RESUMO
BACKGROUND: Breast cancer (BC) is a major health issue threatening women's life. No reliable epidemiological data on BC diagnosed by oncologists/senologists are available in Algeria. METHODS: The BreCaReAl study, a non-interventional prospective cohort study, included adult women with confirmed BC in Algeria. Disease incidence, patients and disease characteristics, treatment patterns, and mortality rate were recorded up to 12 months of follow-up. RESULTS: Overall, 1,437 patients were analysed: median age was 48 [41;57] years and 337 (23.5%) women had a family history of BC. BC incidence was 22.3 (95% CI: 21.5; 23.2) cases per 100,000 inhabitants over 8 months. Delayed diagnosis was reported in 400 (29.2%) patients. First line of treatments were mainly chemotherapy and surgery. Twenty-eight serious adverse events were reported including 10 (37.0%) events which led to death. Mortality rate reached 3.2% at 12 months CONCLUSION: A delayed diagnosis highlights the importance of implementing more effective screening strategies.
Assuntos
Neoplasias da Mama/epidemiologia , Oncologistas/normas , Sorologia/normas , Argélia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.
Assuntos
Antígenos de Protozoários , Glicosilfosfatidilinositóis/metabolismo , Proteína 1 de Superfície de Merozoito/metabolismo , Plasmodium falciparum , Processamento de Proteína Pós-Traducional , Animais , Baculoviridae , Linhagem Celular , Membrana Celular/metabolismo , Expressão Gênica , Vetores Genéticos , Glicosilação , Humanos , Manose , Proteína 1 de Superfície de Merozoito/genética , Polissacarídeos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.
Assuntos
Dolicol Monofosfato Manose/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Manosiltransferases/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Paramecium/metabolismoRESUMO
Prenylated proteins are involved in the regulation of DNA replication and cell cycling and have important roles in the regulation of cell proliferation. Protein farnesyltransferase and protein geranylgeranyltransferase are the two enzymes responsible for catalysing isoprene lipid modifications. Recently these enzymes have been targets for the development of cancer chemotherapeutics. Using metabolic labelling we identified isoprenylated proteins which suggests the presence of protein farnesyltransferase in Toxoplasma gondii. T. gondii protein farnesyltransferase is heat-labile and requires Mg(2+) and Zn(2+) ions for full activity. Peptidomimetic analogues as well as short synthetic peptides were tested in vitro as possible competitors for farnesyltransferase substrates. We found that the synthetic peptide (KTSCVIA) specifically inhibited T. gondiiprotein farnesyltransferase but not mammalian (HeLa cells) farnesyltransferase. Therefore this study suggests the possible development of specific inhibitors of T. gondiiprotein farnesyltransferase as an approach to parasitic protozoa therapy.
Assuntos
Alquil e Aril Transferases/isolamento & purificação , Proteínas de Protozoários/química , Toxoplasma/enzimologia , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Animais , Western Blotting , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Prenilação de Proteína , Toxoplasmose/tratamento farmacológico , Zinco/metabolismoRESUMO
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.
Assuntos
Eucariotos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Animais , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/química , Histocitoquímica , Estrutura Molecular , Paramecium/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismo , Toxoplasma/metabolismoAssuntos
Glicosilfosfatidilinositóis/química , Neospora/química , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Chlorocebus aethiops , Cromatografia em Camada Fina , Glicosilfosfatidilinositóis/metabolismo , Neospora/crescimento & desenvolvimento , Neospora/imunologia , Neospora/metabolismo , Ligação Proteica , Células VeroRESUMO
Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.
Assuntos
Antígenos de Protozoários/química , Antígenos de Superfície/química , Glicoproteínas de Membrana/química , Paramecium/imunologia , Animais , Dicroísmo Circular , Glicosilfosfatidilinositóis/análise , Lipossomos , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Solubilidade , TemperaturaRESUMO
The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.
Assuntos
Antígenos de Superfície/isolamento & purificação , Paramecium/imunologia , Animais , Antígenos de Superfície/química , Membrana Celular/química , Membrana Celular/imunologia , Precipitação Química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/isolamento & purificação , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases , Solubilidade , TemperaturaRESUMO
The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Inositol/metabolismo , Manosídeos/biossíntese , Plasmodium falciparum/metabolismo , Acetilglucosamina/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Coenzima A/farmacologia , Glicolipídeos/análise , Glicolipídeos/biossíntese , Glicosilfosfatidilinositóis/metabolismo , Guanosina Difosfato Manose/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/biossíntese , Fosfatidilinositóis/análiseRESUMO
Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol-phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1-6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide.
Assuntos
Glicoesfingolipídeos/química , Manosefosfatos/análise , Manosídeos/química , Paramecium/química , Animais , Sequência de Carboidratos , Sistema Livre de Células , Cromatografia por Troca Iônica , Cromatografia em Camada Fina , Glucosamina/metabolismo , Glicolipídeos/biossíntese , Glicoesfingolipídeos/biossíntese , Manose/metabolismo , Dados de Sequência MolecularRESUMO
The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.
Assuntos
Acetilgalactosamina/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Manosefosfatos/metabolismo , Paramecium/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Carboidratos , Dolicol Monofosfato Manose/metabolismo , Dolicóis , Retículo Endoplasmático/metabolismo , Glicolipídeos/metabolismo , Glicosilação , Lipídeos de Membrana/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismoRESUMO
Mannose analogues (2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose) have been used to study glycosylphosphatidylinositol (GPtdIns) biosynthesis and GPtdIns protein anchoring in protozoal and mammalian systems. The effects of these analogues on GPtdIns biosynthesis and GPtdIns-protein anchoring of the human malaria parasite Plasmodium falciparum were evaluated in this study. At lower concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D glucose (0.2 and 0.1 mm, respectively), GPtdIns biosynthesis is inhibited without significant effects on total protein biosynthesis. At higher concentrations of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose (1.5 and 0.8 mm, respectively), the incorporation of [3H]glucosamine into glycolipids was inhibited by 90%, and the attachment of GPtdIns anchor to merozoite surface protein-1 (MSP-1) was prevented. However, at these concentrations, both sugar analogues inhibit MSP-1 synthesis and total protein biosynthesis. In contrast to 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose (mannosamine), the formation of new glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-D-glucose. Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mm, but neither an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue. Furthermore, the [3H]mannosamine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling. Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine. This high rate of conversion led us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with mannosamine, which is different to what has been observed for mammalian cells and other parasitic protozoa.
Assuntos
Glicosilfosfatidilinositóis/biossíntese , Manose/análogos & derivados , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Animais , Desoxiglucose/farmacologia , Fluordesoxiglucose F18/farmacologia , Hexosaminas/farmacologia , Manose/farmacologia , Proteína 1 de Superfície de Merozoito/biossíntese , Proteínas de Protozoários/biossínteseRESUMO
The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction, in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. The Saccharomyces cerevisiae GPI8 gene (ScGPI8) encodes a protein which is involved in the GPI transamidation reaction. We have cloned and isolated the Schizosaccharomyces pombe GPI8 homologous gene (SpGPI8). The SpGPI8 gene encodes a protein of 411 amino acids with a calculated molecular weight of about 47 kDa. It shows 53.5% identity with the ScGPI8 and complements a S. cerevisiae GPI8 anchoring mutant.
Assuntos
Aminoaciltransferases/genética , Moléculas de Adesão Celular/genética , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Genes Essenciais , Genes Fúngicos , Teste de Complementação Genética , Inositol/metabolismo , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismoRESUMO
During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Imunoglobulina M/imunologia , Toxoplasma/imunologia , Animais , Chlorocebus aethiops , Feminino , Humanos , Gravidez , Toxoplasma/química , Toxoplasma/citologia , Células VeroRESUMO
Free and protein-bound glycosylphosphatidylinositols (GPIs) of the blood stages of the rodent malarial parasite Plasmodium chabaudi chabaudi AS were identified and characterized. TLC analysis of material extracted by organic solvents from metabolically labelled parasites revealed a distinct set of glycolipids. These glycolipids were identified as GPIs by specific chemical and enzymic treatments and by structural analysis of their glycan and hydrophobic parts. These analyses revealed that P.c.chabaudi AS synthesizes a set of GPI-biosynthesis intermediates and two potential GPI-anchor precursors exhibiting the following structures: ethanolamine-phosphate [(alpha1-2)mannose]mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine - (acyl) inositol-phosphate-diacylglycerol (P.ch. alpha) and ethanolamine-phosphate - mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine-(acyl)inositol-phosphate-diacylglycerol (P.ch. beta). One of these GPI-anchor precursors (P.ch. alpha) possesses the same carbohydrate structure as the GPI membrane anchor of merozoite surface protein-1 from P.c.chabaudi AS.
Assuntos
Glicosilfosfatidilinositóis/química , Plasmodium chabaudi/química , Precursores de Proteínas/química , Proteínas de Protozoários/química , Animais , Antígenos de Protozoários/química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Glucosamina/metabolismo , Glicolipídeos/análise , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Glicosilfosfatidilinositóis/análise , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/isolamento & purificação , Manosídeos/análise , Proteína 1 de Superfície de Merozoito , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligossacarídeos/análise , Oligossacarídeos/isolamento & purificação , Fosfatidilinositóis/análise , Plasmodium chabaudi/metabolismoRESUMO
The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.