Comparative transcriptomic analysis of long noncoding RNAs in Leishmania-infected human macrophages.

It is well established that infection with Leishmania alters the host cell's transcriptome. Since mammalian cells have multiple mechanisms to control gene expression, different molecules, such as noncoding RNAs, can be involved in this process. MicroRNAs have been extensively studied upon Leishmania infection, but whether long noncoding RNAs (lncRNAs) are also altered in macrophages is still unexplored. We performed RNA-seq from THP-1-derived macrophages infected with Leishmania amazonensis (La), L. braziliensis (Lb), and L. infantum (Li), investigating a previously unappreciated fraction of macrophage transcriptome. We found that more than 24% of the total annotated transcripts and 30% of differentially expressed (DE) RNAs in Leishmania-infected macrophage correspond to lncRNAs. LncRNAs and protein coding RNAs with altered expression are similar among macrophages infected with the Leishmania species. Still, some species-specific alterations could occur due to distinct pathophysiology in which Li infection led to a more significant number of exclusively DE RNAs. The most represented classes among DE lncRNAs were intergenic and antisense lncRNAs. We also found enrichment for immune response-related pathways in the DE protein coding RNAs, as well as putative targets of the lncRNAs. We performed a coexpression analysis to explore potential cis regulation of coding and antisense noncoding transcripts. We identified that antisense lncRNAs are similarly regulated as its neighbor protein coding genes, such as the BAALC/BAALC-AS1, BAALC/BAALC-AS2, HIF1A/HIF1A-AS1, HIF1A/HIF1A-AS3 and IRF1/IRF1-AS1 pairs, which can occur as a species-specific modulation. These findings are a novelty in the field because, to date, no study has focused on analyzing lncRNAs in Leishmania-infected macrophage. Our results suggest that lncRNAs may account for a novel mechanism by which Leishmania can control macrophage function. Further research must validate putative lncRNA targets and provide additional prospects in lncRNA function during Leishmania infection.

Differential expression of polyamine biosynthetic pathways in skin lesions and in plasma reveals distinct profiles in diffuse cutaneous leishmaniasis.

Tegumentary leishmaniasis (TL) is a parasitic disease that can result in wide spectrum clinical manifestations. It is necessary to understand host and parasite determinants of clinical outcomes to identify novel therapeutic targets. Previous studies have indicated that the polyamine biosynthetic pathway is critical for Leishmania growth and survival. Despite its importance, expression of the such pathway has not been previously investigated in TL patients. We performed an exploratory analysis employing Systems Biology tools to compare circulating polyamines and amino acid concentration as well as polyamine pathway gene expression in cutaneous lesions patients presenting with distinct TL disease presentations. Diffuse cutaneous leishmaniasis (DCL) was associated with higher concentrations of amino acids, polyamines and its substrate transporters than mucosal cutaneous leishmaniasis or localized cutaneous leishmaniasis. In addition, the RNA expression of polyamine-related genes of patients lesions from two separate cohorts demonstrated that differential activation of this pathway is associated with parasite loads and able to discriminate the clinical spectrum of TL. Taken together, our findings highlight a new aspect of DCL immunopathogenesis indicating that the polyamine pathway may be explored as a novel therapeutic target to control disease burden.

Long Non-Coding RNAs in the Regulation of Gene Expression: Physiology and Disease.

The identification of RNAs that are not translated into proteins was an important breakthrough, defining the diversity of molecules involved in eukaryotic regulation of gene expression. These non-coding RNAs can be divided into two main classes according to their length: short non-coding RNAs, such as microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). The lncRNAs in association with other molecules can coordinate several physiological processes and their dysfunction may impact in several pathologies, including cancer and infectious diseases. They can control the flux of genetic information, such as chromosome structure modulation, transcription, splicing, messenger RNA (mRNA) stability, mRNA availability, and post-translational modifications. Long non-coding RNAs present interaction domains for DNA, mRNAs, miRNAs, and proteins, depending on both sequence and secondary structure. The advent of new generation sequencing has provided evidences of putative lncRNAs existence; however, the analysis of transcriptomes for their functional characterization remains a challenge. Here, we review some important aspects of lncRNA biology, focusing on their role as regulatory elements in gene expression modulation during physiological and disease processes, with implications in host and pathogens physiology, and their role in immune response modulation.

Arginine and Polyamines Fate in Leishmania Infection.

Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg - ). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection.

The dynamics of Brazilian protozoology over the past century.

Brazilian scientists have been contributing to the protozoology field for more than 100 years with important discoveries of new species such as Trypanosoma cruzi and Leishmania spp. In this work, we used a Brazilian thesis database (Coordination for the Improvement of Higher Education Personnel) covering the period from 1987-2011 to identify researchers who contributed substantially to protozoology. We selected 248 advisors by filtering to obtain researchers who supervised at least 10 theses. Based on a computational analysis of the thesis databases, we found students who were supervised by these scientists. A computational procedure was developed to determine the advisors' scientific ancestors using the Lattes Platform. These analyses provided a list of 1,997 researchers who were inspected through Lattes CV examination and allowed the identification of the pioneers of Brazilian protozoology. Moreover, we investigated the areas in which researchers who earned PhDs in protozoology are now working. We found that 68.4% of them are still in protozoology, while 16.7% have migrated to other fields. We observed that support for protozoology by national or international agencies is clearly correlated with the increase of scientists in the field. Finally, we described the academic genealogy of Brazilian protozoology by formalising the "forest" of Brazilian scientists involved in the study of protozoa and their vectors over the past century.

The dynamics of Brazilian protozoology over the past century

Brazilian scientists have been contributing to the protozoology field for more than 100 years with important discoveries of new species such asTrypanosoma cruzi and Leishmania spp. In this work, we used a Brazilian thesis database (Coordination for the Improvement of Higher Education Personnel) covering the period from 1987-2011 to identify researchers who contributed substantially to protozoology. We selected 248 advisors by filtering to obtain researchers who supervised at least 10 theses. Based on a computational analysis of the thesis databases, we found students who were supervised by these scientists. A computational procedure was developed to determine the advisors’ scientific ancestors using the Lattes Platform. These analyses provided a list of 1,997 researchers who were inspected through Lattes CV examination and allowed the identification of the pioneers of Brazilian protozoology. Moreover, we investigated the areas in which researchers who earned PhDs in protozoology are now working. We found that 68.4% of them are still in protozoology, while 16.7% have migrated to other fields. We observed that support for protozoology by national or international agencies is clearly correlated with the increase of scientists in the field. Finally, we described the academic genealogy of Brazilian protozoology by formalising the “forest” of Brazilian scientists involved in the study of protozoa and their vectors over the past century.

Infectiousness of Sylvatic and Synanthropic Small Rodents Implicates a Multi-host Reservoir of Leishmania (Viannia) braziliensis.

BACKGROUND: The possibility that a multi-host wildlife reservoir is responsible for maintaining transmission of Leishmania (Viannia) braziliensis causing human cutaneous and mucocutaneous leishmaniasis is tested by comparative analysis of infection progression and infectiousness to sandflies in rodent host species previously shown to have high natural infection prevalences in both sylvatic or/and peridomestic habitats in close proximity to humans in northeast Brazil. METHODS: The clinical and parasitological outcomes, and infectiousness to sandflies, were observed in 54 colonized animals of three species (18 Necromys lasiurus, 18 Nectomys squamipes and 18 Rattus rattus) experimentally infected with high (5.5 × 10(6)/ml) or low (2.8 × 10(5)/ml) dose L. (V.) braziliensis (MBOL/BR/2000/CPqAM95) inoculum. Clinical signs of infection were monitored daily. Whole animal xenodiagnoses were performed 6 months post inoculation using Lutzomyia longipalpis originating from flies caught in Passira, Pernambuco, after this parasite evaluation was performed at necropsy. Heterogeneities in Leishmania parasite loads were measured by quantitative PCR in ear skin, liver and spleen tissues. RESULTS: All three rodent species proved to establish infection characterized by short-term self-resolving skin lesions, located on ears and tail but not on footpads (one site of inoculation), and variable parasite loads detected in all three tissues with maximum burdens of 8.1 × 10(3) (skin), 2.8 × 10(3) (spleen), and 8.9 × 10(2) (liver). All three host species, 18/18 N. lasiurus, 10/18 N. squamipes and 6/18 R. rattus, also proved infectious to sandflies in cross-sectional study. R. rattus supported significantly lower tissue parasite loads compared to those in N. lasiurus and N. squamipes, and N. lasiurus appeared to be more infectious, on average, than either N. squamipes or R. rattus. CONCLUSIONS: A multi-host reservoir of cutaneous leishmaniasis is indicated in this region of Brazil, though with apparent differences in the competence between the rodent species. The results provide preliminary insights into links between sylvatic and peri-domestic transmission cycles associated with overlaps in the rodent species' ecological niches.

Involvement of pituitary gonadotropins, gonadal steroids and breeding season in sex change of protogynous dusky grouper, Epinephelus marginatus (Teleostei: Serranidae), induced by a non-steroidal aromatase inhibitor.

Two experiments were performed using the aromatase inhibitor (AI) letrozole (100mg/kg) to promote sex change, from female-to-male, in protogynous dusky grouper. One experiment was performed during the breeding season (spring) and the other at the end of the breeding season (summer). During the spring, AI promoted sex change after 9 weeks and the sperm produced was able to fertilize grouper oocytes. During the summer, the sex change was incomplete; intersex individuals were present and sperm was not released by any of the animals. Sex changed gonads had a lamellar architecture; cysts of spermatocytes and spermatozoa in the lumen of the germinal compartment. In the spring, after 4 weeks, 11ketotestosterone (11KT) levels were higher in the AI than in control fish, and after 9 weeks, coincident with semen release, testosterone levels increased in the AI group, while 11KT returned to the initial levels. Estradiol (E2) levels remained unchanged during the experimental period. Instead of decreasing throughout the period, as in control group, 17 α-OH progesterone levels did not change in the AI-treated fish, resulting in higher values after 9 weeks when compared with control fish. fshß and lhß gene expression in the AI animals were lower compared with control fish after 9 weeks. The use of AI was effective to obtain functional males during the breeding season. The increase in androgens, modulated by gonadotropins, triggered the sex change, enabling the development of male germ cells, whereas a decrease in E2 levels was not required to change sex in dusky grouper.

Leishmania amazonensis arginase compartmentalization in the glycosome is important for parasite infectivity.

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.

Axenic Leishmania amazonensis promastigotes sense both the external and internal arginine pool distinctly regulating the two transporter-coding genes.

Leishmania (L.) amazonensis uses arginine to synthesize polyamines to support its growth and survival. Here we describe the presence of two gene copies, arranged in tandem, that code for the arginine transporter. Both copies show similar Open Reading Frames (ORFs), which are 93% similar to the L. (L.) donovani AAP3 gene, but their 5' and 3' UTR's have distinct regions. According to quantitative RT-PCR, the 5.1 AAP3 mRNA amount was increased more than 3 times that of the 4.7 AAP3 mRNA along the promastigote growth curve. Nutrient deprivation for 4 hours and then supplemented or not with arginine (400 µM) resulted in similar 4.7 AAP3 mRNA copy-numbers compared to the starved and control parasites. Conversely, the 5.1 AAP3 mRNA copy-numbers increased in the starved parasites but not in ones supplemented with arginine (p<0.05). These results correlate with increases in amino acid uptake. Both Meta1 and arginase mRNAs remained constant with or without supplementation. The same starvation experiment was performed using a L. (L.) amazonensis null knockout for arginase (arg(-)) and two other mutants containing the arginase ORF with (arg(-)/ARG) or without the glycosomal addressing signal (arg(-)/argΔSKL). The arg(-) and the arg(-)/argΔSKL mutants did not show the same behavior as the wild-type (WT) parasite or the arg(-)/ARG mutant. This can be an indicative that the internal pool of arginine is also important for controlling transporter expression and function. By inhibiting mRNA transcription or/and mRNA maturation, we showed that the 5.1 AAP3 mRNA did not decay after 180 min, but the 4.7 AAP3 mRNA presented a half-life decay of 32.6 +/- 5.0 min. In conclusion, parasites can regulate amino acid uptake by increasing the amount of transporter-coding mRNA, possibly by regulating the mRNA half-life in an environment where the amino acid is not present or is in low amounts.

Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years.

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn2+ center formation of the immobilized enzyme on a Ni2+ resin.

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn2+ applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni2+ resin was directly proportional to the concentration of Mn2+. Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni2+, allowing the following to occur: (1) entrance of Mn2+ and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni2+ after the Mn2+ activation step. The conformational alterations can be summarized as follows: the interaction with the Ni2+ simulates thermal heating in the artificial activation by opening a channel for Mn2+ to enter.

Active transport of glutamate in Leishmania (Leishmania) amazonensis.

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K(m)=0.59+/-0.04 mM, V(max)=0.123+/-0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38+/-4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H(+) but not to Na(+) or K(+). However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K(+), and not H(+) membrane gradient in the transport process.

Successful treatment of cutaneous leishmaniasis with lipid formulations of amphotericin B in two immunocompromised patients.

Pentavalent antimonial drugs are habitually the first choice for treating leishmaniasis, although they possess well-known toxicity and may present some therapeutic failure. Lipid formulations of amphotericin B (LFAB) have been increasingly used for treating several types of leishmaniasis. However, the administration of such lipid formulations specifically to patients with cutaneous leishmaniasis (CL) is still rare, including immunocompromised patients to whom standard treatments are more frequently contraindicated. We describe here two cases of immunocompromised patients with CL, one of them with AIDS, representing the first case of AIDS and CL co-infection treated with LFAB described in the literature. The patient achieved therapeutic success with a total 1.500 mg dose of amphotericin B colloidal dispersion. The other had diabetes mellitus as well as kidney failure and was under dialysis, having obtained the healing of lesion with a total dose of 600 mg of liposomal amphotericin B. Thus, the authors suggest that LFAB can represent a safe, efficient and less toxic therapeutic alternative to pentavalent antimonials, as well as to the so-called second line drugs, pentamidine and amphotericin B deoxycholate.

MT-1 melatonin receptor expression increases the antiproliferative effect of melatonin on S-91 murine melanoma cells.

Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor- and non-receptor-mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S-91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S-91 melanoma cell proliferation in vitro (EC50 = 10-7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT-1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10-10 m) in S-91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT-1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.

New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species.

Glucose-6-phosphate dehydrogenase (G6PD) is one of the multilocus enzymes used to identify Leishmania by zymodeme analysis. The polymorphic pattern revealed by partial characterization of the gene encoding G6PD generated molecular markers useful in the identification of different Leishmania species by PCR. Initially degenerate oligonucleotides were designed on the basis of data on the conserved active center described for other organisms. Primers for reverse transcription-PCR experiments, designed from the nucleotide sequence of the PCR product, enabled us to characterize the 5' and 3' untranslated regions and the G6PD open reading frame of reference strains of Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) amazonensis. Sets of paired primers were designed and used in PCR assays to discriminate between the parasites responsible for tegumentar leishmaniasis of the subgenera Leishmania (Leishmania) and Leishmania (Viannia) and to distinguish L. (Viannia) braziliensis from others organisms of the subgenus Leishmania (Viannia). No amplification products were detected for the DNA of Crithidia fasciculata, Trypanosoma cruzi, or Leishmania (Sauroleishmania) tarentolae or DNA from a healthy human control. The tests proved to be specific and were sensitive enough to detect parasites in human biopsy specimens. The successful discrimination of L. (Viannia) braziliensis from other parasites of the subgenus Leishmania (Viannia) opens the way to epidemiological studies in areas where more than one species of the subgenus Leishmania (Viannia) exist, such as Amazonia, as well as follow-up studies after chemotherapy and assessment of clinical prognoses.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania).

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

Two distinct functional spliced leader RNA gene arrays in Leishmania tarentolae are found in several lizard Leishmania species.

A second distinct array of spliced leader RNA genes has been found in several Leishmania species particular to lizards. This is the first report of two non-allelic arrays of spliced leader RNA genes within a species cell line. The arrays are identical to each other in their transcribed spliced leader RNA gene sequences, but variable in their non-transcribed spacer sequences. In the two arrays from Leishmania tarentolae UC strain the promoter regions are similar, but not identical, at positions shown previously to be critical for spliced leader RNA transcription. These arrays contain similar numbers of genes and are both transcribed in L. tarentolae in vitro transcription extract as well as in vivo. The -66/-58 regions of both genes, which contain an element of the spliced leader RNA gene promoter, bind proteins likely to be transcription factors in a specific manner. A survey of lizard Leishmania spp. revealed a second spliced leader RNA gene array in three of four species. Phylogenetic analyses of these sequences with each other and with the spliced leader RNA gene sequences of non-lizard Leishmania spp. and their near-relatives showed that the lizard groups are more closely related to each other than to arrays from other Leishmania spp. As the transcripts of the two arrays are identical, they may co-exist to fulfil the substantial requirement for spliced leader RNA production; however, they have the potential for differential usage modulated by their distinct promoter elements. The presence of two distinct spliced leader RNA gene arrays within a single cell type may represent dissociated evolution of two redundant loci, or a previously unsuspected level of control in the post-transcriptional gene expression within some kinetoplastids.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982

Genomic organisation and transcription characterisation of the gene encoding Leishmania (Leishmania) amazonensis arginase and its protein structure prediction.

The genomic organisation of the gene encoding Leishmania (Leishmania) amazonensis arginase as well as its flanking regions were characterised. The size of the transcribed RNA was determined, allowing us to map the genomic sites signalling for RNA trans-splicing and putative polyadenylation regions. The general organisation was compared with genes encoding other proteins already described in organisms of the Trypanosomatid family. The complete nucleotide sequence of the arginase open reading frame was obtained and the three-dimensional structure of the enzyme was inferred by a computational analysis of the deduced amino acid sequence, based on the established crystal structure described for Rattus norvergicus arginase. The human liver arginase sequence was analysed in the same way and the comparison of the presumed structure of both the Leishmania and human enzymes identified some differences that may be exploited in chemotherapeutic studies.