Identification of substrates of human protein-tyrosine phosphatase PTPN22

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRzeta, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.     

A protein dynamics study of photosystem II: the effects of protein conformation on reaction center function

Molecular dynamics simulations have been performed to study photosystem II structure and function. Structural information obtained from simulations was combined with ab initio computations of chromophore excited states. In contrast to calculations based on the x-ray structure, the molecular-dynamics-based calculations accurately predicted the experimental absorbance spectrum. In addition, our calculations correctly assigned the energy levels of reaction-center (RC) chromophores, as well as the lowest-energy antenna chlorophyll. The primary and secondary quinone electron acceptors, Q_A and Q_B, exhibited independent changes in position over the duration of the simulation. Q_B fluctuated between two binding sites similar to the proximal and distal sites previously observed in light- and dark-adapted RC from purple bacteria. Kinetic models were used to characterize the relative influence of chromophore geometry, site energies, and electron transport rates on RC efficiency. The fluctuating energy levels of antenna chromophores had a larger impact on quantum yield than did their relative positions. Variations in electron transport rates had the most significant effect and were sufficient to explain the experimentally observed multi-component decay of excitation in photosystem II. The implications of our results are discussed in the context of competing evolutionary selection pressures for RC structure and function.     

Do Current Standards of Practice in Canada Measure What is Relevant to Human Exposure at Contaminated Sites? II: Oral Bioaccessibility of Contaminants in Soil

Human health risk estimates for sites with contaminated soils are often based on the assumption that the outdoor soil sieved to < 250 μm is a reasonable surrogate for predicting exposures via incidental soil ingestion. In vitro bioaccessibility tests are also increasingly used to “improve” ingestion exposure predictions of contaminants at different sites. However, when considered in terms of factors that influence desorption of contaminants from particles and uptake into humans, available studies indicate that current “standards of practice” with respect to assays of oral bioaccessibility have a number of significant shortcomings, at least in Canada. These shortcomings are discussed and various factors that influence the assessment of bioaccessibility of contaminants in soils are examined. We finish with proposing some minimum data submission requirements to support the application and relevance of bioaccessibility assays at contaminated sites.     

The effects of angiotensin II on the coupled microstructural and biomechanical response of C57BL/6 mouse aorta

RationaleAbdominal aortic aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta, the rupture of which is associated with significant morbidity and mortality. Animal models of AAA can be used to study how changes in the microstructural and biomechanical behavior of aortic tissues develop as disease progresses in these animals. We chose here to investigate the effect of angiotensin II (AngII) in C57BL/6 mice as a first step towards understanding how such changes occur in the established ApoE^?/? AngII infused mouse model of AAA.ObjectiveThe objective of this study was to utilize a recently developed device in our laboratory to determine how the microstructural and biomechanical properties of AngII-infused C57BL/6 wildtype mouse aorta change following 14 days of AngII infusion.MethodsC57BL/6 wildtype mice were infused with either saline or AngII for 14 day. Aortas were excised and tested using a device capable of simultaneously characterizing the biaxial mechanical response and load-dependent (unfixed, unfrozen) extracellular matrix organization of mouse aorta (using multiphoton microscopy). Peak strains and stiffness values were compared across experimental groups, and both datasets were fit to a Fung-type constitutive model. The mean mode and full width at half maximum (FWHM) of fiber histograms from two photon microscopy were quantified in order to assess the preferred fiber distribution and degree of fiber splay, respectively.ResultsThe axial stiffness of all mouse aorta was found to be an order of magnitude larger than the circumferential stiffness. The aortic diameter was found to be significantly increased for the AngII infused mice as compared to saline infused control (p=0.026). Aneurysm, defined as a percent increase in maximum diameter of 30% (defined with respect to saline control), was found in 3 of the 6 AngII infused mice. These three mice displayed adventitial collagen that lacked characteristic fiber crimp. The biomechanical response in the AngII infused mice showed significantly reduced circumferential compliance. We also noticed that the ability of the adventitial collagen fibers in AngII infused mice to disperse in reaction to circumferential loading was suppressed.ConclusionsCollagen remodeling is present following 14 days of AngII infusion in C57BL/6 mice. Aneurysmal development occurred in 50% of our AngII infused mice, and these dilatations were accompanied with adventitial collagen remodeling and decreased circumferential compliance.     

Dental topography indicates ecological contraction of lemur communities

Understanding the paleoecology of extinct subfossil lemurs requires reconstruction of dietary preferences. Tooth morphology is strongly correlated with diet in living primates and is appropriate for inferring dietary ecology. Recently, dental topographic analysis has shown great promise in reconstructing diet from molar tooth form. Compared with traditionally used shearing metrics, dental topography is better suited for the extraordinary diversity of tooth form among subfossil lemurs and has been shown to be less sensitive to phylogenetic sources of shape variation. Specifically, we computed orientation patch counts rotated (OPCR) and Dirichlet normal energy (DNE) of molar teeth belonging to 14 species of subfossil lemurs and compared these values to those of an extant lemur sample. The two metrics succeeded in separating species in a manner that provides insights into both food processing and diet. We used them to examine the changes in lemur community ecology in Southern and Southwestern Madagascar that accompanied the extinction of giant lemurs. We show that the poverty of Madagascar's frugivore community is a long-standing phenomenon and that extinction of large-bodied lemurs in the South and Southwest resulted not merely in a loss of guild elements but also, most likely, in changes in the ecology of extant lemurs.     

Consumer-resource coupling in wet-dry tropical rivers

1. Despite implications for top-down and bottom-up control and the stability of food webs, understanding the links between consumers and their diets remains difficult, particularly in remote tropical locations where food resources are usually abundant and variable and seasonal hydrology produces alternating patterns of connectivity and isolation. 2. We used a large scale survey of freshwater biota from 67 sites in three catchments (Daly River, Northern Territory; Fitzroy River, Western Australia; and the Mitchell River, Queensland) in Australia's wet-dry tropics and analysed stable isotopes of carbon (δ(13) C) to search for broad patterns in resource use by consumers in conjunction with known and measured indices of connectivity, the duration of floodplain inundation, and dietary choices (i.e. stomach contents of fish). 3. Regression analysis of biofilm δ(13) C against consumer δ(13) C, as an indicator of reliance on local food sources (periphyton and detritus), varied depending on taxa and catchment. 4. The carbon isotope ratios of benthic invertebrates were tightly coupled to those of biofilm in all three catchments, suggesting assimilation of local resources by these largely nonmobile taxa. 5. Stable C isotope ratios of fish, however, were less well-linked to those of biofilm and varied by catchment according to hydrological connectivity; the perennially flowing Daly River with a long duration of floodplain inundation showed the least degree of coupling, the seasonally flowing Fitzroy River with an extremely short flood period showed the strongest coupling, and the Mitchell River was intermediate in connectivity, flood duration and consumer-resource coupling. 6. These findings highlight the high mobility of the fish community in these rivers, and how hydrological connectivity between habitats drives patterns of consumer-resource coupling.