The antiestrogen ICI 164,384 (ICI) binds the estrogen receptor (ER) with approximately 20% the affinity of estradiol, but without the partial agonistic effects caused by tamoxifen. Investigations into the mechanism of ICI action have used ER molecules expressed in vitro to examine the binding of ER to ICI and the capacity of ICI-ER complexes to dimerize and bind to the estrogen response element (ERE). Our objectives were to study the biological effects, cellular distribution, and ERE-binding capacity of native uterine ICI-ER complexes after ip injection of 1 mg/kg ICI into 10-day castrate adult female mice. Synthesis of DNA and progesterone receptor were measured as end points of agonistic activity. ICI failed to stimulate either DNA or progesterone receptor synthesis above control levels, and pretreatment with ICI for 0.5 h reduced the stimulatory effect of estradiol by 75%. Measurement of uterine nuclear ER and cytosolic levels by exchange binding assay indicated a reduction in total ER levels within 0.5 h after ICI treatment, which remained below 20% for 24 h. Cycloheximide treatment did not block the ICI effect. Western blot analysis, immunohistochemistry, and steroid autoradiography confirmed the loss of ER protein. The ICI effect on ER was also demonstrable in vitro in the mouse TM4 estrogen-responsive cell line. ICI dramatically reduced ER levels to 5% of the control value by 4 h. Northern analysis indicated that ICI did not affect ER message levels, suggesting that the observed reduction in ER did not occur at the level of transcription. Gel shift assays indicated a low, but detectable, amount of ICI-ER binding to the vitellogenin A2 (VitA2) ERE. These results suggest that, although the ICI-ER complex binds weakly to DNA, ICI may cause its antagonistic effect by producing a rapid disappearance of the ER from the target tissue, resulting in an insufficient amount of ER to bind the native ligand and elicit agonist responses.
The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.
Androgens regulate growth of the rat ventral prostate gland. In a search for possible mediators of androgen stimulated growth we have studied c-myc proto-oncogene expression in ventral prostate after androgen withdrawal and replacement. Steady state levels of c-myc mRNA were determined by Northern hybridization and compared with mRNA levels for prostatein, the major androgen dependent protein of ventral prostate. C-myc mRNA in ventral prostate increased nearly 4-fold within 1 day and 6- to 7-fold within 2 days after castration. Administration of androgen at the time of castration prevented this increase in c-myc mRNA levels. Androgen treatment of 4-day castrate rats caused c-myc mRNA levels to decrease within 4 h. Cycloheximide increased c-myc mRNA severalfold within 2 h. The net increase in c-myc mRNA after cycloheximide treatment was greater in the castrate than in the noncastrate or in androgen-treated castrate rats. These results suggest that androgen may influence both transcription and turnover of c-myc mRNA. Prostatein C3 mRNA decreased rapidly after castration and increased after androgen treatment of the castrate but was only slightly influenced by cycloheximide. Steady state levels of c-myc mRNA were higher in the 10-day-old rat and decreased with age while prostatein C3 mRNA increased with age. In situ hybridization demonstrated that both c-myc and prostatein mRNAs are expressed in the epithelial cells of ventral prostate acinar glands. These data indicate that androgens regulate the expression of c-myc in the ventral prostate.
Pseudomonas cepacia utilized penicillin G as the sole source of carbon and energy. We report here an unexplained correlation between lysine auxotrophy and ,8-lactamase deficiency, resulting in loss of capacity to utilize penicillin. Pseudomonas cepacia (P. multivorans) is recognized as the most nutritionally versatile of the pseudomonads (1, 11). As we report here it includes penicillin G (Pen) in its repertoire of utilizable substrates. All wild-type strains of P. cepacia we examined and certain strains of P. marginata and P. caryophylli utilized penicillin G (benzylpenicillin) as the sole source of carbon and energy (see Table 1 for a list of representative strains). The same strains failed to exhibit significant growth with the benzylpenicillin derivatives ampicillin or carbenicillin, but were resistant to these antibiotics. P. cepacia 249, the strain we studied in greatest detail, grew with a mean generation time of 6.5 h in inorganic salts medium containing 2% (wt/vol) penicillin G as the sole carbon source. Neither the closely related P. pickettii (9, 10) strains listed in Table 1 nor the less closely related pseudomonads P. aeruginosa PU21-(RP1) llv-Penr (obtained from G.
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