Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay.

A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.cDNA complexes. The method has comparable sensitivity to 32P-based methods and yields results that are quantitative and highly reproducible. Furthermore, the test can be performed using unfractionated cytoplasm without the need for extraction with organic solvents. This technique provides a rapid and quantitative method for studying changes in cellular mRNA levels, and it is suitable for testing large numbers of samples.

Human apolipoprotein A-I and A-II metabolism.

The kinetics of the major apolipoproteins (apo) of plasma high density lipoproteins (HDL), apoA-I and apoA-II, were examined in a total of 44 individual tracer studies in 22 normal male and female subjects. Following the intravenous injection of radioiodinated HDL, the specific radioactivity decay of apoA-I within HDL (residence time, 5.07 +/- 1.53 days), as determined by column chromatography, was significantly (P < 0.01) faster than that of apoA-II (residence time, 5.96 +/- 1.84 days). The specific radioactivity decay of apoA-I within HDL when labeled on HDL or as apoA-I was found to be almost identical. Similar results were obtained for apoA-II. Analysis of simultaneous paired radiolabeled apoA-I and apoA-II studies revealed that the mean apoA-I plasma residence time (4.46 +/- 1.04 days) was significantly (P < 0.01) shorter than that for apoA-II (4.97 +/- 1.06 days). Females had significantly (P < 0.01) higher apoA-I plasma concentrations (124 +/- 24 mg/dl) and apoA-I synthesis rates (13.58 +/- 2.23 mg/kg. day) than did males (108 +/- 16 mg/dl, and 11.12 +/- 1.92 mg/kg. day, respectively). Plasma apoA-I levels were correlated with plasma apoA-I residence times, but not synthesis rates; and apoA-II concentrations were correlated only with apoA-II whole body residence times. ApoA-I and apoA-II plasma residence times were inversely correlated with plasma triglyceride levels. These data are consistent with the following concepts: 1) labeling of apoA-I and apoA-II as apolipoproteins or on HDL does not affect their specific radioactivity decay within HDL; 2) the mean residence time of apoA-I both in plasma and in HDL is significantly shorter than that of apoA-II; 3) the increased apoA-I levels seen in female subjects are due to increased apoA-I synthesis; and 4) the plasma apoA-I residence time, which is inversely correlated with plasma triglyceride levels, is an important determinant of apoA-I concentration in both males and females.-Schaefer, E. J., L. A. Zech, L. L. Jenkins, T. J. Bronzert, E. A. Rubalcaba, F. T. Lindgren, R. L. Aamodt, and H. B. Brewer, Jr. Human apolipoprotein A-I and A-II metabolism.

Metabolism of high density lipoprotein subfractions and constituents in Tangier disease following the infusion of high density lipoproteins.

The metabolism of apolipoproteins A-I and A-II, as well as other high density lipoprotein (HDL) constituents, was studied in patients with homozygous familial HDL deficiency (Tangier disease) prior to and after plasma exchange or HDL infusion. Mean plasma apoA-I, apoA-II, and HDL cholesterol values in homozygotes (n = 2) were 2.0 mg/dl, 2.7 mg/dl, and 1.5 mg/dl, respectively, and in a normal control subject were 125.1 mg/dl, 23.0 mg/dl, and 53.0 mg/dl, respectively. Based on radioiodinated apoA-I and apoA-II kinetic studies in the baseline state, synthesis rates for apoA-I and apoA-II in mg/kg/day were 3.81 and 1.61, respectively, in one homozygote (patient B) and 11.82 and 1.99, respectively, in the normal subject. ApoA-I and apoA-II plasma residence times in days were 0.22 and 0.81, respectively, in the homozygote, and 4.04 and 4.44, respectively, in the normal subject. These data indicate that this homozygote had both a moderate decrease in the synthetic rates of apoA-I and apoA-II, as well as a marked decrease in the plasma residence times of these two apolipoproteins. In one homozygote (patient A) following a complete plasma exchange during cardiopulmonary bypass, plasma HDL cholesterol, apoA-I, and apoA-II levels were very similar to pre-exchange values within 64 hr after exchange. A second homozygote (patient B) received HDL intravenously as well as 125I-labeled apoA-I and 131I-labeled apoA-II. Following infusion, the residence time in days for HDL subfractions, HDL2b, HDL2a, and HDL3 were 0.1, 0.8, and 2.7, respectively. HDL protein and phospholipid both had a monoexponential decay, with residence times of 0.7 days, while HDL triglyceride disappeared monoexponentially with a residence time of 0.5 days. HDL cholesterol had a biexponential decay, with the residence time of the slow component being 0.7 days. Plasma and HDL apoA-I decayed down to baseline values significantly faster than did plasma and HDL apoA-II. ApoA-II specific radioactivity decreased throughout the course of the infusion study in both plasma and HDL, while apoA-I specific radioactivity decreased slightly, then rose, and subsequently declined in both plasma and HDL. The data indicate that the rapid and altered catabolism of apoA-I and apoA-II in Tangier homozygotes persists despite major increases in the plasma pool size of these proteins. In addition, following HDL infusion, HDL2b and HDL2a disappeared at a faster rate than HDL3, HDL cholesterol and triglyceride were catabolized at a faster rate than HDL protein and phospholipid, and apoA-I disappeared more rapidly than apoA-II. These observations may have important implications with regard to the catabolism of HDL subfractions and constituents in normal man.

Isolation and structure elucidation of a novel griseorhodin.

Three antibiotics possessing cytotoxic properties were isolated from a strain of Streptomyces griseus (FCRC-57). One was found to be identical with griseorhodin A. A second, FCRC-57-U, was found to be identical to griseorhodin C. FCRC-57-G is a new antibiotic structurally related to griseorhodins A and C, and is active against KB cells in vitro. The structure of this new antibiotic was determined using mass spectrometry, proton and carbon nuclear magnetic resonance spectroscopy and synthesis.