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1.
Parasite Immunol ; 23(6): 297-303, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11412382

RESUMO

The understanding of the immunopathology of infections caused by microsporidia has pinpointed the importance of T cell-mediated immunity. The immunopathology caused by the interesting protozoan parasite Encephalitozoon intestinalis, a microsporidium pathogenic in man, is not clearly understood. In this study, we demonstrate that a specific cellular immune response is implicated in the control of microsporidiosis infection in mice. Interferon (IFN)-gamma receptor knockout mice (IFN-gamma R(o/o)) developed a chronic infection with E. intestinalis, whereas a transient infection developed in wild-type mice. Encephalitozoon intestinalis proteins induced proliferation of murine spleen and mesenteric lymph node cells collected from infected mice. The host response to microsporidia infection was regulated by a specific pattern of cytokine protection. Spleen cells derived from resistant 129 Sv/Ev mice inoculated with E. intestinalis secreted significant levels of gamma-interferon and interleukin-2 but cells from highly susceptible IFN-gamma R knockout mice secreted high levels of interleukin-4 (mostly between 2 and 4 weeks post infection). This is the first report in which a specific cellular immune response against E. intestinalis infection is presented.


Assuntos
Encephalitozoon/imunologia , Encefalitozoonose/imunologia , Interferon gama/fisiologia , Interleucina-2/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Interleucina-4/biossíntese , Camundongos , Camundongos Knockout , Coelhos , Baço/metabolismo , Baço/parasitologia
2.
Parasite Immunol ; 23(1): 19-25, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11136474

RESUMO

The dissemination of Encephalitozoon intestinalis, a microsporidium causing intestinal diseases and systemic infection in humans, was investigated in IFN-gamma Ro/o mice. Although lesions were seen in organs of autopsied animals, the parasites were rarely detected using histological examination. Nevertheless, infection of the duodenum, liver, kidneys and lungs was demonstrated by polymerase chain reaction. This method also enabled the detection of the parasite in the brain and the heart. The development of E. intestinalis in RK13 cell cultures to which cell suspensions from liver, kidney, lung or brain of infected IFN-gamma Ro/o mice were added, confirmed the spread of intestinal microsporidiosis to these organs. No dissemination was observed in wild-type mice. These results confirm those of previous studies and emphasize the low morbidity of the infection in IFN-gamma Ro/o mice and confirm the role of IFN-gamma in the control of E. intestinalis infection. These mice infected with E. intestinalis offer important information about this interesting and important parasitic disease of man and animals.


Assuntos
Modelos Animais de Doenças , Encephalitozoon/fisiologia , Encefalitozoonose/imunologia , Encefalitozoonose/parasitologia , Receptores de Interferon/fisiologia , Animais , Anticorpos Antiprotozoários/sangue , DNA de Protozoário/análise , Encephalitozoon/genética , Encephalitozoon/imunologia , Encephalitozoon/isolamento & purificação , Encefalitozoonose/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores de Interferon/genética , Receptor de Interferon gama
3.
Mol Biochem Parasitol ; 111(1): 31-9, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11087914

RESUMO

We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap-1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation. In contrast to the pys21 transcript, which was observed only in infected erythrocytes, all messenger species could be detected in the liver by RT-PCR, peaking at 43 h post infection, a time when parasite burden was maximum, and decreasing markedly thereafter to become hardly visible at 168 h. Some transcripts (pypka, pymap-1, pyrnr1 and pyrnr2) could be detected 12 h after infection, while others (pymrk and pyrnapolII) did not become detectable until 24 h. In addition, we characterised all these messengers by Northern blot of total RNAs extracted from infected erythrocytes. Taken together, these data suggest that a similar set of regulatory genes is expressed during both exoerythrocytic and erythrocytic schizogony.


Assuntos
Perfilação da Expressão Gênica , Genes de Protozoários , Hepatócitos/parasitologia , Malária/parasitologia , Plasmodium yoelii/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Animais , Northern Blotting , Eritrócitos/parasitologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmodium yoelii/metabolismo , RNA Mensageiro/genética , RNA de Protozoário/genética
4.
J Eukaryot Microbiol ; 47(1): 48-56, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10651296

RESUMO

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.


Assuntos
Western Blotting , Imuno-Histoquímica , Proteínas de Protozoários/análise , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Reações Cruzadas , Encephalitozoon/química , Encephalitozoon/crescimento & desenvolvimento , Encephalitozoon/imunologia , Encefalitozoonose/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Hospedeiro Imunocomprometido , Camundongos , Microscopia Confocal , Proteínas de Protozoários/imunologia , Esporos/química , Esporos/imunologia
5.
J Clin Microbiol ; 37(12): 4107-12, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10565939

RESUMO

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Microsporida/imunologia , Microsporidiose/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Western Blotting , Eletroforese em Gel de Poliacrilamida , Fezes/parasitologia , Feminino , Humanos , Enteropatias Parasitárias/diagnóstico , Enteropatias Parasitárias/parasitologia , Masculino , Camundongos , Microscopia Eletrônica , Microsporida/crescimento & desenvolvimento , Microsporida/isolamento & purificação , Microsporidiose/parasitologia , Pessoa de Meia-Idade , Esporos/imunologia
7.
Exp Parasitol ; 89(1): 113-21, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9603496

RESUMO

IFN-gamma receptor knockout mice and wild-type mice were infected per os with Encephalitozoon intestinalis. Both groups developed an infection that was chronic in the mutant mice whereas it was only transient in wild-type mice. The infection of mutant mice was characterized by the continual shedding of spores in feces, splenomegaly, the enlargement of the biliary tract, and the occurrence of numerous nodules in the liver and in the small intestine wall. The humoral response was studied by ELISA, IFA, and Western blotting. ELISA titers of anti-E. intestinalis antibodies of IgG, IgM, and IgA isotypes were higher in IFN-gamma R0/0 mice than in wild-type mice and they increased in time after infection. Levels of IgG2a were inferior to those of IgG1 in mutant mice in contrast to wild-type mice. High levels of parasite specific antibodies were accompanied by an increase in type 2 cytokines (IL-4 and IL-10) secretion in the duodenum of IFN-gamma R0/0 mice. The E. intestinalis spore wall was recognized by IgM, IgG, and IgA from all infected mice whereas the extruded polar tube only reacted with IgG and IgA from IFN-gamma R0/0 mice after 45 days of the infection. IFN-gamma R0/0 mice IgG and IgA reacting with polar tube identified also a series of proteins which could be components of this structure. On the proteins recognized by all infected mice sera, two were first recognized by IgM at day 15 and then by IgG at day 30 in wild-type (WT) mice. The persistent reactivity of all proteins in mutant mice is consistent with the chronicity of the infection in these animals; in contrast, their resorption at day 30 in WT animals corroborates the transient character of the infection in these mice. The correlation between the evolution of the proteic pattern and the development of the infection provides evidence of the validity of this murine model to study human microsporidiosis. Indeed the reported results confirm the potential value of serological methods for diagnosing E. intestinalis infection in immunocompetent and in immunocompromised human subjects, for elucidating the age pattern of the microsporidiosis and also for identifying risk groups.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Anticorpos Antiprotozoários/biossíntese , Encephalitozoon/imunologia , Encefalitozoonose/imunologia , Interferon gama/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Western Blotting , Modelos Animais de Doenças , Duodeno/imunologia , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Imunofluorescência , Imunoglobulinas/biossíntese , Imunoglobulinas/sangue , Imuno-Histoquímica , Interferon gama/biossíntese , Interferon gama/genética , Interleucinas/análise , Interleucinas/biossíntese , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Esporos/isolamento & purificação
8.
J Eukaryot Microbiol ; 44(6): 81S, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9508462

RESUMO

The ultrastructural study of the invasion of cells (THP1 and RK13) by E. intestinalis shows that infective stages enter host cells via a phagocytic process initiated at the contact of the apical part of spores with host cell membrane. The polar tube is extruded within an invagination of the host cell membrane that extends inside a pseudopod containing microfilaments. These observations suggest that microsporidia as well as other intracellular pathogens can induce host cell alterations facilitating the invasion.


Assuntos
Encephalitozoon/patogenicidade , Rim/parasitologia , Macrófagos/parasitologia , Animais , Linhagem Celular , Membrana Celular/parasitologia , Encephalitozoon/ultraestrutura , Humanos , Rim/citologia , Fagocitose , Coelhos , Esporos , Vacúolos/parasitologia
10.
Mol Biochem Parasitol ; 83(2): 201-10, 1996 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-9027753

RESUMO

The cDNA encoding the Toxoplasma gondii microneme protein MIC1 and the corresponding gene have been cloned and sequenced. The MIC1 gene contains three introns. The cDNA encodes a 456 amino acid (aa) sequence, with a typical signal sequence and no other trans-membrane domain. The protein contains a tandemly duplicated domain with conservation of cysteines and presents distant homology with the Plasmodium sp. microneme protein TRAP-SSP2. The MIC1 protein from tachyzoite lysates and a PMAL recombinant expressing the N-terminal duplicated domain of the protein bound to the surface of putative host cells, suggesting a possible involvement of MIC1 in host cell binding/recognition.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Protozoários , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Adesão Celular , Moléculas de Adesão Celular/química , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Genes de Protozoários/genética , Íntrons/genética , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Toxoplasma/metabolismo , Células Vero
11.
Parasite Immunol ; 18(8): 387-92, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9229392

RESUMO

Interferon gamma receptor knockout mice developed a chronic infection when inoculated with spores of Encephalitozoon intestinalis which is a cause of intestinal microsporidiosis in AIDS patients. The infection was evaluated by enumeration of the spores of the parasite shed in the stools, histological examination and follow up over a period of six months. A dose-response was demonstrated since higher numbers of spores were excreted and more infection sites were found in mice which were given an increased quantity of parasites. In infected wild type mice, the number of excreted spores decreased until day 16 post-inoculation, then spores were detected sporadically in low numbers. These data confirmed the role of IFN-gamma in the control of E. intestinalis infection. The infection was not lethal suggesting that other factors are involved in regulation of the parasite infection. This model, with the long survival time of the animals together with the measurable quantity of spores shed in the stools will be useful for testing potential therapeutical agents.


Assuntos
Encefalitozoonose/imunologia , Enteropatias/imunologia , Receptores de Interferon/fisiologia , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Animais , Modelos Animais de Doenças , Encephalitozoon/isolamento & purificação , Encephalitozoon/patogenicidade , Encefalitozoonose/complicações , Encefalitozoonose/parasitologia , Fezes/parasitologia , Humanos , Enteropatias/complicações , Enteropatias/parasitologia , Camundongos , Camundongos Knockout , Receptores de Interferon/genética , Esporos/isolamento & purificação , Esporos/patogenicidade , Receptor de Interferon gama
12.
C R Acad Sci III ; 319(1): 39-43, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8673618

RESUMO

The microsporidia Encephalitozoon intestinalis is an opportunistic pathogen responsible for severe gastrointestinal diseases and disseminated infection in AIDS patients. No light-microscopical method allows the specific detection of this unicellular parasite and up to this date, only electron microscopy could confirm the diagnosis of the species. We propose a method combining the non specific labelling of microsporidian spores by the fluorochrome Uvitex 2B and an indirect immunofluorescent assay with a polyclonal antibody specifically directed against E. intestinalis. Preliminary data demonstrate the specificity of this antibody. This method enables the distinction between E. intestinalis and Enterocytozoon bieneusi an other microsporidian also associated with gastrointestinal infection. Due to the precocious detection of E. intestinalis patients will be treated earlier with albendazole which is potentially active against this species.


Assuntos
Síndrome da Imunodeficiência Adquirida/parasitologia , Encephalitozoon/isolamento & purificação , Animais , Especificidade de Anticorpos , Encephalitozoon/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes , Humanos , Esporos
13.
Mol Biochem Parasitol ; 68(2): 247-57, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7739670

RESUMO

GRA3 is a 30-kDa protein located inside the dense granules of Toxoplasma gondii. Following invasion and exocytosis of dense granules within the parasitophorous vacuole, GRA3 becomes associated with the parasitophorous vacuolar membrane (PVM) and extensions of the PVM which protrude into the cytoplasm. A partial cDNA encoding GRA3 was isolated from a Toxoplasma gondii expression library using polyclonal and monoclonal antibodies to the mature GRA3 protein of tachyzoites. Antibodies affinity purified using the cloned fusion protein reacted with a 30-kDa band on immunoblots and recognized dense granules, the PVM, and PVM extensions by immunofluorescence staining of infected cells. Northern blot analysis indicated the major transcript was of a slightly larger size, and the complete cDNA encoding GRA3 was subsequently obtained. Southern blot analysis suggests that GRA3 is present as a single copy. The cDNA encodes two methionines at the N-terminus followed by an open reading frame with a hydrophobic region of 22 amino acids flanked by charged residues consistent with a signal sequence. Four shorter hydrophobic regions occur but are insufficient to span the membrane. No significant homology was detected to other proteins, including other dense granule proteins. In vitro translation of RNA generated from the cDNA containing either one or two of the N-terminal methionines yielded peptides with apparent M(r) of 35,000 and 37,000 respectively. Translation of RNA from the cDNA containing only the second initiation site in the presence of dog pancreas microsomes resulted in reduction of 4 kDa, sufficient to account for removal of the putative signal sequence.


Assuntos
DNA Complementar/análise , DNA de Protozoário/análise , Proteínas de Protozoários/genética , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Imunofluorescência , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/genética , Toxoplasma/imunologia , Vacúolos/imunologia
14.
Cancer Res ; 54(9): 2372-7, 1994 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-8162583

RESUMO

We previously reported that urokinase (uPA) is produced by the human prostate cancer cell line, PC-3, and could function as a growth factor for cells of the osteoblast phenotype. To examine the role of uPA in metastasis to the skeleton and to extraskeletal sites, we have developed a homologous model of uPA overexpression in a rat prostate cancer cell line. Full length cDNA encoding rat (r) uPA was isolated and subcloned as a 1.4-kilobase XbaI-BspHI fragment in the sense and antisense orientation into the Moloney murine leukemia retroviral vector pYN. The control (pYN) and experimental (pYN-ruPA, pYN-ruPA-AS) plasmids were transfected into Dunning R 3227, Mat LyLu rat prostate carcinoma cells. Experimental clones expressing at least 5-fold higher (pYN-ruPA) or 3-fold lower (pYN-ruPA-AS) than controls were selected, and control and experimental cells were inoculated into the left ventricles of inbred male Copenhagen rats. Animals were sacrificed at timed intervals to examine the evolution of metastatic lesions. Control animals developed metastases to the lumbar vertebrae resulting in spinal cord compression and hind limb paralysis at 20-21 days postinoculation. Animals inoculated with cells overexpressing uPA developed hind limb paralysis significantly earlier (by day 14-15 postinoculation). Additionally, more widespread skeletal (ribs, scapula, and femora) metastases were seen. Serum from experimental animals showed a progressive elevation in alkaline phosphatase levels, and histological examination of lumbar metastases revealed markedly increased osteoblastic activity over that observed in control animals. In contrast to this, animals inoculated with cells underexpressing uPA developed hind limb paralysis significantly later (days 25-29 postinoculation) and displayed decreased tumor metastasis. These studies support a role for the catalytic domain of uPA in enhancing both skeletal and nonskeletal prostate cancer invasiveness and are consistent with a role for the growth factor domain of uPA in mediating an osteoblastic skeletal response.


Assuntos
Neoplasias Ósseas/secundário , Ativadores de Plasminogênio/metabolismo , Neoplasias da Próstata/patologia , Animais , Neoplasias Ósseas/metabolismo , Membro Posterior , Masculino , Paralisia/etiologia , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/urina , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , Ratos , Transfecção , Células Tumorais Cultivadas
15.
Parasitology ; 108 ( Pt 2): 139-45, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7512710

RESUMO

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4-5 kDa. Two different monoclonal antibodies directed against the 4-5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low M(r) antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4-5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/análise , Imunoglobulina M/biossíntese , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Adulto , Animais , Anticorpos Monoclonais/imunologia , Variação Antigênica , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Western Blotting , Epitopos/análise , Epitopos/imunologia , Feminino , Imunofluorescência , Humanos , Camundongos , Microscopia Imunoeletrônica , Peso Molecular , Gravidez , Toxoplasma/ultraestrutura , Células Vero
16.
FEBS Lett ; 338(1): 69-74, 1994 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-8307160

RESUMO

A rat urokinase receptor (uPAR) cDNA fragment was amplified by RT-PCR from RNA of the rat osteoblastic cell line CFK-1. Using this DNA species as a hybridization probe two rat uPAR cDNAs were isolated from a CFK-1 cDNA library. These two cDNAs encode an identical uPAR protein except for a single base mutation which results in the substitution of cysteine to serine at amino acid 71 in one variant. PCR analysis of rat genomic DNA revealed the presence of an additional uPAR arising from alternate splicing which is expressed in a variety of tissues. These studies provide the tools for examining uPAR function in fibrinolysis, tumor invasion and metastasis in the rat and for identifying the mechanism of species specificity in uPA actions.


Assuntos
Osteoblastos/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
17.
Parasitol Res ; 79(5): 402-8, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8415546

RESUMO

The fate of Toxoplasma gondii dense-granule (GRA2, GRA3), rhoptry (ROP1), and surface (SAG1) proteins was followed by immunofluorescence assay (IFA) and immunoelectron microscopy at different stages after infection. Dense-granule exocytosis occurred in the apical area of the tachyzoite within minutes of invasion. Several exocytic events were found simultaneously in the same organism, both by serial sectioning and by freeze-fracture studies. Dense-granule contents were first found as a dense material trapped between parasite and vacuole membranes before either the vacuolar network or the vacuole membrane could be immunolabeled with specific antibodies. The vacuolar network was strongly labeled with dense-granule antibodies but not with the SAG1-specific probe, which suggests that the network is not enriched in membrane proteins. In addition to strongly labeling the vacuole membrane, GRA3 antibodies also labeled strands extending from the parasitophorous vacuoles into the host-cell cytoplasm.


Assuntos
Degranulação Celular , Exocitose , Organelas/metabolismo , Toxoplasma/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Cinética , Camundongos , Microscopia Imunoeletrônica , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Células Vero
18.
Mol Biochem Parasitol ; 56(2): 227-38, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1362450

RESUMO

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.


Assuntos
Anticorpos Antiprotozoários/imunologia , Imunoglobulina A/imunologia , Mucosa Intestinal/imunologia , Proteínas de Protozoários/genética , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Sequência de Bases , Clonagem Molecular , Grânulos Citoplasmáticos/química , Feminino , Genes de Protozoários/genética , Camundongos , Dados de Sequência Molecular , Poli A/genética , Reação em Cadeia da Polimerase , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/imunologia , RNA Mensageiro/genética , Análise de Sequência de DNA , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia
19.
Parasitology ; 103 Pt 3: 321-9, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1780169

RESUMO

The biosynthesis and fate of 4 different dense granule proteins of Toxoplasma gondii were studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; in T. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.


Assuntos
Proteínas de Protozoários/análise , Toxoplasma/química , Vacúolos/química , Animais , Western Blotting , Imunofluorescência , Microscopia Imunoeletrônica , Peso Molecular , Testes de Precipitina , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Toxoplasma/ultraestrutura , Células Vero
20.
Mol Biochem Parasitol ; 47(2): 223-33, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1944419

RESUMO

Three microneme proteins of Toxoplasma gondii have been characterized using 3 monoclonal antibodies and a recombinant protein specific antiserum. In all cases, apical labeling of tachyzoites and bradyzoites was observed by indirect immunofluorescence assay. Immunogold localization on ultrathin sections of bradyzoites or tachyzoites showed a specific labeling of micronemes. The following proteins were characterized using 2-dimensional gel electrophoresis and Western immunoblotting: Mic 1 (60 kDa, Pi 6.5), Mic 2 (120 kDa, Pi 5) and Mic 3 (90 kDa, Pi 6.75). The 90-kDa protein (Mic 3) is a heterodimer of two 38-kDa polypeptides (Pi 6.7 and 6.75 respectively) linked by disulfide bridges. Metabolic labeling and immunoprecipitation assays showed that at least one of the 38-kDa polypeptides was processed from a 40-kDa precursor. No processing was observed during the biosynthesis of the 120- and 60-kDa polypeptides.


Assuntos
Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários , Antígenos de Protozoários/isolamento & purificação , Microscopia Imunoeletrônica , Peso Molecular , Organelas/imunologia , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasma/ultraestrutura
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