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Human cytochrome P450 3A4is recognized as the catalyst for the oxygen-dependent metaboism of a diverse group of medicafly important chemicals, inWuding the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymaticafly active fusion protein (rF450[mHum3A4/mRatOR]Ll) has been expressed at high levels in Escherichia coli and purified to homogeneity.Enzymatic studies show a requirement for lipid, detergent, and cytochrome bs for the 63-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometricafly detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolim of both testosterone and erthromycin. These resuits relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.One ofthe most versatile ofthe cytochrome P450s is the form present in human liver called CYP3A4, which catalyzes the oxidative metabolism of a wide array of different chemicals with markedly different structural characteristics (1). Interest has centered on this P450 since it is reported to be one of the more abundant P450s in human liver (2); it is inducible by a variety of agents including glucocorticoids as well as phenobarbital (3); it appears to play a central role in the metabolism of the immunosuppressive cyclic peptide cyclosporin A as well as macrolide antibiotics, such as erythromycin (4); it also catalyzes the 63-hydroxylation of a number of steroids including testosterone, progesterone, and cortisol (5). Clinical interest relates to the measurement of erythromycin metabolism by a breath test (6) and the presence of6f-hydroxylated steroids in urine (7) as indicators of CYP3A4 function for evaluation of transplant recipients.P450s ofthe 3A family were first characterized by Guzelian and colleagues (8) based on the ability ofthe catabolic steroid pregnenolone-16a-carbonitrile to induce a unique form of P450 (which they called P-450p). They also recognized the ability of the macrolide antibiotic triacetyloleandomycin (TAO) to serve as a powerful inducer of this type of P450-a result of interest because of the known ability of TAO and other compounds to form stable, metabolite-inhibitor complexes with P450 (9). Reconstitution studies using purified P450s of the 3A family have shown the need to include phospholipids, detergents, and cytochrome b5 when testing enzymatic activities (10, 11).

Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp. pallidum was a surface-associated cellular component. Surface binding assays and immunoelectron microscopic studies also suggested that the analogous 47,000-dalton antigenic component of T. pallidum subsp. pertenue may not have been oriented toward the bacterial surface in the same way as the T. pallidum subsp. pallidum antigen or that the relevant antigenic determinant(s) may not have been exposed to the outer surface in the same way. The significance of this antigen relative to its apparent conservation among pathogenic treponemes and its possible diagnostic and vaccinogenic potentials are discussed.

Transformation experiments showed that spontaneous deletions which result in loss of streptomvcin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10 4 in plasnid molecules of R6K. Similar deletions were thus readily selected by conjuLgal transfer of R6K, and their appearance was dependent upon lrecAactivity in either donor or recipient host. The deoxvribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA' strains.In four plasmids examined after transfer to a lrecA host, an inverted repeat of the preexisting TnA element was shown to have been inseited at a similar location and was in two instances associated with deletions which extended from the same TnA terminus and in the same direction as those described above. The deletions are ascribed to the result of recA--dependent recombination between direct repeats of TnA.R6K is a 26-megadalton plasmid that determines resistance to anmpicillin (Ap) and streptomvcin (Sm). Although it is a conjugative plasnmid, it has the unusual property foisuch plasmids that it is relaxed, or multicopv in its replication, under normal conditions, being present in approximately 10 copies per chromosonmal equivalent (20).The tiansfer efficiency of R6K is low compared with derepressed fertility plasmids such as F, so that only about 10 transconjugant (R6K+) cells are produced after overnight mating of a 1:1 mixture of 10' donor and recipient cells (20). Fertility repression may be inferred from the findings of Pinnev and Smith (33), who showed that when R6K and the N-group plasmid R46 coexist in a cell, R6K inhibits both R46 transfer and plaque formation by the bacteriophage IKe, which specifically infects cells carrying N-group plasmids.Ap riesistance of H6K is mediated by the TEM-2 /l-lactamase enzyme (2 7), and in fact R6K was isolated from Escherichia coli TEM, in which this enzyme was first characterized, the plasmid at that time being termed RTf:N (10). The transposable element TnA, which determines Ap resistance through TEM, was later shown to be located on R6K (16). (TnA has been termed alternativelv Tn], Tn2, or Tn3 [3] depending on the plasmid of its oi-igin. However, since the TnA sequence on R6K was not derived by transposition from any of the above, and since this sequence is not transposed to other DNA molecules in these experiments, we will retain the generic term TnA.) The Ap ti-ansposon, Tn3, has been shown to transpose to plasmids such as pSC101 and RSF1010 (21,29,38) at a number of clustered, nonrandom sites, and it is reported that insertion occurs in either orientation with about equal frequency and without influence of the lrecA gene of E. (c-l (21, 29, 38).Deletions resulting from the activity of insert...

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