A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

Relation between the reaction cytochrome oxidase-oxygen and oxygen uptake in cells in vivo. The role of diffusion

Before reaching the oxidase located inside the cell on the mitochondrial membrane, oxygen may be slowed down by diffusion within the cytoplasm. Diffusion or enzymic activity may predominate and this is related to the size and morphology of the organism, the intracellular diffusion coefficient, and the K_m of the oxidizing terminal enzyme. This K_m may be apparently diminished by inhibitors. Different experimental situations can arise in some plants (where oxygen diffusion in bulk tissue is not important in comparison to that in cell): either oxygen diffusion is limiting, or diffusion and an enzymic reaction compete, or diffusion does not slow down the respiratory rate. A mathematical model relates the oxygen concentration in the external medium, to the rate of oxygen uptake at the oxygen cytochrome-oxidase reaction level.     

Individual flowering phenology shapes plant–pollinator interactions across ecological scales affecting plant reproduction

The balance of pollination competition and facilitation among co‐flowering plants and abiotic resource availability can modify plant species and individual reproduction. Floral resource succession and spatial heterogeneity modulate plant–pollinator interactions across ecological scales (individual plant, local assemblage, and interaction network of agroecological infrastructure across the farm). Intraspecific variation in flowering phenology can modulate the precise level of spatio‐temporal heterogeneity in floral resources, pollen donor density, and pollinator interactions that a plant individual is exposed to, thereby affecting reproduction. We tested how abiotic resources and multi‐scale plant–pollinator interactions affected individual plant seed set modulated by intraspecific variation in flowering phenology and spatio‐temporal floral heterogeneity arising from agroecological infrastructure. We transplanted two focal insect‐pollinated plant species (Cyanus segetum and Centaurea jacea, n = 288) into agroecological infrastructure (10 sown wildflower and six legume–grass strips) across a farm‐scale experiment (125 ha). We applied an individual‐based phenologically explicit approach to match precisely the flowering period of plant individuals to the concomitant level of spatio‐temporal heterogeneity in plant–pollinator interactions, potential pollen donors, floral resources, and abiotic conditions (temperature, water, and nitrogen). Individual plant attractiveness, assemblage floral density, and conspecific pollen donor density (C. jacea) improved seed set. Network linkage density increased focal species seed set and modified the effect of local assemblage richness and abundance on C. segetum. Mutual dependence on pollinators in networks increased C. segetum seed set, while C. jacea seed set was greatest where both specialization on pollinators and mutual dependence was high. Abiotic conditions were of little or no importance to seed set. Intra‐ and interspecific plant–pollinator interactions respond to spatio‐temporal heterogeneity arising from agroecological management affecting wild plant species reproduction. The interplay of pollinator interactions within and between ecological scales affecting seed set implies a co‐occurrence of pollinator‐mediated facilitative and competitive interactions among plant species and individuals.     

Adaptive evolution of butterfly wing shape: from morphology to behaviour

Butterflies display extreme variation in wing shape associated with tremendous ecological diversity. Disentangling the role of neutral versus adaptive processes in wing shape diversification remains a challenge for evolutionary biologists. Ascertaining how natural selection influences wing shape evolution requires both functional studies linking morphology to flight performance, and ecological investigations linking performance in the wild with fitness. However, direct links between morphological variation and fitness have rarely been established. The functional morphology of butterfly flight has been investigated but selective forces acting on flight behaviour and associated wing shape have received less attention. Here, we attempt to estimate the ecological relevance of morpho‐functional links established through biomechanical studies in order to understand the evolution of butterfly wing morphology. We survey the evidence for natural and sexual selection driving wing shape evolution in butterflies, and discuss how our functional knowledge may allow identification of the selective forces involved, at both the macro‐ and micro‐evolutionary scales. Our review shows that although correlations between wing shape variation and ecological factors have been established at the macro‐evolutionary level, the underlying selective pressures often remain unclear. We identify the need to investigate flight behaviour in relevant ecological contexts to detect variation in fitness‐related traits. Identifying the selective regime then should guide experimental studies towards the relevant estimates of flight performance. Habitat, predators and sex‐specific behaviours are likely to be major selective forces acting on wing shape evolution in butterflies. Some striking cases of morphological divergence driven by contrasting ecology involve both wing and body morphology, indicating that their interactions should be included in future studies investigating co‐evolution between morphology and flight behaviour.     

Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A,X-linked adrenoleukodystrophy,X-linked hydrocephalus and incontinentia pigmenti loci on Xq28

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89–97% with an allele drop out rate of 2–19 %. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.     

Segmental duplication associated with the human-specific inversion of chromosome 18: a further example of the impact of segmental duplications on karyotype and genome evolution in primates

The human-specific pericentric inversion of chromosome 18 was analysed using breakpoint-spanning BACs from the chimpanzee and human genome. Sequence and FISH analyses disclosed that the breakpoints map to an inverted segmental duplication of 19-kb, which most likely mediated the inversion by intrachromosomal homologous recombination. The 19-kb duplication encompasses the 3 end of the ROCK1 gene and occurred in the human lineage. Only one copy of this segment is found in the chimpanzee. Due to the inversion, the genomic context of the ROCK1 and USP14 genes is altered. ROCK1 flanks USP14 in the long arm of the chimpanzee chromosome 17, which is homologous to human chromosome 18. This order is interrupted by the inversion in humans. ROCK1 is localized close to the pericentromeric region in 18q11 and USP14 is inverted to distal 18p11.3 in direct neighbourhood to LSAU-satellites, -satellites and telomere-associated repeats. Our findings essentially confirm the analysis of Dennehey et al. (2004). Intriguingly, USP14 is differentially expressed in human and chimpanzee cortex as well as fibroblast cell lines determined previously by the analysis of oligonucleotide arrays. Either position effects mediated by the proximity to the telomeric region or nucleotide divergence in regulatory regions might account for the differential expression of USP14. The assignment of the breakpoint region to a segmental duplication underlines the significance of the genomic architecture in the context of genome and karyotype evolution in hominoids.     

Cardiovascular drugs inhibit MMP-9 activity from human THP-1 macrophages

It is now recognized that atherosclerosis complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells, and accumulation of inflammatory cells. Colocalization of macrophages and active matrix metalloproteinases (MMPs) is likely relevant for atherosclerotic lesion disruption. Nevertheless, MMP activity and regulation by cardiovascular drugs remains poorly defined. In this study, we evaluated the effects of avasimibe, fluvastatin, and peroxisome proliferator-activated receptor (PPAR) ligands on 92-kDa gelatinase B (MMP-9) secretion by human THP-1 macrophages. THP-1 macrophages were treated with compounds for 48 h, and secreted MMP-9 protein was quantified by immunoassay. Avasimibe, fluvastatin, and PPARalpha agonists (fenofibric acid and Wy-14643) significantly reduced, in a concentration-dependent manner, MMP-9 protein (up to 67 +/- 5% for fenofibric acid). In these assays, the PPARgamma selective agonist rosiglitazone displayed a lower efficacy than other compounds. Enzymatic activity of MMP-9 was also decreased by all cardiovascular drugs tested. MMP-9 protein/activity inhibition by cardiovascular drugs was due, at least in part, to a decrease in MMP-9 mRNA. These results show that THP-1 macrophages could be an useful cellular model to investigate effects of compounds on plaque vulnerability through MMP-9 activity.     

Grb2 and Nck act cooperatively to promote actin-based motility of vaccinia virus

The Wiskott-Aldrich syndrome protein family member N-WASP is a key integrator of the multiple signalling pathways that regulate actin polymerization via the Arp2/3 complex. Our previous studies have shown that N-WASP is required for the actin-based motility of vaccinia virus and is recruited via Nck and WIP. We now show that Grb2 is an additional component of the vaccinia actin tail-forming complex. Recruitment of Nck and Grb2 to viral particles requires phosphorylation of tyrosine residues 112 and 132 of A36R, the vaccinia actin tail nucleator, respectively. The presence of Grb2 on the virus is also dependent on the polyproline-rich region of N-WASP. The Grb2 pathway alone is therefore unable to nucleate actin tails, as its recruitment requires the prior recruitment of N-WASP by Nck. However, Grb2 does play an important role in actin-based motility of vaccinia, as in its absence, the mean number of actin tails per cell is reduced 2.6-fold. Thus, both Nck and Grb2 act in a cooperative manner to stabilize and/or activate the vaccinia actin-nucleating complex. We suggest that such cooperativity between "primary" and "secondary" adaptor proteins is likely to be a general feature of receptor-mediated signalling.     

A role for Toxoplasma gondii type 1 ser/thr protein phosphatase in host cell invasion

Host cell invasion by Toxoplasma gondii tachyzoites relies on many coordinated processes. The tachyzoite participates in invasion by providing an actomyosin-dependent force driving it into the nascent parasitophorous vacuole as well as by releasing molecules which contribute to the vacuole membrane. Exposure to type 1/2A protein phosphatase inhibitors, okadaic acid (OA) or tautomycin significantly impairs tachyzoite invasiveness. Furthermore, the tachyzoite extract contains a biochemically active type 1, but not a type 2A, serine-threonine protein phosphatase, which is immunologically related to eukaryotic phosphatase type 1 catalytic subunit. When tachyzoite extracts are incubated with a monoclonal antibody reactive to human type 1 catalytic subunit, other T. gondii molecules are coprecipitated among which one competes with the inhibitory toxin OA. Finally, in vitro phosphate labelling assays indicate that the biochemically characterized PP1 activity controls the phosphorylation of several proteins. Taken together, these data strongly suggest that the type 1 phosphatase activity detected in invasive tachyzoites is implicated in the control of the host cell invasion process.     

Eavesdropping on Male Singing Interactions Leads to Differential Allocation in Eggs

Females invest differently in their eggs depending on the quality of their mates. In oscines, female investment is influenced by the quality of male song. In domestic canaries Serinus canaria, as well as in black‐capped chickadees Poecile atricapillus, females pay attention not only to the intrinsic quality of male song but can also gather information, by eavesdropping on male–male singing interactions, on the relative quality of males. During these interactions, overlapping the song of the rival is more threatening than alternating. Moreover eavesdropping female canaries have been shown to prefer the overlapping song rather than the overlapped song. The present study was designed to assess the effect of the information gathered by eavesdropping on female investment in eggs. First, we broadcasted overlapping interactions to female canaries. Then, we broadcasted to each female one of the two songs previously heard and collected eggs. Females exposed to overlapping songs laid eggs with greater yolk ratio than females exposed to overlapped songs. In contrast, yolk testosterone quantity and concentration were not affected by the treatment. Moreover, we found a variation between eggs with regard to the testosterone deposited in yolk: both quantity and concentration increased with laying order. Our results suggest that female canaries use information gathered by eavesdropping to differentially allocate resources into the eggs. They suggest that singing interactions could influence chick quality via female investment.