Rosane Maria Temporal

Proteinase activities during temperature-induced stage differentiation of species complexes of Leishmania

We have examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin, bovine serum albumin (BSA) or human IgG as substrate, proteinase activities in cell lysates from selected species complexes of Leishmania. The inhibition of proteinase activity caused by the reagent L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64), which is known to act only on cysteinyl proteinases, revealed a 31 kDa component of this class of enzymes in soluble, but not in membrane-enriched preparations, of either L. amazonensis or L. major-like parasites from the New World. The proteinase component was detectable in the leishmanial multiplicative promastigote stage (log phase) and its concentration apparently increased during the thermally induced transformation of promastigotes to amastigote-like forms in vitro. Comparative studies revealed that taxonomically distinct species complexes of Leishmania possess high amastigote cysteine proteinase activity. This feature, however, was lacking in other developmental stages of the species (L. braziliensis, L. chagasi, L. aethiopica, and L. donovani) analyzed. Furthermore, lesion amastigotes of L. amazonensis displayed ultrastructurally recognizable megasomes, but megasome-like or large multivesicular body organelles could be detected only in axenic amastigotes of both L. amazonensis and L. major-like species.

Amidine Derivatives and Leishmania amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction

Previous work has demonstrated that N-N′-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infections supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs.

Efficacy of a diarylheptanoid derivative against Leishmania amazonensis

The activity of several diarylheptanoid derivatives (curcuminoids) was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was the [1-(4-methoxy-phenyl)-7-(3,4-methoxy-4-hydroxy-phenyl)-1,6-heptadien-3, 5-dione]. This derivative was chosen to be assayed in vivo in a treatment trial. For these experiments, the curcuminoid compound was used in a concentration equivalent to the IC50/24 h, obtained from the previous study. Balb/c mice were inoculated subcutaneously in the footpad with L. amazonensis infective promastigotes and 4 weeks after the inoculation, the animals were treated with different schemes, varying from 1 to 3 doses. In all the experiments, Pentamidine Isethionate was used as reference drug under the same experimental conditions. The results showed that one dose was not enough to heal the lesion, however, with 2 and 3 doses the efficiency of the assayed compound was clear. On the other hand, treatment with Pentamidine Isethionate using the three different schemes was not satisfactory when compared to the curcuminoid derivative.

Effects of temperature on promastigotes of several species of Leishmania.

ABSTRACT. Six Leishmania species were studied comparatively, in order to determine the influence of temperature “in vitro” on differentiation, infectivily and protein synthesis. Differentiation ocurred in a heterogeneous manner, even in species that produce similar clinical manifestations. Thus, no association could be found between thermosensitivity and disease. The association between expression of proteins and increasing temperatures was analyzed at 34° C by polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS‐PAGE), using different incubation times, and employing a technique involving metabolic incorporation of [35S]‐methionine. Protein synthesis was very similar in all the New World species apart from L. amazonensis, which expressed a protein of approximately 80 kDa when incubated at 34° C for 2 hours. All the tested species had in common the expression of a 70 kDa protein. Differences, however, were observed in relation to the time interval for protein expression. in L. chagasi, synthesis was detected after 30 minutes of incubation at 34° C, while L. braziliensis required 1 hour at the same temperature. The “in vivo” and “in vitro” infectivity of the differentiated forms was also analyzed, but no significant differences were observed.

An Effective Diaryl Derivative Against Leishmania amazonensis and its Influence on the Parasite X Macrophage Interaction

The activity of several diarylheptanoid derivatives (curcuminoids) was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was 5-hydroxy-7- (4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)-1,4,6-heptatrien-3-one. This study was carried out to investigate the influence of this diaryl derivative on the infective promastigotes and Balb/c mice peritoneal macrophage interaction. The potential in vitro toxicity was also evaluated. Promastigotes pretreated for 24 hours with the compound had their infective capacity significantly decreased. When the infection of Balb/c macrophage by L. amazonensis promastigotes was already installed, addition of the drug resulted in a diminishing of the infection rate. It was demonstrated that the compound was not toxic to the host macrophage in a concentration equivalent to the LD50/24 h from the previous in vitro experiment.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center

Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.