Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes.

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro, extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.

Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Environmental factors and features that influence visual search in a 3D WIMP interface

The challenge of moving past the classic Window Icons Menus Pointer (WIMP) interface, i.e. by turning it ‘3D’, has resulted in much research and development. To evaluate the impact of 3D on the ‘finding a target picture in a folder’ task, we built a 3D WIMP interface that allowed the systematic manipulation of visual depth, visual aides, semantic category distribution of targets versus non-targets; and the detailed measurement of lower-level stimuli features. Across two separate experiments, one large sample web-based experiment, to understand associations, and one controlled lab environment, using eye tracking to understand user focus, we investigated how visual depth, use of visual aides, use of semantic categories, and lower-level stimuli features (i.e. contrast, colour and luminance) impact how successfully participants are able to search for, and detect, the target image. Moreover in the lab-based experiment, we captured pupillometry measurements to allow consideration of the influence of increasing cognitive load as a result of either an increasing number of items on the screen, or due to the inclusion of visual depth. Our findings showed that increasing the visible layers of depth, and inclusion of converging lines, did not impact target detection times, errors, or failure rates. Low-level features, including colour, luminance, and number of edges, did correlate with differences in target detection times, errors, and failure rates. Our results also revealed that semantic sorting algorithms significantly decreased target detection times. Increased semantic contrasts between a target and its neighbours correlated with an increase in detection errors. Finally, pupillometric data did not provide evidence of any correlation between the number of visible layers of depth and pupil size, however, using structural equation modelling, we demonstrated that cognitive load does influence detection failure rates when there is luminance contrasts between the target and its surrounding neighbours. Results suggest that WIMP interaction designers should consider stimulus-driven factors, which were shown to influence the efficiency with which a target icon can be found in a 3D WIMP interface.