Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a reaction mixture containing these naphthoquinones and topoisomerase II at an elevated temperature (65°C) resulted in a great reduction in DNA cleavage, suggesting that the mechanism of the topoisomerase II-mediated DNA cleavage induced by these naphthoquinones is through formation of a cleavable complex, as seen with antitumor agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide and demethylepipodophyllotoxin ethylidene-13-glucoside.Lawson and lapacol, which are structurally related plant metabolites with naphthoquinone moieties, could not induce topoisomerase U-mediated DNA cleavage. Plumbagin and shikonin induced a similar DNA cleavage pattern with topoisomerase II which was different from the cleavage patterns induced with other known topoisomerase II-active drugs. A DNA-unwinding assay with T4 DNA ligase showed that shikonin, lawson, and lapacol did not intercalate into DNA, while plumbagin and 2-methyl-1,4-naphthoquinone intercalate into DNA, but to a lower degree than 4'-(9-acridinylamino)methanesulfon-m-anisidide does.DNA topoisomerases are a class of enzymes that alter DNA conformation through a concerted breaking and rejoining of the DNA molecule, thereby controlling the topological state of DNA. They are reported to be involved in many important processes of DNA metabolism including replication, transcription, recombination, and chromosome segregation (38). In addition, topoisomerases have been potential targets for chemotherapy. Bacterial topoisomerase II (DNA gyrase) is well known as the primary target of quinolone antibacterial agents. Mammalian topoisomerase II has also been identified as the primary cellular target for a number of clinically important antitumor agents which include intercalating agents [e.g., 4'-(9-acridinylamino)methanesulfon-manisidide {m-AMSA}, adriamycin, and ellipticine] as well as nonintercalating agents (e.g., VP16 and VM26 [epipodophyllotoxin]) (6,29,34,35). All of these drugs trap topoisomerase II in an intermediary conformation with DNA, termed the "cleavable complex," which can be detected as DNA double-strand breaks upon treatment of the complex with protein denaturants. Structure-activity studies of a large number of acridine derivatives, epipodophyllotoxin congeners, and antitumor quinolones have shown a strong correlation between antitumor activity and the ability to induce the cleavable complex (23,31,39). In addition, there is now good evidence that mammalian topoisomerase I is the cellular target of camptothecin, an alkaloid with antitumor activity which was isolated from the Chinese tree Camptotheca acuminata. Camptothecin derivatives have shown promising activities in clinical studies (12,16 purified mammalian topoisomerases. We found that the plant naphthoquinones plumbagin and shikonin are potent inducers of the cleavable complex formation with topoisomerase II in vitro.In this report, we de...
Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.
Random amplified polymorphic DNA (RAPD) analysis was used to discriminate genotypes in five species of Microcystis cyanobacteria. Strains of each group with the identical allozyme genotype (T. Kato et al., Algol. Stud., 1991, 129-140; M. Watanabe, in "Toxic Microcystis," ed. by M.F. Watanabe et al., CRC Press, Tokyo, 1966, pp. 13-34) gave similar RAPD patterns characterizing the respective group. On the other hand, no similarities in RAPD patterns were observed among strains of which allozyme genotypes were different. A good accordance between the RAPD analysis and allozyme divergence indicated a high reliability of both methods for discrimination of the affiliated groups of Microcystis. Several amplified DNA fragments, which were expected to be markers for a particular taxon with identical allozyme genotype, were also observed on the RAPD patterns. Genetic homogeneities of M. novacekii, M. viridis, and M. wesenbergii were shown by RAPD analysis as well as the allozyme genotype. However, significant variations were observed in M. aeruginosa and M. ichthyoblabe in the levels of DNA and proteins (allozymes).
The CaCCO process (Park et al., 2010), a sugar platform for herbaceous lignocellulosics, was modified and evaluated for fermentable-sugar recovery in solution, using rice straw (RS) as well as stems and leaves of Erianthus arundinaceus (ER), one of the strategic energy crops in Japan. Continuous wet milling of a mixture of feedstock, water and Ca(OH)2, with a weight ratio of 3/7/0.3, was adopted, not only to evaluate the applicability for wet feedstocks, but also for simultaneous Ca(OH)2 mixing with the milled feedstocks. We also applied low temperatures of 95 100 C for the alkali pretreatment to save energy. Enzymatic-saccharification tests were performed for the pretreatment slurries from 10 kg feedstocks for 72 h at 40 C under a CO2-pressurized atmosphere at 0.9 MPa with high-solid loadings of 28.4% (w/w) for RS and 27.8% (w/w) for ER. These tests successfully solubilized 80.6% (RS) and 68.1% (ER) of the total glucose-and xylose residues in the solids. Following centrifugation, the solubilized-sugar concentrations in the recovered solutions were 16.9% (w/v) for RS and 15.5% (w/v) for ER. Thus, we demonstrated that the CaCCO process could be a simple and flexible platform for preparation of dense sugar solutions from dry-and wet feedstocks, which could stimulate a new agricultural bio-industry for vitalizing rural areas.
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