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It was reported before that cells of the trypanosomatid Leptomonas samueli incubated with [14C]glucose synthesized dolichol-P-P-linked Man9GlcNAc2 as the main and largest derivative. It is now reported that this protozoan is deficient in dolichol-P-Glc synthesis as judged from results obtained in a cell-free assay. We have structurally characterized several endo-beta-N-acetylglucosaminidase H sensitive oligosaccharides present in mature glycoproteins of this parasite. The compounds appeared to have the compositions Gal3Man9GlcNAc2, Gal2Man9GlcNAc2, Gal1Man9GlcNAc2, Man9GlcNAc2, Gal1Man8GlcNAc2, Man8GlcNAc2, Gal1Man7GlcNAc2, and Man7GlcNAc2. The galactose residues were in all cases in the furanose form and linked to mannoses in nonreducing ends. In the cases of Gal1Man8GlcNAc2 and Gal1Man7GlcNAc2, the galactose-substituted mannose units were the nonreducing residues originally present in the oligosaccharide transferred from dolichol-P-P (Man9GlcNAc2) and not the nonreducing termini generated by demannosylation of the latter oligosaccharide. Except for Gal3Man9GlcNAc2, the other galactosylated compounds appeared to be mixtures of several isomers.

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Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.

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An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.

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S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

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Xylose and mannose are the main manosaccharide components of Herpetomonas samuelpessoai and Leptomonas samueli promastigotes. Variations in the xylose/mannose ratios are related to the age of cultures. Phenol-aqueous extraction disclosed in both species the presence of carbohydrate-containing fractions which were both soluble and insoluble in chloroform/methanol/water (10:10.3). The xylose enriched, uronic acid-containing glycoconjugates of L. samueli were mainly composed of (1----4)-linked xylose units (Methylation-mass spectrometry and 13C NMR), similar to the glucuronoxylan of H. samuelpessoai (Mendonça-Previato et al., 1979 Biochem 18 149-154). SDS-PAGE and sugar composition analysis disclosed similarities between glycoconjugates of H. samuelpessoai and L. samueli, two species dwelling in the same host, therefore an example of convergent adaptation.

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Kinetoplast DNA of trypanosomatids represents a complex associate composed of mini- and maxicircular molecules, the latter being analogous to mitochondrial DNA of other eukaryotic species. Some new data on the structural organization and transcription of trypanosomatid mitochondrial genome are given. Detailed genetic and transcriptional maps of maxicircular kinetoplast DNAs of trypanosome and leishmania are presented. The replication mechanisms of mini- and maxicircular molecules of kinetoplast DNA are analyzed. Some characteristic features of trypanosomatid mitochondrial genome organization similar and different with respect to mitochondrial genomes of other eukaryotic species are discussed.

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Dolichol-P- and dolichol-P-P-linked saccharides were isolated from several trypanosomatid flagellates incubated with [U-14C]glucose. Formation of Glc-P-dolichol and Man-P-dolichol was observed in Herpetomonas muscarum and Leishmania adleri, whereas only the latter derivative was synthesized in Trypanosoma dionisii and Leptomonas samueli. The main and largest dolichol-P-P-linked oligosaccharide formed in Trypanosoma conorhini, T. dionisii, L. samueli, Herpetomonas samuelpessoai and H. muscarum appeared to be Man9GlcNAc2, whereas in Blastocrithidia culicis it was Man6GlcNAc2. In L. adleri there were two main oligosaccharides linked to dolichol-P-P, Man6GlcNAc2 and Man5GlcNAc2. The The structures of the oligosaccharides were identical with those of the intermediates in the formation of Glc3Man9GlcNAc2-P-P-dolichol in higher eucaryotes. It was concluded that similarly to Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana, no glucosylated derivatives of dolichol-P-P were formed in the additional seven trypanosomatids studied here. The results obtained suggest that the defective step could be in some cases the formation of Glc-P-dolichol and in others, the transfer of glucose residues from the latter compound to dolichol-P-P-linked oligosaccharides.

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Protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were isolated from several trypanosomatids incubated with [U-14C]glucose. Structural analysis of the compounds revealed that Man9GlcNAc2 was the oligosaccharide transferred from dolichol-P-P derivatives to proteins in Trypanosoma dionisii, Trypanosoma conorhini, Leptomonas samueli and Herpetomonas samuelpessoai and Man6GlcNAc2 in Blastocrithidia culicis and Leishmania adleri. In all cases, transiently glucosylated compounds were detected: Glc1Man7-9GlcNAc2 in T. dionisii, T. conorhini, L. samueli; Glc1Man9GlcNAc2 in H. samuelpessoai, Glc1Man6GlcNAc2 in B. culicis and Glc1Man6GlcNAc2 and Glc1Man5GlcNAc2 in L. adleri. The mechanism of protein glycosylation in T. dionisii and T. conorhini appeared to be similar to that described before for Trypanosoma cruzi epimastigotes, although some differences were found between the structures of the main isomers of Man7GlcNAc2 and Man8GlcNAc2 present in T. conorhini and T. cruzi. Differences between the mechanisms of glycosylation occurring in Leishmania mexicana and L. adleri were also found: Man6GlcNAc2 in the latter microorganism was demannosylated to Man5GlcNAc2, a step not detected in the former parasite. A novel substituent in N-linked high mannose-type oligosaccharides was found in L. samueli and H. samuelpessoai: galactose in the furanose configuration. In the latter trypanosomatid, Man9GlcNAc2 was demannosylated only to Man8GlcNAc2, whereas in all other parasites in which the same oligosaccharide was transferred to proteins, Man5-7GlcNAc2 were also detected.

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