Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.     

Endurance exercise training blunts the deleterious effect of high-fat feeding on whole body efficiency

We recently showed that a week-long, high-fat diet reduced whole body exercise efficiency in sedentary men by >10% (Edwards LM, Murray AJ, Holloway CJ, Carter EE, Kemp GJ, Codreanu I, Brooker H, Tyler DJ, Robbins PA, Clarke K. FASEB J 25: 1088-1096, 2011). To test if a similar dietary regime would blunt whole body efficiency in endurance-trained men and, as a consequence, hinder aerobic exercise performance, 16 endurance-trained men were given a short-term, high-fat (70% kcal from fat) and a moderate carbohydrate (50% kcal from carbohydrate) diet, in random order. Efficiency was assessed during a standardized exercise task on a cycle ergometer, with aerobic performance assessed during a 1-h time trial and mitochondrial function later measured using (31)P-magnetic resonance spectroscopy. The subjects then underwent a 2-wk wash-out period, before the study was repeated with the diets crossed over. Muscle biopsies, for mitochondrial protein analysis, were taken at the start of the study and on the 5th day of each diet. Plasma fatty acids were 60% higher on the high-fat diet compared with moderate carbohydrate diet (P < 0.05). However, there was no change in whole body efficiency and no change in mitochondrial function. Endurance exercise performance was significantly reduced (P < 0.01), most probably due to glycogen depletion. Neither diet led to changes in citrate synthase, ATP synthase, or mitochondrial uncoupling protein 3. We conclude that prior exercise training blunts the deleterious effect of short-term, high-fat feeding on whole body efficiency.     

A phylogeny of frugivorous hornbills linked to the evolution of Indian plants within Asian rainforests

Understanding the origin and radiation of modern Asian hornbills and the influential ecological roles they play as seed dispersal agents within Asian rainforests should help reveal the evolution of these roles. We constructed a dated phylogeny of hornbills using mitochondrial DNA sequences of the cytochrome b gene and discovered that all clades leading to frugivorous hornbills originated in the mid-Eocene ～48 Ma. This 'explosive' radiation coincided with a remarkable floral invasion of Asian rainforests from the Indian microcontinent. Analysis of phylogenetic data, in conjunction with palaeontological events, suggests that the invasion of distinctive flora comprised two waves, one during the mid-Eocene, when India was offshore of the Sunda Shelf, and the other late Eocene, when India collided with the Asian mainland. We propose that frugivorous vertebrates, such as hornbills, were present during the first wave and assisted rapid colonization of the Asian flora.     

Increased plasma levels of adipokines and inflammatory markers in older women with persistent HPV infection

We observed diminished lymphoproliferation to multiple stimuli in older women with persistent cervical human papillomavirus (HPV) infection. Adipokines are a class of inflammatory cytokines that are altered in some persistent infections. The objective was to compare the level of adipokines and inflammatory cytokines in heparinized plasma from women with persistent HPV cervical infection (Cases, N=50, oversampled for their weak lymphoproliferation responses) with women with no evidence of persistent HPV cervical infection (Controls, N=50, oversampled for their strong lymphoproliferation responses). Plasma samples were analyzed with multiplex assays for adipokines and inflammatory cytokines. Cases had significantly elevated plasma levels of resistin (p<0.0001) and sFas (p=0.0038) as compared to controls. Risk of persistent HPV infection increased significantly with increasing levels of resistin and 8Fas. This is the first study to demonstrate elevated levels of resistin and sFas in HPV persistently infected, older women with decreased immune function expanding the understanding of the systemic inflammation and immune alterations in individuals persistently infected with HPV. Further studies within a larger cohort are needed to define the generalities of these findings and any role adipokines have in persistent HPV infection.     

Catastrophic mortality on inshore coral reefs of the Florida Keys due to severe low‐temperature stress

Coral reefs of the Florida Keys typically experience seasonal temperatures of 20–31 °C. Deviation outside this range causes physiological impairment of reef‐building corals, potentially leading to coral colony death. In January and February 2010, two closely spaced cold fronts, possibly driven by an unusually extreme Arctic Oscillation, caused sudden and severe seawater temperature declines in the Florida Keys. Inshore coral reefs [e.g., Admiral Reef (ADM)] experienced lower sustained temperatures (i.e., < 12 °C) than those further offshore [e.g., Little Grecian Reef (LG), minimum temperature = 17.2 °C]. During February and March 2010, we surveyed ADM and observed a mass die‐off of reef‐building corals, whereas 12 km away LG did not exhibit coral mortality. We subsequently measured the physiological effects of low‐temperature stress on three common reef‐building corals (i.e., Montastraea faveolata, Porites astreoides, and Siderastrea siderea) over a range of temperatures that replicated the inshore cold‐water anomaly (i.e., from 20 to 16 to 12 °C and back to 20 °C). Throughout the temperature modulations, coral respiration as well as endosymbiont gross photosynthesis and maximum quantum efficiency of photosystem II were measured. In addition, Symbiodinium genotypic identity, cell densities, and chlorophyll a content were determined at the beginning and conclusion of the experiment. All corals were significantly affected at 12 °C, but species‐specific physiological responses were found indicating different coral and/or Symbiodinium cold tolerances. Montastraea faveolata and P. astreoides appeared to be most negatively impacted because, upon return to 20 °C, significant reductions in gross photosynthesis and dark respiration persisted. Siderastrea siderea, however, readily recovered to pre‐treatment rates of dark respiration and gross photosynthesis. Visual surveys of inshore reefs corroborated these results, with S. siderea being minimally affected by the cold‐water anomaly, whereas M. faveolata and P. astreoides exhibited nearly 100% mortality. This study highlights the importance of understanding the physiological attributes of genotypically distinct coral‐Symbiodinium symbioses that contribute to tolerance, recovery, and consequences to an environmental perturbation. These data also document effects of a rarely studied environmental stressor, possibly initiated by remote global climate events, on coral‐Symbiodinium symbioses and coral reef communities.     

Predicting cytotoxic T-cell age from multivariate analysis of static and dynamic biomarkers

Adoptive T-cell transfer therapy relies upon in vitro expansion of autologous cytotoxic T cells that are capable of tumor recognition. The success of this cell-based therapy depends on the specificity and responsiveness of the T cell clones before transfer. During ex vivo expansion, CD8+ T cells present signs of replicative senescence and loss of function. The transfer of nonresponsive senescent T cells is a major bottleneck for the success of adoptive T-cell transfer therapy. Quantitative methods for assessing cellular age and responsiveness will facilitate the development of appropriate cell expansion and selection protocols. Although several biomarkers of lymphocyte senescence have been identified, these proteins in isolation are not sufficient to determine the age-dependent responsiveness of T cells. We have developed a multivariate model capable of extracting combinations of markers that are the most informative to predict cellular age. To acquire signaling information with high temporal resolution, we designed a microfluidic chip enabling parallel lysis and fixation of stimulated cell samples on-chip. The acquisition of 25 static biomarkers and 48 dynamic signaling measurements at different days in culture, integrating single-cell and population based information, allowed the multivariate regression model to accurately predict CD8+ T-cell age. From surface marker expression and early phosphorylation events following T-cell receptor stimulation, the model successfully predicts days in culture and number of population doublings with R2=0.91 and 0.98, respectively. Furthermore, we found that impairment of early signaling events following T cell receptor stimulation because of long term culture allows prediction of costimulatory molecules CD28 and CD27 expression levels and the number of population divisions in culture from a limited subset of signaling proteins. The multivariate analysis highlights the information content of both averaged biomarker values and heterogeneity metrics for prediction of cellular age within a T cell population.