Biology Reference
In-Depth Information
500
400
300
200
100
0
[BYK54826] (
µ
M)
10
6
Lister 427
bloodstream forms) were incubated for 10 min in the presence or absence of BYK54826 at the
concentrations indicated. Incubations were stopped by 30 s centrifugation at 13,000
Fig. 3 Effect of BYK54826 on cAMP levels in
T. b. brucei
. Trypanosomes (2.5
g
followed
by the addition of 150
m
l ice-cold 0.1 M HCl to the cell pellet; cAMP was determined using the
Direct Cyclic AMP ELISA kit (Assay Designs) according to the manufacturer's instructions
18000
No drug
control
15000
[BYK54826] (
µ
M)
12000
100
50
25
12.5
6.25
3.12
1.56
0.78
9000
6000
3000
0
0
2
4
6
8
10
12
14
16
18
Time (h)
Fig. 4 Effect of BYK54826 on plasma membrane permeability in trypanosomes. Bloodstream
form trypanosomes (strain 427, 5
10
6
cells/mL in HMI-9 medium plus 10% FCS) were
incubated at 37
C under 5% CO
2
for 18 h in a FLUOstar OPTIMA fluorimeter in the presence
of 6
m
g/mL propidium iodide (PI). Survival was monitored by measuring fluorescence of PI, which
enters live trypanosomes only extremely slowly and fluoresces after binding to nucleic acids
(Gould et al.
2008
). All traces were recorded simultaneously in a
white
-
bottomed
96-well plate
0.3
m
M caused a 20-fold rise within 20 min and remaining elevated at this level for
hours (not shown); 1
m
M increased cAMP levels up to 50-fold, similar to the
observations with PDEB RNAi (Oberholzer et al.
2007
). In contrast, dipyridamole
(40
m
M) and etazolate (100
m
M), both low-affinity inhibitors of TbrPDEBs (Zoraghi
and Seebeck
2002
), did not affect trypanosomal cAMP levels. However, the rapid
cAMP response did not affect cellular survival for hours. As shown in Fig.
4
,only
extreme concentrations of BYK54286 of 50 or 100
m
M had a rapid effect on parasite
viability. Lower concentrations had no measurable effect on this parameter for up to
