Environmental Engineering Reference
In-Depth Information
buffer (pH 7.2) containing 5 mM DTT, 2 mM EDTA and 0.8 mM PMSF. The homog-
enate was centrifuged at 10000 g for 20 min at 4°C and the resulting supernatant was
ultracentrifuged at 100,000 g for 1 hr at 4°C. The microsomal fraction was then re-
suspended in 0.05 M phosphate buffer and the microsomal protein content was deter-
mined by the Bradford method. Twenty μg microsomal proteins were added to 0.05 M
phosphate buffer (pH = 7.2) containing 0.4 mM 7-ethoxycoumarin (7-Ec, Fluka) and
0.1 mM NADPH for a total reaction volume of 100 μL and incubated at 30°C. After
15 min, the reaction was stopped and the production of 7-hydroxycoumarin (7-OH)
by P450 monooxygenases was evaluated by measuring the fluorescence of each well
(380 nm excitation, 460 nm emission) with a Fluoroskan Ascent spectrofluorimeter
(Labsystems, Helsinski, Finland) in comparison with a scale of 7-OH (Sigma). The
P450 activities were expressed as mean pmoles of 7-OH per mg of microsomal protein
per min ± SE. Statistical comparison of P450 activities between the two strains was
performed by using a Mann and Whitney test (N = 15).
Glutathione S-transferase activities were comparatively measured on 200 μg of
cytosolic proteins from the 100,000 g supernatant (see above) with 1-chloro-2,4-dini-
trobenzene (CDNB, Sigma) as substrate [64]. Reaction mixture contained 2.5 ml of
0.1 M phosphate buffer, 1.5 μM reduced glutathione (Sigma), 1.5 μM CDNB and 200
μg proteins. The absorbance of the reaction was measured after 1 min at 340 nm with
a UVIKON 930 spectrophotometer. Results were expressed as mean nmoles of conju-
gated CDNB per mg of protein per min ± SE. Statistical comparison of GST activities
between the two strains was performed by using a Mann and Whitney test (N = 15).
Carboxylesterases activities were comparatively measured on 30 μg of cytosolic
proteins from the 100,000 g supernatant (see above) according to the method de-
scribed by Van Asperen et al. [65] with α-naphthylacetate and β-naphthylacetate used
as substrates (α-NA and β-NA, Sigma). Thirty μg cytosolic proteins were added to
0.025 mM phosphate buffer (pH 6.5) with 0.5 mM of α-NA or β-NA for a total volume
reaction of 180 μL and incubated at 30°C. After 15 min, reaction was stopped by the
addition of 20 μL 10 mM Fast Garnett (Sigma) and 0.1 M sodium dodecyl sulfate
(SDS, Sigma). The production of α- or β-naphthol was measured at 550 nm with a
Σ960 microplate reader (Metertech, Taipei, Taiwan) in comparison with a scale of
α-naphthol or β-naphthol and expressed as mean μmoles of α- or β-naphthol per mg of
cytosolic protein per min ± SE. Statistical comparison of esterase activities between
the two strains was performed by using a Mann and Whitney test (N = 30).
Kdr Genotyping
Genomic DNA was extracted from whole adult mosquitoes of the Bora-Bora and Vau-
clin strains by grinding tissues with a sterile micro-pestle in DNA extraction buffer
(0.1 M Tris HCl pH 8.0, 0.01 M EDTA, 1.4 M NaCl, 2% cetyltrimethyl ammonium
bromide). The mixture was incubated at 65°C for 5 min. Total DNA was extracted
with chloroform, precipitated in isopropanol, washed in 70% ethanol, and resus-
pended in sterile water. The
kdr
genomic region was amplified by PCR using Dip3
(5'-ATCATCTTCATCTTTGC-3') and Dip2A (5'-TTGTTGGTGTCGTTGTCGGC-
CGTCGG-3') primers. PCR steps included an initial denaturation step at 95°C for 3
min, followed by 45 cycles at 95°C for 30 sec, 48°C for 30 sec, and 72°C for 45 sec,
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