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626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Wong et al.
Eppendorf tubes were incubated in a 30 °C water bath for 2 min.
Then 4 µL of PKC R or PKC protein was added into Eppendorf
tubes to standard or positive control assay, respectively. The
Eppendorf tubes were further incubated in the 30 °C water bath
for 30 min. At the end of the incubation time, the Eppendorf tubes
were placed in a 95 °C heating block for 10 min to stop the reaction.
The Eppendorf tubes were placed on ice and in the dark until
loading began. Then 1 µL of 80% glycerol was added to each
Eppendorf tube before loading onto gel.
For gel preparation/separation, the horizontal agarose gel ap-
paratus was assembled accordingly. An 0.8% agarose solution (1.6
g in 200 mL 50 mM Tris-HCl, pH 8.0) was prepared and heated in
microwave for 2 min and 10 s until all the agarose had been
dissolved. The mixture was allowed to cool to 60 °C and
subsequently poured into a gel tray, ensuring no bubbles were
formed in the process. The gel was allowed to solidify before the
comb was removed. The gel was placed in the electrophoresis
chamber, and 50 mM Tris-HCl (pH 8.0) solution was poured onto
the gel. The PKC assay samples was then loaded into the well (10
µL per well), and the electrophoresis was started at 120 V for about
(12) Edsall, L. C.; Van Brocklyn, J. R.; Cuvillier, O.; Kleuser, B.; Spiegel,
S. N,N-Dimethylsphingosine is a potent competitive inhibitor of
sphingosine kinase but not of protein kinase C: modulation of cellular
levels of sphingosine 1-phosphate and ceramide. Biochemistry 1998,
3
7, 12892–12898.
(
(
(
13) Kono, K.; Tanaka, M.; Mizuno, T.; Kodama, K.; Ogita, T.; Kohama,
T. B-535a, b and c, new sphingosine kinase inhibitors, produced by a
marine bacterium; taxonomy, fermentation, isolation, physico-chemical
properties and structure determination. J. Antibiot. (Tokyo) 2000, 53,
7
53–758.
14) Igarashi, Y.; Hakomori, S.; Toyokuni, T.; Dean, B.; Fujita, S.;
Sugimoto, M.; Ogawa, T.; El-Ghendy, K.; Racker, E. Effect of
chemically well-defined sphingosine and its N-methyl derivatives on
protein kinase C and src kinase activities. Biochemistry 1989, 28,
6
796–6800.
15) Megidish, T.; White, T.; Takio, K.; Titani, K.; Igarashi, Y.; Hakomori,
S.-i. The signal modulator protein 14-3-3 is a target of sphingosine-
or N,N-dimethylsphingosine-dependent kinase in 3T3(A31) cells.
Biochem. Biophys. Res. Commun. 1995, 216, 739–747.
(16) Kim, J.-W.; Kim, Y.-W.; Inagaki, Y.; Hwang, Y.-A.; Mitsutake, S.;
Ryu, Y.-W.; Lee, W. K.; Ha, H.-J.; Park, C.-S.; Igarashi, Y. Synthesis
and evaluation of sphingoid analogs as inhibitors of sphingosine
kinases. Bioorg. Med. Chem. 2005, 13, 3475–3485.
(
17) De Jonghe, S.; Van Overmeire, I.; Poulton, S.; Hendrix, C.; Busson,
R.; Van Calenbergh, S.; De Keukeleire, D.; Spiegel, S.; Herdewijn,
P. Structure-activity relationship of short-chain sphingoid bases as
inhibitors of sphingosine kinase. Bioorg. Med. Chem. Lett. 1999, 9,
3
0 min or until the separation was completed. The gel was then
removed, and the photo of the gel was taken using a Kodax imaging
machine.
3
175–3180.
Acknowledgment. We thank the National University of
Singapore for financial support of this work (ARF Grant R-143-
00-335-112). We also thank H’ng Shiau Chen of the MCI Lab,
Yong Loo Lin of the School of Medicine, National University
of Singapore, for her input and advice on the biological
evaluation studies.
(18) Van Overmeire, I.; Boldin, S. A.; Dumont, F.; Van Calenbergh, S.;
Slegers, G.; De Keukeleire, D.; Futerman, A. H.; Herdewijn, P. Effect
of aromatic short-chain analogues of ceramide on axonal growth in
hippocampal neurons. J. Med. Chem. 1999, 42, 2697–2705.
(19) Garner, P.; Park, J. M.; Malecki, E. A stereodivergent synthesis of
D-erythro-sphingosine and D-threo-sphingosine from L-serine. J. Org.
Chem. 1988, 53, 4395–4398.
0
(
20) Williams, L.; Zhang, Z.; Shao, F.; Carroll, P. J.; Joullie, M. M.
Grignard reactions to chiral oxazolidine aldehydes. Tetrahedron 1996,
Supporting Information Available: Spectral data of the ana-
logues and analytical data of 5c. This material is available free of
charge via the Internet at http://pubs.acs.org.
5
2, 11673–11694.
(21) Bielawska, A.; Hannun, Y. A. Preparation of radiolabeled ceramides
and phosphosphingolipids. Methods Enzymol. 2000, 311, 499–518.
(
22) Cherest, M.; Felkin, H.; Prudent, N. Torsional strain involving partial
bonds. The stereochemistry of the lithium aluminum hydride reduction
of some simple open-chain ketones. Tetrahedron Lett. 1968, 9, 2199–
References
2
204.
(
(
(
(
(
1) Kee, T. H.; Vit, P.; Melendez, A. J. Sphingosine kinase signalling in
immune cells. Clin. Exp. Pharmacol. Physiol. 2005, 32, 153–161.
2) Hannun, Y. A. The sphingomyelin cycle and the second messenger
function of ceramide. J. Biol. Chem. 1994, 269, 3125–3318.
3) Spiegel, S.; Milstien, S. Sphingosine 1-phosphate, a key cell signaling
molecule. J. Biol. Chem. 2002, 277, 25851–25854.
(
23) Herold, P. Synthesis of D-erythro- and D-threo-sphingosine derivatives
from L-serine. HelV. Chim. Acta 1988, 71, 354–362.
(
24) Kaiser, E.; Tam, J. P.; Kubiak, T. M.; Merrifield, R. B. Chlorotrim-
ethylsilane-phenol as a mild deprotection reagent for the tert-butyl
based protecting groups in peptide synthesis. Tetrahedron Lett. 1988,
2
9, 303–306.
4) Haimovitz-Friedman, A.; Kolesnick, R. N.; Fuks, Z. Ceramide
signaling in apoptosis. Br. Med. Bull. 1997, 53, 539–553.
(
25) Mori, A.; Miyakawa, Y.; Ohashi, E.; Haga, T.; Maegawa, T.; Sajiki,
H. Pd/C-catalyzed chemoselective hydrogenation in the presence of
diphenylsulfide. Org. Lett. 2006, 8, 3279–3281.
26) Meyer zu Heringdorf, D.; Lass, H.; Kuchar, I.; Lipinski, M.; Alemany,
R.; Rumenapp, U.; Jakobs, K. H. Stimulation of intracellular sphin-
gosine-1-phosphate production by G-protein-coupled sphingosine-1-
phosphate receptors. Eur. J. Pharmacol. 2001, 414, 145–154.
27) Zhi, L.; Leung, B. P.; Melendez, A. J. Sphingosine kinase 1 regulates
pro-inflammatory responses triggered by TNFalpha in primary human
monocytes. J. Cell. Physiol. 2006, 208, 109–115.
5) Kondo, T.; Matsuda, T.; Kitano, T.; Takahashi, A.; Tashima, M.;
Ishikura, H.; Umehara, H.; Domae, N.; Uchiyama, T.; Okazaki, T.
Role of c-jun expression increased by heat shock- and ceramide-
activated caspase-3 in HL-60 cell apoptosis. Possible involvement of
ceramide in heat shock-induced apoptosis. J. Biol. Chem. 2000, 275,
(
7
668–7676.
(
(
(
(
(
(
(
6) Takabe, K.; Paugh, S. W.; Milstien, S.; Spiegel, S. “Inside-out”
signaling of sphingosine-1-phosphate: therapeutic targets. Pharmacol.
ReV. 2008, 60, 181–195.
7) Kohama, T.; Olivera, A.; Edsall, L.; Nagiec, M. M.; Dickson, R.;
Spiegel, S. Molecular cloning and functional characterization of murine
sphingosine kinase. J. Biol. Chem. 1998, 273, 23722–23728.
8) Melendez, A. J.; Carlos-Dias, E.; Gosink, M.; Allen, J. M.; Takacs,
L. Human sphingosine kinase: molecular cloning, functional charac-
terization and tissue distribution. Gene 2000, 251, 19–26.
28) Shida, D.; Takabe, K.; Kapitonov, D.; Milstien, S.; Spiegel, S.
Targeting SphK1 as a new strategy against cancer. Curr. Drug Targets
2
008, 8, 662–673.
29) Lai, W. Q.; Irwan, A. W.; Goh, H. H.; Howe, H. S.; Yu, D. T.; Valle-
O n˜ ate, R.; McInnes, I. B.; Melendez, A. J.; Leung, B. P. Anti-
inflammatory effects of sphingosine kinase modulation in inflammatory
arthritis. J. Immunol. 2008, 181, 8010–8017.
9) Pitson, S. M.; D’Andrea, R. J.; Vandeleur, L.; Moretti, P. A. B.; Xia,
P.; Gamble, J. R.; Vadas, M. A.; Wattenberg, B. W. Human
sphingosine kinase: purification, molecular cloning and characterization
of the native and recombinant enzymes. Biochem. J. 2000, 350, 429–
(
(
(
30) Lai, W. Q.; Goh, H. H.; Bao, Z.; Wong, W. S.; Melendez, A. J.; Leung,
B. P. The role of sphingosine kinase in a murine model of allergic
asthma. J. Immunol. 2008, 180, 4323–4329.
31) Melendez, A. J. Allergy therapy: the therapeutic potential of targeting
sphingosine kinase signalling in mast cells. Eur. J. Immunol. 2008,
4
41.
(
10) Liu, H.; Sugiura, M.; Nava, V. E.; Edsall, L. C.; Kono, K.; Poulton,
S.; Milstien, S.; Kohama, T.; Spiegel, S. Molecular cloning and
functional characterization of a novel mammalian sphingosine kinase
type 2 isoform. J. Biol. Chem. 2000, 275, 19513–19520.
3
8, 2969–2974.
32) Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal. Biochem. 1976, 72, 248–254.
(11) Buehrer, B. M.; Bell, R. M. Sphingosine kinase: properties and cellular
functions. AdV. Lipid Res. 1993, 26, 59–67.
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