A selective screen reveals discrete functional domains in Drosophila Nanos

The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis.     

Trisomy 8, a Cytogenetic Abnormality in Myelodysplastic Syndromes,Is Constitutional or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS.     

History of disease and the longue durée

This paper summarizes Grmek's theoretical contribution to history of disease and explores to what extent the longue durée could still be a useful concept in order to better understand past perceptions of, and reactions to, diseases. The case of the medical responses to epidemic disease in pre-industrial Europe is synthetically expounded in order to illustrate this issue.     

Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation

Calcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii, for example, a rise in [Ca2+] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca2+ ionophore A23187 and found the affected gene encodes a homologue of Na+/H+ exchangers (NHEs) located on the parasite's plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca2+, which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca2+ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment.     

A Toxoplasma gondii protein with homology to intracellular type Na⁺/H⁺ exchangers is important for osmoregulation and invasion

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na(+)/H(+) exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca(2+) concentration [Ca(2+)](i), and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.     

Targeting of tail‐anchored membrane proteins to subcellular organelles in Toxoplasma gondii

Proper protein localization is essential for critical cellular processes, including vesicle‐mediated transport and protein translocation. Tail‐anchored (TA) proteins are integrated into organellar membranes via the C‐terminus, orienting the N‐terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C‐terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early‐branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow‐up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C‐terminal sequence.      

Toxoplasma gondii: induction of egress by the potassium ionophore nigericin

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. Some of the devastating consequences of toxoplasmosis are in part due to the lysis of the host cell during parasite egress. The process of egress is poorly understood and since it is asynchronous in tissue culture its study has been limited to those conditions that induce it, such as artificial permeabilisation of the host cell and induction of calcium fluxes with ionophores. Given that permeabilisation leads to egress by the activation of motility upon a drop in host cell potassium concentration, we investigated whether the ionophore nigericin, which selectively causes efflux of potassium from the cell without the need for permeabilisation, would cause egress. Nigericin effectively causes intracellular parasites to exit their host cell within 30 min of treatment with the drug. Our results show that nigericin-induced egress depends on an efflux of potassium from the cell and requires phospholipase C function and parasite motility. This novel method of inducing and synchronising egress mimics the effect of artificial permeabilisation in all respects. Nevertheless, since the membrane remains intact during the treatment, in our nigericin-induced egress we are able to detect parasite-dependent permeabilisation of the host cell, a known step in induced egress. In addition, consistent with the model that loss of host cell potassium leads to egress through the activation of intraparasitic calcium fluxes, a previously isolated Toxoplasma mutant lacking a sodium hydrogen exchanger and defective in responding to calcium fluxes does not undergo nigericin-induced egress. Thus, the discovery that nigericin induces egress presents a novel assay that allows for the genetic and biochemical analysis of the signalling mechanisms that lead to the induction of motility and egress.     

A Toxoplasma gondii protein with homology to intracellular type Na/H exchangers is important for osmoregulation and invasion

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na⁺/H⁺ exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca²⁺ concentration [Ca²⁺]_i, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.     

Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii

MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1 , an MSH in the pathogenic parasite, Toxoplasma gondii , confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.