We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( 30 min) caused a profound inhibition of protein synthesis. In each case this was accompanied by dephosphorylation of the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and increased binding of this repressor protein to eIF4E. Binding of 4E-BP1 to eIF4E correlated with loss of eIF4F complexes. Sorbitol and hydrogen peroxide each caused inhibition of the 70-kDa ribosomal protein S6 kinase, while arsenite activated it. The effects of stresses on the phosphorylation of eukaryotic elongation factor 2 also differed: oxidative stress elicited a marked increase in eEF2 phosphorylation, which is expected to contribute to inhibition of translation, while the other stresses did not have this effect. Although all three proteins (4E-BP1, p70 S6 kinase and eEF2) can be regulated through the mammalian target of rapamycin (mTOR), our data imply that stresses do not interfere with mTOR function but act in different ways on these three proteins. All three stresses activate the p38 MAP kinase pathway but we were able to exclude a role for this in their effects on 4E-BP1. Our data reveal that these stress-inducing agents, which are widely used to study stress-signalling in mammalian cells, exert multiple and complex inhibitory effects on the translational machinery.Keywords: stress; initiation; elongation factor; mRNA translation; S6 kinase.The control of mRNA translation in mammalian cells involves the regulation of a range of components of the translational machinery, principally by changes in their phosphorylation, leading to modulation of their activities or their abilities to interact with one another [1,2].Initiation factor 4E (eIF4E) plays a key role in mRNA translation and its control in eukaryotic cells. eIF4E binds to the 5¢ cap structure (containing 7-methylguanosine triphosphate; m 7 GTP) which is present at the 5¢ end of all cellular cytoplasmic mRNAs [3,4]. eIF4E can be regulated by its own phosphorylation (which occurs at a single major site (Ser209) [5,6]; and by binding proteins (4E-BPs) that modulate its availability for initiation complex formation (reviewed in [7]). eIF4E forms a complex termed eIF4F, which also contains the translation factors eIF4G (formerly called p220) and eIF4A. eIF4A has ATP-dependent RNA helicase activity thought to be required to unwind regions of self-complementary secondary structure in the 5¢ UTRs of certain mRNAs [4,8]. Such secondary structure inhibits translation and therefore mRNAs with 5¢ UTRs that contain significant secondary structure are often poorly translated. In contrast to many other cellular mRNAs, translation of heat shock protein mRNAs appears to be relatively cap-independent (reviewed in [9-11]), and translation of the mRNA for the stress-protein BiP/grp78 occ...

The hypertrophic Gq-protein-coupled receptor agonist PE (phenylephrine) activates protein synthesis. We showed previously that activation of protein synthesis by PE requires MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and mTOR (mammalian target of rapamycin). However, it remained unclear whether ERK activation was required and which downstream components were involved in activating mTOR and protein synthesis. Using an adenovirus encoding the MKP3 (MAPK phosphatase 3) to inhibit ERK activity, we demonstrate that ERK is essential for the activation of protein synthesis by PE. Activation and phosphorylation of S6K1 (ribosomal protein S6 kinase 1) and phosphorylation of eIF4E (eukaryotic initiation factor 4E)-binding protein (both are mTOR targets) were also inhibited by MKP3, suggesting that ERK is also required for the activation of mTOR signalling. PE stimulation of cardiomyocytes induced the phosphorylation of TSC2 (tuberous sclerosis complex 2), a negative regulator of mTOR activity. TSC2 was phosphorylated only weakly at Thr1462, but phosphorylated at additional sites within the sequence RXRXX(S/T). This differs from the phosphorylation induced by insulin, indicating that MEK/ERK signalling targets distinct sites in TSC2. This phosphorylation may be mediated by p90RSK (90 kDa ribosomal protein S6K), which is activated by ERK, and appears to involve phosphorylation at Ser1798. Activation of protein synthesis by PE is partially insensitive to the mTOR inhibitor rapamycin. Inhibition of the MAPK-interacting kinases by CGP57380 decreases the phosphorylation of eIF4E and PE-induced protein synthesis. Moreover, CGP57380+rapamycin inhibited protein synthesis to the same extent as blocking ERK activation, suggesting that MAPK-interacting kinases and regulation of mTOR each contribute to the activation of protein synthesis by PE in cardiomyocytes.

Translation elongation consumes a high proportion of cellular energy and can be regulated by phosphorylation of elongation factor eEF2 which inhibits its activity. We have studied the e¡ects of ATP depletion on the phosphorylation of eEF2 in adult rat ventricular cardiomyocytes. Energy depletion rapidly leads to inhibition of protein synthesis and increased phosphorylation of eEF2. Stimulation of the AMP-activated protein kinase also causes increases eEF2 phosphorylation. Only at later times is an e¡ect on mTOR signalling observed. These data suggest that energy depletion leads to inhibition of protein synthesis through phosphorylation of eEF2 independently of inhibition of mTOR signalling.

Recognition of the threats to biodiversity and its importance to society has led to calls for globally coordinated sampling of trends in marine ecosystems. As a step to defining such efforts, we review current methods of collecting and managing marine biodiversity data. A fundamental component of marine biodiversity is knowing what, where, and when species are present. However, monitoring methods are invariably biased in what taxa, ecological guilds, and body sizes they collect.

The L L-adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca 2+ /calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca 2+ /calmodulin-independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP-dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase. ß

Standardised sampling methods are required to meet the Convention on Biological Diversity Aichi Targets to monitor trends in biodiversity. Currently the animals monitored in the marine environment are predominantly vertebrates and surface plankton, but arthropods (especially benthic crustaceans) make up a third of marine species, provide an important link in the food web and are under sampled. Sampling these animals with light traps on the benthos in structurally complex areas would fill a gap in biological monitoring. A survey of the literature of light-trap designs showed they collected at least 12 phyla of benthic and planktonic animals, and 13 orders of crustaceans. These traps can be deployed anywhere from an hour to overnight and on the seabed or in the water column. They can be low cost, have low environmental impact and be used in complex and fragile habitats otherwise difficult to sample. Environmental factors, such as water movement and turbidity, as well as trap design and method of deployment, may affect catch. Experimental field tests are required to develop the use of light-traps as a standard method for marine biodiversity monitoring, including design, deployment, and replication.

Light-traps have been routinely used to sample terrestrial insects for decades. They may provide a similar low cost standard method for sampling and monitoring mobile marine invertebrates. We deployed transparent plastic bottles, with their top inverted to form a funnel, and a chemical light-stick and weight inside, for at least 1 hour on the seabed (< 5 m depth) around Auckland, New Zealand. Traps collected 35 taxa of zooplanktonic and benthic taxa of 6 phyla, 11 classes, 12 orders. Most were Crustacea (26 taxa). Zooplankton included crustacean nauplii, cypris, zoea and megalopa larvae, gelatinous zooplankton, and calanoid Copepoda. In order of abundance, benthos included Amphipoda, Cumacea, Isopoda, Mysidacea, Ostracoda, Harpacticoida, Decapoda, Tanaidacea, Branchiopoda, Siphonostomatoida (caligid fish lice), Stomatopoda, Pycnogonida, and Collembola. Other taxa included Polychaeta, Clitellata (oligochaetes), Turbellaria, Nematoda, Chaetognatha, Gastropoda and Polyplacophora. These taxa are important prey of fish and thus a key link between primary producers and predators but collecting and sorting them from the benthos and plankton nets is time consuming. The light-traps have been efficient for introducing university students to the diversity of mobile macro-fauna and zooplankton. They show promise for surveillance of this component of marine biodiversity as they do for aerial arthropods.

No abstract