Modelling of a metal-containing hepcidin

Hepcidin was originally identified as a liver-expressed antimicrobial peptide but further studies have shown that it also has a key role in iron homeostasis. The NMR structure of the synthetic peptides reveal a distorted beta-sheet containing 4 disulphide bridges, with an unusual vicinal disulphide bridge which has been suggested to be functionally significant. In this study, we report the presence of co-purified iron with the urine-purified 20 and 25 residue hepcidins. Since the published structure does not allow metal binding, the interaction of hepcidin with metals was investigated for other possible structural conformations by threading its primary sequence onto existing 3D folds. Several alignments were obtained and the best scores were used to build a 3D model of hepcidin containing one atom of iron. The new 3D structure, that contains only reduced Cys residues, is completely different from the solved structure of the synthetic peptide. Although the model presented here shows only one metal bound to the peptide, the binding of several metal atoms cannot be excluded from such a short flexible peptide. The co-purification of iron with both peptides, together with our 3D model, suggest a conformational polymorphism for hepcidin, reminiscent of the iron regulatory proteins IRPs.     

Synthesis and antimalarial activities of novel 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes

The oxidative system H2O2/fluorinated alcohol (TFE, HFIP) was used for direct acid- and MeReO3-catalyzed synthesis of 1,2,4,5-tetraoxanes from cyclic (C6, C7, and C12) and acyclic ketones. The influence of ring size and alkyl chain length were studied and antimalarial activities of synthetic 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes were determined. Variations in their antimalarial activities were significant, although they share similar electrochemical properties of the peroxide bond.     

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

Background

STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis.

Methodology/Principal Findings

Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice.

Conclusions/Significance

In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.     

Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer''s disease, and other neurodegenerative disorders.     

Nature of sterols affects plasma membrane behavior and yeast survival during dehydration

The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration-rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.     

Dynamics of microbial planktonic food web components during a river flash flood in a Mediterranean coastal lagoon

Episodic river flash floods, characteristic of Mediterranean climates, are suspected to greatly affect the functioning of microbial food webs. For the first time, the abundance, biomass and diversities of microbial food web components were studied before and during 4 consecutive days after a flash flood that occurred in November 2008, in the surface waters of five stations along a salinity gradient from 20 to 36 in the Thau lagoon. Eukaryotic pico- and nanophytoplankton were discharged from the river into the lagoon and increased by 30- and 70-fold, respectively. Bacteria increased by only 2-fold in the lagoon, from around 4–8 × 10⁶ cells ml⁻¹, probably benefiting from river nutrient input. Chlorophyll a increased 4-fold, and pigment biomarkers showed that the dinophyceae, prasinophyceae and prymnesiophyceae were sensitive to the flood perturbation, whereas the bacillariophyceae, cryptophyceae and chlorophyceae were resistant and/or transported to the lagoon from the river. Predator responses were more complex as total heterotrophic flagellate abundance decreased slightly, whereas those of specific naked ciliates increased, particularly for Uronema sp. The flood also induced a specific change in diversity, from a community dominated by Strobilidium spiralis to a community dominated by Uronema sp. The tintinnid community was particularly sensitive to the flood event as the abundance of all species decreased greatly. The high increases in biomass, mainly brought by the river during the flood, could have eventually sedimented to the benthic layer and/or been transported further into the lagoon, supporting the pelagic food web, or have even been exported to the Mediterranean Sea.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

The role of spiking and bursting pacemakers in the neuronal control of breathing

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I_NaP) and the calcium-activated non-specific cation current (I_CAN), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.