Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause

The oxygen consumption of Nothobranchius korthausae eggs in different developmental stages, including diapause II and III, was measured. Oxygen consumption increases exponentially during embryonic development. In diapause II and III there is a drop in oxygen consumption, which attains a minimal level in diapause II after 3 weeks and in diapause III after 2 weeks. During early development the embryos can escape from hypoxic stress by entering diapause I and II. During late embryogenesis embryos in diapause III can escape from hypoxic stress by hatching. We conclude that survival of annual fish embryos is enhanced during conditions of low oxygen concentration by reduced oxygen consumption rates during diapause.     

T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment

The following study was undertaken to better understand the mechanisms that relate the homeostatic set point of the peripheral T cell population to energy availability in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+T cells notably declined after one week of malnourishment, a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Overall levels of IL-7 were similar in malnourished and control mice. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to quickly adjust the total number of T cells during malnutrition.     

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc₃Man₅GlcNAc₂) and the host 14-sugar precursor (Glc₃Man₉GlcNAc₂) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.Animals, fungi, and plants synthesize Asn-linked glycans (N-glycans) by means of a lipid-linked precursor containing 14 sugars (dolichol-PP-Glc₃Man₉GlcNAc₂) (26). Recently we used bioinformatics and experimental methods to show that numerous protists are missing sets of glycosyltransferases (Alg1 to Alg14) and so make truncated N-glycan precursors containing 0 to 11 sugars (46). For example, Entamoeba histolytica, which causes dysentery, makes N-glycan precursors that contain seven sugars (Man₅GlcNAc₂) (33). Giardia lamblia, a cause of diarrhea, makes N-glycan precursors that contain just GlcNAc₂ (41). N-glycan precursors may be identified by metabolic labeling with radiolabeled mannose (Entamoeba) or glucosamine (Giardia) (46). Unprocessed N-glycans of each protist may be recognized by wheat germ agglutinin 1 (WGA-1) (GlcNAc₂ of Giardia) or by the antiretroviral lectin cyanovirin-N (Man₅GlcNAc₂ of Entamoeba) (2, 33, 41).N-glycans are transferred from lipid-linked precursors to sequons (Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa cannot be Pro) on nascent peptides by an oligosaccharyltransferase (OST) (28). For the most part, transfer of N-glycans by the OST is during translocation, although there are human and Trypanosoma OSTs that transfer N-glycans after translocation (34, 45).N-glycan-dependent quality control (QC) systems for protein folding and endoplasmic reticulum (ER)-associated degradation (ERAD), which are present in most eukaryotes, are missing from Giardia and a few other protists that make truncated N-glycans (5, 26, 53). There is positive Darwinian selection for sequons (sites of N-glycans) that contain Thr in secreted and membrane proteins of organisms that have N-glycan-dependent QC (12). This selection occurs for the most part by an increased probability that Asn and Thr will be present in sequons rather than elsewhere in secreted and membrane proteins. In contrast, there is no selection on sequons that contain Ser, and there is no selection on sequons in the secreted proteins of organisms that lack N-glycan-dependent QC.For numerous reasons, we are interested in the N-glycans of Plasmodium falciparum and Toxoplasma gondii, which cause severe malaria and disseminated infections, respectively.(i) There has been controversy for a long time as to whether Plasmodium makes N-glycans. While some investigators identified a 14-sugar Plasmodium N-glycan resembling that of the human host (29), others identified no N-glycans (6, 22).(ii) There is also controversy concerning whether the N-glycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man₅GlcNAc₂), like Entamoeba (32, 33), or 11 sugars (Man₉GlcNAc₂), like the human host (16, 19, 26). If it is Man₅GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32, 46). If it is Man₉GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16, 19, 26).(iii) Both Plasmodium and Toxoplasma are missing proteins involved in N-glycan-dependent QC of protein folding (5).(iv) We hypothesize that there may be negative selection against N-glycans in Plasmodium and Toxoplasma, because the N-glycans added in the ER lumen during translocation will likely interfere with threading of nucleus-encoded apicoplast proteins into a nonphotosynthetic, chloroplast-derived organelle called the apicoplast (21, 35, 37, 48, 52, 54). Nucleus-encoded apicoplast proteins have a bipartite signal at the N terminus, which targets proteins first to the lumen of the ER and second to lumen of the apicoplast. This bipartite signal has been used in transformed plasmodia where green fluorescent protein (GFP) is targeted to the apicoplast with the bipartite signal of the acyl carrier protein (ACP_leader-GFP), to the secretory system with the signal sequence only (ACP_signal-GFP), and to the cytosol with the organelle-targeting transit peptide only (ACP_transit-GFP) (55). Similar constructs have been used to characterize signals that target nucleus-encoded proteins of Toxoplasma to the apicoplast (11, 25).Here we use a combination of bioinformatic, biochemical, and morphological methods to characterize the N-glycans of Plasmodium and Toxoplasma and to test our hypothesis that there is negative selection against N-glycans in protists with apicoplasts.     

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways^1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca²⁺, a signal that is also critical for host cell invasion^6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress⁹. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches^10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress¹³. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology^11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine^16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype¹³. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites¹¹. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell¹⁹. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed²⁰. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.     

Membrane skeletal association and post‐translational allosteric regulation of Toxoplasma gondii GAPDH1

When Toxoplasma gondii egresses from the host cell, glyceraldehyde‐3‐phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure–function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S‐loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S‐loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N‐terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post‐translational modifications.     

The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole)

The kinetochore is a multi‐protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock‐down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan‐specific motif with a putative role in nuclear localization.     

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly,Apicoplast Division and Cytokinesis

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother''s cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.     

Relationship of cryptorchidism with sex ratios and litter sizes in 12 dog breeds

The aim of this study was to identify the influence of genetic carriership for cryptorchidism on litter sizes and sex ratios in the offspring. Weaning data of 11,230 litters in 12 purebred dog breeds were evaluated. Parents were classified as cryptorchidism 'carriers' (C) when at least one of their offspring was found cryptorchid. Subsequently the effects of 'carrier' and 'non-carrier' (NC) parents on their litters were studied. In litters from C x C parents we found an increased number of males per litter in all breeds, a reduced number of females per litter in 8 breeds and an increased litter size in 11 breeds in comparison with litters from NC x NC parents. Over all breeds the effects on litter size, on number of males per litter and on sex ratio were highly significant. Mixed litters from C x NC and NC x C did not show these effects and were not significantly different from the NC x NC offspring. Our results suggest a general mechanism in the dog species which causes cryptorchidism as well as increased male/female ratios and increased litter sizes. A consequence of such a mechanism is that selection in favor of increasing reproduction output frustrates selective efforts to eliminate cryptorchidism.