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Phosphatidylcholine metabolism and membrane fluidity were studied in microsomes isolated from rabbit lung, which had been exposed to high oxygen tension for 30 min. In these microsomes the incorporation of [3H]-palmitate into phosphatidylcholine increased whereas the incorporation of [14C]-glycerol and [14C]-choline from CDP-[methyl-14C]-choline remained unchanged in comparison to the control microsomes. The enhanced [3H]-palmitate incorporation may be explained by an increase of the specific activity of acyl-CoA:lysophosphatidylcholine acyltransferase which was measured in microsomes from hyperoxic lung. Although microsomal parameters influencing membrane fluidity, such as the cholesterol/phospholipid molar ratio, unsaturation degree of phospholipid acyl chains and lipid/protein ratio, are altered after oxygen treatment in vivo, no change of fluorescence polarization (PDPH) and lipid structural order parameter (SDPH) could be measured. Probably, the membrane maintains its fluidity by counteracting effects on different factors on which the fluidity depends.

792BIOCHEMICAL SOCIETY TRANSACTIONS on to Sephacryl S-1000 as previously described (Nicholls et al., 1987), and the other was labelled with 32P to an activity of around 3 x loy d.p.m./,ug, by random hexadeoxynucletide priming (Feinberg & Vogelstein, 1983). Nucleic acid was extracted from cervical scrapes using a conventional procedure (Wickenden et al., 1985), and denatured by heating. After rapid cooling, the sample was mixed with 200 fmol of fragment A and 5 fmol of fragment B in a screw-cap Eppendorf tube. Hybridization was allowed to proceed by rotation on a Voss wheel overnight at 65"C, in a buffer consisting of 40 mM-Pipes, pH 6.5, 0.6 M-NaCI, 1 mM-EDTA, 0.1% (w/v) SDS, and denatured, sonicated salmon sperm DNA (0.25 mg/ml). After washing by rotation for 3 x 10 min in the same buffer, results were quantified by Cerenkov counting, and the amount of HPV DNA in a sample determined by comparison with a standard curve.The results initially obtained demonstrated that the sandwich technique was capable of distinguishing between HPV types 6, 11, 16 and 18, and that mol ( 6 x lo6 molecules) of viral DNA could be reliably detected (Nicholls, 1989). However, HPV infections which lead to carcinoma have been associated with integration of viral DNA into a host cell chromosome, rather than with persistence of the DNA as a free-replicating episome (Pfister, 1984). Since the number of viral genomes per infected cell is likely to be much lower in the former case, it is essential to identify positive samples reliably, regardless of how little HPV nucleic acid is present. For this reason, we decided to attempt to increase the sensitivity of the sandwich assay by polymerase chain reaction (PCR)-mediated target amplification, before hybridization (Saiki et a [., 1985).Three pairs of HPV-derived PCR primers were synthesized, one pair each for amplifying HPV types 16 and 18, and a single pair for both HPV 6b and HPV 11. Primers were chosen so that the amplified region would have at least 50 base pairs overlap with the appropriate PstI restriction fragments used in the sandwich assay. To simplify the interpretation of results, the primers were designed to amplify a 206 bp region of HPV 6b/HPV 11, a 159 bp region of HPV 16, and a 119 bp region of HPV 18. An additional pair of primers were synthesized, for the amplification of a 119 bp region of the human B-globin gene, in order to confirm that the DNA isolated from a cervical scrape was suitable for PCR amplification.Nucleic acid was extracted from 20 cervical scrapes taken from women with cytological evidence of HPV infection. Approximately 0.5 pg of DNA from each scrape was subjected to amplification with each of the four sets of primers described above, using DNA polymerase from Thermus aquaticus. The conditions of amplification were exactly as described by Saiki et al. (1988). After amplification, 5 pl aliquots from each sample were subjected to sandwich hybridization for the detection of HPV types 6b, 11, 16 and 18, as well as for the detection of the amplified B-globin sequen...

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9–6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non‐specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.