Search

Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circumsporozoite (CS) protein, which contains the well-characterized CD9+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection. [ABSTRACT FROM PUBLISHER]

A new polyketide, cladoic acid, was isolated from a fungus of the genus Cladosporium . The structure of the highly oxygenated trans -decalin ring with an all- E triene side chain was elucidated by extensive spectroscopic analysis. The unique chair/twist-boat conformation of the trans -decalin core and the flexibility of the B-ring were demonstrated by computer-aided conformational analysis. Cladoic acid was active against Trypanosoma cruzi and inhibited the proliferation of amastigotes and epimastigotes with IC 50 values of 27 and 46 μM, respectively, but it did not show any appreciable activity against P388 murine leukemia cells, bacteria, or fungi, indicating it is a potential candidate for drug development against Chagas disease.

Processing bodies (P-bodies, PBs) are cytoplasmic foci formed by condensation of translationally inactivated messenger ribonucleoprotein particles (mRNPs). Infection with the protozoan parasite Trypanosoma cruzi (T. cruzi) promotes PB accumulation in host cells, suggesting their involvement in host mRNA metabolism during parasite infection. To identify PB-regulated mRNA targets during T. cruzi infection, we established a PB-defective human fibrosarcoma cell line by knocking out the enhancer of mRNA decapping 4 (EDC4), an essential component of PB assembly. Next-generation sequencing was used to establish transcriptome profiles for wild-type (WT) and EDC4 knockout (KO) cells infected with T. cruzi for 0, 3, and 24 h. Ingenuity pathway analysis based on the differentially expressed genes revealed that PB depletion increased the activation of several signaling pathways involved in the innate immune response. The proinflammatory cytokine IL-1β was significantly upregulated following infection of PB-deficient KO cells, but not in WT cells, at the mRNA and protein levels. Furthermore, the rescue of PB assembly in KO cells by GFP-tagged wild-type EDC4 (+WT) suppressed IL-1β expression, whereas KO cells with the C-terminal-deleted mutant EDC4 (+Δ) failed to rescue PB assembly and downregulate IL-1β production. Our results suggest that T. cruzi assembles host PBs to counteract antiparasitic innate immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds ( 1 - 5 ) from Peperomia pseudopereskiifolia . The lignan derivatives 1 , 2 , and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1 , 5 , 6 , and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Background: Chagas disease can lead to life-threatening cardiac manifestations. Regional factors, including genetic characteristics of circulating Trypanosoma cruzi (T. cruzi), have attracted attention as likely determinants of Chagas disease phenotypic expression and Chagas cardiomyopathy (CCM) progression. Our objective was to elucidate the differential transcriptomic signatures of cardiomyocytes resulting from infection with genetically discrete T. cruzi strains and explore their relationships with CCM pathogenesis and progression., Methods: HL-1 rodent cardiomyocytes were infected with T. cruzi trypomastigotes of the Colombian, Y, or Tulahuen strain. RNA was serially isolated post-infection for microarray analysis. Enrichment analyses of differentially expressed genes (fold-change ≥ 2 or ≤ 0.5) highlighted over-represented biological pathways. Intracellular levels of reactive oxygen species (ROS) were compared between T. cruzi-infected and non-infected HL-1 cardiomyocytes., Results: We found that oxidative stress-related gene ontology terms (GO terms), 'Hypertrophy model', 'Apoptosis', and 'MAPK signaling' pathways (all with P < 0.01) were upregulated. 'Glutathione and one-carbon metabolism' pathway, and 'Cellular nitrogen compound metabolic process' GO term (all with P < 0.001) were upregulated exclusively in the cardiomyocytes infected with the Colombian/Y strains. Mean intracellular levels of ROS were significantly higher in the T. cruzi-infected cardiomyocytes compared to the non-infected (P < 0.0001)., Conclusions: The upregulation of oxidative stress-related and hypertrophic pathways constitutes the universal hallmarks of the cardiomyocyte response elicited by T. cruzi infection. Nitrogen metabolism upregulation and glutathione metabolism imbalance may implicate a relationship between nitrosative stress and poor oxygen radicals scavenging in the unique pathophysiology of Chagas cardiomyopathy., (© 2023. The Author(s).)

UV absorption spectroscopy-guided fractionation of the culture extract of a marine obligate bacterium of the genus Microbulbifer yielded a novel cyclic hexapeptide, bulbiferamide ( 1 ). NMR spectroscopic and mass spectrometric analyses revealed the structure of 1 to be a cyclic tetrapeptide appending a ureido-bridged two amino acid unit. Notably, Trp is a junction residue, forming on one hand a very rare N -aminoacylated indole linkage for cyclization and on the other hand connecting the ureido-containing tail structure, which is an unprecedented way of configuring peptides. The component amino acids were determined to be l by the advanced Marfey's method. Compound 1 displayed growth inhibitory activity against Trypanosoma cruzi epimastigotes with an IC 50 value of 4.1 μM, comparable to the currently approved drug benznidazole, while it was not cytotoxic to P388 murine leukemia cells at 100 μM.

Background: Since the late twentieth century, Chagas disease gained global attention to suppress the vector burden as a main control strategy in endemic countries. In Central America, multi-national initiative successfully achieved significant reduction in the estimated disease prevalence as well as elimination of the region's principal vector species at the time in 2012. While the last decade has witnessed significant changes in ecosystem-such as urbanization and replacement of the main vector species-that can possibly affect the vector's habitation and residual transmission, the up-to-date vector burden in the region has not been evaluated thoroughly due to the cessation of active vector surveillance. The aim of this study was to update the risk of vector-borne Trypanosoma cruzi infection in El Salvador, the top Chagas disease-endemic country in Central America., Methods: A nationwide vector survey was conducted in the domestic environment of El Salvador from September 2018 to November 2020. The selection of the houses for inspection was based on expert purposeful sampling. Infection for T. cruzi was examined by microscopic observation of the insects' feces, followed by a species confirmation using PCR. The data were analyzed using R software version 4.1.3. Proportion estimates with 95% confidence intervals were inferred using the Jeffrey's method provided under the epiR package., Results: A total of 1529 Triatoma dimidiata was captured from 107 houses (infestation rate, 34.4%; 107/311) in all the fourteen departments of the country visited within the period; prevalence of T. cruzi infection was as high as 10% (153/1529). In the country, domestic T. dimidiata infestation was distributed ubiquitously, while T. cruzi infection rates varied across the departments. Five out of fourteen departments showed higher infection rates than the average, suggesting sporadic high-risk areas in the country., Conclusions: Our comprehensive study revealed substantial T. cruzi infection of T. dimidiata across the country, indicating potential active transmission of the disease. Therefore, strengthened surveillance for both vector and human infection is required to truly eliminate the risk of T. cruzi transmission in Central America., (© 2022. The Author(s).)

Phytohabitols A-C ( 1 - 3 ), new terminally δ-lactonized linear polyketides, were isolated from the culture extract of a rare actinomycete of the genus Phytohabitans . The structures of 1 - 3 , substituted with multiple methyl and hydroxy groups on a conjugated and a skipped diene-containing backbone, were elucidated by NMR and MS spectroscopic analyses. The absolute configuration of 1 was determined by chemical derivatization and chiral anisotropic analysis, coupled with ROESY and J -based configuration analysis. In addition, closely similar 1 H and 13 C NMR data and optical rotations among 1 - 3 supported the same stereochemistry of these polyketides. The related streptomycetes metabolites lagunapyrones B, C, and D have α-pyrone rings on the linear part in place of the δ-lactone, but their chirality at the C19-C21 stereocenters were opposite from those described here, posing a question on the previous assignment made solely by comparison of the optical rotations of four possible diastereomers. Compounds 1 - 3 inhibited migration of cancer cells with IC 50 values of 15, 11, and 8.3 μM, respectively, at noncytotoxic concentrations. In addition, 1 - 3 displayed potent antitrypanosomal activity against Trypanosoma cruzi with IC 50 values of 12, 6.4, and 18 μM, comparable to a commonly used therapeutic drug, benznidazole (IC 50 16 μM).

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and in Central America, it is considered one of the four most infectious diseases. This study aimed to screen the anti-trypanosomal activity of plant species from Salvadoran flora. Plants were selected through literature search for plants ethnobotanically used for antiparasitic and Chagas disease symptomatology, and reported in Museo de Historia Natural de El Salvador (MUHNES) database. T. cruzi was incubated for 72 h with 2 different concentrations of methanolic extracts of 38 species, among which four species, Piper jacquemontianum, Piper lacunosum, Trichilia havanensis, and Peperomia pseudopereskiifolia, showed the activity (≤ 52.0% viability) at 100 µg/mL. Separation of the methanolic extract of aerial parts from Piper jacquemontianum afforded a new flavanone (4) and four known compounds, 2,2-dimethyl-6-carboxymethoxychroman-4-one (1), 2,2-dimethyl-6-carboxychroman-4-one (2), cardamomin (3), and pinocembrin (5), among which cardamomin exhibited the highest anti-trypanosomal activity (IC 50  = 66 µM). Detailed analyses of the spectral data revealed that the new compound 4, named as jaqueflavanone A, was a derivative of pinocembrin having a prenylated benzoate moiety at the 8-position of the A ring., (© 2021. The Author(s).)

A series of quinone derivatives with a variety of side chains were synthesized. These synthetic quinone compounds were evaluated for in vitro antitrypanosomal activity against trypomastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas disease. Measurement of solubility of quinones and their ability to permeate cell membranes were assessed to address their possible use as oral drugs. Some synthesized compounds exhibited potent antitrypanosomal activity. However, most compounds with a promising activity showed poor solubility that did not seem suitable for oral usage. Meanwhile, compound 5a, an N-tert-butoxycarbonylpiperidine derivative, exhibited good antitrypanosomal activity, ability to permeate membranes, and good solubility.

Objective: We aimed to establish correlations for the levels of follicle-stimulating hormone (FSH), estrone (E1) and estradiol (E2) between urine and serum in premenopausal and postmenopausal women using immunoassays., Methods: In this study of 92 women (61 postmenopausal, 31 premenopausal), both urine and blood specimens were collected on the same day and stored at 4 °C for analysis by chemiluminescent immunoassay, radioimmunoassay and/or electrochemiluminescent immunoassay., Results: There were correlations in the levels of FSH, E1 and E2 between urine and serum in both postmenopausal (r = 0.96 for FSH, r = 0.91 for E1, r = 0.80 for E2) and premenopausal (r = 0.98 for FSH, r = 0.92 for E1, r = 0.90 for E2) women. It is indicated that the correlations were stronger in the premenopausal group compared with the postmenopausal group, especially for FSH., Conclusion: The levels of FSH, E1 and E2 in urine correlated with those in the serum in premenopausal and postmenopausal women. Urine samples could be used instead of serum samples to measure hormone levels, which would reduce the difficulty of conducting large survey studies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The associations between ABO blood type and risk of diseases including cancer have been reported from epidemiological studies. Self-reporting is one of the most widely used methods of collecting the ABO blood type information. Verifying the accuracy of self-reporting is important to consider measurement errors. We aimed to conduct validation of self-reported ABO blood types in the Japan Nurses’ Health Study (JNHS), which is a large prospective cohort study. Methods: The concordance rate between self-reported and serologically or genetically inferred ABO blood groups was investigated for a subsample of 41 subjects from the Gunma Nurses’ Health Study, which was a pilot cohort study that preceded the JNHS. The presence of antibodies to A or B antigens in serum (serological test) and allele types of the ABO gene (genotyping test) were determined by using frozen blood samples that were preserved for approximately 7 years. ABO blood types were determined from these tests and compared with self-reported data. Results: All of the nurses reported that their ABO blood groups were concordant with those determined by a serological and/or genotyping test. Self-reported ABO blood types of 35 of 38 (92.1%) participants were consistent with the results from serological typing, while the answers of three participants were not. In these three participants, ABO genotypes that were inferred from genotyping of three single nucleotide polymorphisms in ABO loci perfectly matched with their self-reported ABO types, and all of these were O-type. Conclusions: Japanese health professionals report their blood type with a high degree of accuracy. Special attention should be paid to the O-type group in serological analysis of blood samples that have been preserved for several years in longitudinal studies., (Creative Commons Attribution License)

Results of studies on the associations of soy food intake with urinary estrogen levels in premenopausal women and in postmenopausal women have been inconsistent. We examined the associations of urinary isoflavone levels as well as soy food intake with estrone (E1) and estradiol (E2) in pre- and postmenopausal women. In addition, we compared the levels of isoflavones, E1 and E2 across current hormone users such as those receiving hormone replacement therapy and those using oral contraceptives and non-users among both pre- and postmenopausal women. Urinary levels of isoflavones, E1 and E2 in 498 women (36 hormone users and 462 non-users) were analyzed. Premenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, but there were no significant associations between E1 and E2 levels and urinary isoflavone levels. Levels of E1 and E2 in hormone users were significantly lower than those in hormone non-users among premenopausal women, but levels of E1 and E2 in hormone users were significantly higher than those in hormone non-users among postmenopausal women. Postmenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, and postmenopausal women with high urinary isoflavone levels had significantly higher E1 and E2 levels. In conclusion, the associations of urinary isoflavone levels with urinary estrogen levels differed with menopausal status. Urinary levels of E1 and E2 were high in postmenopausal women with high urinary isoflavone levels but not in premenopausal women with high urinary isoflavone levels., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The change in follicle-stimulating hormone (FSH) during the menopausal transition and associations of FSH with various diseases have been assessed by using blood samples. We examined cross-sectionally the variation of FSH levels, associations of estrone and estradiol with FSH, and associations of BMI with these hormones by using urinary samples from peri- and postmenopausal women in Japan. Of 4472 participants in the Urinary Isoflavone Concentration Survey of the Japan Nurses' Health Study, we analyzed urinary levels of estrone, estradiol and FSH in 547 women aged from 45 to 54 years. Urinary FSH levels varied widely in postmenopausal women and the pattern of change in urinary FSH levels seems to be similar to that in blood FSH levels in previous studies. There were no significant differences in age, body mass index (BMI), estradiol, estrone and estradiol/estrone ratio among three groups according to the tertile of FSH. In postmenopausal women, there were significant associations of BMI with levels of estrone and estradiol, but there was no significant association of BMI with FSH. Studies using urinary samples will allow us to establish a study project as a large-scale population-based study to determine associations between FSH and various diseases after menopause. J. Med. Invest. 66 : 297-302, August, 2019.

Equol is one of the most active soy isoflavones. When the association between soy food intake in daily life and health outcomes is examined in epidemiological studies, it is important to define the equol-producing status of each individual. However, few studies have assessed equol-producing status without a soy challenge test. To determine a robust cutoff criterion for equol producer classification in observational studies, we conducted a urinary isoflavone concentration survey in daily life among women. Furthermore, we examined the association between eating habits regarding soy foods and equol-producing status. A total of 4,412 participants were included in the analyses. Urinary isoflavones were analyzed using a high-performance liquid chromatography method. We examined the distribution of the log10 equol/daidzein ratios, finding a mixture of two normal distributions, corresponding to equol producer and non-producer subpopulations. Applying a finite mixture model, we estimated the means, standard deviations, and mixing proportions of these two distributions. The estimation was carried out using the SAS NLIN procedure. The optimal cutoff point for the log10 equol/daidzein ratio in the study population was determined to be -1.42, according to the estimated parameters of the mixture distribution. Based on this criterion, 1,830 (41.5%) of the participants were identified as equol producers. Compared with non-consumers of soy foods, consumers of soy foods had significantly higher odds of being equol producers. Using log10-transformed equol/daidzein ratios ≥ -1.42 to define equol producers among Japanese women is reasonable and suitable for determining equol-producing status in epidemiological studies. We found that soy food eating habits were associated with equol-producing status. Further investigation is required to evaluate associations between equol-producing status in daily life and health outcomes. The results of this study suggest the best cutoff point to use in the definition of equol-producing status in daily life., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TU and SU are employees of Otsuka Pharmaceutical Co., Ltd. YI, KH, JNS, YO, and MK. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Although several studies have shown that blood type O is associated with increased risk of peptic ulcer, few studies have investigated these associations in Japan. We sought to investigate the association between the ABO blood group and risk of gastroduodenal ulcers (GDU) using combined analysis of both retrospective and prospective data from a large cohort study of Japanese women, the Japan Nurses' Health Study (JNHS; n = 15,019)., Methods: The impact of the ABO blood group on GDU risk was examined using Cox regression analysis to estimate hazard ratios (HRs) and 95% confidence intervals (CI), with adjustment for potential confounders., Results: Compared with women with non-O blood types (A, B, and AB), women with blood type O had a significantly increased risk of GDU from birth (multivariable-adjusted HR 1.18; 95% CI, 1.04-1.34). Moreover, the highest cumulative incidence of GDU was observed in women born pre-1956 with blood type O. In a subgroup analysis stratified by birth year (pre-1956 or post-1955), the multivariable-adjusted HR of women with blood type O was 1.22 (95% CI, 1.00-1.49) and 1.15 (95% CI, 0.98-1.35) in the pre-1956 and post-1955 groups, respectively., Conclusion: In this large, combined, ambispective cohort study of Japanese women, older women with blood type O had a higher risk of developing GDU than those with other blood types.

Autophagy has emerged as an essential component of the defense system against intracellular pathogens. We demonstrated that Trypanosoma cruzi, an intracellular protozoan parasite, was not eliminated by the host's autophagic machinery despite exposure to the host cell cytoplasm. Puncta of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and LC3-II, a lipidated form of LC3, were significantly increased after infection with T. cruzi, indicating that the parasite activated the early steps of host autophagy and induced autophagosome formation. However, autolysosomes were not observed in the infected cells. In addition, T. cruzi was not enwrapped by autophagosomes, suggesting that the parasite has mechanisms to allow it to evade autophagic capture. The results of this study indicate that host autophagy is incomplete following T. cruzi infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities, including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship between the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan-1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities at 1 µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 µM significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.

Processing bodies (PBs) are cytoplasmic granules containing mRNAs and proteins involved in translation and degradation of mRNAs. PBs are constitutively present in cells and are induced to accumulate when external stressors including microbial infection are applied to cells, followed by a rapid translational arrest. We have examined the impact of Trypanosoma cruzi (T. cruzi, Tc) infection on host cytoplasmic PB assembly. Within 24h post-infection, we found the average number of PB foci per cell increased by more than 2-fold. Protein levels of PB components were unaltered during infection. These results indicated that Tc infection caused accumulation of PBs by changing the localization pattern of PB protein components. To elucidate the role of the accumulated PBs on Tc infection, we knocked down PBs using a siRNA specific for PB components EDC4 and Lsm14A, which are involved in mRNA decapping and translational repression, respectively. We observed that the inhibition of PB accumulation significantly enhanced the infectivity and growth of intracellular amastigotes. Depletion of PBs did not affect nitric oxide (NO) production during Tc infection, indicating that the growth promotion was not caused by modulation of NO-mediated killing of Tc. Our results suggest that the accumulated PBs partially contribute to anti-parasitic responses by manipulating the host's mRNA metabolism., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Current chemotherapy drugs for Chagas' disease are insufficient due to their limited efficacy; however, anti-trypanosomal agents have recently shown promise. As such, synthetic intermediates of komaroviquinone were evaluated for anti-trypanosomal activity. Based on the results, a series of novel quinone derivatives were screened for anti-trypanosomal activity and mammalian cytotoxicity. Several quinone derivatives displayed higher antiprotozoal activity against Trypanosoma cruzi trypomastigotes than the reference drug benznidazole, without concomitant toxicity toward the host cell., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals., (© 2014 Saegusa, Sato, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Previous intervention studies have shown that the most effective agents used in the treatment of malaria were isolated from natural sources. Plants consumed by non-human primates serve as potential drug sources for human disease management due to the similarities in anatomy, physiology and disease characteristics. The present study investigated the antiplasmodial properties of the primate-consumed plant, Schima wallichii ( S. wallichii) Korth. (family Theaceae ), which has already been reported to have several biological activities. The ethanol extract of S. wallichii was fractionated based on polarity using n -hexane, ethyl acetate and water. The antiplasmodial activity was tested in vitro against chloroquine-resistant Plasmodium falciparum ( P. falciparum ) at 100 μg/ml for 72 h. The major compound of the most active ethyl acetate fraction was subsequently isolated using column chromatography and identified by nuclear magnetic resonance. The characterized compound was also tested against chloroquine-resistant P. falciparum in culture to evaluate its antiplasmodial activity. The ethanol extract of S. wallichii at 100 μg/ml exhibited a significant parasite shrinkage after 24 h of treatment. The ethyl acetate fraction at 100 μg/ml was the most active fraction against chloroquine-resistant P. falciparum . Based on the structural characterization, the major compound isolated from the ethyl acetate fraction was kaempferol-3- O -rhamnoside, which showed promising antiplasmodial activity against chloroquine-resistant P. falciparum with an IC 50 of 106 μM after 24 h of treatment. The present study has provided a basis for the further investigation of kaempferol-3- O -rhamnoside as an active compound for potential antimalarial therapeutics.

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72μM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100μM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM) dependent serine/threonine protein phosphatase and plays important role in several cellular functions in both higher and lower eukaryotes. Here we report inhibition of CN by linear alkylbenzene sulfonate. The clue to the finding was obtained while identifying the inhibitory material leaching from acrylonitrile butadiene rubber used for packing. Using standard dodecylbenzene sulfonate (C12-LAS), we obtained strong inhibition of CN with a half maximal inhibitory concentration of 9.3 µM, whereas analogs such as p-octylbenzene sulfonate and SDS hardly or only slightly affected CN activity. Three alkaline phosphatases, derived from shrimp, bacteria, and calf-intestine, which exhibit similar enzymatic activities to CN, were not inhibited by C12-LAS at concentrations of up to 100 µM. Furthermore, C12-LAS did not inhibit Ca(2+)/CaM-dependent myosin light chain kinase activity when tested at concentrations of up to 36 µM. The results indicate that C12-LAS is a potent selective inhibitor of CN activity.

Plasmodium falciparum has for some time been developing resistance against known anti-malarial drugs, and therefore a new drug is urgently needed. Selenium (Se), an essential trace element, in the form of inorganic Se, selenite (SeO32-), has been reported to have an anti-plasmodial effect, but its mechanism is still unclear. In the present study, we evaluated the anti-plasmodial effect of several Se compounds against P. falciparum in vitro. The anti-plasmodial effect of several Se compounds was analysed and their apoptosis-inducing activity was evaluated by morphological observation, DNA fragmentation assay and mitochondrial function analysis. SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial effects with 50% inhibition concentration at 9, 10, 45, and 65 μm, respectively, while selenate and methylselenocysteine up to 100 μm have no effect on parasite growth. The effective Se compounds caused the parasites to become shrunken and pyknotic and significantly increased mitochondrial damage against P. falciparum compared to the untreated control. In conclusion, SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial activities that induce apoptosis-like cell death in P. falciparum, and the anti-plasmodial effects of Se seem to be based on its chemical forms. The apoptosis-like cell-death mechanism in P. falciparum can be beneficial to respond to the growing problem of drug resistance.

A novel potent trypanocidal diterpene, komaroviquinone, was reduced by Trypanosoma cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. Since TcOYE is expressed throughout the T. cruzi life cycle, komaroviquinone is an interesting candidate for developing new antichagasic drugs.

To determine the molecular mechanism by which apoptosis is inhibited in Trypanosoma cruzi-infected host cells, we used human cDNA apoptosis chips to compare the gene expression profiles in response with 'death ligands target' (Fas) stimulation in infected and uninfected cells. Of the 164 apoptosis-related genes examined, 20, including those encoding both pro- and anti-apoptotic proteins, were highly up-regulated in the infected group. Genes encoding caspases and apoptosis inhibitors were optimally expressed 10-30 min after induction of apoptosis, whereas genes involved in transcriptional regulation and cell proliferation were up-regulated after 2-24 h. These results suggest that host anti-apoptotic gene(s) may play a crucial role in the inhibition of Fas-mediated apoptosis in T. cruzi-infected cells.

Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically up-regulates cellular FLICE inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, posttranscriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T. cruzi-infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T. cruzi posttranscriptionally up-regulates and exploits host c-FLIP for the inhibition of death-inducing signal, a mechanism that may allow parasites to persist in host cells.

Trypanocidal constituents of dried leaves of Laurus nobilis L. (Lauraceae) were examined. Activity-guided fractionation of the methanol extract resulted in the isolation of two guaianolides, dehydrocostus lactone (1) and zaluzanin D (2), and a new p-menthane hydroperoxide, (1R,4S)-1-hydroperoxy-p-menth-2-en-8-ol acetate (3). The minimum lethal concentrations of these compounds against epimastigotes of Trypanosoma cruzi were 6.3, 2.5, and 1.4 microM, respectively.

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.

The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating. In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling. Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response. Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration. In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone. These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway. Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway [Iida et al., (1990) J. Biol. Chem., 265: 13391-13399] Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed., (Copyright 1999 Academic Press.)

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

Aequorin is a Ca2(+)-binding protein that emits light upon reacting with Ca2+ and has been used as a probe for monitoring changes in the intracellular free Ca2+ concentration, [Ca2+]i. The protein consists of three components: apoaequorin (apoprotein), molecular oxygen and a chromophore. The present study was designed to conditionally express the apoaequorin cDNA of the jellyfish Aequorea victoria under the control of the GAL1 promoter in the yeast Saccharomyces cerevisiae and to investigate whether apoaequorin can be accumulated in high enough concentration in the cells to detect a Ca2+ signal in vitro. The results showed that the cells accumulated sufficient amounts of recombinant apoaequorin when incubated in the galactose-based medium and that the protein was active and not toxic to the cells, suggesting that the recombinant apoaequorin may be applicable to monitoring changes in [Ca2+]i in intact yeast cells.