Wanda D McCormick

Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mm increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mm extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.

Increased receptor stimulation elicits differential calcium-sensing receptort888 dephosphorylation

The calcium-sensing receptor (CaR) elicits oscillatory Ca2+i mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaRT888. While modest CaR stimulation elicits Ca2+i oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mm Ca2+o, 10 min) increased CaRT888 phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3–5 mm Ca2+o treatments were without apparent effect. Treatment with 2 mm Ca2+o caused sustained CaRT888 phosphorylation (≥20 min) and oscillatory Ca2+i mobilization. However, 5 mm Ca2+o increased CaRT888 phosphorylation only briefly while eliciting sustained Ca2+i mobilization, suggesting that greater CaR activation induces rapid CaRT888 dephosphorylation, thus permitting sustained Ca2+i responses. Indeed, 5 mm Ca2+o stimulated protein phosphatase 2A activity and induced CaRT888 dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and l-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca2+o-stimulated protein kinase C acts in concert with high Ca2+o-induced phosphatase activity to generate and maintain CaR-induced Ca2+i oscillations via the dynamic phosphorylation and dephosphorylation of CaRT888.

A preliminary assessment of how zoo visitors evaluate animal welfare according to enclosure style and the expression of behavior

Abstract Visitors to zoos make judgements about animal welfare on the basis of what they see during their visit. There has been a considerable amount of research and debate surrounding the use of enclosure style and/or animal behavior to act as indicators of animal welfare. There are assumptions, supported by some studies but contradicted by others, that naturalistic enclosures and the expression of “wild behavior” inherently promote good welfare. These assumptions also appear to be used by the public to judge the welfare of zoo-housed animals. The aim of this study was to evaluate whether visitors to Paignton Zoo Environmental Park, UK, were using these assumptions to judge the welfare of zoo-housed animals. Visitors (n=42) were shown two series of photographs (n=8 in each series), one representing different primate enclosure styles and one depicting different tiger behaviors. The visitors were asked to rank the photographs in response to a series of questions (n=4 primate enclosure; n=5 tiger behavior). The public were consistent in their assessment of enclosure styles, which confirmed they held the assumption that naturalistic enclosures are good; all respondents rated the greenest enclosure highly and thought its inhabitants would have the best welfare. The interpretation of captive tiger behavior was also consistent across respondents, but this did not clearly indicate that they thought wild behavior was good. Most respondents thought that tigers in captivity and in the wild performed similar behavior. However, they did not think that expression of wild-type behavior was indicative of good welfare in captivity.

Prevalence, risk factors and vaccination efficacy of contagious ovine ecthyma (orf) in England

Orf is a viral disease found in English sheep flocks which can cause economic losses. It is a zoonosis with little epidemiological research available in the UK. In 2012, 3000 questionnaires were sent to English sheep farms in order to investigate the prevalence of orf, determine vaccination efficacy and to identify some of the potential risk factors. The usable response rate was 25.4 per cent. The usable farms (N=762 in the years 2011 and 2012) were used to model the percentage of animals affected on the farm, and the probability of a farm being found with the disease. The disease prevalence (DP) was standardised for the year and calculated as 1.88 per cent for ewes and 19.53 per cent for lambs. The disease risk ratio (RR) for the use of the vaccine was calculated as 2.04 for ewes and 0.75 for lambs, and therefore, the study found that lamb vaccination was beneficial (RR <1). Weed infestation and an increased number of orphan lambs were associated with increased cases of orf. We conclude that the DP in ewes and lambs affect each other, though the impact is higher for lambs in the presence of increasing prevalence in ewes. A short lambing season lowers the probability of a farm experiencing cases of orf. Vaccination was effective in lambs but not in ewes, though lambs benefitted when ewes were vaccinated (reduced orf prevalence in lambs born from vaccinated ewes), probably because any unvaccinated ewes may have been carriers that could spread the virus to the new-born lambs.

The degree of acceptability of swine blood values at increasing levels of hemolysis evaluated through visual inspection versus automated quantification

The pronounced fragility that characterizes swine erythrocytes is likely to produce a variable degree of hemolysis during blood sampling, and the free hemoglobin may then unpredictably bias the quantification of several analytes. The aim of this study was to evaluate the degree of acceptability of values obtained for several biochemical parameters at different levels of hemolysis. Progressively increased degrees of physical hemolysis were induced in 3 aliquots of 30 nonhemolytic sera, and the relative effects on the test results were assessed. To define the level of hemolysis, we used both visual estimation (on a scale of 0 to 3+) and analytical assessment (hemolytic index) and identified the best analytical cutoff values for discriminating the visual levels of hemolysis. Hemolysis led to a variable and dose-dependent effect on the test results that was specific for each analyte tested. In mildly hemolyzed specimens, C-reactive protein, haptoglobin, β1-globulin, β2-globulin, α1-globulin, γ-globulin, sodium, calcium, and alkaline phosphatase were not significantly biased, whereas α2-globulin, albumin, urea, creatinine, glucose, total cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, nonesterified fatty acids, bilirubin, phosphorus, magnesium, iron, zinc, copper, lipase, triglycerides, lactate dehydrogenase, unbound iron-binding capacity, and uric acid were significantly biased. Chloride and total protein were unbiased even in markedly hemolyzed samples. Analytical interference was hypothesized to be the main source of this bias, leading to a nonlinear trend that confirmed the difficulty in establishing reliable coefficients of correction for adjusting the test results.