Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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P30, also referred to as SAG-1, is now recognized as a major Toxoplasma gondii antigen potentially important for both diagnosis and immunoprophylaxis of toxoplasmosis. By using predictive algorithms, five synthetic peptides (48-67, 82-102, 213-230, 238-256 and 279-285) derived from P30, were investigated for B- and T-cell determinants in mouse and rat experimental models. Antibody recognition appeared more broadly distributed along the P30 sequence, whereas T-cell recognition was mainly targeted on the 238-256 peptide. In the absence of any carrier protein, this peptide induced a B- and T-cell immune response independent of the route of immunization (oral route or subcutaneous injection). This peptide (238-256) induced multiple antibody isotypes. In contrast with the 238-256 peptide, the 48-67 peptide, either free or in the form of a multiple antigenic peptide (MAP) construct or the 279-295 peptide, elicited antibodies associated with a TH2 response. This study reports for the first time the analysis of the antigenic and immunogenic properties of P30-derived peptides and are potentially useful for vaccinal strategies incorporating the P30 Toxoplasma gondii antigen.
Mol Immunol 1994 Dec
PMID:Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis. 799 47

Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30 kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen. Immunoelectron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in bradyzoites only, not in tachyzoites.
Mol Microbiol 1995 Jun
PMID:Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. 857 55

Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarities and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis.
Mol Biochem Parasitol 1996 Jan
PMID:Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene. 899 15

Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the dense granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.
Mol Biochem Parasitol 1998 Mar 15
PMID:Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii. 956 17

As an initial effort to dissect the signaling pathways responsible for pathogenesis of Toxoplasma gondii infection, we report the cloning and in vitro functional studies of TPK3 (Toxoplasma protein kinase-3), a homologue of shaggy/glycogen synthase kinase-3 (GSK-3) kinases. The shaggy/GSK-3 family of kinases are highly conserved protein kinases that play important roles in cell fate determination, nuclear signaling and hormonal regulation. The TPK3 gene was isolated by RT-PCR with degenerate primers corresponding to highly conserved regions of serine/threonine protein kinases. The complete sequences of genomic and cDNA clones indicated the open reading frame, 1185 bp in size, is interrupted by five introns. The predicted protein sequence of TPK3 shows 54% identity to shaggy/GSK-3 over the catalytic domains. Southern analysis revealed TPK3 is a single copy locus in the Toxoplasma genome. Antisera to other GSK-3 proteins from other species recognized GST-TPK3 and a protein of the predicted size in parasites lysates. In vitro kinase assays with GST-TPK3 indicated that TPK3 autophosphorylates and phosphorylates protein phosphatase inhibitor-2 (I-2), a specific substrate of GSK-3 kinase.
Mol Biochem Parasitol 1998 Jun 01
PMID:Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases. 966 11

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.
Mol Microbiol 1999 Aug
PMID:Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. 1044 80

Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone against T. gondii and to understand how resistance might arise, drug-resistant mutants were generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant strains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene of atovaquone-resistant parasites (M129L and I254L) and represent alterations in two different regions of the ubiquinol-binding pocket (Q(o) domain) of cytochrome b, suggesting that atovaquone interferes with electron transport at the cytochrome bc(1) complex in T. gondii. A structural model for how this hydroxynaphthoquinone is binding within the Q(o) domain is presented. Further analysis of the cytochrome b gene suggested that the protein may differ from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate protein to be described for T. gondii.
Mol Biochem Parasitol 2000 Apr 30
PMID:Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance. 1080 14

The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals. We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223-235]. Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage. The two transcripts are encoded by two different genes, which are stage-specifically regulated. The deduced amino-acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite-specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast. Interestingly, the putative protein encompasses an N-terminal extension that is approximately 40 amino-acids longer than that of PtdIns synthases from other organisms. Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase-deficient mutant (PIS1/pis1:kanMX4) showed that only the T. gondii putative PtdIns synthase truncated at its N-terminal extension is able to restore the viability of the cells. We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity. To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.
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PMID:Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii. 1105 8

The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.
Mol Biol Evol 2001 Mar
PMID:Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. 1208 39

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.
J Mol Biol 2001 Feb 16
PMID:The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication. 1123 91


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