóides em ratos. Animais depletados desses carotenóides receberam, durante quinze dias, os isômeros puros todo-trans, 9-cis e 13-cis do beta-caroteno. Ao final deste período, verificou-se a ocorrência de re-isomerização in vivo desses isômeros, a partir da quantificação dos mesmos depositados no fígado dos animais. Foi observada re-isomerização do 9-cis em todo-trans, do todo-trans em 9-cis, do 13-cis em 9-cis e todo-trans. O 13-cis foi mais susceptível à isomerização que o 9-cis, pois este último passou a todo-trans e nunca a 13-cis. Já o 13-cis, tanto pode se transformar em 9-cis quanto em todo-trans.

Identificação de bergenina e carotenóides no fruto de uchi (Endopleura uchi, Humiriaceae)

O extrato obtido em metanol da polpa de frutos Endopleura uchi foi submetido a fracionamento utilizando-se técnicas cromatográficas convencionais levando ao isolamento de bergenina. Na análise da composição dos carotenóides foi evidenciada a predominância de beta-caroteno (16,57 mig.g-1). Os isômeros do beta-caroteno foram detectados, trans-beta-caroteno (89,3%), 13-cis-beta-caroteno (8%) e 9-cis-beta-caroteno (3%). Considerando a importância do papel nutricional dos carotenóides e a bioatividade do glicosídeo bergenina, esse estudo sugeriu o potencial desse fruto como alimento funcional.

Role of sensory nervous system vasoactive peptides in hypertension

The goal of the present research was to elucidate the roles and mechanisms by which the sensory nervous system, through the actions of potent vasodilator neuropeptides, regulates cardiovascular function in both the normal state and in the pathophysiology of hypertension. The animal models of acquired hypertension studied were deoxycorticosterone-salt (DOC-salt), subtotal nephrectomy-salt (SN-salt), and Nomega-nitro-L-arginine methyl ester (L-NAME)-induced hypertension during pregnancy in rats. The genetic model was the spontaneously hypertensive rat (SHR). Calcitonin gene-related peptide (CGRP) and substance P (SP) are potent vasodilating neuropeptides. In the acquired models of hypertension, CGRP and SP play compensatory roles to buffer the blood pressure (BP) increase. Their synthesis and release are increased in the DOC-salt model but not in the SN-salt model. This suggests that the mechanism by which both models lower BP in SN-salt rats is by increased vascular sensitivity. CGRP functions in a similar manner in the L-NAME model. In the SHR, synthesis of CGRP and SP is decreased. This could contribute to the BP elevation in this model. The CGRP gene knockout mouse has increased baseline mean arterial pressure. The long-term synthesis and release of CGRP is increased by nerve growth factor, bradykinin, and prostaglandins and is decreased by alpha2-adrenoreceptor agonists and glucocorticoids. In several animal models, sensory nervous system vasoactive peptides play a role in chronic BP elevation. In the acquired models, they play a compensatory role. In the genetic model, their decreased levels may contribute to the elevated BP. The roles of CGRP and SP in human hypertension are yet to be clarified.

CONFIRMAÇÃO DA IDENTIDADE DA alfa-CRIPTOXANTINA E INCIDÊNCIA DE CAROTENÓIDES MINORITÁRIOS PROVITAMÍNICOS A EM VERDURAS FOLHOSAS VERDES

Numerosos trabalhos comprovaram que os carotenóides principais de folhas verdes são invariavelmente luteína, beta-caroteno, violaxantina e neoxantina. No entanto, há discordância em torno dos carotenóides minoritários. Portanto, a espectrometria de massas por impacto de elétrons e cromatografia líquida de alta eficiência com detector de arranjo de diodos foram utilizados para confirmar a identidade de carotenóides minoritários com atividade provitamínica A em verduras folhosas brasileiras. Os carotenóides pró-vitamínicos A, incluindo os isômeros cis e trans de beta-caroteno, foram separados em coluna de C18 polimérica, Vydac 201TP54, com metanol/água (98:2) como fase móvel. Os espectros UV-visível e de massas confirmaram o carotenóide monoidroxilado como sendo alfa-criptoxantina e não beta-criptoxantina como aponta a literatura internacional. Todas as onze folhas analisadas (agrião, alface crespa, alface lisa, almeirão, caruru, chicória, couve, espinafre, rúcula, salsinha e taioba) apresentaram alfa-criptoxantina, 13-cis-beta-caroteno e 9-cis-beta-caroteno, enquanto que alfa-caroteno foi encontrado em apenas quatro folhas (caruru, couve, salsinha e taioba).

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni.

Candidate gene analysis of ocular toxoplasmosis in Brazil: evidence for a role for toll-like receptor 9 (TLR9)

Toxoplasma gondii infection is an important mediator of ocular disease in Brazil more frequently than reported from elsewhere. Infection and pathology are characterized by a strong proinflammatory response which in mice is triggered by interaction of the parasite with the toll-like receptor (TLR)/MyD88 pathway. A powerful way to identify the role of TLRs in humans is to determine whether polymorphisms at these loci influence susceptibility to T. gondii-mediated pathologies. Here we report on a small family-based study (60 families; 68 affected offspring) undertaken in Brazil which was powered for large effect sizes using single nucleotide polymorphisms with minor alleles frequencies > 0.3. Of markers in TLR2, TLR5 and TLR9 that met these criteria, we found an association Family Based Association Tests [(FBAT) Z score = 4.232; p = 1.5 x 10-5; p corrected = 1.2 x 10-4] between the C allele (frequency = 0.424; odds ratio = 7; 95% confidence interval 1.6-30.8) of rs352140 at TLR9 and toxoplasmic retinochoroiditis in Brazil. This supports the hypothesis that direct interaction between T. gondii and TLR9 may trigger proinflammatory responses that lead to severe pathologies such as the ocular disease that is associated with this infection in Brazil.

Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum). Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9) in transgenic tomato plants from the T0 and T1 generations.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Polimorfismos do gene NRAMP1 em indivíduos com reações hansênicas, atendidos em dois Centros de Referência no Recife, nordeste do Brasil

INTRODUÇÃO: Para investigar susceptibilidade às reações hansênicas, três polimorfismos do gene natural resistance-associated macrophage protein (NRAMP1), foram determinados em 201 indivíduos, atendidos em dois centros de referência no Recife, entre 2007 e 2008, sendo 100 paucibacilares e 101 multibacilares. MÉTODOS: A determinação dos polimorfismos 274C/T, D543N e 1729+55del4 do gene NRAMP1 foi realizada utilizando a técnica do polimorfismo de fragmento de restrição em DNA extraído de sangue periférico e as estimativas das freqüências alélicas e genotípicas foram feitas por contagem direta. RESULTADOS: Os genótipos predominantes foram: CC (51,8%) para 274C/T, GG (86,6%) para D543N e +-TGTG (59,9%) para 1729+55del4. O genótipo mutante 274 TT predominou na negatividade da reação reversa (p=0,03) e na positividade do eritema nodoso (p=0,04). CONCLUSÕES: Nossos resultados sugerem que o polimorfismo 274 C/T do gene NRAMP1 pode auxiliar na determinação da susceptibilidade à reação tipo II em indivíduos com hanseníase.

Distribution of QPY and RAH haplotypes of granzyme B gene in distinct Brazilian populations

INTRODUCTION: The cytolysis mediated by granules is one of the most important effector functions of cytotoxic T lymphocytes and natural killer cells. Recently, three single nucleotide polymorphisms (SNPs) were identified at exons 2, 3, and 5 of the granzyme B gene, resulting in a haplotype in which three amino acids of mature protein Q48P88Y245 are changed to R48A88H245, which leads to loss of cytotoxic activity of the protein. In this study, we evaluated the frequency of these polymorphisms in Brazilian populations. METHODS: We evaluated the frequency of these polymorphisms in Brazilian ethnic groups (white, Afro-Brazilian, and Asian) by sequencing these regions. RESULTS: The allelic and genotypic frequencies of SNP 2364A/G at exon 2 in Afro-Brazilian individuals (42.3% and 17.3%) were significantly higher when compared with those in whites and Asians (p < 0.0001 and p = 0.0007, respectively). The polymorphisms 2933C/G and 4243C/T also were more frequent in Afro-Brazilians but without any significant difference regarding the other groups. The Afro-Brazilian group presented greater diversity of haplotypes, and the RAH haplotype seemed to be more frequent in this group (25%), followed by the whites (20.7%) and by the Asians (11.9%), similar to the frequency presented in the literature. CONCLUSIONS: There is a higher frequency of polymorphisms in Afro-Brazilians, and the RAH haplotype was more frequent in these individuals. We believe that further studies should aim to investigate the correlation of this haplotype with diseases related to immunity mediated by cytotoxic lymphocytes, and if this correlation is confirmed, novel treatment strategies might be elaborated.

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence

Many lines of Plasmodium falciparum undrgo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Sex pheromone and period gene characterization of Lutzomyia longipalpis sensu lato (Lutz & Neiva) (Diptera: Psychodidae) from Posadas, Argentina

Lutzomyia longipalpis s.l. is the primary vector of Leishmania (L.) infantum in the New World. In this study, male Lutzomyia longipalpis specimens from Posadas, Argentina were characterized for two polymorphic markers: the male sex pheromone and the period (per) gene. The male sex pheromone was identified as (S)-9-methylgermacrene-B, the same compound produced by Lu. longipalpis from Paraguay and many populations from Brazil. The analysis of per gene sequences revealed that the population from Argentina is significantly differentiated from previously studied Brazilian populations. Marker studies could contribute to the understanding of the distribution and spread of urban American visceral leishmaniasis, thus aiding in the design of regional surveillance and control strategies.

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species

A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.