Real-time PCR identification of lake whitefish Coregonus clupeaformis in the Laurentian Great Lakes.

The purpose of this study was to develop a real-time PCR assay to specifically identify lake whitefish Coregonus clupeaformis in larval fish assemblages based on a 122 bp amplicon from the mitochondrial genome. The efficiency of the reaction, as calculated from the standard curve, was 90.77% with the standard curve having an r(2) value of 0.998. Specificity of the assay provided single melt peak in a melt-curve analysis and amplification of only the target species. The assay successfully identified target DNA in as low as 0.1% proportion of a DNA mixture. This assay was designed on the portable Smart Cycler II platform and can be used in both field and laboratory settings to successfully identify C. clupeaformis.

Spironolactone- and canrenone-induced changes in hepatic (Na+,K+)ATPase activity, surface membrane cholesterol and phospholipid, and fluorescence polarization in the rat.

We studied changes in hepatic membrane (Na+,K+)ATPase activity and membrane lipids induced by canrenoate, the water-soluble congener of canrenone, the active metabolite of spironolactone. (Na+,K+)ATPase activity was decreased after canrenoate in a dose- and time-dependent manner. Decreased activity was demonstrated at the lowest dose (91% of control after 5 mumoles per 100 gm body weight per day X 3 days); the maximum dose (30 mumoles per 100 gm body weight per day X 3 days) resulted in activity 38% of untreated control values. A 20 mumoles per 100 gm body weight per day dose decreased enzyme activity to 89 and 55% of control after 24 and 72 hr, respectively. The nonionic detergent Triton WR-1339 partially reversed drug-induced inhibition, suggesting that the enzyme changes may be related to altered membrane lipids. Membrane cholesterol increased 17% after 3 days of 30 mumoles canrenoate per 100 gm body weight per day; phospholipids decreased by 12%. The cholesterol to phospholipid molar ratio increased from 0.419 to 0.555. Membrane fluidity, as measured by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene decreased after treatment with 20 mumoles canrenoate per 100 gm body weight per day for 3 days. These results describe in vivo and in vitro inhibition of hepatic (Na+,K+)ATPase activity. Increased membrane cholesterol with decreased phospholipid alters membrane fluidity and may be partially responsible for the change in (Na+,K+)ATPase activity.

The effect of ethanol on hepatic sodium plus potassium activated adenosine triphosphatase activity in the rat.

We studied the acute an chronic effects of ethanol on hepatic Na+, K+-ATPase activity. Graded doses of ethanol given 1 h before death acutely increased Na+, K+-ATPase activity in a dose-related manner to a maximum of 138% of control with a simultaneous serum ethanol of 35.4 mM (163 mg%). Rats chronically fed 10% ethanol as their only fluid for 5 days had normal Na+, K+-ATPase activity, however, when ethanol was withdrawn 14 h before death Na+, K+-ATPase activity fell to 60% of control. The in vitro addition of ethanol to membrane preparations from untreated rats caused an increase in Na+, K+-ATPase activity with ethanol concentrations less than 81 mM with decreased activity at higher concentrations. In membranes from rats treated chronically with ethanol Na+, K+-ATPase activity did not respond to in vitro ethanol while the decreased activity noted after ethanol withdrawal was reversed by in vitro ethanol. These results suggest hepatocytes respond to the acute effects of ethanol by membrane adaptation which restores Na+, K+-ATPase activity to normal in the presence of ethanol. This adaptive response is probably due to changes in membrane lipids modulating membrane fluidity.