Nucleolar accumulation of RNA binding proteins induced by Actinomycin D is functional in Trypanosoma cruzi and Leishmania mexicana but not in T. brucei

We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs), involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD) treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2) nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1 was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage.     

Studies on Trypanosomatid Actin I. Immunochemical and Biochemical Identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai , actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Studies on trypanosomatid actin. I. Immunochemical and biochemical identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.     

Biochemical properties of trypanosomatid lactate dehydrogenases

Lactate dehydrogenase (LDH, E.C.1.1.1.27) was found in supernatant (cytoplasmic enzyme) fractions of the trypanosomatid flagellates Trypanosoma conorhini and Crithidia fasciculata if 10 mm cysteine was present in the homogenizing medium. The T. conorhini LDH activity with pyruvate as substrate was increased 35% if 5 mm cysteine was also included in reaction mixtures. K(m) values for the T. conorhini enzyme were 3.3 x 10(-4)m with pyruvate, and 1.6 x 10(-4)m with alpha-ketobutyrate. Cysteine inhibited alpha-ketobutyrate reduction. Comparison of trypanosomatid and human serum LDH enzymes with respect to K(m), substrate activity and inhibition, pH optima, and K(i) values for oxalate and oxamate indicated that the trypanosomatid isoenzymes differed significantly from serum LDH. C. fasciculata LDH was extremely labile, since 59% of the activity was lost 90 min after isolation. The role of LDH enzymes in trypanosomatid metabolism is discussed, and the results are related to other trypanosomatid LDH enzymes. The comparison of homologous enzymes in host and parasite is discussed with regard to metabolic function and a possible model system for chemotherapy.     

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera

Abstract Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same abtisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.     

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 μM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.     

The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.     

Dyes,trypanosomiasis and DNA: a historical and critical review

Abstract

Trypanosomiasis, a group of diseases including sleeping sickness in humans and Nagana in cattle in Africa, and Chagas’ disease in South America, remains a considerable problem in the 21^st century. The therapies that are available, however, usually have their roots in the “dye therapy” of a century ago, knowledge gained at the microscope from parasite staining procedures and converted to chemotherapy based on compounds closely related to the laboratory reagents. Dyes such as trypan red and trypan blue led to the development of suramin, while cationic nitrogen heterocyclic dyes furnished examples of the phenanthridinium class, such as ethidium (homidium) and isometamidium. Both suramin and isometamidium remain in use. Owing to mutagenicity issues, the presence of ethidium among the phenanthridinium dyes has led to concerns over the clinical use of related derivatives. There are several mechanisms for dye-DNA interaction, however, including possible hydrogen bonding of dye to the polymer, and these are discussed together with structure-activity relations and cellular localization of the phenanthridine and isomeric acridines involved. Better understanding of nucleic acid binding properties has allowed the preparation of more effective phenanthridinium analogues intended for use as anticancer/antiviral therapy.     

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.     

ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.     

Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species, Endotrypanum monterogeii and Trypanosoma cruzi.

The relative roles of acetate and leucine in the provision of a carbon source for fatty acid and sterol biosynthesis in several trypanosomatid species were investigated using 14C- and 13C-labelled acetate, glucose and leucine as substrates. Promastigotes of Leishmania species synthesized a large proportion of their sterol from leucine. L. major (LV39), L. amazonensis and L. mexicana were the most efficient utilizers of leucine, producing at least 70-77% of their sterol from leucine; L. braziliensis, L. donovani and L. tropica apparently produced less sterol from leucine (23-36%) and L. major (LV561), L. adleri and L. panamamensis were intermediate, utilizing leucine to provide 51-58% of their sterol. In all the cases the balance of the sterol produced was apparently synthesized from carbon arising from acetate. The related trypanosomatid Endotrypanum monterogeii also produced a large amount (77%) of its sterol from leucine rather than acetate. By contrast Trypanosoma cruzi elaborated only 8% of its sterol from leucine and used acetate far more effectively than the Leishmania species for sterol biosynthesis. The fatty acid moieties of the triacylglycerols and phospholipids were produced from acetate. Leucine was also incorporated into the fatty acids to varying extents in the different organisms showing that leucine can also be metabolized in trypanosomatids to generate acetyl-CoA.     

Protein kinases as drug targets in trypanosomes and Leishmania

Protein kinases represent promising drug targets for a number of human and animal diseases. The recent completion of the sequenced genomes of three human-infective trypanosomatid protozoa, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi, has allowed the kinome for each parasite to be defined as 179, 156 and 171 eukaryotic protein kinases respectively, that is about one third of the human complement. The analysis revealed that the trypanosomatids lack members of the receptor-linked or cytosolic tyrosine kinase families, but have an abundance of STE and CMGC family protein kinases likely to be involved in regulating cell cycle control, differentiation and response to stress during their complex life-cycles. In this review, we examine the prospects for exploiting differences between parasite and mammalian protein kinases to develop novel anti-parasitic chemotherapeutic agents.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.     

On the production of glycerol and l-alanine during the aerobic fermentation of glucose by trypanosomatids

Abstract Glucose consumption and catabolite production by thick suspensions of Trypanosoma cruzi, Leishmania mexicana and Crithidia fasciculata were similar under aerobic and anaerobic conditions, indicating lack of Pasteur effect. Succinate was the main product for L. mexicana and C. fasciculata ; the latter also produced similar amounts of ethanol. T. cruzi produced succinate and l -alanine to a similar extent. l -Alanine was also a major product of L. mexicana , but was neither produced, nor consumed, by C. fasciculata . Small amounts of glycerol were produced by L. mexicana and C. fasciculata , but not by T. cruzi , which had no detectable NAD-dependent sn -glycerol-3-phosphate dehydrogenase activity.     

A developmentally regulated cysteine proteinase gene of Leishmania mexicana

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.     

Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors.Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1–26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, ¹H-, and ¹³C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200–0.350 μM) of NTPDase-1, twelve (Ki = 0.071–1.060 μM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700–4.03 μM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ± 0.007, 6.39 ± 0.89 and 1.180 ± 0.002 μM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.