Pokaż cały numer - FPN - Farmaceutyczny PrzeglÄ d Naukowy

Farm Przegl Nauk, 2009,8
dried residues were dissolved in 1 ml of 2M HCl and submitted
to chromatography on a column (1 x 9 cm), containing
Dowex 50W, equilibrated and eluted with 2M HCl. Such
a procedure made possible to separate [ 3 H]-hydroxyproline
from [ 3 H]-proline [16]. The amounts of radioactive hydroxyproline
were counted in a scintillation counter.
Pulse-chase experiment
The confluent cell cultures were incubated for 48 h with
radioactive proline as described in the Experimental Procedure.
The medium was removed, the cell layer was washed
with PBS and submitted to incubation in high and low glucose
medium (without serum), containing non-labelled 10
mM proline (“0” time) and incubated for 4 h. After this time
the cells were lysed, cell lysates were submitted to filtration
assay (to measure total protein) or to the determination of
collagenase-sensitive proteins. The decrease in the amounts
of total and collagen- sensitive proteins was calculated.
Detection of gelatinolytic activity
Gelatinolytic activity in cell culture lysates was studied
with zymographic method described by Woessner [18].
In order to activate zymogen forms of metalloproteinases
some lysates were submitted to the action of p-aminophenylmercuric
acetate (APMA). The lysates were mixed with
Laemmli sample buffer [17] containing 2.5% SDS (without
reducing agent). 30 μg of protein were submitted to electrophoresis
on 10% polyacrylamide gels containing gelatin at a
concentration of 1 mg/ml, under non-reducing conditions.
After electrophoresis, the gels were incubated first in 2%
Triton X-100 at 37°C for 30 min to remove SDS and then in
substrate buffer (50 mM Tris-HCl, pH 8.0 containing 5mM
CaCl 2
) at 37°C for 24 h. After staining with 1% Coomassie
Brilliant Blue R 250, the gelatine-degrading enzymes
present in cell culture lysates were identified as clear zones
in a blue background.
Statistical analysis
The mean values for six assays ± standard deviations
(SD) were calculated. Statistical analysis was performed using
Student’s ‘t’-test.
Results
It can be seen from Fig. 1A that fibroblasts incubated
in high glucose medium incorporated significant amounts
of radioactive proline into high molecular weight proteins.
Sodium dodecyl sulphate /Polyacrylamide gel electrophoresis
(SDS/PAGE)
The cell cultures were washed with cold PBS and solubilised
in 200 μl lysis buffer per well. The lysates (containing
both cells and extracellular matrix lysis products) were
centrifuged at 10,000 x g, at 4 O C, for 10 min. The samples of
lysates containing 30 μg of protein were subjected to SDS-
PAGE, as described by Laemmli [17]. The following Bio-Rad’s
unstained standards were used: myosin (200 kDa), galactosidase
(116.2 kDa), phosphorylase b (97.4 kDa), bovine serum
albumin (66.2 kDa), ovalbumin (45 kDa). The electrophoresis
was run for 40–45 minutes. In each experiment 7.5 % polyacrylamide
gel and constant current (25 mA) were used.
Immunoblotting
The proteins were transferred to nitrocellulose membranes
and then pre-treated with Tris-buffered saline (TBS)
containing 0.05% Tween 20 (TBS-T) and 5% non fat dry
milk, at room temperature, for 2 hours. Membranes were
probed with a mixture containing anti-ORP150 antibody (1:
1000) in 5% dried milk in TBS-T at 4 O C for 16 h. Then,
the alkaline phosphatase conjugated antibody against mouse
IgG (whole molecule) was added at concentration 1:7500
in TBS-T for 1 h with slow shaking. The nitrocellulose was
washed with TBS-T (five times for 5 min) and exposed to
Sigma-Fast BCIP/NBT reagent.
Fig. 1. The effect of glucose deprivation on the incorporation of [ 3 H]-
L-proline into: (A) total proteins, (B) collagenase-sensitive proteins,
(C) hydroxyproline-containing proteins by human skin fibroblasts incubated
in high glucose (white bars) and low glucose (black bars)
DMEM for 12 h, 24 h and 48 h. Mean values from 3 experiments ±
SD are presented. * p< 0.001.
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