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Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein., Author summary The cell cycle is a complex and highly regulated cellular process in which different checkpoints are coordinated and a unique pattern of protein activities is present. In trypanosomatids, this process is even more complex because of the presence of the kinetoplast, a network of circular DNA inside a large mitochondrion that coordinates its division and segregation with the nuclear mitosis. Aurora kinases comprise a family of enzymes that regulate key steps during the cell cycle by varying their subcellular localization. In this work, we identified three Aurora kinase genes (TcAUK1, -2 and -3) in T. cruzi, the agent of Chagas disease, and established that TcAUK1 has the typical behavior of a mammalian Aurora B kinase, localizing inside the nucleus and associating with the mitotic spindle during mitosis. On the other hand, during interphase TcAUK1, localizes at each side of the kinetoplast, showing a novel localization that has not been described before. Finally, here we present evidence for the relationship between the novel localization of TcAUK1 and its participation in kinetoplast division.

Significance Mitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail, much less is known about the molecular machinery required to distribute the replicated genomes. Using superresolution microscopy in combination with molecular biology and biochemistry, we show in which order the segregation machinery is assembled and that it is likely assembled de novo rather than in a semiconservative fashion in the single-celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes; however, the molecular components are highly diverse., In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. Results The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2181-x) contains supplementary material, which is available to authorized users.

Trypanosomatida parasites, such as Trypanosoma and Leishmania, are the cause of deadly diseases in many third world countries. The three dimensional structure of their mitochondrial DNA, known as kinetoplast DNA (kDNA), is unique since it is organized into several thousands of minicircles that are topologically linked. How and why the minicircles form such a network have remained unanswered questions. In our previous work we have presented a model of network formation that hypothesizes that the network is solely driven by the confinement of minicircles. Our model shows that upon confinement a percolation network forms. This network grows into a space filling network, called saturation network, upon further confinement of minicircles. Our model also shows, in agreement with experimental data, that the mean valence of the network (that is, the average number of minicircles topologically linked to any minicircle in the network) grows linearly with minicircle density. In our previous studies however we disregarded DNA flexibility and used rigid minicircles to model DNA, here we address this limitation by allowing minicircles to be flexible. Our numerical results show that the topological characteristics that describe the growth and topology of the minicircle networks have similar values to those observed in the case of rigid minicircles suggesting that these properties are robust and therefore a potentially adequate description of the networks observed in Trypanosomatid parasites.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

Trypanosoma cruzi is the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti- T. cruzi activity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC 50 ) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K 1 cells, exhibiting a 50% cytotoxic concentration (CC 50 ) of 13.47 ± 0.37 μM and an IC 50 of 0.14 ± 0.12 μM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 μM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (T. cruzi .

The current intraspecific nomenclature in Trypanosoma cruzi describes two major lineages, named T. cruzi I and T. cruzi II, and five sublineages within T. cruzi II, named IIa, IIb, IIc, IId and IIe. The polymorphism of minicircle hypervariable regions (mHVRs) of T. cruzi has been used in many studies for the molecular characterization of parasite populations directly from biological samples. However, the molecular bases that allow strain typing by these markers are still unclear. In this work we examined forty cloned mHVRs sequences of CL-Brener reference strain (IIe sublineage), and we found a predominant group of sequences, with 40% of frequency in this strain, with a 97% of identity among them. Out of the forty clones analyzed, we identified other less representative types, and a few unique ones. This predominant sequence is also present in different reference strains belonging to the other main T. cruzi lineages and sublineages (TcI, IIa, IIb, IIc and IId) although in a many thousand times lower frequency than in the CL-Brener strain, as shown by semiquantitative PCR. Similarly, predominant mHVR sequences previously described for TcIId strains, were clearly more frequent (many thousand times higher) in the IId reference strain analyzed by us (Mncl2) than within the reference strains belonging to the other lineages and sublineages. The analysis of the cloned sequences shows that more sequences than just the major one contribute to define the global pattern of mHVRs RFLP in the CL-Brener strain. The possible usefulness of these predominant sequences for typing TcIId and TcIIe sublineages by semiquantitative PCR, as well as the possible role of these sequences in genotype identification by mHVR probes are discussed. Fil: Velázquez, Melisa María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; Argentina Fil: Diez, Cristina Noemí. Universidad Nacional del Litoral; Argentina Fil: Mora, C.. Universidad Nacional del Litoral; Argentina Fil: Diosque, Patricio. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Marcipar, Iván Sergio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization., Author Summary Proper segregation of the mitochondrial genome during cell division is a prerequisite of healthy eukaryotic cells. However, the mechanism underlying the segregation process is only poorly understood. We use the single celled parasite Trypanosoma brucei, which, unlike most model organisms, harbors a single large mitochondrion with a single mitochondrial genome, also called kinetoplast DNA (kDNA), to study this question. In trypanosomes, kDNA replication and segregation are tightly integrated into the cell cycle and thus can be studied alongside cell cycle markers. Furthermore, previous studies using electron microscopy have characterized the tripartite attachment complex (TAC) as a structural element of the mitochondrial genome segregation machinery. Here, we characterize TAC102, a novel trypanosome protein localized to the TAC. The protein is essential for proper kDNA segregation and cell growth. We analyze the presence of this protein using super resolution microscopy and show that TAC102 is a mitochondrial protein localized between the kDNA and the basal body of the cell’s flagellum. In addition, we characterize different parts of the protein and show that the C-terminus of TAC102 is important for its proper localization. The data and resources presented will allow a more detailed characterization of the dynamics and hierarchy of the TAC in the future and might open new avenues for drug discovery targeting this structure.

Background Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence. Methods Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani. The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured. Results L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU−/−) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU−/+) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU−/+ amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU−/+ parasites (LdU−/+AB). Conclusion The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU−/+ parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0072-z) contains supplementary material, which is available to authorized users.

La leishmaniasis es un problema creciente de salud pública a nivel mundial, en Colombia la situación es preocupante debido al incremento de casos de leishmaniasis cutánea que se ha registrado en los últimos años, y al cambio en el patrón epidemiológico dado por la aparición de nuevos focos, el proceso creciente de domiciliación y urbanización del ciclo de transmisión, problemática que afecta especialmente las Fuerzas Militares debido a la misión que cumplen en áreas endémicas de esta zoonosis, actualmente reportan en promedio 4000 pacientes por año con un porcentaje del 13% que presentan recaídas y recidivas de la enfermedad aparentemente por falla en la adherencia al tratamiento sin tener como evaluar la respuesta temprana al tratamiento. Viridans group streptococci (VGS) are a heterogeneous group of saprophytic facultative anaerobic bacteria that are part of the normal flora of the oral cavity. They are also major pathogens in different orofacial infections. The increased resistance to mu Magíster en Ciencias Biológicas Maestría

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Different molecular-genetic methods were used to identify a cohort of Leishmania strains from natural foci of zoonotic cutaneous leishmaniasis located in Central Asia, on the former USSR territory. The results obtained using isoenzymes, PCR, restriction fragment length polymorphisms of kDNA and molecular hybridization techniques are discussed in terms of their applicability, discrimination power and feasibility for answering questions related to molecular epidemiological research and for detecting mixed Leishmania infections.

Após mais de um século da descoberta da doença de Chagas, cujo agente etiológico é o parasito denominado Trypanosoma cruzi, muito ainda há para ser desvendado sobre essa doença. O polimorfismo, a forma de reprodução, a correlação entre cepa e forma clínica, métodos sorológicos e moleculares, transferência gênica e suas consequências clínicas, tratamento e cura são tópicos que envolvem grandes questionamentos ainda não totalmente elucidados pelos pesquisadores desta enigmática doença. O controle de doadores em bancos de sangue e a diminuição nos índices de transmissão vetorial fizeram com que a transmissão congênita ganhasse uma maior importância. No presente estudo, o monitoramento das mulheres durante a gestação possibilitou o acompanhamento do recém-nascido até os nove meses de idade, fase na qual se espera que os anticorpos maternos tenham desaparecido por completo. Para se conhecer a população de gestantes infectadas por Trypanosoma cruzi, no serviço da Maternidade de um Hospital Universitário, 1.979 prontuários foram analisados num intervalo de três anos (2010 a 2012). Perfis socioeconômicos e demográficos, assim como as características reprodutivas e sorológicas foram estudados. Tiveram sorologia positiva para Tripanossomíase Americana 3,1% das mulheres (61/1.979) e poucas relataram aborto. Estudos sobre o aborto demonstraram que em mães infectadas que não transmitiram sua infecção ao feto não houve maior frequência de aborto, prematuridade e mortalidade perinatal, porém, houve uma maior tendência à natimortalidade nas mães que transmitiram a infecção aos seus filhos. Trinta e oito gestantes infectadas por T. cruzi (duas com gestações gemelares) e seus conceptos (quarenta) participaram de uma pesquisa para verificar a integração de minicírculos de kDNA deste protozoário em seus filhos. Para o diagnóstico sorológico as três técnicas convencionais de ELISA, IFI e HAI foram realizadas em todas as amostras. Todas as mães confirmaram a positividade da doença de Chagas pela sorologia. Duas crianças tiveram diagnóstico sorológico, parasitológico e molecular positivos para Trypanosoma cruzi, caracterizando a transmissão congênita, além da transferência gênica horizontal. As reações de polimerização em cadeia (PCR) foram realizadas visando identificar a presença de DNA nuclear (nDNA) e mitocondrial (kDNA) nas amostras estudadas. As amplificações foram realizadas em triplicata com cada par de primers, para confirmação diagnóstica. A amplificação do material nuclear de T. cruzi ocorreu em 92,1% das mães e em 10% das crianças (4/40). Em 70% das crianças e em 92,1% das mães houve amplificação do DNA do cinetoplasto do parasito. A maior parte dos eventos de integração de minicírculos de kDNA ocorreu por meio de elementos móveis, sendo a maioria deles o retrotransposon LINE-1. O maior número de integrações foi observado no cromossomo X. After more than a century the discovery of Chagas disease, which etiological agent parasite is called Trypanosoma cruzi, there is still much to be revealed about this disease. Polymorphism, how to play, the correlation between strain and the clinical, serological and molecular methods, gene transfer and its clinical consequences, treatment and cure are topics that involve major issues still not completely understood by researchers of this enigmatic disease. The control of donors in blood banks and the reduction of vector transmissions rates caused the congenital transmission to gain greater importance. In this study, monitoring women during pregnancy allowed the accompaniment of the newborn to nine months of age, at which stage it is expected that maternal antibodies have disappeared completely. To know the population of pregnant women infected by Trypanosoma cruzi, in the service of a Maternity Hospital, 1979 records were analyzed at an interval of three years (2010 to 2012). Socioeconomic and demographic profiles, as well as reproductive and serological features were studied. Had positive serology for American trypanosomiasis 3.1% of women (61/1.979) and a few of them reported abortions. Studies have shown that abortion in infected mothers who failed to transmit their infection to the fetus had no greater frequency of miscarriage, prematurity and perinatal mortality, but there was a greater tendency to stillbirth in mothers who transmitted the infection to their children. Thirty-eight infected by T. cruzi pregnant women (two with twin pregnancies) and their fetuses (forty) participated in a survey to verify integration of kDNA minicircle of this protozoan in their children. For serological diagnosis the three conventional techniques of ELISA, IFA and HAI were performed on all samples. All mothers confirmed the positivity of Chagas disease by serology. Two children had positive serological, parasitological and molecular diagnosis of Trypanosoma cruzi, featuring congenital transmission, in addition to horizontal gene transfer. The polymerase chain reactions (PCR) were performed to identify the presence of nuclear DNA (nDNA) and mitochondrial (kDNA) in the samples studied. The amplifications were performed in triplicate with each primer pair for diagnostic confirmation. Amplification of nuclear material of T. cruzi occurred in 92.1% of mothers and 10% of children (4/40). In 70% of children and 92.1% of the mothers was no amplification of kinetoplast DNA of the parasite. Most integration events of minicircle kDNA occurred through mobile elements, with most of them the LINE-1 retrotransposon. The largest number of integrations was observed on chromosome X. Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG

A flagellte trypanosomatid was isolated in culture from the digestive tract of the mosquito Culex saltanensis Dyar, 1928. It grows exuberantly in liver infusion in the form of epimastigotes and choanomastigotes typical of the genus Crithidia. The trypanosomatid was compared to C. deanei, C. fasciculata, C. luciliae, C. oncopelti and C. guilhermei. The techniques used for comparison were electron transmission microscopy, isoenzymes and kDNA restriction profiles. No endosymbionts were found at electron microscopy. Results for the biochemical methods employd indicate that the trypanosomatid isolated from C. saltanensis is a new species of Crithidia. The name C. ricardoi sp. n. is proposed for thes new species.

A investigação da possibilidade de diagnóstico para a Leishmaniose Tegumentar Americana (LTA), a partir de sangue venoso e através da Reação em Cadeia da Polimerase (Polymerase Chain Reaction = PCR), foi realizada em duas amostras de populações de área endêmica do Estado de Pernambuco, Brasil. Foi coletado um total de 119 amostras de sangue venoso, sendo 63 do município do Cabo de Santo Agostinho e 56 do município de Triunfo. Inicialmente, realizaram-se alguns experimentos para padronização da técnica de PCR, e os primers escolhidos amplificaram o fragmento de 750 pares de bases pertencente ao minicírculo do DNA do cinetoplasto (kDNA) da Leishmania Viannia braziliensis, espécie identificada com esta patologia em Pernambuco. Os resultados revelaram 48,7% de indivíduos positivos para a PCR, dos quais 31% tinham sido tratados e considerados clinicamente curados por apresentarem a lesão cicatrizada. Esses achados demonstram que: a) o sangue venoso pode ser utilizado como ferramenta para diagnóstico; b) a ausência de lesão nem sempre corresponde a uma PCR negativa; c) a existência de DNA circulante de parasita em indivíduos tratados, chama atenção para a possível presença do mesmo e conseqüente risco de recidiva; d) o tratamento preconizado não parece ser capaz de eliminar completamente os parasitas

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

The ability of the anti-trypanosomal drug berenil to interfere with the activities of eukaryotic type II topoisomerases is evaluated using two different types of reactions catalysed by the enzyme: the relaxation of naturally supercoiled DNA and the decatenation of kinetoplast DNA (kDNA). The results indicate that berenil acts as an inhibitor of the catalytic activity, but the inhibiting ability appears to be smaller than for other minor-groove binding ligands.

Phytomonas sp. which belongs to the Trypanosomatidae family, is a pathogen of plants. Its kinetoplast DNA consists of a huge network of about 7000 catenated minicircles of 2880 ± 30 base pairs each. It represents 30–33% of the total cellular DNA. An AT composition of 65% has been evaluated from the buoyant density xxx = 1.694 g ml . The restriction endonuclease HpaII was the only one able to cleave the minicircles into a single fragment. The other enzymes so far tested (EcoRI, HindIII, HaeIII, BamHI) do not cleave these minicircles significantly. Analysis of the kDNA by Southern blot hybridization shows that minicircles of Phytomonas sp. have little sequence homology with minicircles of other species of trypanosomes. Maxicircles, present in low proportions, have been identified. Our data indicate that the kinetoplast DNA of Phytomonas sp. presents biochemical characteristics which could be used to identify the Phytomonas genus.

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy. Inhibição do crescimento de um subespécie de Leishmania por exometabólitos de outra subespécie, um fenômeno ainda não notificado, é sugerido em nossas recentes observações em experimentos de clonagem celular com Leishmania mexicana mexicana e Leishmania mexicana amazonensis. Os clones foram identificados usando a técnica de análise de esquizodemas. O fenômeno observado é claramente relevante em estudos de isolamento parasitário, metabolismo, imunidade cruzada e quimioterapia.

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.