Manipulation of major membrane lipid synthesis and its effects on sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

A new perspective on phylogeny and evolution of tetraodontiform fishes (Pisces: Acanthopterygii) based on whole mitochondrial genome sequences: Basal ecological diversification?

Background  

The order Tetraodontiformes consists of approximately 429 species of fishes in nine families. Members of the order exhibit striking morphological diversity and radiated into various habitats such as freshwater, brackish and coastal waters, open seas, and deep waters along continental shelves and slopes. Despite extensive studies based on both morphology and molecules, there has been no clear resolution except for monophyly of each family and sister-group relationships of Diodontidae + Tetraodontidae and Balistidae + Monacanthidae. To address phylogenetic questions of tetraodontiform fishes, we used whole mitochondrial genome (mitogenome) sequences from 27 selected species (data for 11 species were newly determined during this study) that fully represent all families and subfamilies of Tetraodontiformes (except for Hollardinae of the Triacanthodidae). Partitioned maximum likelihood (ML) and Bayesian analyses were performed on two data sets comprising concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y]), 22 transfer RNA and two ribosomal RNA genes (total positions = 15,084).     

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.     

A thermostable dolichol phosphoryl mannose synthase responsible for glycoconjugate synthesis of the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

Dolichol phosphoryl mannose synthase (DPM synthase) is an essential enzyme in the synthesis of N- and O-linked glycoproteins and the glycosylphosphatidyl-inositol anchor. An open reading frame, PH0051, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes a DPM synthase ortholog, PH0051p. A full-length version of PH0051p was produced using an E. coli in vitro translation system and its thermostable activity was confirmed with a DPM synthesis assay, although the in vitro productivity was not sufficient for further characterization. Then, a yeast expression vector coding for the N-terminal catalytic domain of PH0051p was constructed. The N-terminal domain, named DPM(1-237), was successfully expressed, and turned out to be a membrane-bound form in Saccharomyces cerevisiae cells, even without its hydrophobic C-terminal domain. The membrane-bound DPM(1-237) was solubilized with a detergent and purified to homogeneity. The purified DPM(1-237) showed thermostability at up to 75 degrees C and an optimum temperature of 60 degrees C. The truncated mutant DPM(1-237) required Mg(2+) and Mn(2+) ions as cofactors the same as eukaryotic DPM synthases. By site-directed mutagenesis, Asp(89) and Asp(91) located at the most conserved motif, DXD, were confirmed as the catalytic residues, the latter probably bound to a cofactor, Mg(2+). DPM(1-237) was able to utilize both acceptor lipids, dolichol phosphate and the prokaryotic carrier lipid C(55)-undecaprenyl phosphate, with Km values of 1.17 and 0.59 muM, respectively. The DPM synthase PH0051p seems to be a key component of the pathway supplying various lipid-linked phosphate sugars, since P. horikoshii could synthesize glycoproteins as well as the membrane-associated PH0051p in vivo.     

A chemically modified glass surface that facilitates transglutaminase-mediated protein immobilization

An amino-modified glass surface for enzymatic protein immobilization by microbial transglutaminase (MTG) was developed. Diamine substrates with secondary amino groups in the linker moiety, like triethylenetetramine (TETA), exhibited at most a 2-fold higher reactivity in the MTG-catalyzed reaction compared to those with the alkyl linker. A 96-well glass plate was subsequently modified with selected diamine substrates. Validation of the modified surface by enzymatic immobilization of enhanced green fluorescent protein tagged with a glutamine donor-substrate peptide (LLQG) of MTG revealed that the protein loading onto the TETA-modified glass surface was approximately 15-fold higher than that on the unmodified one.     

Enhancement of spermidine content and antioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in response to salinity and hyperosmosis

In our previous work, an apple spermidine synthase (SPDS)-overexpressing transgenic European pear (Pyrus communis L. 'Ballad'), line no. 32 (#32), demonstrated attenuated susceptibility to stress treatment. In the current paper, changes in enzymatic and non-enzymatic antioxidant capacity of the transgenic pear (line #32) were investigated in response to NaCl or mannitol stress. Under non-stressed conditions (before stress treatment), spermidine (Spd) contents and SPDS activity of line #32 were higher than those of the non-transformant (wild type). However, no significant differences were detected between line #32 and the wild type as regards contents of malondialdehyde (MDA) and H2O2, and activities of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). When exposed to NaCl or mannitol stress, both the wild type and line #32 exhibited accumulation of Spd with the latter accumulating more. The transgenic line contained higher antioxidant enzyme activities, less MDA and H2O2 than the wild, implying it suffered from less injury. These results suggested that increase of Spd content in the transgenic line could, at least in part, lead to enhancing enzymatic and non-enzymatic antioxidant capacity.     

Anthocyanins from red flowers of Camellia cultivar 'Dalicha'

Five anthocyanins, cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(Z)-p-coumaroyl)-β-galactopyranoside (2), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-p-coumaroyl)-β-galactopyranoside (3), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-caffeoyl)-β-galactopyranoside (4), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-galactopyranoside (5), and cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-glucopyranoside (6), together with the known cyanidin 3-O-(2-O-β-xylopyranosyl)-β-galactopyranoside (1), were isolated from red flowers of Camellia cultivar ‘Dalicha’ (Camellia reticulata) by chromatography using open columns. Their structures were subsequently determined on the basis of spectroscopic analyses, i.e., ¹H NMR, ¹³C NMR, HMQC, HMBC, HR ESI-MS and UV-vis.