Development and operation of a trickling biofilter system for continuous treatment of gas-phase trichloroethylene

A parallel trickling biofilter (TBF) system that consists of two TBFs units in parallel, one for biodegradation of trichloroethylene (TCE) and the other for reactivation of an inactivated biofilm, was developed and operated for continuous treatment of gas-phase TCE by Burkholderia cepacia G4. For inlet loadings below 8.6 mg TCE l^–1 d^–1, complete removal of TCE was achieved. The maximal TCE elimination capacity was 17 mg l^–1 d^–1.     

Enhanced production of recombinant B-domain deleted factor VIII from Chinese hamster ovary cells by propionic and butyric acids

Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities.     

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.     

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.     

Application of waste activated bleaching earth containing rapeseed oil on riboflavin production in the culture of Ashbya gossypii

Waste activated bleaching earth (ABE) that contained 40% rapeseed oil and was discharged by an oil refinery plant, was used for riboflavin production in a culture of Ashbya gossypii. When 125 g/L waste ABE that contained 50 g/L rapeseed oil was added into the culture, the riboflavin concentration was 1.12 g/L, which was almost 1.6-fold as high as that of pure rapeseed oil. However, in waste ABE concentration higher than 125 g/L, the produced riboflavin concentration decreased, which was due to the difficulty in mixing due to the presence of a high amount of solid material in the culture. The surface of the waste ABE was smooth without a hitch, because of being covered with rapeseed oil. However, after the culture, the surface of the waste ABE seemed like that of new one, and the oil content was nearly zero grams per liter. The waste ABE, oily clay, and its black color gradually fade and yellow little by little, and finally the waste ABE changed to yellow powder. Of the riboflavin produced during the culture, 70% was adsorbed in the oil free waste ABE. With diluted alkali solution, extraction only two times yielded 90% recovery of riboflavin adsorbed in the waste ABE. The waste ABE containing waste vegetable oil was suitable for raw material for production of the value-added useful bioproducts, which might be a good model for reuse of the waste resource.     

Nanoparticulate DNA packaging using terpolymers of poly(lysine-g-(lactide-b-ethylene glycol))

Terpolymers of poly(lysine-g-(lactide-b-ethylene glycol)) (pK-pLL-pEG) were synthesized by using ring-opening polymerization and functional end-group grafting. Synthesis was characterized with gel permeation chromatography, proton nuclear magnetic resonance spectroscopy, and a trinitrobenzene sulfonic acid binding assay. Polymer association behavior with DNA was investigated using an ethidium bromide exclusion assay, static light scattering, and scanning electron microscopy. Polylactide molecular weight was varied to investigate its impact on DNA association and resulting complex characteristics. Polylysine ( = 8800, DP = 42) modified with either 7400 or 10 870 pLL-pEG reduced the minimum amount of primary amines necessary for complete condensation by 23% and 48%, respectively, compared to unmodified polylysine (pK42). Complexes formed with the highest molecular weight terpolymer demonstrated significantly (p < 0.1) greater resistance to DNase I than lyophilized pK42-DNA particles. This study suggests that modification of pK42 with pLL-pEG diblock copolymers impacts polylysine's associative and binding behavior to DNA and resulting particle characteristics. Modulation of terpolymer composition in complexes can enable control over intracellular plasmid dissociation rates to improve transfection efficiency.     

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.