Trypomastigote Forms Research Articles

Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease

To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease. IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T.cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n=72), cardiac (CARD, n=47) and digestive/cardiodigestive (DIG/CARD-DIG, n=12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n=30) and patients with ischaemic cardiomyopathy (ISCH, n=15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations. Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P=0.042) and myosin (P=0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r=0.9508, P=0.0001) between the production of troponin T and myosin autoantibodies. These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy.

NATURAL INFECTION BY Trypanosoma cruzi IN ONE DOG IN CENTRAL WESTERN BRAZIL: A CASE REPORT

SUMMARY It is estimated that about 10 million people are infected with Trypanosoma cruzi worldwide, mostly in Latin America and more than 25 million are at risk of acquiring this infection in endemic areas. Dogs are an important reservoir for this pathogen and thus, considered a risk factor for human populations. This report describes one case of Chagas disease in a dog from Cuiabá, Mato Grosso State, Brazil. The diagnosis was obtained by direct examination of trypomastigote forms in blood smears. Amastigotes forms were visualized in microscopy of the bone marrow, lymph nodes, kidneys, liver and brain. The T. cruzi (ZIII) infection was confirmed by Polymerase Chain Reaction, and sequencing. The animal presented multisystemic failure and died. Although acute Chagas disease in humans is not reported in Cuiabá, this is the first report of a canine case in this region. This case represents a warning, to health professionals and authorities, to the possibility of transmission of this zoonosis in Cuiabá.

Molecular design, synthesis and biological evaluation of 1,4-dihydro-4-oxoquinoline ribonucleosides as TcGAPDH inhibitors with trypanocidal activity

The 1,4-dihydro-4-oxoquinoline ribonucleoside, Neq135, is the first low micromolar trypanosomatidae inhibitor to show good ligand efficiency (0.28kcalmol−1atom−1) and good ligand lipophilicity efficiency (0.37kcalmol−1atom−1) when acting against Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase (TcGAPDH). This and other six ribonucleosides were synthesized using our in-house technology, and assayed against the GAPDH NAD+ site using isothermal titration calorimetry (ITC). Compound Neq135 had acceptable in vitro cytotoxicity, inhibited TcGAPDH with a Kiapp value of 16μM and killed the trypomastigote form of Trypanosoma cruzi Tulahuen strain with a concentration similar to that displayed by the control drug benznidazole. Neq135 is tenfold lower kinetic affinity against hGAPDH and does not kill Balb-c fibroblast nor spleen mouse cells. These results emphasize the possibility of integrating ligand- and target-based designs to uncover potent and selective TcGAPDH inhibitors that expands the opportunity for further medicinal chemistry endeavor towards NAD+ TcGAPDH site.

Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease

It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10−5M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10−6M ShPI-I, in the same periods of time, reduced parasite viability about 91–95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.

Medicinal Plants of Chile: Evaluation of their Anti-Trypanosoma cruzi Activity

The extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 51.2 microg/mL) was purified and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of T. cruzi with IC50 values of 27.9 and 25.1 microM, respectively.

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K+ cycling and to active Na+ extrusion

BackgroundOrthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. Methods32Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na+, H+ and K+ fluxes were also investigated. The transport capacities of different evolutive forms were compared. ResultsEpimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na+. We found that the parasites express TcPho84, a H+:Pi-symporter, and TcPho89, a Na+:Pi-symporter. Both Pi influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na+-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K+ ionophore) or SCH28028 (inhibitor of (H++K+)ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H+ gradient energizes uphill Pi entry and that K+ recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, decreased only the Na+-dependent Pi uptake, indicating that this Na+ pump generates the Na+ gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. ConclusionsPi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na+ or H+/K+ fluxes. General significanceThis study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

Morphological polymorphism of Trypanosoma copemani and description of the genetically diverse T. vegrandis sp. nov. from the critically endangered Australian potoroid, the brush-tailed bettong (Bettongia penicillata (Gray, 1837))

BackgroundThe trypanosome diversity of the Brush-tailed Bettong (Bettongia penicillata), known locally as the woylie, has been further investigated. At a species level, woylies are critically endangered and have declined by 90% since 1999. The predation of individuals made more vulnerable by disease is thought to be the primary cause of this decline, but remains to be proven.MethodsWoylies were sampled from three locations in southern Western Australia. Blood samples were collected and analysed using fluorescence in situ hybridization, conventional staining techniques and microscopy. Molecular techniques were also used to confirm morphological observations.ResultsThe trypanosomes in the blood of woylies were grouped into three morphologically distinct trypomastigote forms, encompassing two separate species. The larger of the two species, Trypanosoma copemani exhibited polymorphic trypomastigote forms, with morphological phenotypes being distinguishable, primarily by the distance between the kinetoplast and nucleus. The second trypanosome species was only 20% of the length of T. copemani and is believed to be one of the smallest recorded trypanosome species from mammals. No morphological polymorphism was identified for this genetically diverse second species. We described the trypomastigote morphology of this new, smaller species from the peripheral blood of the woylie and proposed the name T. vegrandis sp. nov. Temporal results indicate that during T. copemani Phenotype 1 infections, the blood forms remain numerous and are continuously detectable by molecular methodology. In contrast, the trypomastigote forms of T. copemani Phenotype 2 appear to decrease in prevalence in the blood to below molecular detectable levels.ConclusionsHere we report for the first time on the morphological diversity of trypanosomes infecting the woylie and provide the first visual evidence of a mixed infection of both T. vegrandis sp. nov and T. copemani. We also provide supporting evidence that over time, the intracellular T. copemani Phenotype 2 may become localised in the tissues of woylies as the infection progresses from the active acute to chronic phase. As evidence grows, further research will be necessary to investigate whether the morphologically diverse trypanosomes of woylies have impacted on the health of their hosts during recent population declines.

Potential Mechanism of the Anti-trypanosomal Activity of Organoruthenium Complexes with Bioactive Thiosemicarbazones

In the search for new metal-based drugs against diseases produced by trypanosomatid parasites, four organoruthenium(II) compounds [Ru2(p-cymene)2(L)2]X2, where L are bioactive 5-nitrofuryl-containing thiosemicarbazones and X = Cl or PF6, had been previously obtained. These compounds had shown activity on Trypanosoma brucei, the etiological agent of African trypanosomiasis. Because of genomic similarities between trypanosomatides, these ruthenium compounds were evaluated, in the current work, on Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease). Two of them showed significant in vitro growth inhibition activity against the infective trypomastigote form of T. cruzi (Dm28c clone, IC50 = 11.69 and 59.42μM for [Ru2(p-cymene)2(L4)2]Cl2 and [Ru2(p-cymene)2(L1)2]Cl2, respectively, where HL4 = 5-nitrofuryl-N-phenylthiosemicarbazone and HL1 = 5-nitrofurylthiosemicarbazone), showing fairly good selectivities toward trypanosomes with respect to mammalian cells (J774 murine macrophages). Moreover, [Ru2(p-cymene)2(L2)2]Cl2, where HL2 = 5-nitrofuryl-N-methylthiosemicarbazone, was synthesized in order to evaluate the effect of improved solubility on biological behavior. This new chloride salt showed higher activity against T. cruzi than that of the previously synthesized hexafluorophosphate one (Dm28c clone, IC50 = 14.30μM for the former and 231.3μM for the latter). In addition, the mode of antitrypanosomal action of the organoruthenium compounds was investigated. The complexes were not only able to generate toxic free radicals through bioreduction but they also interacted with two further potential parasite targets: DNA and cruzipain, a cysteine protease which plays a fundamental role in the biological cycle of these parasites. The results suggest a "multi-target" mechanism of trypanosomicidal action for the obtained complexes.

Article Synthesis and Trypanocidal Activity of Novel 2,4,5-Triaryl-N-Hydroxylimidazole Derivatives

Herein, we report the design, synthesis and trypanocidal activity of some novel trisubstituted imidazole derivatives. These heterocyclic derivatives were structurally planned by exploring the concept of molecular hybridisation between two arylhydrazones derived from megazol, which has potent trypanocidal activity. The trypanocidal activity of these triarylimidazole derivatives was evaluated against infective trypomastigote forms of T. cruzi and the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol showed moderate biological activity (IC50 = 23.9 µM) when compared to benznidazole, a standard trypanocidal drug. These compounds did not present cytotoxic effects at concentrations near the trypanocidal IC50, being considered a good starting point for the development of new anti-Chagas drug candidates.

Chemical Composition and Anti-Trypanosoma cruzi Activity of Essential Oils Obtained from Leaves of Xylopia frutescens and X. laevigata (Annonaceae)

Essential oils from leaves of Xylopia frutescens (XFMJ) and two specimens of Xylopia laevigata (XLMC and XLSI) were obtained by hydrodistillation using a Clevenger-type apparatus, and analyzed by GC-MS and GC-FID. Sesquiterpenes dominated the essential oils. The main constituents of XFMJ were (E)-caryophyllene (24.8%), bicyclogermacrene (20.8%), germacrene D (17.0%), beta-elemene (7.9%), and (E)-beta-ocimene (6.8%). XLMC contained significant quantities of germacrene D (18.9%), bicyclogermacrene (18.4%), beta-elemene (9.5%), delta-selinene (9.2%), (E)-caryophyllene (8.5%), germacrene B (5.7%) and gamma-muurolene (5.7%), while germacrene D (27.0%), bicyclogermacrene (12.8%), (E)-caryophyllene (8.6%), gamma-muurolene (8.6%), delta-cadinene (6.8%), and germacrene B (6.0%) were the main components of XLSI. The essential oils had trypanocidal activity against the Y strain of Trypanosoma cruzi, with IC50 values lower than 30 microg x mL(-1) and 15 microg x mL(-1) against epimastigote and trypomastigote forms of T. cruzi, respectively, and were also able to reduce the percentage in vitro of T. cruzi-infected macrophages and the intracellular number of amastigotes at concentrations that were non-cytotoxic to macrophages.

Caspase-8 and caspase-9 mediate thymocyte apoptosis in Trypanosoma cruzi acutely infected mice

Trypanosoma cruzi acute infection leads to thymic atrophy, largely as a result of death of immature DP T cells. In a second vein, the glucocorticoid hormone imbalance promotes DP T cell apoptosis in infected mice. Herein, we assessed the involvement of caspase signaling in thymocyte death during T. cruzi acute infection. BALB/c mice were infected i.p. with 10(2) trypomastigote forms of T. cruzi and analyzed from 7 to 19 dpi. Thymocyte apoptosis was observed in early stages of infection, increasing along with time postinfection. Immature DN and DP as well as CD4(+) and CD8(+) thymocytes from infected mice showed increased activation of caspase-8, -9, and -3. In vitro treatment of thymocytes from infected mice with a general caspase inhibitor or the combination of caspase-8- and caspase-9-specific inhibitors increased the number of living thymocytes. Intrathymic injection of the general caspase inhibitor, but not caspase-8 or -9 inhibitors individually, prevented thymic atrophy and thymocyte depletion in infected mice. Moreover, blockade of glucocorticoid receptor activity with RU486 prevented DP thymocyte apoptosis, together with caspase-8 and -9 activation. These findings indicate that DP T cell apoptosis following experimental T. cruzi acute infection is dependent on glucocorticoid stimulation, promoting caspase-8 and -9 activation.

Trypanosoma cruzi: Effects of heat shock on ecto-ATPase activity

In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.

Trypanocidal Activity of Thioamide-Substituted Imidazoquinolinone: Electrochemical Properties and Biological Effects

Three thioamide-substituted imidazoquinolinone, which possess a heterocyclic center similar to tryptanthrin and are named C1, C2, and C3, were studied regarding (a) their in vitro anti-Trypanosoma cruzi activity, (b) their cytotoxicity and electrochemical behaviour, and (c) their effect on cell viability, redox state, and mitochondrial function. The assayed compounds showed a significant activity against the proliferative forms, but only C1 showed activity on the trypomastigote form (for C1, IC50 epi = 1.49 μM; IC50 amas = 1.74 μM; and IC50 try = 34.89 μM). The presence of an antioxidant compound such as ascorbic acid or dithiotreitol induced a threefold increase in the antiparasitic activity, whereas glutathione had a dual effect depending on its concentration. Our results indicate that these compounds, which exhibited low toxicity to the host cells, can be reduced inside the parasite by means of the pool of low molecular weight thiols, causing oxidative stress and parasite death by apoptosis. The antiparasitic activity of the compounds studied could be explained by a loss of the capacity of the antioxidant defense system of the parasite to keep its intracellular redox state. C1 could be considered a good candidate for in vivo evaluation.

Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi

We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

Effect of thiazolidine LPSF SF29 on the growth and morphology of Trypanosoma cruzi

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic illness in Latin America. Efforts have been made by several groups to develop new effective and safe anti-T. cruzi drugs. In the present work, we show that thiazolidine LPSF SF29 inhibited growth of the epimastigote and amastigote forms and caused lysis in the trypomastigote form of T. cruzi, leading to death of the protozoan. Mitochondrial dysfunction was also observed. The thiazolidine induced ultrastructural alterations such as detachment of the flagellar membrane, intense mitochondrial swelling, formation of myelin-like figures and the appearance of autophagosomes. Taken together, these results suggest that this new thiazolidine is active against T. cruzi and constitutes a promising drug for the therapy of Chagas disease.

In vitro Metabolism of Grandisin, a Lignan with Anti-chagasic Activity

Tetrahydrofuran lignans represent a well-known group of phenolic compounds capable of acting as antiparasitic agents. In the search for new medicines for the treatment of Chagas disease, one promising compound is grandisin which has shown significant activity on trypomastigote forms of Trypanosoma cruzi. In this work, the in vitro metabolism of grandisin was studied in the pig cecum model and by biomimetic phase I reactions, aiming at an ensuing a preclinical pharmacokinetic investigation. Although grandisin exhibited no metabolization by the pig microbiota, one putative metabolite was formed in a biomimetic model using Jacobsen catalyst. The putative metabolite was tested against T. cruzi revealing loss of activity in comparison to grandisin.

Solid Dispersion of Ursolic Acid in Gelucire 50/13: a Strategy to Enhance Drug Release and Trypanocidal Activity

Solid dispersions (SDs) are an approach to increasing the water solubility and bioavailability of lipophilic drugs such as ursolic acid (UA), a triterpenoid with trypanocidal activity. In this work, Gelucire 50/13, a surfactant compound with permeability-enhancing properties, and silicon dioxide, a drying adjuvant, were employed to produce SDs with UA. SDs and physical mixtures (PMs) in different drug/carrier ratios were characterized and compared using differential scanning calorimetry, hot stage microscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size, water solubility values, and dissolution profiles. Moreover, LLC-MK2 fibroblast cytotoxicity and trypanocidal activity evaluation were performed to determine the potential of SD as a strategy to improve UA efficacy against Chagas disease. The results demonstrated the conversion of UA from the crystalline to the amorphous state through XRD. FTIR experiments provided evidence of intermolecular interactions among the drug and carriers through carbonyl peak broadening in the SDs. These findings helped explain the enhancement of water solubility from 75.98μg/mL in PMs to 293.43μg/mL in SDs and the faster drug release into aqueous media compared with pure UA or PMs, which was maintained after 6months at room temperature. Importantly, improved SD dissolution was accompanied by higher UA activity against trypomastigote forms of Trypanosoma cruzi, but not against mammalian fibroblasts, enhancing the potential of UA for Chagas disease treatment.

Trypanocidal activity of guaianolide obtained from Tanacetum parthenium (L.) Schultz-Bip. and its combinational effect with benznidazole

In the present study, we evaluated the in vitro antiprotozoal activity of a guaianolide (11,13-dehydrocompressanolide) isolated from Tanacetum parthenium against Trypanosoma cruzi and investigated the possible combinational effect of guaianolide and benznidazole. The isolated compound was shown to be effective against T. cruzi, with IC50 values of 18.1±0.8 and 66.6±1.3μM against the multiplicative epimastigote and amastigote forms, respectively. The best results were obtained against trypomastigotes, with an EC50 of 5.7±0.7μM. The guaianolide presented no toxicity in LLCMK2 cells (CC50 of 93.5μM) and was 16.4-fold more selective for trypomastigotes. The study of the combinational effect of benznidazole and guaianolide revealed the presence of a synergistic effect against the epimastigote form and marginal additive effect against the trypomastigote form. Striking morphological changes were observed in epimastigotes treated with guaianolide, such as thinning and stretching of the cell body and flagellum and changes in the format of the cell body with apparent leakage of the cytoplasmic content in trypomastigote forms. The ultrastructural analysis of epimastigotes revealed the presence of membranes that involved organelles and formation of myelin-like figures. Flow cytometry revealed a cell volume reduction and decrease in mitochondrial membrane potential. However, no major changes in cell membrane integrity were found in the epimastigote form treated with guaianolide.

Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.

Biochemistry detection of acetylcholinesterase activity in Trypanosoma evansi and possible functional correlations

Several chemical and immunohistochemical techniques can be used for the detection of acetylcholinesterase (AChE) activity. In this experiment we aimed to detect AChE activity in Trypanosoma evansi. For this, the parasites were isolated from the blood of experimentally infected rats using a DEA-cellulose column. Enzymatic activity was determined in trypomastigote forms at 0, 0.2, 0.4, 0.8 and 1.2 mg/mL of protein concentrations by a standard biochemical protocol. At all concentrations tested, the study showed that T. evansi expresses the enzyme AChE and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.70 μmol of AcSCh/h. Therefore, we concluded that it is possible to biochemically detect AChE in T. evansi, an enzyme that may be associated with vital functions of the parasite and also can be related to chemotherapy treatments, as further discussed in this article.