Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor

The application of omega-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20% (w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70 degrees C. A plot of the natural logarithm of the frequency factor, In ka0, vs. the activation energy, Ea, demonstrated that In ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.     

Biotransformation of phenanthrene and 1-methoxynaphthalene with Streptomyces lividans cells expressing a marine bacterial phenanthrene dioxygenase gene cluster

The phdABCD gene cluster in a marine bacterium Nocardioides sp. strain KP7 codes for the multicomponent enzyme phenanthrene dioxygenase. phdA encoding an iron-sulfur protein large subunit alpha, phdB encoding its small subunit beta, phdC encoding ferredoxin, and phdD encoding ferredoxin reductase, were replaced in such a way that the termination codons of the preceding open reading frames were overlapped with the initiation codons of the following genes. This manipulated phdABCD gene cluster was positioned downstream of the thiostrepton-inducible promoter PtipA in a high-copy-number vector pIJ6021, and introduced into the gram-positive, soil-inhabiting, filamentous bacterium Streptomyces lividans. The recombinant S. lividans cells converted phenanthrene into a cis-diol form, which was determined to be cis-3,4-dihydroxy-3,4-dihydrophenanthrene by its UV spectral data as well as HPLC property, using the authentic sample for comparison. This biotransformation proceeded very efficiently; 200 microM and 2 mm of phenanthrene were almost completely converted to its cis-diol form in 6 h and 32 h, respectively. In addition, the S. lividans cells carrying the phdABCD gene cluster were found to transform 1-methoxynaphthalene to two products, which were identified to be 8-methoxy-2-naphthol in addition to 8-methoxy-1,2-dihydro-1,2-naphthalenediol by their EI-MS, 1H- and 13C-NMR spectral data.     

Algorithms and Complexity Analyses for Control of Singleton Attractors in Boolean Networks

A Boolean network (BN) is a mathematical model of genetic networks. We propose several algorithms for control of singleton attractors in BN. We theoretically estimate the average-case time complexities of the proposed algorithms, and confirm them by computer experiments. The results suggest the importance of gene ordering. Especially, setting internal nodes ahead yields shorter computational time than setting external nodes ahead in various types of algorithms. We also present a heuristic algorithm which does not look for the optimal solution but for the solution whose computational time is shorter than that of the exact algorithms.     

Involvement of Crosstalk between Oct4 and Meis1a in Neural Cell Fate Decision

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6–12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.     

5′-Phosphodiesterase Formation by Cultured Plant Cells

5′-Phosphodiesterase (5′-PDase) which degrades RNA to nucleoside-5′-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5 mg of 2,4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5′-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.

The 5′-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5′-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5′-PDase activity as the mother plant on dry cell weight basis.     

A new, convenient cell-based screening method for small-molecule glycolytic inhibitors

To counteract active glycolysis in tumors, we developed a new, convenient cell-based screening system to identify an inhibitor of glycolysis. Using this system, we searched for an inhibitor in the synthetic Carbasugar library and found two candidates. It was found that both inhibited glycolysis by suppressing the glucose uptake step in tumor cells.     

Structure-based design of dipeptide derivatives for the human neutral endopeptidase

Neutral endopeptidase (NEP) plays a key role in the metabolic inactivation of various bioactive peptides such as atrial natriuretic peptide (ANP), endothelins, and enkephalins. Furthermore, NEP is known to work as elastase in skin fibroblast. Therefore, effective inhibitors of NEP offer significant therapeutic interest as antihypertensives, analgesics, and skin anti-aging agents. Recently, the X-ray crystal structure of human NEP complexed with phosphoramidon has been reported and provided insights into the active site specificity of NEP. Here, we designed new inhibitors by using in silico molecular modeling and synthesized them by short steps. Expectedly, we found highly effective inhibitors with sub-nanomolar levels of IC(50) values. These results indicate that our structure-based molecular designing program is useful for obtaining novel NEP inhibitors. Furthermore, these inhibitors may be attractive leads for the generation of new pharmaceuticals for NEP-related diseases.     

Karyotype of a Japanese salamander Hynobius katoi and its implication on breeding ecology (Amphibia: Caudata)

Karyotype of a Japanese small salamander, Hynobius katoi, was first described. All individuals examined had 2n=58 chromosomes, consisting of nine pairs of biarmed macrochromosomes, four pairs of biarmed medium-sized chromosomes, six pairs of biarmed microchromosomes, and 10 pairs of uniarmed microchromosomes, although distinction of the second and the third groups of chromosome pairs was not clear. All pairs appeared homologous and no sexual dimorphism was found. Possession of 2n=58 chromosomes in H. katoi strongly suggests its lotic-breeding habits as was expected from the number and size of eggs and adult morphology. When compared morphology of chromosomes among lotic-breeders with 2n=58 chromosomes, metacentric nature of No. 10 seems to characterize the karyotype of H. katoi.