BIOCHEMISTRY: NIRENBERG AND MATTHAEI PROC. N. A. S.template RNA. Other explanations, however, are fully plausible, and it is not possible at this state to rule out alternative interpretations. In the following paper, further experiments on amino acid incorporation using the system described here are presented. It will be shown that in addition to the usual requirements, the system is stimulated by template RNA. Summary.-Cell-free E. coli extracts have been obtained which actively incorporate amino acids into protein. Methods were devised whereby these extracts could be dialyzed and stored for long periods of time at -150 without undue loss of activity. The characteristics of amino acid incorporation by such stored extracts were strongly suggestive of de novo protein synthesis, for incorporation required both ribosomes and 105,000 X g supernatant fractions, ATP and an ATPgenerating system, was stimulated by a mixture of other L-amino acids, and was markedly inhibited by puromycin, chloramphenicol, and RNAase. The initial rate of amino acid incorporation was not inhibited by DNAase; subsequent incorporation was greatly inhibited. The possible relationship of the DNAase inhibition of amino acid incorporation into protein to the synthesis of "messenger" RNA was briefly discussed.* Supported by a NATO Postdoctoral Research Fellowship.1 Tissikres, A., D. Schlessinger, and F. Gros, these PROCEEDINGS, 46, 1450. 2 Matthaei, J. H., and M. W. Nirenberg, Fed. Proc., 20, 391 (1961).3 Kameyama, T., and G. D. Novelli, Biochem. Biophys. Res. Comm., 2, 393 (1960). 4 Kirsch, J. F., P. Siekevitz, and G. E. Palade, J. Biol. Chem., 235, 1419 (1960). 5 Mora, P. T., E. Merler, and P. Maury, J. Am. Chem. Soc., 81, 5449 (1959). 6 Anson, M. L., J. Gen. Physiol., 22, 79 (1938). 7 Sevag, M. G., D. B. Lackmann, and J. Smolens, J. Biol. Chem., 124, 425 (1938). 8 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, ibid., 193, 265 (1951). 9 Allfrey, V. G., and A. E. Mirsky, these PROCEEDINGS, 44, 981 (1958).10 Hurwitz, J., A. Bresler, and R. Diringer, Biochem. Biophys. Res. Comm., 3, 15 (1960).11 Stevens, A., ibid., 3, 92 (1960).12 Weiss, S. B., and T. Nakamoto, J. Biol. Chem., 236, PC18 (1961). 13 Siekevitz, P., ibid., 195, 549 (1952

BackgroundGraft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection.Methods and findingsTraditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy-proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%–41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%–3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%–10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)–positive, rejection-free patients. LFTs had low overall correlations (r = 0.28–0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%–98.0%) and 92.9% (95% CI 89.3%–95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%–100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits.ConclusionsIn this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further r...

The low bioavailability of the anti-migraine drug sumatriptan is partially caused by first-pass hepatic metabolism. In this study, we analyzed the impact of the hepatic organic cation transporter OCT1 on sumatriptan cellular uptake, and of OCT1 polymorphisms on sumatriptan pharmacokinetics. OCT1 transported sumatriptan with high capacity and sumatriptan uptake into human hepatocytes was strongly inhibited by the OCT1 inhibitor MPP(+) . Sumatriptan uptake was not affected by the Met420del polymorphism, but was strongly reduced by Arg61Cys and Gly401Ser, and completely abolished by Gly465Arg and Cys88Arg. Plasma concentrations in humans with two deficient OCT1 alleles were 215% of those with fully active OCT1 (P = 0.0003). OCT1 also transported naratriptan, rizatriptan, and zolmitriptan, suggesting a possible impact of OCT1 polymorphisms on the pharmacokinetics of other triptans as well. In conclusion, OCT1 is a high-capacity transporter of sumatriptan and polymorphisms causing OCT1 deficiency have similar effects on sumatriptan pharmacokinetics as those observed in subjects with liver impairment.

Fenoterol is a widely used anti-asthmatic and tocolytic agent, but high plasma concentrations of fenoterol may lead to severe and even fatal adverse reactions. We studied whether heritable deficiency of the liver organic cation transporter 1 (OCT1), a trait observed in 3% of Europeans and white Americans, affects fenoterol plasma concentrations and toxicity. OCT1 transported fenoterol with high affinity, and OCT1 inhibition in human hepatocytes reduced fenoterol uptake threefold. After administration of 180 µg of fenoterol to 39 healthy individuals, the OCT1-deficient individuals (zero active OCT1 alleles; n = 5) showed 1.9-fold greater systemic fenoterol exposure (P = 4.0 × 10 ) and 1.7-fold lower volume of distribution (P = 8.0 × 10 ). Correspondingly, the OCT1-deficient individuals had a 1.5-fold stronger increase in heart rate (P = 0.002), a 3.4-fold greater increase in blood glucose (P = 3.0 × 10 ), and significantly lower serum potassium levels. In conclusion, heritable OCT1 deficiency significantly increases plasma concentrations of fenoterol and may be an important factor underlying the excess mortality associated with fenoterol.

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