Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.
(25/7816)
Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin. (+info)
Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.
(26/7816)
Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen. (+info)
Seroconversion to circumsporozoite antigen of Plasmodium falciparum demonstrates a high risk of malaria transmission in travelers to East Africa.
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Circumsporozoite (CS) antibodies have been shown to be reliable indicators of malaria transmission in endemic areas. Their prevalence in travelers can indicate the degree of exposure to plasmodial infection. Two hundred sixty-two short-term travelers to Kenya were recruited to a prospective study to determine the incidence of CS antibody conversion. All travelers were receiving malaria chemoprophylaxis. Serum samples were drawn before departure and 4-6 weeks after their return to Germany. Sera from 310 volunteers who did not leave Germany served as controls. Serum specimens from 13 (4.96%) of the 262 travelers were found to be positive after return. None of the travelers developed symptoms of clinical malaria or antibodies against the blood stages of Plasmodium falciparum. All 310 control samples tested negative. These data demonstrate a considerable risk of malaria transmission for short-term travelers to East Africa. (+info)
Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell.
(28/7816)
The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite. (+info)
The conjusome: a novel structure in Tetrahymena found only during sexual reorganization.
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A unique structure, the conjusome, has been identified and initially characterized in Tetrahymena thermophila. The conjusome appears only during a specific phase of conjugation. Immunofluorescence microscopy reveals that the conjusome is strongly labeled by antibodies to the protein Pdd1p. Pdd1p is a chromodomain protein and participates in the formation of chromatin-containing structures in developing macronuclear anlagen. Recent studies suggest that Pdd1p is physically associated with the elimination of specific germ-line sequences from developing macronuclei (anlagen) and may play a role in heterochromatin assembly. The conjusome contains Pdd1p, but it is devoid of any detectable DNA. The conjusome appears before DNA elimination begins in the developing anlagen and after Pdd1p is found in the parental macronucleus. Transmission electron microscopic observations reveal that the conjusome is not a membrane-bounded structure. The conjusome ranges in size from about 1 microm to sizes approaching 7 microm, depending on its maturity. It is composed of a coarse reticulum of a fibrous, electron dense material, interspersed with apparent background cytoplasm. Our initial characterization does suggest a number of possible functions for what may be a new, transient organelle. (+info)
Virulence and transmission success of the malarial parasite Plasmodium falciparum.
(30/7816)
Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages. (+info)
An insertional mutagenesis approach to Dictyostelium cell death.
(31/7816)
Programmed cell death (PCD) in Dictyostelium shows a pattern of ordered degeneration similar to that observed in higher eukaryotes but somewhat different from the most studied form of PCD, i.e. apoptosis. To contribute to a genetic definition of this process, Dictyostelium HMX44A cells have been subjected to insertional mutagenesis, followed by selection based on several rounds of differentiation/regrowth to recover only cells resistant to death. We describe here the approach used, a partial characterization of the first mutant thus obtained called C5 showing some dissociation of cell death signs, and, in this case where plasmid rescue was not possible, as a first step towards identification of the gene at play recovery of genomic flanking sequences via genomic recircularization and PCR. This work demonstrates the feasibility of an insertional mutagenesis approach to obtain death-resistant mutants in Dictyostelium. (+info)
Up-regulation of Duffy antigen receptor expression in children with renal disease.
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BACKGROUND: The Duffy antigen chemokine receptor (DARC) is a promiscuous chemokine receptor that binds chemokines from the C-X-C and C-C families. DARC was initially described on red blood cells, but subsequent studies have demonstrated DARC protein expression on renal endothelial and epithelial cells, even in Duffy-negative individuals whose red cells lack DARC. Because approximately 68% of African Americans lack the Duffy/DARC on their red cells, we carried out experiments to identify the specific renal cells expressing DARC protein and mRNA in African American children and to define whether DARC expression was altered in renal inflammatory processes. METHODS: Immunohistochemistry and in situ hybridization studies were done in 28 renal sections from children with each of the following diagnoses: HIV nephropathy (HIVAN), HIV-associated hemolytic uremic syndrome (HIV-HUS), HIV infection without renal disease, HIV-negative children without renal disease, and Argentinean children with classic HUS. RESULTS: The predominant localization of DARC mRNA and protein was found in endothelial cells underlying postcapillary renal venules in all patients studied. However, DARC mRNA and protein were significantly up-regulated in peritubular and glomerular capillaries, collecting duct epithelial cells, and interstitial inflammatory cells in children with HIVAN, HIV-HUS, and classic HUS. CONCLUSION: These findings support the notion that the renal DARC is linked to the inflammatory cascade and that African American children may be at risk of accumulating chemokines in renal tissues. (+info)