Protein kinase A regulation of P2X4 receptors: Requirement for a specific motif in the C-terminus

AbstractThe P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X4R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X4R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X4R expressing HEK-293 cells. To define which region of the P2X4R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X4R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X4R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\ P2X4-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca2+ signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X4R, markedly enhances ATP-stimulated P2X4R currents and hence cytosolic Ca2+ signals. This may occur at least in part, by altering the trafficking of a population of P2X4R present at the plasma membrane

How can we demonstrate the economic value of Precision Agriculture (PA) practices to New Zealand Agriculture service providers and arable farmers?

The amount of data collected has become a major challenge to the uptake of PA practices in New Zealand. There is a lack of clear value propositions around some PA practices, e.g. variable rate seeding (VRS). The importance of calibrating yield monitors, collecting yield data and mapping results has not been realised by farmers. The goal of the study is to provide economic evidence through yield data mining to encourage the adoption of PA

CCK antagonists reveal that CCK-8 and JMV-180 interact with different sites on the rat pancreatic acinar cell CCKA receptor

The ability of CCKA antagonists to inhibit full and partial CCK agonists of the rat pancreatic acinar cell CCKA receptor has been studied. When isolated rat pancreatic acini were superfused with CCK-8 (10 pM-1 nM) or CCK-4 (1 [mu]M), an increase in [Ca2+]i signal was initiated. Concurrent superfusion of either L-364,718 (0.1 [mu]M) or lorglumide (10 [mu]M), chemically distinct, specific, potent antagonists of the CCKA receptor, resulted in a rapid inhibition of the [Ca2+]i signal initiated by all concentrations of CCK-8. In contrast, Ca2+ oscillations, initiated by JMV-180 (25 nM-1 [mu]M), a partial agonist analogue of CCK-8, were essentially unaffected by concurrent superfusion of either L-364,718 or lorglumide. When JMV-179, an analogue of JMV-180 that exhibits characteristics of a pure antagonist, was superfused concurrently with either CCK-8 or JMV-180, Ca2+ oscillations were inhibited, even in the presence of 0.1 [mu]M L-364,718. In a similar fashion, amylase secretion stimulated by CCK-8 was markedly attenuated by L-364,718, lorglumide, and JMV-179, whereas secretion stimulated by JMV-180 was only inhibited by JMV-179. A model is proposed to reconcile this data, based on the assumption that JMV-180 and CCK-8 interact with discrete sites on the CCKA receptor, which are differentially affected by the binding of antagonists. This model may also explain how a single receptor may transduce multiple signals in response to different agonists.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31948/1/0000901.pd

Agonist-Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK-N-MCIXC Neuroepithelioma Cells

Fura-2 digital imaging microfluorimetry was used to evaluate the Ca 2+ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca 2+ i ) but of different magnitudes (ET-1 = ATP> NE). The Ca 2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca 2+ influx. The Ca 2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca 2+ signal, which was characteristic of the response to Oxo-M, was observed in 94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were ∼74 and ∼48%. Sequential addition of agonists to cells maintained in a Ca 2+ -free buffer indicated that each ligand mobilized Ca 2+ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four- to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET-1 or ATP resulted in larger increases in the net formation of inositol 1,4,5-trisphosphate than did either Oxo-M or NE. These results indicate that, in SK-N-MCIXC cells, the characteristics of both Ca 2+ signaling and inositol phosphate production are agonist specific.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66414/1/j.1471-4159.1994.63062099.x.pd

Targeting the ATP-dependent formation of herpesvirus ribonucleoprotein particle assembly as an antiviral approach

Human herpesviruses are responsible for a range of debilitating acute and recurrent diseases, including a number of malignancies. Current treatments are limited to targeting the herpesvirus DNA polymerases, however with emerging viral resistance and little efficacy against the oncogenic herpesviruses, there is an urgent need for new antiviral strategies. Herein we describe a mechanism to inhibit the replication of the oncogenic herpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV), by targeting the ATP-dependent formation of viral ribonucleoprotein particles (vRNPs). We demonstrate that small molecule inhibitors which selectively inhibit the ATPase activity of the cellular human transcription/export complex (hTREX) protein UAP56, result in effective inhibition of vRNP formation, viral lytic replication and infectious virion production. Strikingly, as all human herpesviruses utilize conserved mRNA processing pathways involving hTREX components, we demonstrate the feasibility of this approach for pan-herpesvirus inhibition

Safe Eye Surgery : Non-technical aspects

Peer reviewedPostprin

Towards Computing Inferences from English News Headlines

Newspapers are a popular form of written discourse, read by many people, thanks to the novelty of the information provided by the news content in it. A headline is the most widely read part of any newspaper due to its appearance in a bigger font and sometimes in colour print. In this paper, we suggest and implement a method for computing inferences from English news headlines, excluding the information from the context in which the headlines appear. This method attempts to generate the possible assumptions a reader formulates in mind upon reading a fresh headline. The generated inferences could be useful for assessing the impact of the news headline on readers including children. The understandability of the current state of social affairs depends greatly on the assimilation of the headlines. As the inferences that are independent of the context depend mainly on the syntax of the headline, dependency trees of headlines are used in this approach, to find the syntactical structure of the headlines and to compute inferences out of them.Comment: PACLING 2019 Long paper, 15 page

Conscious monitoring and control (reinvestment) in surgical performance under pressure.

Research on intraoperative stressors has focused on external factors without considering individual differences in the ability to cope with stress. One individual difference that is implicated in adverse effects of stress on performance is "reinvestment," the propensity for conscious monitoring and control of movements. The aim of this study was to examine the impact of reinvestment on laparoscopic performance under time pressure

Scaling properties of protein family phylogenies

One of the classical questions in evolutionary biology is how evolutionary processes are coupled at the gene and species level. With this motivation, we compare the topological properties (mainly the depth scaling, as a characterization of balance) of a large set of protein phylogenies with a set of species phylogenies. The comparative analysis shows that both sets of phylogenies share remarkably similar scaling behavior, suggesting the universality of branching rules and of the evolutionary processes that drive biological diversification from gene to species level. In order to explain such generality, we propose a simple model which allows us to estimate the proportion of evolvability/robustness needed to approximate the scaling behavior observed in the phylogenies, highlighting the relevance of the robustness of a biological system (species or protein) in the scaling properties of the phylogenetic trees. Thus, the rules that govern the incapability of a biological system to diversify are equally relevant both at the gene and at the species level.Comment: Replaced with final published versio