U2800a spectrophotometer

The detection of lipolysis rate was performed as previously described by Berger and Barnard [41 (link)]. Briefly, samples (0.2 g perirenal adipose tissues) were minced, and then mixed in 2 mL of 25 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (pH 7.4) containing 1 μM isoproterenol at 37°C. A glycerol assay kit (Randox Laboratories, Antrim, UK) was used to measure the glycerol levels after 1, 2, and 3 h of incubation. A Hitachi U2800A spectrophotometer was used to detect the absorbance at 520 nm. An equation of micromoles of glycerol released per gram of adipose tissue per hour indicated the lipolysis rate.
The activity of LPL in the adipose tissues was analyzed as previously described by Kusunoki et al. [42 (link)]. Briefly, samples (0.1 g adipose tissues) were minced, and then mixed in Krebs–Ringer bicarbonate buffer (pH 7.4) containing 10 units/mL heparin for 60 min at 37 °C. The sample solution was then reacted with an equal volume of p-nitrophenyl butyrate (2 mM). A Hitachi U2800A spectrophotometer was used to measure the absorbance at 400 nm. The amount of p-nitrophenol formation over the 10 min incubation indicated the LPL activity.

To anneal the DNA duplexes (Tables 1 and 2), solutions of complementary single-stranded oligonucleotides in a buffer consisting of 10 mM Tris-HCl (pH 7.8), 150 mM KCl, and 10 mM MgCl₂ were heated at 90°C for 5 min and then were slowly cooled down to the room temperature. Thermal stability of the duplexes (0.4–0.5 μM) was determined from the dependence of the solution’s optical density on temperature. The measurements were performed in triplicate on a U-2800A spectrophotometer (Hitachi, Japan) equipped with an SPR-10 temperature regulator, in 1 cm quartz cuvettes (Hellma, Germany) at 260 nm. DNA duplexes were incubated at 15°C for 10 min and then heated to 65°C during 100 min. Melting temperatures of the DNA duplexes were calculated as a maximum of f'(T) = ΔA₂₆₀/(ΔT); the standard error did not exceed 1°C.