Sex differences in the excretion of fecal glucocorticoid metabolites in the Syrian hamster

We verified the relevance of measuring fecal glucocorticoid metabolites (FGM) to assess the stress response of the Syrian hamster. Male and female hamsters (n = 10 each) were submitted to an adrenocorticotropic hormone (ACTH) challenge test, whereas animals in the control group received 0.5 mL of sterile isotonic saline solution. All feces voided by each animal were collected at 4 h intervals from 24 h before (baseline) until 48 h after injections. FGM were quantified using an 11-oxoetiocholanolone enzyme immunoassay (EIA). Basal concentrations of FGM were almost four times higher in males than in females. Following ACTH administration, FGM levels started rising from 8 h onwards, reaching peak concentrations 20 or 28 h post injection in males and females, respectively. Despite the much higher absolute concentrations present in males, the relative increase (500%) in response to the ACTH stimulation was similar in both sexes. Sex differences in FGM levels are in accordance with results reported by others regarding the hamster adrenal physiology. The comparison of the adrenocortical response of males and females to an ACTH challenge provided new information about the amplitude and the timing of such a response and the excretion of glucocorticoids in both sexes. We demonstrated for the first time in the Syrian hamster that adrenocortical activity can be monitored in fecal samples in a noninvasive way. Our study provides a humane, practical, and noninvasive alternative to blood removal and therefore a powerful tool for stress-related studies in a species frequently used as an animal model in medical research.     

The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes

Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA⁺ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA⁺ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.Mitochondria not only supply cells with the bulk of their ATP but also contribute to the fine regulation of metabolism, calcium homeostasis, and apoptosis (27). Coordination of these functions is dependent on the dynamic nature of mitochondria (5). These organelles constantly fuse and divide to form small spheres, short rods, or long tubules and are actively transported to specific subcellular locations. These processes are essential for mammalian development, and defects can lead to degenerative diseases and cancers (9, 17). In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function (30, 45). The best-understood mitochondrial process is the fusion and fission pathways, which rely on conserved GTPases, and their binding partners to regulate organelle connectivity (10, 18, 45). There are also evidences that dynamic interactions between the outer membrane (OM) and inner membrane (IM) exist for coordinated fusion and fission, channeling of metabolites, and protein transport, but proteins playing a role in these interactions have yet to be identified (34). In the present study, we provide a detailed biochemical and functional characterization of the mitochondrial AAA⁺ ATPase ATAD3A protein that is present exclusively in multicellular eukaryotes and which participates in the control of mitochondrial dynamics at the interface between the IMs and OMs. Proteins related to the Atad3A genes have been previously identified in proteomic surveys of mouse brain mitochondria (28) and liver mitochondrial inner membrane (8), as mitochondrial DNA-binding proteins (4, 21, 44) and as nuclear mRNA-associated proteins (6). The Atad3A protein has also been identified as a cell surface antigen in some human tumors (16). Functional genomics identified the Drosophila Atad3A ortholog (bor) as a major gene positively regulated by the TOR (for target of rapamycin) signaling pathway involved in cell growth and division (19). In our laboratory, we identified ATAD3A as a specific target for the Ca²⁺/Zn²⁺-binding S100B protein (B. Gilquin et al., unpublished data). We here show that ATAD3A is anchored into the mitochondrial IM at contact sites with the OM. The N-terminal domain of ATAD3A interacts with the inner surface of the OM and its C-terminal AAA ATPase domain localizes in a specific matrix compartment. Thanks to its simultaneous interaction with two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the IMs and OMs and controls diverse cell responses ranging from cell growth, channeling of cholesterol, and mitochondrial fission.     

Specific host genes required for the killing of Klebsiella bacteria by phagocytes

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.     

Contribution of magnetic resonance imaging to the knowledge of CNS malformations related to chromosomal aberrations

Summary Twelve patients presenting with various clinicopathological syndromes related to chromosomal diseases have been evaluated using magnetic resonance imaging. They include patients with trisomy 21, trisomy 18, trisomy 13, 4p-syndrome, 5p-syndrome, and 7p-syndrome. In all these patients karyotype studies were performed demonstrating the chromosomal aberrations. All patients were examined using magnetic resonance imaging to evaluate the head and neck malformations which may be specifically associated with their chromosomal anomaly. We were particularly interested in brain abnormalities and the morphological findings correlated with some pathologic anatomical findings. A review of the literature on neuropathological data is reported and compared with the in vivo anatomical results obtained using this highly anatomical non-ionising and non-invasive investigative procedure. Particular interest is paid to trisomy 21 in which all recognizable stereotyped morphological skull and brain malformations are depicted with magnetic resonance and some other malformations demonstrated such as the excessive forward bending and ascension of the brainstem which correlated well with a simian cephalic organization.     

In vitro induction of microglial and endothelial cell apoptosis by cerebrospinal fluids from patients with human African trypanosomiasis

In human African trypanosomiasis, trypanosomes first develop in the blood and lymph (Stage 1), then spread to the central nervous system (CNS) (Stage 2). Disruption of the blood-brain barrier of unknown mechanism occurs in Stage 2 disease. The hypothesis that cerebrospinal fluids (CSF) from African trypanosomiasis patients might contain factor(s) able to induce apoptosis in endothelial cells led us to evaluate this effect by two methods, the TdT-mediated dUTP nick end labelling (TUNEL) method and the measurement of soluble nucleosomes released by apoptotic cells in culture supernatant by ELISA. Apoptosis induction by CSF was also studied with microglial cells, the resident macrophages in the brain, which participate in the blood-brain barrier in the perivascular area. In contrast with control CSF, African trypanosomiasis patients' CSF induced apoptosis in both microglial and endothelial cells. The results obtained with the two methods correlated well, and showed that Stage 2 CSF induced apoptosis at higher levels in microglial cells, whereas the disease stage was not decisive for apoptosis induction in endothelial cells. We measured soluble Fas ligand (sFasL) and anti-Fas antibodies levels, two potent inducers of the Fas signalling pathway leading to apoptosis, in CSF from African trypanosomiasis patients and controls. CSF from African trypanosomiasis patients contained sFasL, and anti-Fas antibodies at higher levels than in controls. Stage 2 CSF contained more sFasL than Stage 1 CSF, and anti-Fas antibodies were detected only in Stage 2 CSF. Caspase-8 inhibitor effect and statistical data suggest that other pro-apoptotic factors may be involved in some CSF-induced apoptosis. Apoptosis induction may participate in the pathogenesis during African trypanosomiasis, and the presence of sFasL and anti-Fas antibodies may provide new tools for diagnosis and prognosis of the disease.     

Permanent expression of a cyclin B homologue in the cell cycle of the dinoflagellate Karenia brevis

The eukaryotic cell cycle is driven by a set of cyclin-dependent kinases associated with their regulatory partners, the cyclins, which confer activity, substrate specificities and proper localization of the kinase activity. We describe the cell cycle of Karenia brevis and provide evidence for the presence of a cyclin B homologue in this dinoflagellate using two antibodies with different specificities. This cyclin B homologue has an unusual behavior, since its expression is permanent and it has a cytoplasmic location throughout the cell cycle. There is no evidence for translocation to the nucleus during mitosis. However, it appears also to be specifically bound to the nucleolus throughout the cell cycle. The permanent expression and the cytoplasmic localization during mitosis of this cyclin B homologue is similar to p56, a cyclin B homologue previously described in a different species of dinoflagellate, Crypthecodinium cohnii. Here we discuss this unusual behavior of the cyclin B homologue in dinoflagellates, its relationship to the unusual characteristics of dinomitosis, and its potential implications regarding the evolution of cell cycle regulation among eukaryotes.     

Colocalization of the cyclin B homologue P56 and β-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During motosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti β-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and β-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-β-tubulin antibodies confirm that p56 antigens colocalize with β-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.     

Autumnal nitrogen nutrition affects the C and N storage and architecture of young peach trees

Nitrogen fertilisation is a regular practice in orchards. Its effect on tree development, N and C acquisition and allocation were evaluated simultaneously, while coupling on the same trees in situ measurements of N uptake and shoot development and destructive determinations of organ composition in N and Total Non structural Carbohydrates (TNC). An hydroponic set-up was designed that could grow young peach trees at constant NO₃ concentration while measuring N uptake. Forty-eight trees were raised outdoors under excessive N supply. Between October 2 and December 7, half of them were then N-limited to reduce N uptake by 75%. Organ N concentrations remained stable in the controls but were halved in N-limited trees. Growth (390 vs. 353 g DW tree⁻¹) was less affected by the treatment than N uptake (10.6 vs. 2.7 g N tree⁻¹). Growth was affected only in terms of axillary bud development, which was restricted to the median and upper crown parts. The number of buds which transformed into elongating axes (44 vs. 84 tree⁻¹) was halved, thus reducing leaf area by one-third (10,464 vs. 15,568 cm²). Tree TNC content was not impacted. The difference in C acquisition likely balanced the C costs of N uptake. In N-limited trees, more TNC was stored as starch (73 vs. 56%), and the allocation patterns of TNC and N were altered in favour of the roots. Our results provide deeper insights into the tree integrated response to autumnal N fertilisation, focusing on an alteration of the balance between storage and growth.     

Analysis and modelling of effects of leaf rust and Septoria tritici blotch on wheat growth

A model to predict Septoria tritici blotch (STB) and leaf rust effects on wheat growth was constructed and evaluated in two steps. At the leaf scale, Bastiaans' approach that predicts the relative photosynthesis of a wheat leaf infected with a single disease, was extended to the case of two diseases, one biotrophic and one necrotrophic by considering the leaf rust-STB complex. A glasshouse experiment with flag leaves inoculated either singly with one disease or with two diseases combined was performed to check the leaf damage model. No interaction of the two diseases on photosynthesis loss was observed when they occurred simultaneously on the same leaf. In a second step, the single-leaf model was extended to the canopy scale to model the effects of the leaf rust-STB complex on the growth of a wheat crop. The model predicts the effects of disease on the growth of an affected crop relative to the growth of a healthy crop. The canopy model accounted for different contributions to photosynthetic activity of leaf layers, derived from their position in the canopy and their natural leaf senescence. Treatments differing in nitrogen fertilization, microclimatic conditions, and wheat cultivars were implemented in a field experiment to evaluate the model. The model accurately estimated the effect of disease on crop growth for each cultivar, with differences from experimental values lower than 10%, which suggests that this model is well suited to aid an understanding of disease effects on plant growth. A reduction in green leaf area was the main effect of disease in these field experiments and STB accounted for more than 70% of the reduction in plant growth. Simulations suggested that the production of rust spores may result in a loss of biomass from diseased crops and that stem photosynthesis may need to be considered in modelling diseased crop growth.