Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins--PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata--were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P(T7)) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P(T7) in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P(T7) and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.     

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml^–1. A maximum production of 4.6 g recombinant tumstatin (10⁷ cells)^–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO₄.     

Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats

Extracts of Phellinus linteus (EPB), grown on germinated brown rice, protected rats from liver injury induced by carbon tetrachloride (CCl4). Peroxidation products in the liver were decreased to 10% by EPB. Catalase and superoxide dismutase activities were significantly decreased to 55% and 39% by CCl4 administration, but EPB blocked this effect, resulting in enzyme activities at control levels. Expression of cytochromeP450 2E1 (CYP2E1) protein was significantly decreased to 88% in CCl4-treated rats but remained at control levels when EPB was also administered. EPB did not affect the altered fatty acid composition induced by CCl4. The hepatoprotective effect of EPB may be mediated by EPB's prevention of CCl4-induced CYP2E1 degradation.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

Effects of 20-hydroxyecdysone and serotonin on neurite growth and survival rate of antennal lobe neurons in pupal stage of the silk moth Bombyx mori in vitro

Effects of 20-hydroxyecdysone and serotonin on the morphological development and the survival of antennal lobe neurons from day-2 pupal brains of the silk moth Bombyx mori were investigated in vitro. Four morphologically distinct neuronal types could be identified in the cultured antennal lobe neurons: unipolar, bipolar, multi-polar and projection neurons. Antennal lobe neurons in culture with 20-hydroxyecdysone and serotonin showed different patterns of the morphological development from those described in Manduca sexta. Projection neurons extend their neurites remarkably by 20-hydroxyecdysone in B. mori, but there is no extension from antennal lobe neurons in M. sexta. Multi-polar neurons conspicuously increase only formation of new branches from their primary neurites by serotonin in B. mori, but there are both extension and branching of the neurites in M. sexta. On day-5, antennal lobe neurons in lower titers of 20-hydroxyecdysone had significantly higher survival rates than those in higher titers. Neurons cultured for 7 days at different levels of 20-hydroxyecdysone generally showed significantly lower survival rates than neurons cultured for 5 days under the same conditions.     

Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.     

Tunable Internal and Surface Structures of the Bifunctional Oxygen Perovskite Catalysts

Perovskite oxide ceramics attracts significant attention as a strong candidate of bifunctional oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) electrocatalyst for the metal‐air batteries. Numerous approaches to the viability of bifunctional perovskite electrocatalyst represent that the electro­chemical performance is highly correlated with defect chemistry, surface structure, and overall polycrystalline perovskite structure. By making use of the intrinsic flexibility of internal structure and high nonstoichiometry in perovskite oxide, the heat treatment effect of the complex Ba_0.5Sr_0.5Co_xFe_1‐xO_3‐δ (x = 0.2 and 0.8) perovskites in argon atmosphere at 950 °C (Ar‐BSCF5582 and Ar‐BSCF5528) on the surface structure/defect chemistry and electrocatalytic performance is intensively investigated. Upon heat‐treatment in argon atmosphere, the amorphous thickness layer increases from ≈20 to 180–200 nm in BSCF5582, while there is little change in BSCF5528 with ≈20 nm. The electrocatalytic performance of BSCF5582 catalyst both in ORR and OER deteriorates seriously, while Ar‐BSCF5528 demonstrates a significant increase of electro­chemical performance in ORR. This study demonstrates that the electrochemical performances of a perovskite catalyst can be significantly determined by the simultaneous modification of both surface structure and internal defect chemistry, which are explained with transmission electron microscopy and atomic‐selective X‐ray absorption fine structure analyses, respectively.     

Structural Brain Changes after Traditional and Robot-Assisted Multi-Domain Cognitive Training in Community-Dwelling Healthy Elderly

The purpose of this study was to investigate if multi-domain cognitive training, especially robot-assisted training, alters cortical thickness in the brains of elderly participants. A controlled trial was conducted with 85 volunteers without cognitive impairment who were 60 years old or older. Participants were first randomized into two groups. One group consisted of 48 participants who would receive cognitive training and 37 who would not receive training. The cognitive training group was randomly divided into two groups, 24 who received traditional cognitive training and 24 who received robot-assisted cognitive training. The training for both groups consisted of daily 90-min-session, five days a week for a total of 12 weeks. The primary outcome was the changes in cortical thickness. When compared to the control group, both groups who underwent cognitive training demonstrated attenuation of age related cortical thinning in the frontotemporal association cortices. When the robot and the traditional interventions were directly compared, the robot group showed less cortical thinning in the anterior cingulate cortices. Our results suggest that cognitive training can mitigate age-associated structural brain changes in the elderly.

Trial Registration

ClnicalTrials.gov NCT01596205