No abstract

No abstract

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

Carbazomycins G (I) and H (II), new congeners of the carbazomycin complex, have been isolated from the culture broth of Streptoverticillium ehimense. They have proved to contain a unique quinol moiety in the molecule. Their structures have been elucidated by mass and NMR spectrometries and X-ray crystallographic analysis. Carbazomycin G showed moderate antifungal activity against Trichophyton species.In a previous paper1}, we have reported the isolation of carbazomycins C, D, E and F as the minor components of carbazomycin complex. The structures of carbazomycins C (IV) and D (V) were determined by spectroscopic and chemical means, and in the present paper are confirmed by an X-ray crystallographic analysis of IV.In our successive search for other active substances produced by the same microorganism, we have found two new components named carbazomycins G (I) and H (II) which are shown to possess a unique quinol moiety in the molecule. In this paper, we report the isolation, physico-chemical and antimicrobial properties, and structure elucidation of I and II by mass,^-H and 13C NMRspectrometries and X-ray crystallographic analysis. The confirmation of the structures of IV and V by X-ray crystallography of IV is also reported.Results and Discussion Isolation of Carbazomycins G and H Streptoverticillium ehimense was cultured in the same wayas reported previously0. The broth filtrate was extracted with ethyl acetate and the extract was fractionated by silica gel columnchromatography using the solvent composed of «-hexane and ethyl acetate (7 : 1~1 : 1). The last fraction eluted with «-hexane and ethyl acetate (1 : 1) was concentrated and further purified by repeated preparative TLCon precoated Silica gel plates (Merck Art. No. 5715) developed with «-hexane and ethyl acetate (2 : 3). Under a UV-light (365 nm), I gave strong blue fluorescence and n gave dark yellow one, and both components gave the same brownish purple color on silica gel TLCby heating after spraying with 10 % sulfuric acid.

Four diarylheptanoid monoglycosides (2a), (2b), (3), and (4), in addition to the hydroxy ketone ( I ) , were isolated from the femate flowers of Alnus serrulatoides. On the basis of the physicochemical and the X-ray crystallographic analyses, these were characterized as (5s) -1,7-bis(3,4-di hydroxyphenyl) -5hydroxyheptan-3-one(1 ),5s) -1,7bis (3,4-d i hydroxyp hen y I) -5-( p -Dxylopyranosyloxy )heptan-3-one (3), and (5s) -1,7-bis( 3,4-di hydroxyphenyl) -5-(p-D-gl ucopyranosyloxy) heptan -3-one (4), respectively. Of these compounds, the glycosides (2a), (2b), and (4) are new.(2 b) ,In our previous studies we elucidated the structures of five novel C3 ,-dammarane-type triterpenoids '-' from the male flowers of Anus serrufatoides Call. (Japanese name: Kawara-hannoki) and also those of four novel C3 ,-secodammarane-type L a x . (log ~) / n m ' 221 (4.07), 283 (3.75) 222 (4.07), 283 (3.75) 228 (4.07), 280 (3.69) 220 (4.00), 283 (3.67) 229 (4.13), 280 (3.70) 230 (4.32), 286 (3.89) 225 (4.24), 287 (3.70), 284 (3.65) 225 (4.26), 278 (3.71), 284 (3.66) 228 (4.14), 280 (3.73) 229 (4.25), 280 (3.80) 227 (4.24), 279 (3.73) 231 (4.25), 287 (3.79) v, , , .

No abstract

No abstract

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.