Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe

We have succeeded to produce a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we solved the problem by identifying plg7, encoding phospholipase A2, as a multicopy suppressor. Characterization of the RA-tolerant strains suggested that the removal of RA moieties from phospholipids would be the suppression mechanism by plg7. In this study, we extended our analysis and report our new discovery that the overexpression of plg7 enabled cells to secrete free RA into culture media. When the FAH12 integrant in the absence of the overexpressed plg7 was grown at 20 °C for 11 days, the amount of intracellular RA reached 200.1 μg/ml of culture and only 69.3 μg/ml of RA was detected in culture media. On the other hand, the FAH12 integrant harboring the plg7 multicopy plasmid secreted RA in the media (184.5 μg/ml) without decreasing the amount in the cells, i.e., a significantly higher total secretion and a lead to making RA by its secretory production in S. pombe.     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

In vitro selection of peptide aptamers with affinity to single-wall carbon nanotubes using a ribosome display

A ribosome display from a diverse random library was applied for selecting peptide aptamers with high binding affinity to single-wall carbon nanotubes (SWCNTs). The selected peptide aptamer bound to and solubilized SWCNTs more strongly than did the peptide aptamer selected by a phage display method reported previously, and more strongly than other commonly used organic surfactants. The fluorescence spectrum of this aptamer showed a red shift upon interaction with SWCNTs but circular dichroism spectroscopy did not show any significant difference between the presence or absence of SWCNT binding.     

Neurons innervating the lamina in the butterfly, Papilio xuthus

The butterfly Papilio xuthus has compound eyes with three types of ommatidia. Each type houses nine spectrally heterogeneous photoreceptors (R1–R9) that are divided into six spectral classes: ultraviolet, violet, blue, green, red, and broad-band. Analysis of color discrimination has shown that P. xuthus uses the ultraviolet, blue, green, and red receptors for foraging. The ultraviolet and blue receptors are long visual fibers terminating in the medulla, whereas the green and red receptors are short visual fibers terminating in the lamina. This suggests that processing of wavelength information begins in the lamina in P. xuthus, unlike in flies. To establish the anatomical basis of color discrimination mechanisms, we examined neurons innervating the lamina by injecting Neurobiotin into this neuropil. We found that in addition to photoreceptors and lamina monopolar cells, three distinct groups of cells project fibers into the lamina. Their cell bodies are located (1) at the anterior rim of the medulla, (2) between the proximal surface of the medulla and lobula plate, and (3) in the medulla cell body rind. Neurobiotin injection also labeled distinct terminals in medulla layers 1, 2, 3, 4 and 5. Terminals in layer 4 belong to the long visual fibers (R1, 2 and 9), while arbors in layers 1, 2 and 3 probably correspond to terminals of three subtypes of lamina monopolar cells, respectively. Immunocytochemistry coupled with Neurobiotin injection revealed their transmitter candidates; neurons in (1) and a subset of neurons in (2) are immunoreactive to anti-serotonin and anti-γ-aminobutyric acid, respectively.     

The Molecular Evolution of the Pif Family Proteins in Various Species of Mollusks

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.     

Isolation and Characterization of Prothoracicotropic Hormone from Silkworm,Bombyx mori

Members of the small family of Arabidopsis PSEUDO-RESPONSE REGULATORS (PRR1/TOC1, PRR3, PRR5, PRR7, and PRR9) play roles close to the circadian clock in Arabidopsis thaliana. We have reported that the rice (Oryza sativa) genome also encodes a set of PRR counterparts (designated OsPRR1, OsPRR37, OsPRR59, OsPRR73, and OsPRR95 respectively). To gain new insight into the molecular functions of OsPRRs, we carried out genetic complementation analyses by introducing two representative rice genes, OsPRR1 and OsPRR37, into the corresponding Arabidopsis loss-of-function mutants (toc1 and prr7 respectively). The results showed that these OsPRR and AtPRR genes are genetically interchangeable at least in part, suggesting the conserved clock-associated function of these OsPRRs.     

Chemical Studies on Amino––Carbonyl Reaction

The 3-deoxy-n-pentosone (I) was isolated from the browning degradation mixture of N-n-xy1osy1-n-butylamine by the action of acetic acid at 55°C. The 3-deoxy-d-erythrohexosone (IIa) and the 3-deoxy-n-threohexosone (IIb) were also prepared by degradation of the corresponding N-glycosyl-n-butylamine. The 3-deoxy-d-pentosone was characterized as the 2,4-dinitrophenylosazone and its diacetate, and the p-nitrophenylosazone. The two 3-deoxy-d-hexosones were also characterized as the analogous derivatives. The three 3-deoxyosones gave positive color reactions with 2-thiobarbituric acid.

As one of the intermediates in 3-deoxyosone formation from N-glycoside, 1,2-eno1 form of 1-deoxy-1-n-butylamino-2-ketose (IV) was proposed.     

Isolation and Identification of Tubaic Acid and β-Tubaic Acid from Derris Roots

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10^-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco plants.     

Effects of a Daisaikoto Extract on the Biliary Constituents in Mice and Rats

The effects of an extract of Daisaikoto (a traditional Chinese medicine) on biliary constituents was studied in mice fed with a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate (the control diet) and in rats fed with a cholesterol-free diet. The Daisaikoto extract was added to the control diet at a level of 0.5%. A high incidence of cholesterol gallstones were found in the control mice, but not in the mice given the Daisaikoto extract. This difference was concluded to have been due to the absolute concentration of bile acid in the bile being significantly higher in the mice given the Daisaikoto extract than in the control mice. The result from rats fed with the cholesterol-free diet also demonstrated that the Daisaikoto extract caused an increase in the absolute concentration of bile acid in the bile.