Internalization and sequestration of the human prostacyclin receptor

Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for sequestration. Internalization was unaltered by cotransfection of cells with G protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hIP is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.     

Pesticide exposure: the hormonal function of the female reproductive system disrupted?

Some pesticides may interfere with the female hormonal function, which may lead to negative effects on the reproductive system through disruption of the hormonal balance necessary for proper functioning. Previous studies primarily focused on interference with the estrogen and/or androgen receptor, but the hormonal function may be disrupted in many more ways through pesticide exposure. The aim of this review is to give an overview of the various ways in which pesticides may disrupt the hormonal function of the female reproductive system and in particular the ovarian cycle. Disruption can occur in all stages of hormonal regulation: 1. hormone synthesis; 2. hormone release and storage; 3. hormone transport and clearance; 4. hormone receptor recognition and binding; 5. hormone postreceptor activation; 6. the thyroid function; and 7. the central nervous system. These mechanisms are described for effects of pesticide exposure in vitro and on experimental animals in vivo. For the latter, potential effects of endocrine disrupting pesticides on the female reproductive system, i.e. modulation of hormone concentrations, ovarian cycle irregularities, and impaired fertility, are also reviewed. In epidemiological studies, exposure to pesticides has been associated with menstrual cycle disturbances, reduced fertility, prolonged time-to-pregnancy, spontaneous abortion, stillbirths, and developmental defects, which may or may not be due to disruption of the female hormonal function. Because pesticides comprise a large number of distinct substances with dissimilar structures and diverse toxicity, it is most likely that several of the above-mentioned mechanisms are involved in the pathophysiological pathways explaining the role of pesticide exposure in ovarian cycle disturbances, ultimately leading to fertility problems and other reproductive effects. In future research, information on the ways in which pesticides may disrupt the hormonal function as described in this review, can be used to generate specific hypotheses for studies on the effects of pesticides on the ovarian cycle, both in toxicological and epidemiological settings.     

Health of infants in an Imperial Roman skeletal sample: perspective from dental microstructure

This study examines general health in the first year of life of a population of 127 subadults from the Imperial Roman necropolis of Isola Sacra (2nd-3rd century ACE). Health status was determined by analyzing 274 deciduous teeth from these children for Wilson bands (also known as accentuated striae), microscopic defects caused by a disruption to normal enamel development arising from some generalized external stressor. While macroscopic enamel defects, or hypoplasias, have long been used as proxies of general population health, we believe that this is the first population-wide study of microscopic defects in deciduous teeth. We used microstructural markers of enamel to attach very precise chronologies to Wilson band formation that allowed us to calculate maximum prevalence (MAP) and smoothed maximum prevalence (SMAP) distributions to portray what we believe to be a realistic risk profile for a past population of children. There appear to be two periods of high prevalence, the first beginning around age 2 months and continuing through month 5, and the second higher period beginning around month 6 and continuing through month 9. These results are discussed in light of historical records of Roman childhood rearing practices.     

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.     

In Vitro and In Vivo Effects of Palmaria palmata Derived Peptides on Glucose Metabolism

International Journal of Peptide Research and Therapeutics - Three synthetic peptides, ILAP, LLAP and MAGVDHI, derived from a Palmaria palmata protein hydrolysate were assessed for their...     

The anatomy of choice: dopamine and decision-making

This paper considers goal-directed decision-making in terms of embodied or active inference. We associate bounded rationality with approximate Bayesian inference that optimizes a free energy bound on model evidence. Several constructs such as expected utility, exploration or novelty bonuses, softmax choice rules and optimism bias emerge as natural consequences of free energy minimization. Previous accounts of active inference have focused on predictive coding. In this paper, we consider variational Bayes as a scheme that the brain might use for approximate Bayesian inference. This scheme provides formal constraints on the computational anatomy of inference and action, which appear to be remarkably consistent with neuroanatomy. Active inference contextualizes optimal decision theory within embodied inference, where goals become prior beliefs. For example, expected utility theory emerges as a special case of free energy minimization, where the sensitivity or inverse temperature (associated with softmax functions and quantal response equilibria) has a unique and Bayes-optimal solution. Crucially, this sensitivity corresponds to the precision of beliefs about behaviour. The changes in precision during variational updates are remarkably reminiscent of empirical dopaminergic responses—and they may provide a new perspective on the role of dopamine in assimilating reward prediction errors to optimize decision-making.     

Enzymatic debittering of food protein hydrolysates

Protein hydrolysates have a range of applications in the food and allied healthcare sectors. Bitterness is a negative attribute associated with most food protein hydrolysates. The development of biotechnological solutions for hydrolysate debittering is ongoing. Specific enzymatic debittering strategies have focused on the application of proline specific exo- and endopeptidases given the contribution of proline residues to peptide/hydrolysate bitterness. Hydrolysate manufacturing conditions may also play an important role in bitterness development. Practical solutions to hydrolysate debittering are likely to involve judicious choice of enzymatic processing conditions in conjunction with the use of peptidase activities having targeted hydrolytic specificity.