Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.Succinic acid is one of the fermentation products of anaerobic metabolism as well as an intermediate of the tricarboxylic acid cycle. It has been used as a precursor for various chemicals, a food additive, an ion chelator, and a supplement to pharmaceuticals (34). Succinic acid has mostly been produced chemically from maleic anhydride. Recently, fermentative production of succinic acid has been receiving much research attention, as several bacteria can produce succinic acid as a major fermentation product. The naturally isolated obligate anaerobe Anaerobiospirillum succiniciproducens (6) and facultative anaerobes belonging to the family Pasteurellaceae, such as Actinobacillus succinogenes (10) and Mannheimia succiniciproducens (16), have been shown to be able to produce succinic acid efficiently.Escherichia coli also produces succinic acid but as a minor fermentation product. E. coli prefers to produce much more acetic acid, formic acid, lactic acid, and ethanol rather than succinic acid during anaerobic fermentation. Thus, it is necessary to redirect metabolic fluxes for increasing succinic acid production as well as reducing formation of other metabolites. Toward this goal, an ldhA and pfl double mutant E. coli strain NZN111 was developed to block the formation of lactic, acetic, and formic acids (2). However, cell growth was relatively slow, possibly due to the inactivation of enzymes involved in pyruvate dissimilation (3). Chatterjee and coworkers (4) reported that an additional ptsG gene mutation recovered the growth of NZN111 to some extent. However, acetic acid, formic acid, and ethanol were still...

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DNA oligonucleotides were covalently immobilized to prepatterned single-walled carbon nanotube (SWNT) multilayer films by amidation. SWNT multilayer films were constructed via consecutive condensation reactions creating stacks of functionalized SWNT layers linked together by 4,4'-oxydianiline. Aminated- or carboxylated-DNA oligonucleotides were covalently immobilized to the respective carboxylated or aminated SWNT multilayer films through amide bond formation using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. UV-vis-NIR spectroscopic analysis indicated that the SWNT film surface density increased uniformly according to the number of reaction cycles. Scanning electron microscopy and contact angle measurements of the SWNT multilayer film revealed a uniform coverage over the substrate surface. The covalent attachment of DNA oligonucleotides to the SWNT multilayer films and their subsequent hybridization with complementary oligonucleotides were verified using X-ray photoelectron spectroscopy and fluorescence-based measurements. This is the first report demonstrating that DNA oligonucleotides can be covalently attached to immobilized SWNT multilayer films. The anchored DNA oligonucleotides were shown to exhibit excellent specificity, realizing their potential in future biosensor applications.

Aim: In this study, we investigated the regulatory effects of honokiol on various inflammatory events mediated by monocytes/macrophages (U937/RAW264.7 cells) and lymphocytes (splenic lymphocytes and CTLL-2 cells) and their putative action mechanism. Methods: In order to investigate the regulatory effects, various cell lines and primary cells (U937, RAW264.7, CTLL-2 cells, and splenic lymphocytes) were employed and various inflammatory events, such as the production of inflammatory mediators, cell adhesion, cell proliferation, and the early signaling cascade, were chosen. Results: Honokiol strongly inhibited various inflammatory responses, such as: (i) the upregulation of nitric oxide (NO), prostaglandin E 2 and TNF-α production and costimulatory molecule CD80 induced by lipopolysaccharide (LPS); (ii) the functional activation of β1-integrin (CD29) assessed by U937 cell-cell and cell-fibronectin adhesions; (iii) the enhancement of lymphocytes and CD8+CTLL-2 cell proliferation stimulated by LPS, phytohemaglutinin A (PHA), and concanavalin A or interleukin (IL)-2; and (iv) the transcriptional upregulation of inducible NO synthase, TNF-α, cyclooxygenase-2, IL-12, and monocyte chemoattractant protein (MCP)-1. These anti-inflammatory effects of honokiol seem to be mediated by interrupting the early activated intracellular signaling molecule phosphoinositide 3-kinase (PI3K)/Akt, but not Src, the extracellular signal-regulated kinase, and p38, according to pharmacological, biochemical, and functional analyses. Conclusion: These results suggest that honokiol may act as a potent anti-inflammatory agent with multipotential activities due to an inhibitory effect on the PI3K/Akt pathway. Key wordsho nok i ol; ma g nolo l; a nti -infla m ma to ry effe cts; m a croph a ges a nd lym phocyt es; phosphoinositide 3-kinase/Akt pathway