Ion and metabolite transport in the chloroplast of algae: lessons from land plants

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.     

Two-photon laser spectroscopy of antiprotonic helium and the antiproton-to-electron mass ratio

Physical laws are believed to be invariant under the combined transformations of charge, parity and time reversal (CPT symmetry). This implies that an antimatter particle has exactly the same mass and absolute value of charge as its particle counterpart. Metastable antiprotonic helium (pHe(+)) is a three-body atom consisting of a normal helium nucleus, an electron in its ground state and an antiproton (p) occupying a Rydberg state with high principal and angular momentum quantum numbers, respectively n and l, such that n?≈?l?+?1?≈?38. These atoms are amenable to precision laser spectroscopy, the results of which can in principle be used to determine the antiproton-to-electron mass ratio and to constrain the equality between the antiproton and proton charges and masses. Here we report two-photon spectroscopy of antiprotonic helium, in which p(3)He(+) and p(4)He(+) isotopes are irradiated by two counter-propagating laser beams. This excites nonlinear, two-photon transitions of the antiproton of the type (n, l)?→?(n?-?2, l?-?2) at deep-ultraviolet wavelengths (λ = 139.8, 193.0 and 197.0?nm), which partly cancel the Doppler broadening of the laser resonance caused by the thermal motion of the atoms. The resulting narrow spectral lines allowed us to measure three transition frequencies with fractional precisions of 2.3-5 parts in 10(9). By comparing the results with three-body quantum electrodynamics calculations, we derived an antiproton-to-electron mass ratio of 1,836.1526736(23), where the parenthetical error represents one standard deviation. This agrees with the proton-to-electron value known to a similar precision.     

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI ⁺] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.     

中国古代军事中的计和算——以开方术在布营中的运用为例

中国古代军事家对数学在军事上的具体运用是十分重视的.《孙子》中“计”和“庙算”等概念,反映了古代军事家在进行军事运筹活动时十分重视运用具体精密的定量分析方法.《武编》中的下营算法则提供了较复杂的数学方法应用于军事的一个实例.下营算法不仅反映了军事活动中对准确计算的要求,而且表明在保证准确的前提下,通过在一定程度上降低精度来简化计算、提高效率也是同样重要的.因此,在计算精度和计算效率不能兼顾时,保持二者的平衡可能是军事数学知识的一个特点.     

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo

We previously showed that, in vitro, hyperforin from St. John’s wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B₄ formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.     

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.     

Large-scale prediction and testing of drug activity on side-effect targets

Discovering the unintended 'off-targets' that predict adverse drug reactions is daunting by empirical methods alone. Drugs can act on several protein targets, some of which can be unrelated by conventional molecular metrics, and hundreds of proteins have been implicated in side effects. Here we use a computational strategy to predict the activity of 656 marketed drugs on 73 unintended 'side-effect' targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 μM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a drug-target-adverse drug reaction network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic oestrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme cyclooxygenase-1. The clinical relevance of this inhibition was borne out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.