3746
K. Dolezˇal et al. / Bioorg. Med. Chem. 15 (2007) 3737–3747
performed as previously described,7–9 albeit with slight
modifications as detailed below. The E. coli strains were
grown overnight at 25 ꢁC in M9 media enriched with
0.1% casamino acids to OD600 ꢀ 1. The preculture
was diluted 1:600 in 200 lL M9 medium containing
0.1% casamino acids and 1 lL stock solution of either
the tested compound or solvent control was added.
The cultures were incubated for further periods at
25 ꢁC. Incubation times of 17 and 28 h were found to
be optimal for CRE1/AHK4 and AHK3, respectively.
The cultures were centrifuged and 50 lL aliquots of
the supernatant were transferred to microtitre plates
in which each well contained 2 lL of 50 mM 4-methyl-
umbelliferyl b-D-galactoside. The plates were subse-
quently incubated for 1 h at 37 ꢁC, and the reaction
was stopped by adding 100 lL of 0.2 M Na2CO3. Fluo-
rescence was measured using a Fluoroskan Ascent
(Labsystems, Finland) at excitation and emission wave-
lengths of 365 and 460 nm, respectively. The OD600 of
the remainder of each culture was determined and
b-galactosidase activity was calculated as nanomole
assistance, Alexander Popa for his help with NMR mea-
surements and David Morris for his helpful suggestions
and critical reading of the manuscript. K.C is thankful
to Laboratory of Growth Regulators, Palacky Univer-
sity & Institute of Experimental Botany ASCR, Olo-
mouc, Czech Republic, for providing her stay in Czech
Republic.
Supplementary data
Supplementary data associated with this article can be
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Acknowledgments
This work was supported by the Grant Agency of the
Czech Republic (GA 206/07/0570, GA 522/06/0108)
and Czech Ministry of Education (MSM 6198959216,
MSM 6198959218).
´
We thank Jarmila Balonova, Eva Friesnerova, Olga
Hustakova and Miloslava Subova for skilful technical
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