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1.
J Biol Chem ; 272(14): 9464-73, 1997 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-9083086

RESUMO

Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Leishmania mexicana/patogenicidade , Macrolídeos , Sequência de Aminoácidos , Aminoquinolinas/farmacologia , Cloreto de Amônio/farmacologia , Animais , Antibacterianos/farmacologia , Sequência de Bases , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Cricetinae , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Indicadores e Reagentes/metabolismo , Ionomicina/farmacologia , Ionóforos/farmacologia , Leishmania mexicana/enzimologia , Macrófagos/parasitologia , Mesocricetus , Dados de Sequência Molecular , Nigericina/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Transdução de Sinais
2.
Braz J Med Biol Res ; 27(2): 177-84, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8081227

RESUMO

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.


Assuntos
Retículo Endoplasmático/metabolismo , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Leishmania major/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/metabolismo , Fosfolipases Tipo C/biossíntese , Animais , Compartimento Celular , Citoplasma/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/metabolismo , Leishmania major/genética , Mamíferos , Linfócitos T/metabolismo
3.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;27(2): 177-84, Feb. 1994. ilus
Artigo em Inglês | LILACS | ID: lil-138282

RESUMO

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots, gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma


Assuntos
Humanos , Retículo Endoplasmático , Fosfatidilinositóis/biossíntese , Glicolipídeos/biossíntese , Hemoglobinúria Paroxística/metabolismo , Leishmania tropica , Trypanosoma brucei brucei , Fosfolipases Tipo C , Linhagem Celular , Fosfatidilinositóis/metabolismo , Glicolipídeos/metabolismo , Mamíferos , Glicoproteínas Variantes de Superfície de Trypanosoma
4.
J Biol Chem ; 265(21): 12240-7, 1990 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-2373691

RESUMO

The extent of protein N-glycosylation in Leishmania mexicana amazonensis has been proposed to be a factor in the virulence of the parasite. The N-linked oligosaccharides of gp63, the major surface glycoprotein of L. mexicana amazonensis, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P-4 yielded four fractions, and the oligosaccharides present were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis, and methylation analysis. Four major structures were found and were biantennary oligomannose type with compositions of Glc1Man6GlcNAc2 (La), Man6GlcNAc2 (Lb), Man5GlcNAc2 (Lc), and Man4GlcNAc2 (Ld). The largest oligosaccharide (La) was shown to contain a terminal glucopyranosyl residue on the alpha (1----3) arm. The biantennary oligomannose structures (Lb and Lc) and the glucosylated structure Glc1Man6GlcNAc2 (La) have not previously been reported as a component of a mature glycoprotein from any source.


Assuntos
Antígenos de Protozoários/análise , Leishmania mexicana/análise , Glicoproteínas Variantes de Superfície de Trypanosoma/análise , Animais , Sequência de Carboidratos , Cromatografia em Gel , Metilação , Dados de Sequência Molecular , Oligossacarídeos/análise , Glicoproteínas Variantes de Superfície de Trypanosoma/isolamento & purificação
5.
J Biol Chem ; 264(13): 7483-9, 1989 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-2708373

RESUMO

Acid proteinase activity is associated with the major surface glycoprotein (gp63) of both extracellular promastigotes and intracellular amastigotes of the parasitic protozoan, Leishmania mexicana. The enzyme purified by monoclonal affinity chromatography from promastigotes is strongly inhibited by metal ion chelators, which is reversible by the addition of Zn(II). This proteinase loses its activity after dialysis against 1,10-phenanthroline. The apoenzyme thus prepared is reactivated substantially by Zn(II) and partially by Cu(II), Cd(II), Co(II), or Ni(II). From the recently published structure of the gene encoding gp63, we identify hitherto unrecognized sequences, which can be aligned to the consensus zinc-binding sites of other known metalloproteinases. Anti-gp63 polyclonal antibodies, but not the monoclonals, precipitate similar molecules from amastigotes. These molecules differ slightly from gp63 in electrophoretic mobility but have similar endopeptidase activity. Phagolysosomal degradation by macrophages of proteins entrapped in liposomes is prevented by coating them with native gp63. This protection is lost with heat denaturation of gp63 to kill its enzymatic activity. The proteolytic activity of the metalloenzyme on the surface of these parasites may thus protect their membrane from cytolytic damages during their survival, differentiation, and multiplication in the phagolysosomes of macrophages.


Assuntos
Leishmania mexicana/enzimologia , Macrófagos/fisiologia , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Animais , Western Blotting , Diferenciação Celular , Quelantes/farmacologia , Concentração de Íons de Hidrogênio , Leishmania mexicana/citologia , Lisossomos/parasitologia , Macrófagos/parasitologia , Metaloendopeptidases/antagonistas & inibidores , Fagossomos/parasitologia
6.
J Biol Chem ; 263(7): 3418-24, 1988 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-2449440

RESUMO

Tunicamycin-resistant variants of Leishmania mexicana were found to contain elevated activity of N-acetylglucosamine-1-phosphate transferase and amplified DNA (Kink, J. A., and Chang, K.-P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1253-1257). Complete digestion of their DNA with restriction endonucleases produced discrete ethidium bromide-staining bands after agarose gel electrophoresis. All four BamHI fragments of the amplified DNA were cloned separately into pBR322 and found to share no substantial sequence homology. DNA complementary to each of the cloned fragments is 64-128-fold more abundant in the variants than in the wild type cells. The amplified DNA appears to originate from a single chromosomal region of 63 kilobases. Individual copies of the 63 kilobases are each circularized at the newly formed junction site producing multiple extrachromosomal supercoiled molecules in the drug-resistant cells. There is overproduction of RNA ranging in size from 1.9 to 6.6 kilobases complementary to the amplified DNA in these cells.


Assuntos
DNA Super-Helicoidal/genética , Amplificação de Genes , Leishmania mexicana/genética , Transferases (Outros Grupos de Fosfato Substituídos) , Tunicamicina , Animais , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Recombinante , Resistência a Medicamentos/genética , Eletroforese em Gel de Ágar , Concentração de Íons de Hidrogênio , Hibridização de Ácido Nucleico , Fosfotransferases/metabolismo , RNA/genética , Homologia de Sequência do Ácido Nucleico
7.
Mol Biochem Parasitol ; 27(2-3): 181-90, 1988 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-2830512

RESUMO

Promastigotes of Leishmania mexicana amazonensis grown in vitro under different conditions showed variable degrees of virulence, as determined quantitatively by the size of the lesions and the number of intracellular parasites produced in mice and in cultured macrophages, respectively. Promastigotes newly transformed from amastigotes gave the highest degree of virulence, which decreased progressively with periods of their continuous in vitro cultivation. This loss of virulence was prevented by making virulent wildtype promastigotes resistant to tunicamycin, an inhibitor of N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of protein glycosylation. In the wildtype cells, the progressive loss of virulence during a period of two years was marked by a gradual decrease in the activity of the glycosyltransferase, incorporation of 2-D-[3H]mannose and the expression of a surface glycoprotein (gp63). Virulent and avirulent wildtype cells differed in these activities by 3-4 fold. In contrast, during equivalent periods of in vitro cultivation, tunicamycin-resistant cells were found to consistently maintain the biochemical phenotypes of the virulent wildtype, except for a disproportional elevation of the glycosyltransferase activity. Thus, only a portion of the over-produced enzyme is relevant to leishmanial virulence in the drug-resistant variants. The bulk of its activity in these cells serves to overcome the inhibitory effect of tunicamycin responsible for their resistance to this drug, as shown previously. A role of N-glycosylation in leishmanial virulence is now indicated by studying cells of this phenotype selected by two independent methods. It is inferred that leishmanial virulence may be regulated by the level of the glycosyltransferases for N-glycosylation of proteins, e.g. gp63 in relation to their expression as virulent determinants.


Assuntos
Leishmania mexicana/patogenicidade , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Células Cultivadas , Glicoproteínas/metabolismo , Glicosilação , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Macrófagos/parasitologia , Camundongos , Fosfotransferases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tunicamicina/farmacologia , Virulência
8.
Mol Biochem Parasitol ; 27(1): 43-52, 1988 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-3278222

RESUMO

A unique protease with activity optimal at pH 4.0 and trailing toward the alkaline pH spectrum was detected with intact glutaraldehyde-fixed promastigotes of Leishmania mexicana amazonensis, indicating surface localisation of the enzyme. That this surface protease may be a virulence factor is suggested by its apparent roles in multiple steps during leishmanial infections of macrophages. Indeed, its specific activity was 2-2.5 fold higher on virulent cells than on avirulent cells. Several lines of evidence indicate that this acid protease activity is expressed by the major surface glycoprotein (gp63) of L. m. amazonensis. Monoclonal antibody affinity purified gp63 degraded serum albumin, hemoglobin, complement C3, immunoglobulin G and purified rat liver lysosomal proteins in their native forms. The specific activity is about 20-fold higher at pH 4.0 than at pH 7.5 and is about four-fold higher at the body temperature of the mammalian host (37 degrees C) than at that of the insect host (27 degrees C). The protease activity is sodium dodecyl sulphate-sensitive. Among various protease inhibitors tested, only heavy metal ions (1 mM), 1,10-orthophenanthroline (1 mM) and bestatin (100 ng ml-1) significantly inhibited gp63 acid protease activity by up to 80%. N-linked oligosaccharides of gp63 appear to be important for the stability of this molecule, possibly by preventing its autodegradation. Purified gp63 effected limited proteolysis of human complement C3 molecules at the physiological serum pH of 7.5 in a manner, which supports the idea of its participation in complement-receptor mediated endocytosis of promastigotes by macrophages.


Assuntos
Antígenos de Protozoários/metabolismo , Endopeptidases/metabolismo , Leishmania mexicana/enzimologia , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases , Animais , Ácido Aspártico Endopeptidases , Complemento C3/metabolismo , Eletroforese em Gel de Poliacrilamida , Hemoglobinas/metabolismo , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Leishmania mexicana/patogenicidade , Lisossomos , Proteínas/metabolismo , Soroalbumina Bovina/metabolismo , Temperatura , Virulência
9.
Infect Immun ; 55(7): 1692-700, 1987 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3036710

RESUMO

A parasitic protozoan, Leishmania mexicana amazonensis, was previously made resistant to tunicamycin (J.A. Kink and K.-P. Chang, Proc. Natl. Acad. Sci. USA 84:1253-1257, 1987). In the present study, six different tunicamycin-resistant variants were biologically and biochemically compared with their parental wild type to further delineate the mechanism of tunicamycin resistance and that of their virulence observed. In contrast to their parental wild type, all tunicamycin-resistant variants were found to grow and differentiate in tunicamycin-containing medium. The 50% lethal doses of tunicamycin for variants resistant to 10 or 80 micrograms of tunicamycin per ml were 20- and 100-fold higher, respectively, than that of the wild type. Specific activity of the microsomal N-acetylglucosamine-1-phosphate transferase was 4- to 12-fold higher in the tunicamycin-resistant cells than in their parental wild type and tunicamycin-sensitive revertants. The level of the enzyme activity is proportional to the degree of drug resistance. Inhibition kinetics studies showed that the enzyme from all groups was equally sensitive to the drug, with a 50% effective concentration of 1 to 1.3 micrograms of tunicamycin per ml. Thus, tunicamycin resistance of the variants is caused primarily by an increased level of their enzyme without alteration of its structure. Protein glycosylation determined by the incorporation of 2-D-[3H]mannose was about twofold higher in the tunicamycin-resistant variants than in their parental wild type. The increased glycosyltransferase activity in the latter apparently renders their protein glycosylation insensitive to the inhibition by tunicamycin. A major membrane glycoprotein of 63 kilodaltons (gp63) on the leishmania surface was found to be about threefold higher in the tunicamycin-resistant variants than in the wild type, as determined by immunoprecipitation with a monoclonal antibody specific for this antigen. Tunicamycin treatment of the wild type and tunicamycin-resistant variants caused changes in the electrophoretic mobility of this molecule, indicating a higher degree of its glycosylation in the latter cells. The tunicamycin-resistant variants parasitized macrophages in vitro more effectively than did the wild type, accounting for their virulence seen in mice. Thus, a high level of the glycosyltransferase enables the tunicamycin-resistant cells not only to overcome the inhibitory effect of tunicamycin on protein glycosylation but also to express their virulence, possibly by regulating N glycosylation of leishmanial proteins critical for leishmanias to establish intracellular parasitism.


Assuntos
Leishmania mexicana/efeitos dos fármacos , Leishmaniose/parasitologia , Transferases (Outros Grupos de Fosfato Substituídos) , Tunicamicina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Resistência a Medicamentos , Glicoproteínas/metabolismo , Glicosilação , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/patogenicidade , Camundongos , Microssomos/enzimologia , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional
10.
Proc Natl Acad Sci U S A ; 84(5): 1253-7, 1987 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2950522

RESUMO

Tunicamycin at 10 micrograms/ml inhibits the growth and infectivity of the parasitic protozoan Leishmania mexicana amazonensis. Tunicamycin-resistant variants of this parasite were produced by gradual acclimatization of cells to increasing concentrations of the drug up to 80 micrograms/ml and a single-step selection of ethyl methanesulfonate-pretreated or differentiating leishmanias with the drug at 10 micrograms/ml. Prolonged exposure to the drug increases stability of drug resistance of those resistant to 10 micrograms/ml. Tunicamycin-resistant cells contain amplified DNA, which hybridizes in proportion to the cells' degree of drug resistance with Alg 7, a cloned DNA probe apparently encoding yeast N-acetylglucosaminyltransferase. This enzyme from all variants remained sensitive to inhibition by tunicamycin, but its specific activity was up to 15-fold higher than that of the wild type. Thus, amplification of the gene encoding this enzyme appears to result in its overproduction in the variants, accounting for their resistance to tunicamycin. The tunicamycin-resistant cells are more virulent to mice than their parental wild type. Thus, leishmanial virulence may be related to amplification or expression of gene(s) encoding enzymes involved in the regulation of N-glycosylation of parasite proteins.


Assuntos
Amplificação de Genes , Genes , Glucosiltransferases/genética , Leishmania mexicana/genética , N-Acetilglucosaminiltransferases , Tunicamicina/farmacologia , Animais , Resistência a Medicamentos , Variação Genética , Glucosiltransferases/metabolismo , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/patogenicidade , Leishmaniose/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico , Virulência
11.
Mol Biochem Parasitol ; 18(2): 197-210, 1986 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-3515177

RESUMO

Our previous work by immunoprecipitation with a specific monoclonal antibody showed multiple, closely apposed electrophoretic bands of a major surface antigen specific to the promastigote stage of Leishmania mexicana amazonensis. Here, we analyzed the antigen during growth and transformation of this parasite with particular emphasis on the origin of the multiple bands. Immunobinding assays revealed the presence of the antigen throughout all phases of growth of cloned and uncloned promastigotes in various media for different number of generations. More antigen is expressed by promastigotes grown in Medium 199 plus fetal bovine serum than those in serum-supplemented Schneider's medium or a defined medium; however, this is clone-dependent. Purified monoclonal antibody coupled to Affi-Gel 10 gave a high capacity of antigen binding, resolving four electrophoretic bands of 60-66 kDa. A 63 kDa membrane protein, representing one of the four bands, became predominant after [35S]methionine label and chase. Pretreatment of promastigotes with 10 micrograms ml-1 tunicamycin reduces the antigen to a single band of 54 kDa. Treatment of the antigen bound to the affinity gel with endoglycosidase-H produces similar, but less complete effect. These results indicate glycosylation of this antigen with asparagine-linked oligosaccharides, which appears to account at least in part for its expression as multiple, closely apposed bands during biosynthesis. Binding of fluorescein isothiocyanate-labeled 6H12 monoclonal IgG or Fab to the promastigotes showed an even distribution of the antigen over the cell surface and its capping upon the addition of rabbit anti-mouse IgG. Additional hybridomas prepared against amastigotes yielded monoclonal antibodies which recognized surface antigens common to both stages of the parasite.


Assuntos
Antígenos de Protozoários/análise , Antígenos de Superfície/análise , Glicoproteínas/análise , Leishmania mexicana/crescimento & desenvolvimento , Animais , Anticorpos Monoclonais , Técnicas de Imunoadsorção , Leishmania mexicana/imunologia , Peso Molecular , Oligossacarídeos/metabolismo , Tunicamicina/farmacologia
13.
Mol Biochem Parasitol ; 16(3): 267-76, 1985 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-4058483

RESUMO

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg-1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2400 pmol h-1 mg-1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10(-9)-10(-3) M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.


Assuntos
Heme/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Porfirinas/farmacologia , Animais , Heme/biossíntese , Leishmania mexicana/efeitos dos fármacos , Macrófagos/citologia , Relação Estrutura-Atividade
15.
Proc Natl Acad Sci U S A ; 81(18): 5782-6, 1984 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6592587

RESUMO

The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.


Assuntos
Genes , Leishmania/crescimento & desenvolvimento , RNA Mensageiro/genética , Tubulina (Proteína)/genética , Animais , Leishmania/genética , Hibridização de Ácido Nucleico , RNA Mensageiro/isolamento & purificação
16.
Proc Natl Acad Sci U S A ; 79(23): 7366-70, 1982 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6961414

RESUMO

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.


Assuntos
Antígenos de Superfície/análise , Leishmania/imunologia , Animais , Anticorpos Monoclonais , Diferenciação Celular , Leishmania/citologia
17.
Infect Immun ; 36(1): 430-1, 1982 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7076306

RESUMO

Antigenic changes during the intracellular transformation of Leishmania mexicana subsp. amazonensis from promastigotes to amastigotes in macrophages of J774G8 line were noted mostly among protein bands of 24 to 68 kilodaltons in apparent molecular weight. In this region, six were identified as common antigens of both stages, six to seven were identified as promastigote specific, and three to five were identified as amastigote specific. At the higher-molecular-weight region (greater than 68 kilodalton) were two bands, one being predominant in amastigotes and the other in promastigotes. There may be a transformation-specific band (apparent molecular weight = 20 kilodaltons). The transition of these stage-specific antigens varies considerably with different protein species and may play important roles in intracellular leishmanial differentiation.


Assuntos
Antígenos/análise , Leishmania/imunologia , Macrófagos/parasitologia , Animais , Linhagem Celular , Leishmania/crescimento & desenvolvimento , Camundongos , Peso Molecular
18.
Proc Natl Acad Sci U S A ; 78(12): 7624-8, 1981 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6950404

RESUMO

Cytodifferentiation in the transition cycle of the parasitic protozoan Leishmania mexicana amazonensis was studied in vitro. The flagellated motile promastigotes transform into the nonmotile amastigotes in 7 days at 35 degrees C intracellularly in the murine macrophage line J774G8. In medium 199 plus fetal bovine serum, the reverse transformation occurs extracellularly at 27 degrees C in 2 days. Slab gel electrophoresis of leishmanias labeled with [35S]methionine during transformation revealed changes in protein banding patterns. The intensity of two protein species with apparent molecular weights of approximately equal to 55,000 increased in the amastigote-to-promastigote differentiation and decreased during the reverse transformation. These two protein species comigrated approximately with alpha- and beta-tubulin of Chlamydomonas flagella in two-dimensional gel electrophoresis. The lower band was further identified as beta-tubulin by immunoprecipitation using rabbit antiserum specific to the beta-tubulin of Chlamydomonas axonemes. The biosynthetic change of tubulin was found to correlate with the morphological change of microtubules is leishmanial flagella and cytoskeleton during transformation.


Assuntos
Leishmania/metabolismo , Tubulina (Proteína)/biossíntese , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Leishmania/citologia , Leishmania/genética , Microtúbulos/metabolismo , Peso Molecular , Movimento , Fatores de Tempo
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