Randal C. Jaffe

Corticosterone concentrations in mice during ethanol drinking and withdrawal

Consumption of an ethanol‐containing diet by mice resulted in a significant increase in circulating concentrations of corticosterone which was maintained for 8 days. There were no changes in the concentrations of plasma corticosterone binding globulin. Ethanol withdrawal symptoms followed the removal of ethanol from the diet and circulating corticosterone concentrations were further increased. There was no correlation between blood ethanol and glucocorticoid concentrations during the chronic ethanol treatment. Stress related to ethanol consumption may be of greater importance than the circulating ethanol concentrations in producing the elevation in plasma glucocorticoids.

Regulation of Insulin-Like Growth Factor Binding Protein-1 Promoter Activity by FKHR and HOXA10 in Primate Endometrial Cells

Abstract Insulin-like growth factor binding protein-1 (IGFBP-1) is abundantly expressed in the liver and decidualized endometrium. FKHR, a FOXO forkhead transcription factor, stimulates IGFBP-1 promoter activity in liver cells through the insulin response sequences (IRSs). HOXA10, a homeobox transcription factor, is important in the decidualization process. Here we show that FKHR and HOXA10 are expressed in baboon endometrium during the menstrual cycle and pregnancy. Levels are lowest during the follicular phase and highest in pregnancy. Reporter gene studies reveal that FKHR stimulates both baboon and human IGFBP-1 promoter activity, whereas HOXA10 alone has a relatively weak effect. When FKHR and HOXA10 are expressed together, promoter activity is markedly up-regulated, which is indicative of cooperativity. A DNA binding-deficient FKHR mutant fails to stimulate promoter activity, even in the presence of HOXA10, and deletion or mutation of IRSs also disrupts the effect of FKHR and cooperativity with HOXA10. Conversely, the IRS region placed upstream of the 31 base pair IGFBP-1 minimal promoter is sufficient to mediate effects of FKHR and cooperativity with HOXA10. Pull-down studies reveal physical association between GST-FKHR and 35S-HOXA10. These studies show that FKHR and HOXA10 interact directly and can function cooperatively to stimulate IGFBP-1 promoter activity in endometrial cells and perhaps in other settings.

Plasma concentration of corticosterone during Rana catesbeiana tadpole metamorphosis.

Abstract Plasma corticosterone concentrations have been studied in Rana catesbeiana tadpoles during metamorphosis. Corticosterone concentrations, determined by radioimmunoassay, in tadpole plasma were low prior to the onset of metamorphic climax (stage XX). The concentration of corticosterone in tadpole plasma from animals stages XXI to XXIV were significantly elevated above the concentration seen in animals of earlier stages. Adrenocorticotropin (ACTH) was able to stimulate an increase in plasma corticosterone in tadpoles of stages I to X. Triiodothyronine (T 3 ) treatment of tadpoles for 7 days caused a slight but insignificant increase in plasma corticosterone concentration over the concentration observed in control animals. The T 3 -treated animals had, however, a significantly greater elevation of plasma corticosterone in response to ACTH than control animals.

The impact of ovulation on fallopian tube epithelial cells: Evaluating three hypotheses connecting ovulation and serous ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy affecting American women. Current hypotheses concerning the etiology of ovarian cancer propose that a reduction in the lifetime number of ovulations decreases ovarian cancer risk. Advanced serous carcinoma shares several biomarkers with fallopian tube epithelial cells, suggesting that some forms of ovarian carcinoma may originate in the fallopian tube. Currently, the impact of ovulation on the tubal epithelium is unknown. In CD1 mice, ovulation did not increase tubal epithelial cell (TEC) proliferation as measured by bromodeoxyuridine incorporation and proliferating cell nuclear antigen staining as compared to unstimulated animals. In superovulated mice, an increase in the number of pro-inflammatory macrophages was detected in the oviduct. Ovulation also increased levels of phospho-γH2A.X in TEC, indicating that these cells were susceptible to double-strand DNA breakage following ovulation. To determine which components of ovulation contributed to DNA damage in the fallopian tube, an immortalized baboon TEC cell line and a three-dimensional organ culture system for mouse oviduct and baboon fallopian tubes were developed. TEC did not proliferate or display increased DNA damage in response to the gonadotropins or estradiol alone in vitro. Oxidative stress generated by treatment with hydrogen peroxide or macrophage-conditioned medium increased DNA damage in TEC in culture. Ovulation may impact the fallopian tube epithelium by generating DNA damage and stimulating macrophage infiltration but does not increase proliferation through gonadotropin signaling.

Prolactin and tadpole metamorphosis evidence of prolactin receptors in premetamorphic Rana catesbeiana liver and tail fin

The binding characteristics of ovine prolactin (OPRL) to a particulate fraction from liver and tail fin of Rana catesbeiana tadpoles were studied. The specific binding of [125I]oPRL to both tissues was found to be a saturable process with a single class of binding sites in each tissue. Although the dissociation constants were similar for each tissue, the tail fin demonstrated a 10-fold higher binding capacity than the liver tissue. Pretreatment of the liver and tail fin particulate fractions with degradative enzymes revealed that trypsin and phospholipase C reduced the subsequent specific [125I]oPRL binding in both tissues. However, neuraminidase treatment decreased the prolactin binding in the liver while having no effect on the tail fin. The binding of prolactin to the amphibian tissues was found to be specific for prolactin and growth hormones. [125I]oPRL binding to both tissues was a reversible process although the dissociation rate was faster for the tail fin than for the liver. Therefore, prolactin receptors are associated with both a prolactin responsive tissue, the tail, and an unresponsive tissue, the liver, in the tadpole.

Cytosol and nuclear progesterone receptor in cat uterus and oviduct.

Abstract Binding of progesterone (P) to a specific cytosol receptor and the in vivo translocation of the cytosol receptor complex to the nucleus was studied in the cat uterus and oviduct. Cytosol and nuclear fractions were prepared from the reproductive tracts of ovariectomized animals treated for 14 days with estradiol (E 2 ) and from those which were subsequently infused with a P solution for 15 min. In vitro cytosol incubation with [ 3 H]-P included cortisol to eliminate binding to CBG. The specific binding of [ 3 H]-P reached equilibrium within 90 min at 0°C and was stable for at least 4 h in both tissues. Competitive binding studies revealed the following binding affinities for the cytosol receptor; R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) = P ⪢ corticosterone = E 2 = testosterone. Scatchard analysis of [ 3 H] P binding in cytosols from E 2 -treated animals yielded a K D of 3.2 × 10 −8 M in the uterus and 2.5 × 10 −8 M in the oviduct. The P binding protein had a sedimentation coefficient of 6.8 S. The content of nuclear receptor was assessed by an exchange assay using R5020. Equilibrium was reached within 4 h at 22°C and remained unchanged for another 2h. The relative binding affinities for the nuclear receptor were; R5020 > P > corticosterone > testosterone > E 2 . The K D was 8.2 × 10 −9 M in uterine nuclei and 6.0 × 10 −9 M in oviduct nuclei from E 2 + P treated animals. Following P-infusion the decrease in the quantity of specifically bound [ 3 H]-P in the cytosol, and the increase in the quantity of specifically bound [ 3 H]-R5020 in the nuclei, were significant in both tissues.

Partial characterization of frog (Rana catesbeiana) hepatic glucocorticoid receptor

The binding of the synthetic glucocorticoid dexamethasone to Rana catesbeiana frog hepatic cytosol has been characterized partially. Specific binding of [3H]dexamethasone at 0° reached a maximum after 2 hr of incubation. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 5.51 × 10−9M and a binding capacity of 1.99 × 10−13 mol dexamethasone/mg protein. The binding was a reversible process (t12 = 5.10 hr). Binding of [3H]dexamethasone was not affected by pretreatment of cytosol with ribonuclease A or deoxyribonuclease but was reduced drastically when cytosol was pretreated with trypsin. The receptor was specific for glucocorticoids as [3H]dexamethasone was displaced by dexamethasone, corticosterone, and cortisol to a greater extent than by aldosterone, progesterone, 17β-estradiol, and testosterone. On sucrose gradients in low salt, two areas of [3H]dexamethasone were found (3 and 6.8 S), while in 0.3 M KCl only a single peak with a sedimentation coefficient of 3 S was noted.

Characteristics of receptors and enzymes in brains of human alcoholics

Acute and chronic ethanol administration to animals has been shown to produce changes in the turnover of numerous neurotransmitters, as well as to change the characteristics of certain neurotransmitter receptors. In the present study, brains obtained from human alcoholics and matched control subjects were examined for similar changes. In frontal cortex, the affinity of opiate receptors for dihydromorphine was significantly reduced in brains of alcoholics. Judging from animal studies, this change may reflect alterations in opiate receptor-effector coupling processes. No changes were observed in muscarinic cholinergic or beta-adrenergic receptors in humans, in contrast to animals, possibly because of the protracted abstention from alcohol among the alcoholic patients prior to death. Similarly, no changes in the activities of choline acetyltransferase, tyrosine hydroxylase or MAO-B were observed in brains of alcoholics as compared to the control population. Our studies suggest that many of the alterations witnessed during alcohol intoxication and withdrawal in animals may be more subtle in humans, or may be reversible with abstinence.

Changes in specific prolactin binding in Rana catesbeiana tadpole tissues during metamorphosis and following prolactin and thyroid-hormone treatment ☆

The prolactin-binding affinity (KD) and number of binding sites *N) in Rana catesbeiana tadpole liver, tail fin and kidney tissues were studied during metamorphosis and following administration of oPRL and L-T3 to premetamorphic tadpoles. With increasing developmental stage there was an increase in N; a maximum was found at stage XVIII followed by a gradual decrease in N through metamorphic climax for all 3 tissues. No change in KD was noted. L-T3 treatment of premetamorphic tadpoles for 7 days caused a significant decrease in tail length and height and body length and an increase in hindlimb length with a concurrent increase in N of approximately 3-fold while treatment for 1 or 3 days was without effect on tadpole morphology or oPRL binding. OPRL treatment for 7 days caused a significant increase in tail length and height and body length with no significant changes in hindlimb length and a 3-5-fold increase in N. Treatment with both L-T3 and oPRL for 7 days resulted in an inhibition of the T3-induced decrease in tail length and height and body length and no inhibition of the hindlimb length increase. N increased in all tissues similar to that found with eight treatment alone. No change in KD was noted in any of these studies. Therefore, oPRL and L-T3 are able to regulate the numbers of specific oPRL-binding sites in amphibian tissues. The change in N with development parallels the reported change in tadpole pituitary capacity to stimulate growth but occurs prior to the reported surge of endogenous T3 during metamorphosis. Thus, the variation in the number of oPRL-binding sites may be due to the changes in endogenous PRL levels during development.

Histone deacetylase inhibition and progesterone act synergistically to stimulate baboon glycodelin gene expression

During the late luteal phase of the menstrual cycle and early pregnancy, the major secretory product of the uterine glandular epithelial cells in humans and non-human primates is glycodelin. Previous studies using Ishikawa cells, a human endometrial cell line, have shown that a chimeric plasmid containing the baboon glycodelin promoter responds to progestins but the response is modest compared with the induction of glycodelin seen in vivo and in gene array analysis. A recent report indicating that the histone deacetylase inhibitor trichostatin A (TSA) promoted glycodelin expression prompted us to examine its mechanism of action. In Ishikawa cells transfected with the baboon glycodelin promoter, TSA and the synthetic progestin medroxyprogesterone acetate both stimulated expression of the reporter and the combined treatment produced a synergistic effect. The effect of TSA and progestin was absent when the same promoter constructs were transfected into COS-1 cells, a kidney cell line, and a TSA effect but no progestin effect was observed in T47D cells, a mammary cell line. Through deletion analysis, the TSA action was localized to the -67/-52 region of the baboon glycodelin promoter, a region which contains the proximal Sp1 site. Deletions of this same region had no effect on progestin responsiveness. Our findings indicate that at least two regions of the glycodelin promoter are important for the normal induction of glycodelin expression. Non-target cells may lack factors which act on the response elements resulting in the restriction of expression to the appropriate target tissue.