An ER‐resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

Nicotinic acetylcholine receptors (nAChRs) are homo‐ or heteropentameric ligand‐gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell‐surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA‐2 or NRA‐4 (n icotinic r eceptor a ssociated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype‐specific agonists of these nAChRs is altered differently, as demonstrated by whole‐cell voltage‐clamp of dissected adult muscle, when applying exogenous agonists or after photo‐evoked, channelrhodopsin‐2 (ChR2) mediated acetylcholine (ACh) release, as well as in single‐channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra‐2 mutants. Cell‐surface expression of different subunits of the ‘levamisole‐sensitive’ nAChR (L‐AChR) is differentially affected in the absence of NRA‐2 or NRA‐4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.     

Structural studies in solution and in the solid state on the zinc chelate of 2-hydroxy-(4-methylthio)butanoic acid, an effective mineral supplement in animal feeding

The bis-chelate complex of Zn²⁺ with 2-hydroxy-(4-methylthio)butanoate (MHA_-H the anion derived from the so-called methionine hydroxy-analogue, MHA) is an effective, bioavailable mineral supplement for animal feeding. It can be obtained in two solid forms: the anhydrous [Zn(OC(O)CH₂CH(OH)CH₂CH₂SCH₃)₂] and the corresponding dihydrate species, both well distinguishable by IR spectroscopy and powder X-ray diffraction. The crystal and molecular structure of the dihydrate form has been solved by single-crystal X-ray diffraction. It consists of dinuclear bis-chelate species with a bridging carboxylate group, both zinc atoms displaying hexacoordination involving all the hydroxyl and carboxyl groups from the four MHA_-H anions and three oxygens from different water molecules. The fourth water molecule does not participate in coordination. Therefore, the dihydrate complex must be formulated as [Zn₂(OC(O)CH₂CH(OH)CH₂CH₂SCH₃)₄(H₂O)₃] · H₂O (1). A molecular computational analysis has been carried out by density functional theory (DFT) on three possible MHA_-H zinc chelates, i.e. the dinuclear bis-chelate observed in the solid state, the mononuclear bis-chelate diaquo-complex, and the monochelate tetraaquo-complex. Calculations have suggested that between the dinuclear and mononuclear bis-chelates, the preferred form in aqueous solution may be the second one. Moreover, both ¹H (chemical shifts and relaxation rates) and ¹³C NMR data provide further evidence for the formation of Zn/MHA_-H chelates in solution.     

Micropropagation of adult Stone Pine (Pinus pinea L.)

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees. Paloma Moncaleán and Ricardo Javier Ordás contributed equally.     

Metabolomic analysis reveals a common pattern of metabolic re-programming during invasion of three host plant species by Magnaporthe grisea

The mechanisms by which biotrophic and hemi-biotrophic fungal pathogens simultaneously subdue plant defences and sequester host nutrients are poorly understood. Using metabolite fingerprinting, we show that Magnaporthe grisea , the causal agent of rice blast disease, dynamically re-programmes host metabolism during plant colonization. Identical patterns of metabolic change occurred during M. grisea infections in barley, rice and Brachypodium distachyon . Targeted metabolite profiling by GC-MS confirmed the modulation of a conserved set of metabolites. In pre-symptomatic tissues, malate and polyamines accumulated, rather than being utilized to generate defensive reactive oxygen species, and the levels of metabolites associated with amelioration of redox stress in various cellular compartments increased dramatically. The activity of NADP-malic enzyme and generation of reactive oxygen species were localized to pathogen penetration sites, and both appeared to be suppressed in compatible interactions. Early diversion of the shikimate pathway to produce quinate was observed, as well as accumulation of non-polymerized lignin precursors. These data are consistent with modulation of defensive phenylpropanoid metabolism by M. grisea and the inability of susceptible hosts to mount a hypersensitive reaction or produce lignified papillae (both involving reactive oxygen species) to restrict pathogen invasion. Rapid proliferation of M. grisea hyphae in plant tissue after 3 days was associated with accelerated nutrient acquisition and utilization by the pathogen. Conversion of photoassimilate into mannitol and glycerol for carbon sequestration and osmolyte production appear to drive hyphal growth. Taken together, our results suggest that fungal pathogens deploy a common metabolic re-programming strategy in diverse host species to suppress plant defence and colonize plant tissue.     

Polycations. 17. Synthesis and properties of polycationic derivatives of carbohydrates

In our continuing investigation of polycationic salts for purposes of antimicrobial action, ion-channel blocking, and construction of ionic liquids, we have prepared several series of polycationic salts derived from carbohydrate precursors. These salts are currently being investigated for optimal efficacy as antibacterials and antifungals, as well as for other applications. The syntheses of such series of salts are described here along with preliminary antibacterial testing results and a discussion of their properties indicating their potential utility for several purposes.     

Nitric oxide accumulation is required to protect against iron-mediated oxidative stress in frataxin-deficient Arabidopsis plants

Frataxin is a mitochondrial protein that is conserved throughout evolution. In yeast and mammals, frataxin is essential for cellular iron (Fe) homeostasis and survival during oxidative stress. In plants, frataxin deficiency causes increased reactive oxygen species (ROS) production and high sensitivity to oxidative stress. In this work we show that a knock-down T-DNA frataxin-deficient mutant of Arabidopsis thaliana (atfh-1) contains increased total and organellar Fe levels. Frataxin deficiency leads also to nitric oxide (NO) accumulation in both, atfh-1 roots and frataxin null mutant yeast. Abnormally high NO production might be part of the defence mechanism against Fe-mediated oxidative stress.     

Patterns of resistance and resilience of the stress-tolerant coral Siderastrea radians (Pallas) to sub-optimal salinity and sediment burial

The coastal lagoons of south Florida, U.S., experience fluctuating levels of sedimentation and salinity and contain only a subset of the coral species found at the adjacent reefs of the Florida Reef Tract. The dominant species within these habitats is Siderastrea radians, which can reach densities of up to 68 colonies m^- 2 and is commonly exposed to salinity extremes (< 10 psu to > 37 psu) and chronic sediment burial. In this study, we document the patterns of resistance and resilience of S. radians to sub-optimal salinity levels and sediment burial in a series of short-term, long-term, acute, chronic, single-stressor, and sequential-stressor experiments.S. radians displayed remarkable patterns of resistance and resilience and mortality was documented only under prolonged (≥ 48 h) continuous exposure to salinity extremes (15 and 45 psu) and chronic sediment burial. A reduction in photosynthetic rates was documented for all salinity exposures and the decrease in photosynthesis was linearly related to exposure time. Negative impacts on photosynthetic rates were more severe under low salinity (15 psu) than under high salinity (45 psu). Corals exposed to intermediate, low-salinity levels (25 psu), exhibited initial declines in photosynthesis that were followed by temporary increases that may represent transient acclimatization patterns. The impacts of sediment burial were influenced by the duration of the burial period and ranged from a temporary reduction in photosynthesis to significant reductions in growth and tissue mortality. The maintenance of P/R ratios > 1 and the rapid (< 24 h) recovery of photosynthetic rates after burial periods of 2-24 h indicates that S. radians is able to resist short-term burial periods with minor physiological consequences. However, as burial periods increase and colonies become covered at multiple chronic intervals, sediment burial resulted in extended photosynthetic recovery periods, reduced growth, and mortality. Under normal conditions (i.e., no salinity stress), S. radians was very effective at clearing sediments, and > 50% of the colonies' surface area was cleared within 1 h. However, clearing rates were influenced by physiological status, and prior exposure to sub-optimal salinity significantly reduced the clearing rates of stressed colonies.The response of S. radians to disturbance documented in this study characterizes this species as highly stress-tolerant and provides an explanation for its present high abundance in both reef and marginal environments. Moreover, the key life-history attributes of S. radians, such as brooding reproductive strategy, small colony size, high stress-tolerance, and high recruitment rates, suggest the potential for this species to replace reef-building taxa under increased disturbance scenarios in Florida and elsewhere in the region.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.