Journal of Natural Products
Article
flow rate, 1.0 mL/min; detection at 215 nm; solvent H O; retention
times (min) of components: Pro (3.2), N-Me-Val (3.7), Leu (4.8), N-
Me-Phe (12.5)].
96-wells plates (Iwaki) and cultured overnight. HL60 cells were seeded
at 1 × 10 cells/well in 96-well plates. Various concentrations of
compounds were then added, and cells were incubated for 72 h. Cell
proliferation was measured by the MTT assay.
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Each fraction was dissolved in H O (50 μL) and analyzed by chiral-
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phase HPLC, and the retention times were compared to those of
authentic standards [column, DAICEL CHIRALPAK MA(+) (ϕ 4.6 ×
Trypan Blue Dye Exclusion Assay. HeLa cells were seeded at 4
× 10 cells/well in 24-well plates (Iwaki), cultured overnight, and then
preincubated with or without 50 μM Z-VAD-FMK (Promega) for 30
min. The cells were then treated with various concentrations of
compounds for 48 h. They were then stained with 0.8 mg/mL trypan
blue (Sigma-Aldrich), and the cell viability was determined by
counting the number of stained (killed) cells.
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50 mm) or DAICEL CHIRALPAK WH (ϕ 4.6 × 50 mm); flow rate,
1.0 mL/min; detection at 254 nm; solvent, several conditions]. With
DAICEL CHIRALPAK MA(+) by using 2 mM CuSO as a solvent,
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the retention times for authentic standards were 2.5 min (N-Me-D-Val)
and 3.6 min (N-Me-L-Val). The retention time of N-Me-Val in the
hydrolysate was 3.6 min, indicating the presence of N-Me-L-Val. With
Caspase 3 Activity Assay. Caspase 3 (DEVDase) activity of the
cell lysate was measured using the CaspACE assay system, colorimetric
(Promega). Exponentially growing HeLa cells were seeded on a 60
DAICEL CHIRALPAK WH by using 3.0 mM CuSO , the retention
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times for authentic standards were 18.0 min (D-Pro) and 10.5 min (L-
Pro). The retention time of Pro in the hydrolysate was 10.5 min,
indicating the presence of L-Pro. With DAICEL CHIRALPAK WH by
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mm dish at 2 × 10 cells. After cells were incubated for 12 h at 37 °C,
compounds were added. After 24 h, cells were washed with ice-cold
phosphate-buffered saline (PBS) and collected. The cell pellet was
resuspended in cell lysis buffer (included in the above kit, 30 μL).
After a freeze−thawing process (three times) and incubation on ice for
15 min, the supernatant was collected by centrifugation (15000g, 4 °C,
20 min). The lysate was used for enzymatic assays (37 °C, 4 h) with
Ac-Asp-Glu-Val-Asp-pNA (Ac-DEVD-pNA) as a substrate, according
to the manufacturer’s protocol. The liberated p-nitroaniline was
measured using a spectrophotometer at 405 nm. The relative activity
was calculated by comparison with a control experiment (1% MeOH)
based on the average of three assays.
using 10.0 mM CuSO −MeCN (80:20), the retention times for
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authentic standards were 7.4 min (N-Me-D-Phe) and 24.6 min (N-Me-
L-Phe). The retention time of N-Me-Phe in the hydrolysate was 24.6
min, indicating the presence of N-Me-L-Phe.
The Leu fraction was dissolved in H O (50 μL). A 1.0% 1-fluoro-
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,4-dinitrophenyl-5-L-leucineamide (L-FDLA) solution in acetone (100
μL) and 25 μL of 1 M NaHCO were added, and the mixture was
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heated at 80 °C for 3 min. The solution was cooled to room
temperature, neutralized with 1 M HCl, and evaporated to dryness.
The residue was resuspended in 50 μL of MeCN−H O (50:50), and
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the solution was analyzed by reversed-phase HPLC [Cosmosil
Cell Culture of SOAT1- and SOAT2-CHO Cells. Two cell lines,
Cholester (ϕ 4.6 × 250 mm); flow rate, 1.0 mL/min; detection at
CHO cells expressing SOAT1 and SOAT2 isozymes of African green
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40 nm; solvent 0.02 M NaOAc−MeCN (60:40)]. The retention
monkey (SOAT1- and SOAT2-CHO cells, respectively), were kind
times for authentic standards were 9.0 min (D-Leu) and 4.3 min (L-
Leu). The retention time of Leu in the hydrolysate was 4.3 min,
indicating the presence of L-Leu.
gifts from Dr. L. L. Rudel (Wake Forest University). Briefly, both cell
lines were maintained at 37 °C in 5% CO in Ham’s F-12 medium
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supplemented with MEM vitamins, Geneticin (300 μg/mL), and 10%
heat-inactivated FBS (hereafter referred to as medium A).
Determination of the Configurations of the Amino Acids in
Biseokeaniamides B (2) and C (3). Biseokeaniamides B (2, 0.3 mg)
and C (3, 0.3 mg) were treated with 6 M HCl (100 μL) for 24 h at
Assay for Neutral Lipid Synthesis in SOAT1- and SOAT2-
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CHO Cells. The assay for the synthesis of neutral lipids ([ C]-
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10 °C, respectively. Each hydrolyzed product was evaporated to
cholesteryl ester (CE), [ C]triglyceride (TG), and [ C]phospholipid
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dryness and could be separated into each component [conditions for
(PL)) from [ C]oleic acid in SOAT1- or SOAT2-CHO cells was
carried out by our established method. Briefly, SOAT1- or SOAT2-
CHO cells (1.25 × 10 cells in 250 μL of medium A) were cultured in
a 48-well plastic microplate in the culture medium described above and
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HPLC separation: column, Cosmosil 5C -PAQ (ϕ 4.6 × 250 mm);
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flow rate, 1.0 mL/min; detection at 215 nm; solvent H O; retention
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times (min) of components: Pro (3.2), Val (3.4), N-Me-Val (3.7), Leu
(
4.8), N-Me-Phe (12.5)].
Each fraction was dissolved in H O (50 μL). A 1.0% L-FDLA
allowed to recover overnight at 37 °C in 5% CO . The assays were
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done with cells that were at least 80% confluent. Following the
overnight recovery, test sample (2.5 μL; 0, 0.01, 0.1, and 1 mg/mL in
solution in acetone (100 μL) and 25 μL of 1 M NaHCO were added,
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and the mixture was heated at 80 °C for 3 min. The solution was
cooled to room temperature, neutralized with 1 M HCl, and
evaporated to dryness. The residue was resuspended in 50 μL of
MeCN−H O (50:50), and the solution was analyzed by reversed-
phase HPLC [Cosmosil 5C -AR-II (ϕ 4.6 × 250 mm); flow rate, 1.0
MeOH) and [1- C]oleic acid (5 μL of 10% EtOH−PBS solution, 1
nmol, 1.85 KBq) were added to each culture. After a 6 h incubation at
37 °C in 5% CO , the medium was removed, and the cells in each well
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were washed twice with PBS. The cells were lysed by adding 0.25 mL
of 10 mM Tris-HCl (pH 7.5) containing 0.1% (w/v) sodium dodecyl
sulfate, and the cellular lipids were extracted by the method of Bligh
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mL/min; detection at 340 nm; solvent 0.02 M NaOAc−MeCN
(
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65:35) or 0.02 M NaOAc−MeCN (75:25)]. With 0.02 M NaOAc−
and Dyer. After concentrating the organic solvent, the total lipids
were separated on a thin-layer chromatography plate (silica gel F254,
0.5 mm thick, Merck, Darmstadt, Germany) and analyzed with an
MeCN (65:35), the retention times for authentic standards were 5.9
min (D-Pro), 3.8 min (L-Pro), 16.3 min (D-Val), 4.2 min (L-Val), 16.7
min (N-Me-D-Val), 5.5 min (N-Me-L-Val), 26.0 min (D-Leu), and 4.9
min (L-Leu). The retention times of the amino acids in the hydrolysate
were 3.8, 4.2, 5.5, and 4.9 min, indicating the presence of L-Pro, L-Val,
N-Me-L-Val, and L-Leu in each hydrolysate of both compounds 2 and
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FLA7000 analyzer (Fuji Film). In this cell-based assay, [ C]CE was
produced by the reaction of SOAT1 or SOAT2. SOAT-inhibitory
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activity (%) is defined as ([1- C]CE-drug/[ C]CE-control) × 100.
The IC50 value is defined as the drug concentration causing 50%
inhibition of a biological activity and is calculated from two times
duplicated experiments (n = 3).
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. With 0.02 M NaOAc−MeCN (75:25), the retention times for
authentic standards were 4.7 min (N-Me-D-Phe) and 3.6 min (N-Me-
L-Phe). The retention time of the amino acid in the hydrolysate was
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Preparation of Crude Enzymes from SOAT1- and SOAT2-
CHO Cells. Crude enzyme from SOAT1- and SOAT2-CHO cells
using a Potter-type homogenizer was prepared by our established
.6 min, indicating the presence of N-Me-L-Phe in each hydrolysate of
both compounds 2 and 3.
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Cell Growth Analysis. HeLa cells were cultured at 37 °C with 5%
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method. Briefly, the SOAT1 or SOAT2-CHO cells (2.0 × 10 cells)
were homogenized in 5 mL of cold buffered sucrose solution (pH 7.2,
100 mM sucrose, 50 mM KCl, 40 mM KH PO , and 30 mM EDTA,
CO in Dulbecco’s modified Eagle’s medium (Nissui) supplemented
with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL
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penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300
hereafter referred to as buffer B) including protease inhibitors
(Complete mini (Roche)) in a Potter-type homogenizer. The
microsomal fraction was pelleted by centrifugation at 100000g for
1.0 h at 4.0 °C (TLA110, Beckman Coulter), resuspended in the same
buffer at a concentration of 5.0 mg protein/mL, and stored at −80 °C
until use.
μg/mL L-glutamine, and 2.25 mg/mL NaHCO . HL60 cells were
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cultured at 37 °C with 5% CO in RPMI (Nissui) supplemented with
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0% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL
streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL L-glutamine, and
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.25 mg/mL NaHCO . HeLa cells were seeded at 2 × 10 cells/well in
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J. Nat. Prod. XXXX, XXX, XXX−XXX
Iwasaki, Arihiro
