Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction

Traub-Csekö, Yara M.; Duboise, Monroe; Boukai, Linda Khalili; McMahon-Pratt, Diane

doi:10.1016/0166-6851(93)90248-v

Cited by 72 publications

(32 citation statements)

References 38 publications

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“…panamensis promastigotes and axenic amastigotes was assessed by PCR amplification of transcripts encoding a cystein protease 480-bp region, which has been shown to be overexpressed in amastigotes. 21,22 As presented in Figure 6b, a high level of transcripts was detected, mostly in L. (V). panamensis axenic amastigotes compared with promastigotes.…”

Section: Biological and Molecular Assessment Of Axenic Amastigotes Amentioning

confidence: 92%

“…To amplify the cysteine protease, two oligonucleotides flanking a 0.48-kb region were used: LCP-1 5Ј-AAG GCG CGT GCG GGT CGT-3Ј and LCP-2 5Ј-CGA GTT CTT GAT CAC CCA GTA3-Ј. 21,22 Amplification was run as described above and ␤-tubulin was used as housekeeping gene. The products were analyzed by electrophoresis on a 1% agarose gel.…”

Section: Macrophagesmentioning

confidence: 99%

“…Several amastigote-specific genes have been described in different Leishmania species, including hsp83, hsp100, histone H1, cpb cysteine proteinase, the A2 gene family, the LmcDNA16 gene family, the spliced leader RNA gene, and the Ca 2ϩ -ATPase gene. [1][2][3][4][5][15][16][17][18][19][20][21][22] Amastigote isolation, from either lesions or infected macrophages, usually produces low parasite numbers, contaminated by host components. 23,24 Axenic cultures have been obtained from several Leishmania species through changes in temperature and pH.…”

Section: Introductionmentioning

confidence: 99%

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Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

Puentes¹,

Díaz²,

Hoya³

et al. 2000

The American Journal of Tropical Medicine and Hygiene

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Abstract. The study of the differential regulation of several genes, in both Leishmania parasite life cycle forms, has been simplified by the development of in vitro axenic amastigote culture. Different reports have described extracellular amastigote production and maintenance from several Leishmania spp. A general approach to induce amastigote-like transformation includes progressive pH and temperature changes. Production of axenic amastigotes in continuous cultures using amastigotes recovered from macrophages is described in this report. Leishmania (Viannia) panamensis (M/HOM/PA/71/LS94) and Leishmania (V). guyanensis (M/HOM/BR/75/M4147) intracellular amastigotes were recovered from the human macrophage-like U937 cell line previously infected with promastigotes. The parasites were immediately adapted for growth and kept as axenic amastigotes at 34ЊC and acidic pH. These organisms were able to infect macrophage cell lines, maintain amastigote morphologic features, and express stage-specific transcripts. The relevance of axenic amastigotes in characterizing virulence factors in American leishmaniasis is discussed.

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Section: Biological and Molecular Assessment Of Axenic Amastigotes Amentioning

confidence: 92%

Section: Macrophagesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

Puentes¹,

Díaz²,

Hoya³

et al. 2000

The American Journal of Tropical Medicine and Hygiene

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“…Probes and hybridization assays -The following probes were used: a 3.8 Kb fragment of the L. major β-tubulin gene -clone pLT-1 (Huang et al 1984), the Lpcys2 cysteine proteinase gene of L. pifanoi (Traub-Cseko et al 1993), a 0.4 Kb EcoRI fragment containing T. cruzi hsp 70 coding sequence (De Carvalho et al 1990) and a sequence of the T. cruzi actin gene (Paixão 1995). These probes were radiolabeled with [α-32P]dCTP by the random priming method (Rediprime kit®, Amersham, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

Toaldo

Steindel

Sousa³

et al. 2001

Mem. Inst. Oswaldo Cruz

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“…Trypanosome cysteine proteases are synthesized as precursor proteins with a hydrophobic signal peptide, a 100 -122-amino acid prodomain, a 200 -210-amino acid catalytic domain, and, in most cases, a 100 -130-amino acid carboxyl-terminal domain (7)(8)(9)(10)(11)(12). The function of the carboxyl-terminal domain is as yet unknown.…”

mentioning

confidence: 99%

Protease Trafficking in Two Primitive Eukaryotes Is Mediated by a Prodomain Protein Motif

Huete-Pérez¹,

Engel²,

Brinen

et al. 1999

Journal of Biological Chemistry

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Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.Sorting of newly translated lysosomal proteases may occur by two different mechanisms. In mammalian cells, the predominant is through the addition of phosphomannosyl residues and targeting to the lysosome pathway by binding to M6P 1 receptors within the Golgi (1). However, transport of lysosomal enzymes in many cells is unaffected by a deficiency in the phosphotransferase, which is required for M6P synthesis (2). M6P-receptor-independent membrane association has been reported for several lysosomal proteins (3). Confirmation and further analysis of a M6P-independent sorting pathway in mammalian cells has been complicated by both the presence of the M6P pathway itself and difficulty in distinguishing effects on protein folding versus protein trafficking when deletional mutants of the protease precursors were analyzed (4, 5). Trypanosoma cruzi and Leishmania mexicana are protozoa (Trypanosoma) representing one of the earliest lineages of eukaryotic cells. They nevertheless express proteases homologous to cathepsins L and B and, unlike yeast, have an organelle ultrastructure more reminiscent of mammalian cells (6). While the cathepsin L-like protease prodomains of these organisms have significant homology to mammalian procathepsin L (e.g. 45% identity for cruzain versus mouse cathepsin L), there are no carbohydrate modifications, so they are unique experimental models for analyzing M6P-independent protein sorting.Trypanosome cysteine proteases are synthesized as precursor proteins with a hydrophobic signal peptide, a 100 -122-amino acid prodomain, a 200 -210-amino acid catalytic domain, and, in most cases, a 100 -130-amino acid carboxyl-terminal domain (7-12). The function of the carboxyl-terminal domain is as yet unknown. Hypotheses that it plays a role in protease inactivation, or in facilitating folding of the catalytic domain, have been ruled out by expression of fully active recombinant proteases without this domain (10). Other proposed functions have included mediating intracellular trafficking of the protease (13), immune evasion (14), and facilitating activity against specific macromolecular substrates (15).The pr...

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Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction

Cited by 72 publications

References 38 publications

Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

Protease Trafficking in Two Primitive Eukaryotes Is Mediated by a Prodomain Protein Motif

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