Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

BACKGROUND: Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. METHODS: Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. RESULTS: Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. CONCLUSION: Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines

What’s special about the ethical challenges of studying disorders with altered brain activity?

Where there is no viable alternative, studies of neuronal activity are conducted on animals. The use of animals, particularly for invasive studies of the brain, raises a number of ethical issues. Practical or normative ethics are enforced by legislation, in relation to the dominant welfare guidelines developed in the UK and elsewhere. Guidelines have typically been devised to cover all areas of biomedical research using animals in general, and thus lack any specific focus on neuroscience studies at the level of the ethics, although details of the specific welfare recommendations are different for invasive studies of the brain. Ethically there is no necessary distinction between neuroscience and other biomedical research in that the brain is a final common path for suffering, irrespective of whether this involves any direct experience of pain. One exception arises in the case of in vitro studies, which are normally considered as an acceptable replacement for in vivo studies. However, to the extent sentience is possible, maintaining central nervous system tissue outside the body naturally raises ethical questions. Perhaps the most intractable challenge to the ethical use of animals in order to model neuronal disorder is presented by the logical impasse in the argument that the animal is similar enough to justify the validity of the experimental model, but sufficiently different in sentience and capacity for suffering, for the necessary experimental procedures to be permissible

Analysis of the Candida albicans Phosphoproteome.

Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins

Analysis of the Candida albicans Phosphoproteome.

Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins

Effects of SKF-83566 and haloperidol on performance on progressive ratio schedules maintained by sucrose and corn oil reinforcement: quantitative analysis using a new model derived from the Mathematical Principles of Reinforcement (MPR)

Rationale Mathematical models can assist the interpretation of the effects of interventions on schedule-controlled behaviour and help to differentiate between processes that may be confounded in traditional performance measures such as response rate and the breakpoint in progressive ratio (PR) schedules. Objective The effects of a D1-like dopamine receptor antagonist, 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide (SKF-83566), and a D2-like receptor antagonist, haloperidol, on rats’ performance on PR schedules maintained by sucrose and corn oil reinforcers were assessed using a new model derived from Killeen’s (Behav Brain Sci 17:105–172, 1994) Mathematical Principles of Reinforcement. Method Separate groups of rats were trained under a PR schedule using sucrose or corn oil reinforcers. SKF-83566 (0.015 and 0.03 mg kg−1) and haloperidol (0.05 and 0.1 mg kg−1) were administered intraperitoneally (five administrations of each treatment). Running and overall response rates in successive ratios were analysed using the new model, and estimates of the model’s parameters were compared between treatments. Results Haloperidol reduced a (the parameter expressing incentive value) in the case of both reinforcers, but did not affect the parameters related to response time and post-reinforcement pausing. SKF-83566 reduced a and k (the parameter expressing sensitivity of post-reinforcement pausing to the prior inter-reinforcement interval) in the case of sucrose, but did not affect any of the parameters in the case of corn oil. Conclusions The results are consistent with the hypothesis that blockade of both D1-like and D2-like receptors reduces the incentive value of sucrose, whereas the incentive value of corn oil is more sensitive to blockade of D2-like than D1-like receptors