Reconstruction and verification of a genome-scale metabolic model for <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803

In terms of generating sustainable energy resources, the prospect of producing energy and other useful materials using cyanobacteria has been attracting increasing attention since these processes require only carbon dioxide and solar energy. To establish production processes with a high productivity, in silico models to predict the metabolic activity of cyanobacteria are highly desired. In this study, we reconstructed a genome-scale metabolic model of the cyanobacterium Synechocystis sp. PCC6803, which included 465 metabolites and 493 metabolic reactions. Using this model, we performed constraint-based metabolic simulations to obtain metabolic flux profiles under various environmental conditions. We evaluated the simulated results by comparing these with experimental results from ¹³C-tracer metabolic flux analyses, which were obtained under heterotrophic and mixotrophic conditions. There was a good agreement of simulation and experimental results under both conditions. Furthermore, using our model, we evaluated the production of ethanol by Synechocystis sp. PCC6803, which enabled us to estimate quantitatively how its productivity depends on the environmental conditions. The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities, and prediction of the metabolic characteristics, of Synechocystis sp. PCC6803.     

Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase

G9a is one of the well-characterized histone methyltransferases. G9a regulates H3K9 mono- and dimethylation at euchromatic region and consequently plays important roles in euchromatic gene regulation. Mammalian G9a contains several distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET and PostSET. These domains are highly conserved between mammals and Drosophila. Although mammalian G9a has nuclear localization signal (NLS) in its N-terminal region, the amino acid sequences of this region are not conserved in Drosophila. Here we have examined the subcellular localization of a series of truncated forms of Drosophila G9a (dG9a). The identified region (aa337-aa470) responsible for nuclear localization of dG9a contains four short stretches of positively charged basic amino acids (NLS1, aa334-aa345; NLS2, aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472). Each of NLS1, NLS3 and NLS4 is sufficient for the nuclear localization when they are fused with the enhanced green fluorescent protein. These NLSs of dG9a are distinct from those of mammalian G9a in their positions and amino acid sequences.     

Microbe-I: fungal biota analyses of the Japanese experimental module KIBO of the International Space Station before launch and after being in orbit for about 460 days

In addition to the crew, microbes also find their way aboard the International Space Station (ISS). Therefore, microbial monitoring is necessary for the health and safety of the crew and for general maintenance of the facilities of this station. Samples were collected from three sites in the Japanese experimental module KIBO on the ISS (air diffuser, handrail, and surfaces) for analysis of fungal biota approximately 1 year after this module had docked with the ISS. Samples taken from KIBO before launch and from our laboratory were used as controls. In the case of KIBO, both microbe detection sheet (MDS) and swab culture tests of orbital samples were negative. The MDS were also examined by field emission-scanning electron microscopy; no microbial structures were detected. However, fungal DNAs were detected by real-time PCR and analyzed by the clone library method; Alternaria sp. and Malassezia spp. were the dominant species before launch and in space, respectively. The dominant species found in specimens from the air conditioner diffuser, lab bench, door push panel, and facility surfaces on our laboratory (ground controls) were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. The fungi in the KIBO were probably derived from contamination due to humans, while those in our laboratory came from the environment (e.g., the soil). In conclusion, the cleanliness in KIBO was equivalent to that in a clean room environment on the ground.     

B-transferase with a Pro234Ser substitution acquires AB-transferase activity

A/B-Transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen, which is responsible for the synthesis of glycoprotein- and glycolipid-conjugates termed A/B-antigens. One polymorphism that causes the Pro234Ser substitution in B-transferase was recently found in a genotyping study, and might be cis-AB. In the present study, we analyzed the phenotypes arising from the enzymatic specificity of B-transferase with the Pro234Ser mutation. To evaluate the effect of the P234S mutation on enzymatic specificity, we generated an expression plasmid for B-transferase with Pro234Ser as well as A-transferase with Leu266Met, which is frequently found in cis-ABs. Transfection of B-transferase/P234S or A-transferase/L266M cDNA into HeLa cells, an O-blood group cell line, resulted in an AB-phenotype by absorption-elution testing and immunostaining, whereas A- and B-transferase-expressing HeLa cells exhibited only their own activity. Molecular simulation indicated that the P234S mutation causes a conformational change in the substrate pocket making it suitable for N-acetylgalactosamine.     

Reversible swelling of the cell wall of poplar biomass by ionic liquid at room temperature

Time-resolved autofluorescence, Raman microspectroscopy, and scanning microprobe X-ray diffraction were combined in order to characterize lignocellulosic biomass from poplar trees and how it changes during treatment with the ionic liquid 1-n-ethyl-3-methylimidazolium acetate (EMIMAC) at room temperature. The EMIMAC penetrates the cell wall from the lumen, swelling the cell wall by about a factor of two towards the empty lumen. However, the middle lamella remains unchanged, preventing the cell wall from swelling outwards. During this swelling, most of the cellulose microfibrils are solubilized but chain migration is restricted and a small percentage of microfibrils persist. When the EMIMAC is expelled, the cellulose recrystallizes as microfibrils of cellulose I. There is little change in the relative chemical composition of the cell wall after treatment. The action of EMIMAC on the poplar cell wall at room temperature would therefore appear to be a reversible swelling and a reversible decrystallization of the cell wall.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.     

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag"

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.     

Semaphorin 3A induces CaV2.3 channel-dependent conversion of axons to dendrites

Polarized neurites (axons and dendrites) form the functional circuitry of the nervous system. Secreted guidance cues often control the polarity of neuron migration and neurite outgrowth by regulating ion channels. Here, we show that secreted semaphorin 3A (Sema3A) induces the neurite identity of Xenopus spinal commissural interneurons (xSCINs) by activating Ca(V)2.3 channels (Ca(V)2.3). Sema3A treatment converted the identity of axons of cultured xSCINs to that of dendrites by recruiting functional Ca(V)2.3. Inhibition of Sema3A signalling prevented both the expression of Ca(V)2.3 and acquisition of the dendrite identity, and inhibition of Ca(V)2.3 function resulted in multiple axon-like neurites of xSCINs in the spinal cord. Furthermore, Sema3A-triggered cGMP production and PKG activity induced, respectively, the expression of functional Ca(V)2.3 and the dendrite identity. These results reveal a mechanism by which a guidance cue controls the identity of neurites during nervous system development.     

Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of [(13)C(5)]glutamate derived from [(13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [(13)C(5)]glutamate using [(13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of [(13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of [(13)C(5)]glutamate formed from [(13)C(5)]glutamine using amidophosphoribosyltransferase.     

Mitochondria and viruses

Mitochondria are involved in a variety of cellular metabolic processes, and their functions are regulated by extrinsic and intrinsic stimuli including viruses. Recent studies have shown that mitochondria play a central role in the primary host defense mechanisms against viral infections, and a number of novel viral and mitochondrial proteins are involved in these processes. Some viral proteins localize in mitochondria and interact with mitochondrial proteins to regulate cellular responses. This review summarizes recent findings on the functions and roles of these molecules as well as mitochondrial responses to viral infections.