Atividades biológicas e composição química dos extratos brutos de Chresta exsucca

As atividades tripanocida, leishmanicida, antibacteriana e antifungica dos extratos brutos de Chresta exsucca foram investigadas. Formas tripomastigotas do Trypanosoma cruzi, formas amastigotas de Leishmania amazonensis e vinte cepas de microrganismos, incluindo bactérias Gram-positivas, Gram-negativas e leveduras, foram utilizadas nos ensaios biológicos. Os melhores resultados foram obtidos para a atividade leishmanicida. A composição química dos extratos hexânicos e etanólicos dessa espécie foi determinada empregando-se técnicas cromatográficas como HRGC e HPLC-ESI-MS, respectivamente. Esteróides, triterpenos e flavonóides foram identificados.Crude extracts of Chresta exsucca were investigated for their in vitro trypanocidal, leishmanicidal, antibacterial and antifungal activities. Trypomastigote forms of Trypanosoma cruzi, amastigote-like forms of Leishmania amazonensis and twenty strains of microorganisms including Gram-positive and Gram-negative bacteria and yeasts were utilized in the bioassays. The best results were found for the leishmanicidal activity. The chemical composition of hexanic and ethanolic extracts of this species was determinate using chromatographic techniques as HRGC and HPLC-ESI-MS, respectively. Steroids, triterpenes and flavonoids were identified

Disposable versus reusable hypodermic syringes: study of feasible effects upon the virus of the live, attenuated measles vaccine

The study was performed in the State of S.Paulo, SP, Brazil, with the purpose of finding out whether reusable (glass) and disposable hypodermic syringes used for administration of live attenuated measles vaccines would interfere with their virus. At least six different experiments using two distinct lots of vaccines were carried out. Each time, a lot was reconstituted with reusable and disposable syringes in parallel to form separate pools from which samples were collected hourly from zero to the sixtieth hour after reconstitution for virus titration in monolayers of Vero cells. The straight line regression system chosen for the analysis of the results demonstrated that although vaccines reconstituted with both types of syringes presented a statistically significative titer decrease as time went by, there was a more pronounced decrease for vaccines manipulated with the glass syringes. The fact that the disposable syringes affected the titer of the virus present in the live, attenuated measles vaccine less confirmed that the preconization and routine usage of this type of syringe by the Health Department of the State of S.Paulo, Brazil, is ideal and highly recommended because it preserves the vaccine from reconstitution to administration better, and thus, its efficacy in preventing the infection.Objetivou-se verificar entre seringas hipodérmicas descartáveis e reutilizáveis qual interfere mais com o vírus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituídas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos à temperatura de +2 a+8°C e protegidos da luz. De cada lote foram realizadas, no mínimo, seis titulações em paralelo, com amostragem a cada hora, de zero a seis horas após reconstituição. A análise estatística dos resultados obtidos nas titulações, feita pelo sistema de retas de regressão demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decréscimo de título estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituídas com seringas reutilizáveis. A menor interferência das descartáveis no título da vacina viva, atenuada contra o sarampo, demonstrou que a preconização e uso desse tipo de seringas pela Secretaria de Estado da Saúde de São Paulo é o ideal e recomendável, por preservar mais a vacina desde a reconstituição até sua administração e, conseqüentemente, a sua eficácia na prevenção dessa infecção

Seringas hipodérmicas descartáveis versus reutilizáveis: estudo de possíveis efeitos sobre o vírus da vacina viva, atenuada contra o sarampo Disposable versus reusable hypodermic syringes: study of feasible effects upon the virus of the live, attenuated measles vaccine

Objetivou-se verificar entre seringas hipodérmicas descartáveis e reutilizáveis qual interfere mais com o vírus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituídas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos à temperatura de +2 a+8°C e protegidos da luz. De cada lote foram realizadas, no mínimo, seis titulações em paralelo, com amostragem a cada hora, de zero a seis horas após reconstituição. A análise estatística dos resultados obtidos nas titulações, feita pelo sistema de retas de regressão demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decréscimo de título estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituídas com seringas reutilizáveis. A menor interferência das descartáveis no título da vacina viva, atenuada contra o sarampo, demonstrou que a preconização e uso desse tipo de seringas pela Secretaria de Estado da Saúde de São Paulo é o ideal e recomendável, por preservar mais a vacina desde a reconstituição até sua administração e, conseqüentemente, a sua eficácia na prevenção dessa infecção.The study was performed in the State of S.Paulo, SP, Brazil, with the purpose of finding out whether reusable (glass) and disposable hypodermic syringes used for administration of live attenuated measles vaccines would interfere with their virus. At least six different experiments using two distinct lots of vaccines were carried out. Each time, a lot was reconstituted with reusable and disposable syringes in parallel to form separate pools from which samples were collected hourly from zero to the sixtieth hour after reconstitution for virus titration in monolayers of Vero cells. The straight line regression system chosen for the analysis of the results demonstrated that although vaccines reconstituted with both types of syringes presented a statistically significative titer decrease as time went by, there was a more pronounced decrease for vaccines manipulated with the glass syringes. The fact that the disposable syringes affected the titer of the virus present in the live, attenuated measles vaccine less confirmed that the preconization and routine usage of this type of syringe by the Health Department of the State of S.Paulo, Brazil, is ideal and highly recommended because it preserves the vaccine from reconstitution to administration better, and thus, its efficacy in preventing the infection

Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruziepimastigotes

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle

The glutamine synthetase of <i>Trypanosoma cruzi</i> is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

<div><p><i>Trypanosoma cruzi</i>, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the <i>T</i>. <i>cruzi</i> parasite, but their consumption produces an accumulation of NH₄⁺ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene <i>Tc</i>GS from <i>T</i>. <i>cruzi</i> encodes a functional glutamine synthetase; it can complement a defect in the <i>GLN1</i> gene from <i>Saccharomyces cerevisiae</i> and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH₄⁺ production is predictable. During host-cell invasion, <i>Tc</i>GS is required for the parasite to escape from the parasitophorous vacuole, a process <i>sine qua non</i> for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.</p></div

MS as a GS inhibitor.

<p>(A) The extract of epimastigotes (filled circles) and the recombinant enzyme <i>Tc</i>GS (filled squares) were both incubated with different MS concentrations for 30 min. Then, the specific activity was measured and the inhibition percentage was calculated. The values were adjusted to a dose-response curve (IC₅₀ for cell-free extracts: 19.66 ± 0.37 μM, IC₅₀ for <i>Tc</i>GS: 14.70 ± 0.54 μM). The data refer to three independent experiments. (B) GS activity was measured with different MS concentrations for 30 min; at the same time, it was measured in different glutamate concentrations. The data were adjusted to a Michaelis-Menten equation. (C) The K_M values for each MS concentration were compared. (D) The relationship between K_M/V_max per MS concentration revealed the inhibitory constant value (K_i = 4.12 ± 0.21). Statistical analysis were made using one-way ANOVA / Dunnet’a Multiple Comparision Test. *p<0.05, **p<0.01, ***p<0.001. Error bars represent standard deviation (n = 3).</p

Yeast functional complementation assay.

<p><i>Saccharomyces cerevisiae</i> SAH35 yeast with an endogenous glutamine synthetase gene controlled by the <i>GAL1</i> gene promoter was transformed with an empty plasmid, a copy of <i>S</i>. <i>cerevisiae</i> GS gene or the <i>T</i>. <i>cruzi</i> gene (p416, p416-<i>Sc</i>GLN1 and p416-<i>Tc</i>GS, respectively) and plated at different dilutions (a and e: 10⁴ yeasts; b and f: ≈10³ yeasts; c and g: ≈100 yeasts d and h: ≈10 yeasts). The endogenous <i>ScGLN1</i> gene is not transcribed in a defined medium with glutamate as a <i>ScGLN1</i> non-repressible nitrogen source and with glucose as a carbon source. Furthermore, when glutamine is supplied, compensation of the glutamine biosynthesis pathway occurs; GS is not required in this case, and it is not essential for yeast clones (line 1—a to d). However, the empty vector transformed yeast do not grow in a medium without glutamine (line 1—e to h). The yeast recover the capacity to proliferate when they are transformed with the same gene (<i>ScGLN1</i>) or with GS from <i>T</i>. <i>cruzi</i>.</p