Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0128-y) contains supplementary material, which is available to authorized users.
Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role.
Neocarzinostatin is a potent enediyne antibiotic chromoprotein, secreted by Streptomyces carzinostaticus. It consists of an 11 kDa all‐beta protein that is non‐covalently associated with a very labile 659 Da enediyne antibiotic chromophore. Although the production and purification of neocarzinostatin were reported in detail earlier, execution of the reported methods in our laboratory frequently results in very poor antibiotic yield because of its instability. Crude culture filtrates containing active neocarzinostatin antibiotic from fermentation of S. carzinostaticus (ATCC15945 and ATCC15944) were prepared using the reported procedures. Here we describe a slight modification to replace the clay‐involved precipitation step in the reported method of purifying the chromoprotein antibiotic neocarzinostatin. The modified procedure employs a quick concentrating step by involving hollow fiber cartridges to avoid extensive degradation of the labile antibiotic during the prolonged purification steps. A combination of 500 kDa (or 50 kDa) and 3 kDa cutoff cartridges is used to exclude large insoluble materials and to retain fractions containing active neocarzinostatin. Followed by quick concentrating step, buffer exchange with samples is then carried out using the 3 kDa cutoff cartridge. This allows the semi‐purified samples to be applied directly to ion‐exchange chromatographic purification. This work is funded by National Science Council and National Health Research Institute of Taiwan, R.O.C.
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