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Endometriosis is a benign, though aggressive, disease of the female reproductive tract that consists of endometrial stromal and epithelial cells growing at an extrauterine site. Although it is widely accepted that the majority of cases of endometriosis result from the ectopic implantation of refluxed menstrual tissue, the precise mechanisms by which this disease becomes established are not well understood. Matrix metalloproteinases (MMPs), enzymes which are important for extracellular matrix turnover, have recently been implicated in the development of endometriosis. MMPs appear to be overexpressed in endometriotic lesions, but expression levels decrease following successful medical therapy. Intriguingly, although transforming growth factor-beta (TGF-beta) mediates progesterone suppression of specific endometrial MMPs, this growth factor is overexpressed in women with endometriosis. In the current study, we used an established experimental model of endometriosis to explore MMP regulation by TGF-beta. Our findings indicate that blocking the action of TGF-beta opposes progesterone-mediated suppression of MMPs and blocks the ability of this steroid to prevent experimental endometriosis. However, we also show that the action of TGF-beta does not lead to a sustained suppression of MMPs as observed following progesterone treatment. Taken together, our data suggest that in the absence of a normal progesterone response, common in ectopic lesions of endometriosis, sensitivity to TGF-beta may be altered, resulting in a failure to regulate MMPs.

The human mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily, which includes receptors for retinoic acid, vitamin D, and other steroids, such as the glucocorticoids (which bind the glucocorticoid receptor, GR). MR and GR, the corticosteroid receptors, share significant homology and are activated by steroid binding, resulting in a conformational change, nuclear translocation, and DNA binding. Despite these similarities with GR, the MR remains less well characterized. However, protein components known to be present in the unliganded GR are also likely to be components of the heteromeric MR complex. In the current study, we investigated whether or not hsp70, hsp90, and the immunophilin FKBP-52 are present in the nonsteroid-bound MR complex, because these proteins are known to be present in the unliganded GR complex. The unliganded MR complex was assembled in vitro using reticulocyte lysate and in vivo using the baculovirus overexpression system and Spodoptera frugiperda (Sf9) cells. Western blot analysis revealed the presence of hsp70, hsp90, and FKBP-52 in the unliganded complexes, but hsp90 and FKBP-52 were not detected following exposure to aldosterone. Electrophoretic mobility shift analysis demonstrated that DNA binding of MR occurred only after treatment with aldosterone. These studies indicate that proteins associated with the unliganded GR are also present in the unliganded MR complex, and that hsp90 and FKBP-52 dissociate prior to DNA binding in a manner similar to that described for GR. Finally, the stoichiometric analysis of the proteins present within the heteromeric MR complex suggests a divergence between this receptor and the GR.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.

Electrophoretic separation of oligonucleotides in denaturing polyacrylamide gels is primarily a function of length-dependent mobility. The 3' terminal nucleotide sequence of the oligonucleotide is a significant, secondary determinant of mobility and separation. Oligomers with 3'-ddT migrate more slowly than expected on the basis of length alone, and thus are better separated from the preceding, shorter oligomers in the sequencing ladder. Oligomers with 3'-ddC are relatively faster than expected, and are therefore less separated. At the 3' penultimate position, -dC- increases and -dT- reduces separation. Purines at the 3' terminal or penultimate positions of oligonucleotides affect separation less than the pyrimidines. These results suggest specific interactions among neighboring nucleotides with important effects on the conformation of oligonucleotides during electrophoresis. These interactions are compared to compression artifacts, which represent more extreme anomalies of length-dependent separation of oligonucleotides. Knowledge of base-specific effects on electrophoretic behavior of DNA oligomers supplements the usual information available for determination of sequences; additionally it provides an avenue to thermodynamic and hydrodynamic investigations of DNA structure.