Targeting EDEM protects against ER stress and improves development and survival in C. elegans

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.     

The indris of Anjanaharibe-Sud,northeastern Madagascar

During a short field trip to the Special Reserve of Anjanaharibe-Sud in northeastern Madagascar, data concerning pelage coloration, behavior (especially vocalization), and ecology of indris were collected. Anjanaharibe-Sud is the northernmost locality of indri distribution. In comparison to the better-known indris from the southern part of their distribution, the indris in this region show different pelage coloration. Several types of loud vocalizations are analyzed, based on a small sample of tape recordings. Their song structure is more complicated than previously reported, containing distinct sequences of duetting. Data on behavior and ecology were collected by interviewing guides and local inhabitants. Some information contrasts with reports on the more southern indri populations. The conservation status of indris in Anjanaharibe-Sud and the future of the reserve are outlined.     

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.     

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.     

Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.     

Synthesis and preliminary conformational analysis of TOAC spin‐labeled analogues of the medium‐length peptaibiotic tylopeptin B

A set of analogues of the 14‐residue peptaibol tylopeptin B, containing the stable free‐radical 4‐amino‐1‐oxyl‐2,2,6,6,‐tetramethylpiperidine‐4‐carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid‐phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane‐mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α‐helix or the 3₁₀‐helix type depending upon the nature of the solvent. In aqueous environment, the spin‐labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N‐terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC⁸‐ and TOAC¹³‐tylopeptin B, showed that the labeled peptides exhibit membrane‐modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium‐length peptaibiotics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Epigenetic learning in non-neural organisms

Learning involves a usually adaptive response to an input (an external stimulus or the organism℉s own behaviour) in which the input-response relation is memorized; some physical traces of the relation persist and can later be the basis of a more effective response. Using toy models we show that this characterization applies not only to the paradigmatic case of neural learning, but also to cellular responses that are based on epigenetic mechanisms of cell memory. The models suggest that the research agenda of epigenetics needs to be expanded.