RESUMO
PURPOSE: Trypanosoma caninum exhibits atypical epimastigote forms under axenic conditions. This study aimed to analyze this evolutionary form under different cultivation conditions and provide more information about this evolutionary form. METHODS: We selected a T. caninum isolate with a high percentage of aflagellar epimastigote forms in axenic cultures. Two separate growth curves were generated for T. caninum cultured in Schneider axenic medium and co-cultured with the DH82 cell line, followed by analysis and quantification of evolutionary forms using bright field microscopy. In addition, ultrastructural analysis of T. caninum was performed under both cultivation conditions. RESULTS: The growth curves of T. caninum under axenic and co-cultivation conditions exhibited similar profiles. However, in the axenic culture, the number of parasites was three times higher at the peak of the exponential phase than in the co-culture. In contrast to that in the axenic culture, in which only the epimastigote forms were observed along the entire curve, during co-cultivation with the DH82 cell line, differentiation was observed for the trypomastigote and spheromastigote forms in low proportions. These results demonstrated that when cultured alone, the T. caninum isolate preserved the aflagellar epimastigote form, but in the presence of DH82 canine macrophages, they differentiated into evolutionary forms, particularly trypomastigote forms. Moreover, this study is the first to describe the presence of lipid bodies, structure described as the parasite's nutritional reserve, throughout the body of T. caninum. CONCLUSIONS: These findings describe biological and ultrastructural aspects of epimastigote aflagellar and suggest that this evolutionary form may be involved in the biological cycle of T. caninum, still unknown.
Assuntos
Trypanosoma cruzi , Animais , Cultura Axênica , Linhagem Celular , Meios de Cultura , Cães , Macrófagos/parasitologiaRESUMO
The present study evaluated the use of the probiotic Lacticaseibacillus paracasei DTA-83 as a nitrite-reducing agent to produce potentially probiotic or postbiotic pre-converted nitrite from celery. The results obtained were compared to those achieved by direct addition of sodium nitrite for the typical reddish color formation in cooked pork sausages and the inhibitory potential against the growth of target microorganisms, including the clostridia group. Regarding the sausages color, similar findings were observed when comparing the use of pre-converted nitrite from celery produced by L. paracasei DTA-83 and the direct addition of sodium nitrite. Additionally, it presented an inhibitory effect against Salmonella spp., which was not observed with the direct addition of nitrite, revealing a potential strategy to control salmonellosis in the matrix. However, a non-equivalent preservative effect against Clostridium perfringens (INCQS 215) was determined. The results highlight a promising alternative to produce probiotic or postbiotic meat ingredients; however, further studies should be conducted to investigate doses that achieve microbial control.
Assuntos
Lactobacillaceae , Produtos da Carne/análise , Nitritos/química , Probióticos , Animais , Apium/química , Cultura Axênica , Clostridium perfringens/efeitos dos fármacos , Cor , Produtos da Carne/microbiologia , Salmonella/efeitos dos fármacos , Nitrito de Sódio/química , SuínosRESUMO
Lentinus crinitus is a medicinal basidiomycete, little studied regarding the basic cultivation conditions, which is used in bioremediation and consumed by native Indians from the Brazilian Amazon. Also, it produces a fungal secondary metabolite panepoxydone that has been described as an essential regulator of the inflammatory and immune response. This study aimed to evaluate basic conditions of temperature, pH, and nitrogen concentration and source in the cultivation of L. crinitus mycelial biomass. In order to evaluate fungal growth temperature, 2% malt extract agar (MEA) medium, pH 5.5, was utilized from 19 to 40 °C. For pH, MEA had pH adjusted from 2 to 11 and cultivated at 28 °C. Urea or soybean meal was added to MEA to obtain final concentration from 0.5 and 16 g/L of nitrogen, pH of 5.5, cultivated at 28 °C. The best temperature growth varies from 31 to 34 ºC and the optimal one is 32.7º C, and the best pH ranges from 4.5 to 6.5 and the optimal one is 6.1. Protein or non-protein nitrogen concentration is inversely proportional to the mycelial biomass growth. Nitrogen concentrations of 2.0 g/L soybean meal and urea inhibit mycelial biomass growth in 11% and 12%, respectively, but high concentrations of 16.0 g/L nitrogen inhibit the growth in 46% and 95%, respectively. The fungus is robust and grows under extreme conditions of temperature and pH, but smaller adaptation with increasing nitrogen concentrations in the cultivation medium, mainly non-protein nitrogen.
Lentinus crinitus é um basidiomiceto medicinal consumido por índios nativos da Amazônia brasileira. Este fungo tem sido estudado quanto ao potencial de biorremediação de metais, mas ainda carece de estudos sobre às condições básicas de crescimento. L. crinitus produz panepoxidona - um metabólito secundário fúngico - descrito como regulador da resposta inflamatória e imune em células animais. Este trabalho teve como objetivo avaliar as condições básicas de temperatura, pH e concentração e fonte de nitrogênio para o crescimento micelial de L. crinitus. O fungo foi crescido em meio agar extrato de malte a 2% (MEA), pH 5,5 e mantido entre 19 e 40 °C. Para a avaliação de pH o MEA teve o pH ajustado de 2 a 11 e o crescimento foi realizado a 28 °C. As fontes de nitrogênio estudadas foram a uréia e o farelo de soja adicionado ao MEA para obter entre 0,5 a 16 g/L de nitrogênio, pH de 5,5, cultivado a 28 ° C. A melhor faixa temperatura para o crescimento micelial foi de 31 a 34 ºC com ótimo a 32,7 º C; a melhor faixa de pH de 4,5 a 6,5 e com ótimo de 6,1. A concentração de nitrogênio proteico ou não proteico é inversamente proporcional ao crescimento do fungo. Concentrações de nitrogênio de 2,0 g/L reduzem o crescimento da biomassa micelial em 11% e 12%, respectivamente e meios com nitrogênio de 16,0 g/L reduzem o crescimento em 46% e 95%, respectivamente. O fungo é robusto e cresce sob condições extremas de temperatura e pH, mas menor adaptação em meios com alta concentração de nitrogênio, principalmente não proteico.
Assuntos
Biomassa , Lentinula , Cultura Axênica , Micelas , UreiaRESUMO
Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Animais , Cultura Axênica , Caseínas/análise , Linhagem Celular , Cães , Genótipo , Humanos , Células Madin Darby de Rim Canino , Trofozoítos/metabolismoRESUMO
Microalgae are versatile sources of bioproducts, a solution for many environmental problems. However, and despite its importance, one of the main problems in large-scale cultures-the presence of contaminants-is rarely systematically approached. Contamination, or the presence of undesirable organisms in a culture, is deleterious for the culture and frequently leads to culture crashes. To avoid contamination, closed systems can be used; however, for very large-scale open systems, contamination is unavoidable and remediation procedures are necessary-ranging from physicochemical treatment to addition of biocidal substances. In all cases, early detection and culture monitoring are paramount. This article describes the biological contaminants, contamination mechanisms, and control systems used in open and closed cultures, discussing the latest advances and techniques in the area. It also discusses the complex interactions of algae with other microorganisms that can be expected in cultivation systems.
Assuntos
Técnicas de Cultura de Células/normas , Microalgas/microbiologia , Cultura Axênica , Biomassa , Reatores Biológicos , Técnicas de Cocultura , Meios de Cultura/análise , Interações MicrobianasRESUMO
The rupestrian Triatoma costalimai species has been found infected by Trypanosoma cruzi in wild, peridomicile, and intradomicile environments in the municipality of Aurora do Tocantins, Tocantins, Brazil. Proximity between rock outcrops increases the risk of vector transmission of Chagas disease via this species. This work describes a focus of colonization by T. costalimai specimens infected by T. cruzi in rock outcrops located in an urban area in this municipality. Parasitological examination of feces from the collected specimens, axenic cultivation of T. cruzi-positive samples, and genetic characterization of the isolates were performed. Nymph and adult specimens were collected with a high infection prevalence (64.5%) for T. cruzi discrete type unit (DTU I). Participation of the T. costalimai species in the wild cycle of T. cruzi in rock outcrops located in an urban area demonstrates the need for entomological surveillance and control of vector transmission of Chagas disease in the municipality of Aurora do Tocantins, Tocantins.
Assuntos
Doença de Chagas/parasitologia , Insetos Vetores/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/genética , Animais , Animais Domésticos/parasitologia , Cultura Axênica , Brasil , Fezes/parasitologia , Genótipo , Ninfa/parasitologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.
Assuntos
Estágios do Ciclo de Vida/genética , Transcriptoma/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Cultura Axênica , Western Blotting , Polirribossomos/genética , Análise de Sequência de RNA , Trypanosoma cruzi/genéticaRESUMO
Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.
Assuntos
Produtos Biológicos/metabolismo , Biotecnologia/métodos , Engenharia Metabólica/métodos , Compostos Fitoquímicos/biossíntese , Células Vegetais/metabolismo , Plantas/metabolismo , Artemisininas/isolamento & purificação , Artemisininas/metabolismo , Cultura Axênica , Berberina/isolamento & purificação , Berberina/metabolismo , Produtos Biológicos/isolamento & purificação , Técnicas de Cultura de Células , Naftoquinonas/isolamento & purificação , Naftoquinonas/metabolismo , Paclitaxel/biossíntese , Paclitaxel/isolamento & purificação , Compostos Fitoquímicos/isolamento & purificação , Células Vegetais/química , Plantas/química , Plantas/genética , Metabolismo Secundário , Técnicas de Cultura de TecidosRESUMO
BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.
Assuntos
Humanos , Análise de Sequência de RNA , Transcriptoma/genética , Cultura Axênica , Estágios do Ciclo de Vida/genéticaRESUMO
Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."
Assuntos
Ananas/microbiologia , Bactérias/isolamento & purificação , Endófitos/isolamento & purificação , Orchidaceae/microbiologia , Ananas/crescimento & desenvolvimento , Cultura Axênica , Bactérias/classificação , Bactérias/genética , Biodiversidade , DNA Bacteriano/genética , DNA Ribossômico/genética , Endófitos/classificação , Endófitos/genética , Orchidaceae/crescimento & desenvolvimento , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Cyanobacteria are commonly found in association with other microorganisms, which constitutes a great challenge during the isolation of cyanobacterial strains. Although several methods have been published for obtaining axenic cyanobacterial cultures, their efficiency is usually evaluated by observing the growth of non-cyanobacteria in culture media. In order to verify whether uncultured bacteria should be a concern during cyanobacterial isolation, this work aimed to detect by molecular methods sequences from cyanobacteria and other bacteria present before and after a technique for obtaining axenic cultures from plating and exposure of Fischerella sp. CENA161 akinetes to the Extran detergent and sodium hypochlorite. Solutions containing 0.5, 1, and 2% sodium hypochlorite were able to remove contaminant bacterial CFUs from the culture. However, qPCR pointed that the quantity of sequences amplified with universal bacteria primers was higher than the number of cyanobacteria-specific sequences before and after treatments. The presence of uncultured bacteria in post-hypochlorite cultures was confirmed by high-throughput Illumina sequencing. These results suggest that culturing may overlook the presence of uncultured bacteria associated to cyanobacterial strains and is not sufficient for monitoring the success of cyanobacterial isolation by itself. Molecular methods such as qPCR could be employed as an additional measure for evaluating axenity in cyanobacterial strains.
Assuntos
Cultura Axênica/métodos , Cianobactérias/genética , Cianobactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Meios de Cultura/química , Cianobactérias/crescimento & desenvolvimento , Primers do DNA , DNA Bacteriano/genética , Metagenômica/métodos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , SimbioseRESUMO
Shiitake mushroom consumption is increasing in Brazil. In addition to the implementation of new production methods, it is also important to increase productivity, quality and reduce production costs. In this study, six commercial Lentinula edodes strains were characterized for genetic diversity (rep-PCR analysis) and mushroom production (yield, number and weight of individual mushrooms) using different substrates and cultural conditions. All strains showed genetic differences by repetitive element palindromic based-polymerase chain reaction (rep-PCR). The richest substrate resulted in the greatest production under both environmental conditions. Strains LE4 and LE6 produced the majority of their mushrooms earlier than the other strains. The highest number of mushrooms was observed in the LE6 strain while the highest weights of individual mushrooms were observed in the LE4 strain. Controlled environmental conditions resulted in superior production for all strains, except for LE4, which had empirically greater yield in the semi-controlled environmental condition.
Assuntos
Cultura Axênica/métodos , Cogumelos Shiitake/crescimento & desenvolvimento , Cogumelos Shiitake/genética , Agricultura/métodos , Brasil , DNA Fúngico/análise , Variação Genética , Reação em Cadeia da Polimerase/métodos , Locos de Características Quantitativas , Cogumelos Shiitake/classificaçãoRESUMO
The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri.
Assuntos
Acanthamoeba castellanii/microbiologia , Arcobacter/fisiologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/ultraestrutura , Aerobiose , Cultura Axênica , Reservatórios de Doenças , Microscopia de Contraste de Fase , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Microbiologia da ÁguaRESUMO
This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.
Assuntos
Antifúngicos/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Cultura Axênica , Burseraceae/metabolismo , Cymbopogon/metabolismo , Substituição de Medicamentos , Furanos/administração & dosagem , Cromatografia Gasosa-Espectrometria de Massas , Compostos Heterocíclicos com 2 Anéis/administração & dosagem , Concentração Inibidora 50 , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Microsporum/efeitos dos fármacos , Piper/metabolismo , Plantago/metabolismo , Sesquiterpenos/administração & dosagem , Trichophyton/efeitos dos fármacosRESUMO
The etiologic agent of Chagas disease is Trypanosoma cruzi, a protozoan whose life cycle involves obligatory passage through vertebrate and invertebrate hosts in a series of stages. The aim of this study was to explore the transferability of mixed discrete typing units (DTUs) of T. cruzi present in chronic chagasic patients when passed through an invertebrate host during xenodiagnosis (XD) and then when transferred to axenic cultures to obtain T. cruzi isolates. DTUs of T. cruzi present in these two hosts and axenic cultures were identified by kDNA PCR amplification and subsequent hybridization with DTU-specific probes. Mixtures of Tc I, Tc II, Tc V and Tc VI DTUs were detected in blood samples. However as a result of XD and axenic cultures it was possible to identify mostly Tc V. We conclude that the transferability of an isolate of T.cruzi derived from mixed DTUs present in human blood depends upon the starved invertebrate host used for xenodiagnosis.
Assuntos
Doença de Chagas/parasitologia , Insetos Vetores/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Animais , Cultura Axênica , Doença de Chagas/transmissão , Chile , Sondas de DNA , DNA de Cinetoplasto/genética , Genótipo , Humanos , Filogenia , Reação em Cadeia da Polimerase , Trypanosoma cruzi/classificação , XenodiagnósticoRESUMO
Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor.
Assuntos
Proteínas de Protozoários/metabolismo , RNA Helicases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Cultura Axênica , Domínio Catalítico , Núcleo Celular/enzimologia , Sequência Conservada , Citoplasma/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Poro Nuclear/enzimologia , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Helicases/química , RNA Helicases/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Giardia lamblia/efeitos dos fármacos , Omeprazol/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Triose-Fosfato Isomerase/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Albendazol/farmacologia , Cultura Axênica , Cisteína/química , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/isolamento & purificação , Humanos , Metronidazol/farmacologia , Mutação , Nitrocompostos , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Tiazóis/farmacologia , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimentoRESUMO
Leishmaniasis, a complex of diseases caused by protozoa of the genus Leishmania, is endemic in 98 countries, affecting approximately 12 million people worldwide. Current treatments for leishmaniasis have many disadvantages, such as toxicity, high costs, and prolonged treatment, making the development of new treatment alternatives highly relevant. Several studies have verified the antileishmanial activity of ß-carboline compounds. In the present study, we investigated the in vitro antileishmanial activity of N-butyl-[1-(4-methoxy)phenyl-9H-ß-carboline]-3-carboxamide (ß-CB) against Leishmania amazonensis. The compound was active against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis, exhibiting high selectivity for the parasite. Moreover, ß-CB did not exhibit hemolytic or mutagenic potential. Promastigotes treated with the alkaloid presented rounding of the body cell, cell membrane projections, an increase in the number of promastigotes presenting two flagella, and parasites of abnormal phenotype, with three or more flagella and/or nuclei. Furthermore, we observed an increase in the subpopulation of cells in the G2/M stage of the cell cycle. Altogether, these results suggest that ß-CB likely prevents cytokinesis, although it does not interfere with the duplication of cell structures. We also verified an increase in O2(·-) production and the accumulation of lipid storage bodies. Cell membrane integrity was maintained, in addition to the absence of phosphatidylserine externalization, DNA fragmentation, and autophagosomes. Although the possibility of an apoptotic process cannot be discarded, ß-CB likely exerts its antileishmanial activity through a cytostatic effect, thus preventing cellular proliferation.
Assuntos
Antiprotozoários/farmacologia , Carbolinas/farmacologia , Citocinese/efeitos dos fármacos , Citostáticos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Estágios do Ciclo de Vida/efeitos dos fármacos , Animais , Antiprotozoários/síntese química , Cultura Axênica , Carbolinas/síntese química , Linhagem Celular , Citostáticos/síntese química , Eritrócitos/efeitos dos fármacos , Feminino , Flagelos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVES: To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). MATERIALS AND METHODS: Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey's test, using the Statistix 8.0 software. RESULTS: Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. CONCLUSIONS: Although the technique of Chelex100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents.
Assuntos
Cultura Axênica , DNA de Cinetoplasto/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Trypanosoma cruzi/genética , Parasitologia/métodosRESUMO
Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil.