Differential Roles of the PKC Novel Isoforms, PKCδ and PKCε, in Mouse and Human Platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI

Computational Neural Models of Spatial Integration in Perceptual Grouping

Recent developments in the neural computational modeling of perceptual grouping are described with reference to a newly proposed taxonomy to formalize mechanisms of spatial integration. This notational framework and nomenclature is introduced in or-der to clarify key properties common to all or most models, while permitting unique attributes of each approach to be independently examined. The strength of spatial integration in the models that are considered is always some function of the distances and relative alignments in perceptual space of the centers of units representing orien-tational features or energy in a visual scene. We discuss the signicance of variations of the constituents of an activation function for spatial integration, and also consider the larger modeling framework in which this function is applied in each approach. We also discuss the relationship of feedforward and feedback mechanisms and the issues of self-organization as core principles underlying the establishment of spatial integra-tion mechanisms. The relationship of the grouping models to models of other visual competencies is considered with respect to prospects for future research. 354 From Fragments to Object

Investigating the mechanisms of platelet spreading

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Distribution and trophic importance of anthropogenic nitrogen in Narragansett Bay: An assessment using stable isotopes

Narragansett Bay has been heavily influenced by human activities for more than 200 years. In recent decades, it has been one of the more intensively fertilized estuaries in the USA, with most of the anthropogenic nutrient load originating from sewage treatment plants (STP). This will soon change as tertiary treatment upgrades reduce nitrogen (N) loads by about one third or more during the summer. Before these reductions take place, we sought to characterize the sewage N signature in primary (macroalgae) and secondary (the hard clam, Mercenaria mercenaria) producers in the bay using stable isotopes of N (δ15N) and carbon (δ13C). The δ15N signatures of the macroalgae show a clear gradient of approximately 4‰ from north to south, i.e., high to low point source loading. There is also evidence of a west to east gradient of heavy to light values of δ15N in the bay consistent with circulation patterns and residual flows. The Providence River Estuary, just north of Narragansett Bay proper, receives 85% of STP inputs to Narragansett Bay, and lower δ15N values in macroalgae there reflected preferential uptake of 14N in this heavily fertilized area. Differences in pH from N stimulated photosynthesis and related shifts in predominance of dissolved C species may control the observed δ13C signatures. Unlike the macroalgae, the clams were remarkably uniform in both δ15N (13.2±0.54‰ SD) and δ13C (-16.76±0. 61‰ SD) throughout the bay, and the δ15N values were 2-5‰ heavier than in clams collected outside the bay. We suggest that this remarkable uniformity reflects a food source of anthropogenically heavy phytoplankton formed in the upper bay and supported by sewage derived N. We estimate that approximately half of the N in the clams throughout Narragansett Bay may be from anthropogenic sources. © 2007 Coastal and Estuarine Research Federation

Metagenomic assessment of the diversity and ubiquity of antimicrobial resistance genes in Bangladeshi aquaculture ponds

In Bangladesh, fish provide over 60% of animal-source food with 56.2% of this coming from aquaculture produced predominantly in rural freshwater ponds. Increasing demand for fish products is driving intensification and resulting in higher disease prevalence, posing a risk to food security. Biosecurity is often absent in rural aquaculture practices in Bangladesh and antibiotics are commonly used to treat and prevent disease outbreaks. Antibiotics are often administered incorrectly - a key factor associated with the development of antimicrobial resistance (AMR). AMR can be disseminated rapidly within microbial ecosystems via mobile genetic elements, posing a risk for humans and animals infected with AMR pathogens as treatments with antibiotics become ineffective. Early AMR detection and understanding of the spread of antimicrobial resistance genes (ARGs) in rural aquaculture practices is critical for both food security, human health protection and food safety. Here, we apply a metagenomic approach to assess the ARG composition in pond water from six finfish (tilapia and pangasius) farms in the Mymensingh division of North-central Bangladesh. We found microbial communities within the ponds had similar alpha and beta diversities, with multiple ARGs predicted to confer resistance to eighteen different classes of antimicrobials. The most common ARGs conferred resistance to aminoglycosides and sulphonamides and were present in taxa associated with both fish and human pathogens. This ARG diversity potentially confers resistance to a wide variety of antibiotic classes and questions the effectiveness of current and future treatment of diseases with antibiotics in earthen aquaculture ponds. The microbial and ARG compositions between fish ponds within each farm were similar, which may relate to parallels in farming practices creating similar microbial selection pressures and thus comparable microbial populations. Without a more controlled approach towards antibiotic usage, we will inevitably further exacerbate the challenges in treating and preventing disease outbreaks as aquaculture production intensifies in Bangladesh

Effect of Ro 31-8220 (Ro) on PKCδ tyrosine phosphorylation.

<p>Human washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were treated with (A) 1 U/ml thrombin or (B) 3 µg/ml convulxin in the presence or absence of Ro 31-8220 (10 µM, 1 min) for the times shown. Immunoprecipitations and reprobes were carried out as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histograms show mean density of bands, normalised according to the basal value. *signifies statistically significant from basal, ^#signifies statistically significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

(A) Effect of PP2 on PKCδ tyrosine phosphorylation.

<p>Human washed platelets were treated with 0.1 or 1 U/ml thrombin for 1 min in the presence or absence of PP2 (10 µM, 5 min). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (B) Effect of PP2 on pleckstrin phosphorylation in thrombin-stimulated platelets. Washed human platelets were labelled with radioactive ³²P-orthophosphate and stimulated with 1 U/ml thrombin for 1 min. PP2 (10 µM, 5 min) or Ro31-8220 (10 µM, 1 min) were added where indicated. After separation by SDS-PAGE, the resulting gel was analysed using a phosphoimager to obtain radioactivity levels. Histogram shows the level of pleckstrin phosphorylation compared to basal levels. Data from one experiment, representative of two. (C) Tyrosine phosphorylation of PKCδ in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin in the absence or presence of Ro31-8220 (10 µM, 1 min) (left), or with 1 U/ml thrombin for the times shown (right) in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (D) Tyrosine phosphorylation of PKCε in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin for 1 min. Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. All studies described in this figure were performed in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Histograms show mean density of bands, normalised according to the basal value: *signifies values significant from basal, ^#signifies significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Presence of novel PKC isoforms in human and mouse platelets.

<p>Washed human (Hu) or mouse (Mo) platelets were lysed and samples separated by SDS-PAGE. The resulting membranes were blotted with antibodies raised against each isoform. Alongside each sample, the positive control (+) was run, as recommended by the antibody manufacturer. The results are representative of three experiments.</p

Platelet function in PKCε-null mice.

<p>(A) Aggregation and ATP secretion. Washed platelets from PKCε-deficient (PKCε^−/−) mice or wild-type (WT) littermate controls were stimulated with the indicated concentrations of CRP, collagen or PAR4 peptide. Aggregation and dense granule secretion were investigated using light-scattering aggregometry and luciferin-luciferase luminescence, respectively. Traces are representative of between 3–7 mice. (B) Spreading. PKCε^−/−and wild-type (WT) washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were exposed to surfaces of 100 µg/ml fibrinogen (FG), 1 U/ml thrombin (Thr), or 100 µg/ml collagen (COLL) for 45 min before fixing and mounting. Representative images from each condition are shown. Histograms depict levels of platelet adhesion to each surface and platelet mean surface area (spreading). The results are from one experiment that is representative of three. (C) Expression levels of surface glycoproteins. PKCε^−/− and WT washed platelets were incubated with FITC-labelled antisera against (upper panels) GPVI and integrin α_IIbβ₃ and (lower panels) integrin α₂β₁ and expression levels analysed by flow cytometry. The shaded area represents the signal from non-specific IgG control.</p

Platelet adhesion to collagen under flow in PKCε ^−/− mice.

<p>Blood from PKCε^−/− mice or wild-type (WT) littermate controls was flowed over collagen for 4 min at a shear rate of 1000 s⁻¹, then rinsed with Tyrode's buffer for 5 min. A. Adherent platelets and aggregates were imaged by DIC microscope and representative images are shown. B. The surface coverage was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a> and is shown as the mean±s.e.m. from four experiments.</p