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P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
scatter and green fluorescence) were acquired using the
Cell Quest software (Becton Dickinson, USA). Analysis
was performed by applying appropriate gates with refer-
ence to the autofluorescence measured under similar
conditions. Platelet isolated as described earlier was sus-
4.1.14. Confocal microscopy. To 1 ml of PRP, 20 ll com-
pound (1) (100 lM), 20 ll arginine (50 lM), and 10 ll
DCFH-DA (20 lM) were added. The contents were
incubated for 30 min at 37 ꢁC and centrifuged at 1000g
for 15 min at 4 ꢁC. The pellet obtained was washed
and finally suspended in 0.5 ml PBS. Hundred microli-
ters of the cell suspension was placed on microscopic
slide with coverslip for observation under the confocal
microscope (Model LSM 510, Carl, Zeis, Germany).
The images were visualized using 40· oil objective and
the green DCF fluorescence was observed following
excitation at 488 nm from argon laser with the help of
long pass filter LP 505. The fluorescence was confirmed
from intensity profile measurement with the built in soft-
ware provided by the manufacturer.
6
pended in PBS and platelet counts were adjusted to 10 /
ml using electronic particle counter (SYSMEX, Model
No. FA20). DCFH-DA (20 lM) and L-arginine at vari-
ous concentrations were then added to the cell suspen-
sion and preincubated for 37 ꢁC for 30 min while
rotating (10 rpm) to prevent adherence during assay.
To stop the assay, samples were placed on ice for
1
0 min in dark. Relative green DCF fluorescence was
measured. Similarly, various PA (1–5) were preincubat-
ed separately with L-arginine and their relative fluores-
cence was measured thereafter.
4
.1.12. Demonstration of TAase catalyzed acetylation of
Acknowledgments
NOS by 7,8-diacetoxy-4-methylcoumarin. Anti-acetyl ly-
sine as primary antibody was used to show the acetylation
of NOS. For this purpose purified TAase (50 lg) was
incubated with eNOS (50 lg), compound (1) (200 lM),
and 10 mM phosphate buffer (pH 7.2), and incubated
for 30 min at 37 ꢁC. After completion of the reaction,
samplebuffer(loadingdye) was added tothe reaction mix-
ture to stop the reaction. This reaction mixture was used
to detect the acetylated NOS by Western blot.
The financial assistance from the Department of Bio-
technology, Government of India, is gratefully acknowl-
edged. R.K. is a recipient of Senior Research Fellowship
from C.S.I.R, Government of India. Purified eNOS was
generously provided by Prof. Bettie Masters, University
of Texas, Health science Center, Galveston, TX, USA.
References and notes
For Western blot, electrophoretically separated proteins
were transferred onto nitrocellulose membrane at
1
2
. Toda, N.; Okamura, T. Pharmacol. Rev. 2003, 55, 271.
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3
00 mA for 3 h at 4 ꢁC. Non-specific sites on the nitro-
cellulose sheet were blocked with 5% blocking buffer.
Primary antibody dilution (1:1000) was prepared in
TBST containing 1% BSA and incubated overnight at
4
5
. Cardena, G. G.; Fan, R.; Stern, D. F.; Liu, J.; Sessa, W. C.
J. Biol. Chem. 1996, 271, 27237.
. Raj, H. G.; Parmar, V. S.; Jain, S. C.; Kohli, E.; Goel, S.;
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. Raj, H. G.; Kohli, E.; Tyagi, Y. K.; Parmar, V. S.; Olsen,
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. Kohli, E.; Gaspari, M.; Raj, H. G.; Parmar, V. S.; Greef,
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4
ꢁC. The nitrocellulose membrane was extensively
washed with TBST (TBS with 0.05% Tween). Goat
anti-rabbit–HRPO (horseradish peroxidase)-conjugated
secondary antibody, appropriately diluted (1:1000) in
TBST, was then added and an incubation of 1 h at room
temperature was carried out. The membrane was
washed extensively and antibody-reactive moieties were
visualized with DAB (diaminobenzidine) system.
6
7
4.1.13. Aggregation studies. PRP was prepared by the
above-mentioned technique. Platelet count was adjusted
to 250,000/ll with homologous platelet-poor plasma
8
. Kohli, E.; Gaspari, M.; Raj, H. G.; Parmar, V. S.;
Sharma, S. K.; Greef, J. V.; Kumari, R.; Gupta, G.;
Seema, ; Khurana, P.; Tyagi, Y. K.; Watterson, A. C.;
Olsen, C. E. Biochim. Biophys. Acta 2004, 1698, 55.
. Parmar, V. S.; Bisht, K. S.; Jain, R.; Singh, S.; Sharma, S.
K.; Gupta, S.; Malhotra, S.; Tyagi, O. D.; Vardhan, A.;
Pati, H. N. Indian J. Chem. 1996, 35B, 220.
(
PPP). PPP was prepared by centrifugation of the
remainder of blood at 2500g for 10 min. Platelet counts
PC) were adjusted according to the following
9
(
formula:
1
0. Gadre, J. N.; Audi, A. A.; Karambelkar, N. P. Indian J.
Chem. 1996, 35 B, 60.
PC ðPRPÞ ꢂ ml PRP ꢃ 250; 000 ¼ ml PRPð250; 000Þ
Various PA (in appropriate concentrations) were preincu-
bated with PRP to make the final volume of 0.18 ml. Plate-
let aggregation was induced by the addition of 5 lM ADP.
Platelet aggregation studies were performed using BIO-
DATA Corporation, Platelet Aggregation Profiler, Model
No. PAP-4. Platelet aggregation was expressed as the
maximum percentage of light transmittance change (%
max) from the baseline at the end of the recording time,
using PPP as a reference. Platelet aggregation curves were
recorded for 6 min and analyzed according to interna-
11. Kelly, T. R.; Kim, M. H. J. Org. Chem. 1992, 57, 1593.
12. Lowry, O. H.; Roebrough, N. J.; Farr, A. T.; Randall, R.
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1
1
1
1
3. Zhang, J.; Vanhaesebrock, B.; Rittenhouse, S. E. Biochem.
Biophys. Res. Commun. 2002, 96, 178.
4. Habig, W. H.; Pabst, M. J.; Jacoby, W. B. J. Biol. Chem.
1
974, 249, 7130.
5. Masters, B. S. S.; Williams, C. H.; Kamin, H. Methods
Enzymol. 1967, 10, 565.
6. Raj, H. G.; Parmar, V. S.; Jain, S. C.; Goel, S.; Singh, A.;
Tyagi, Y. K.; Jha, H. N.; Olsen, C. E.; Wengel, J. Bioorg.
Med. Chem. 1999, 7, 369.
1
8
tionally established standards.