Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.

Endometrial and vaginal effects of low-dose estradiol delivered by vaginal ring or vaginal tablet

Aims The major aims of the study were to compare the safety of a continuous low-dose estradiol-releasing vaginal ring (ESTring) to that of a vaginal estradiol tablet (Vagifem®) on the endometrium and the relief of subjective symptoms and signs of urogenital estrogen deficiency. Quality of life and acceptability of treatment delivery were also assessed. Study design A prospective, randomized study in which women were assigned in a 2: 1 ratio to ESTring and Vagifem and followed for 12 months. The primary endpoint was endometrial safety, based on the results of ultrasound measurement of endometrial thickness and a progestogen challenge test at baseline and week 48. Efficacy was determined by subjective assessment of urogenital estrogen deficiency symptoms at baseline and weeks 3, 12, 24, 36 and 48 and assessment of signs of vaginal epithelial atrophy by the clinician at baseline, 12 and 48 weeks. In addition, pelvic floor strength, vaginal cytological evaluation and pH, bacteruria and patient acceptability were assessed. Quality of life was assessed using a menopause-specific quality-of-life questionnire and a 2-day bladder diary at baseline and 12 and 48 weeks. The comparability of the two groups was assessed using ANOVA, χ(2) or Fisher's exact tests. Results A total of 126 women were randomized to ESTring and 59 to Vagifem. There was no statistical difference between the groups in the alleviation of symptoms and signs of urogenital estrogen deficiency. Maturation indices increased in both groups, from generally atrophic at baseline to proliferative or highly proliferative at 48 weeks. After 48 weeks of treatment, there was no statistically significant difference in endometrial thickness between the two groups. A statistically smaller proportion of bleeding/spotting occurred in the ESTring group (n = 0) compared to the Vagifem users (n = 4). Estradiol and total estrone serum levels increased during treatment in both groups but remained within the normal postmenopausal range. General health status in both groups was unchanged but the urogenital component of health burden was significantly improved in both groups. Bladder diary variables showed no differences between treatment groups. Conclusion Equivalent endometrial safety and efficacy in the relief of the symptoms and signs of urogenital estrogen deficiency were demonstrated for the 12 months' use of a low-dose estradiol-releasing vaginal ring and a vaginal estradiol tablet.

Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d (2 x 2 x 2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E-2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8) M, beta-E-2 significantly slowed the decrease in volume fraction of myofilaments (V(v)myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E-2 On spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E-2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2-3 (synthetic state), where beta-E-2-treated cells replicated significantly faster than untreated cells. beta-E-2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10) to 10-8 M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic stare. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation however, once cells have modulated to the synthetic phenotype, it promotes their replication. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

In vitro human epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 from a range of solvents

Purpose. To study epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 (BP) from a range of single solvent vehicles and evaluate solvent effects on permeability parameters. Methods. The solubility of BP was measured in a number of solvents. Penetration of BP across human epidermis and high density polyethylene (HDPE) membranes was studied from 50% saturated solutions in each solvent. Results. Maximal BP fluxes from the solvents across the two membranes varied widely. Highest fluxes were observed from 90% ethanol (EtOH) for epidermis and from isopropyl myristate (IPM) and C12-15 benzoate alcohols (C12-15 BA) for HDPE membrane. Both the flux and estimated permeability coefficient and skin-vehicle partitioning of BP appeared to be related to the vehicle solubility parameter (delta(v)). The major effects of solvents on BP flux appear to be via changes in BP diffusivity through the membranes. Conclusions. Minimal penetration of sunscreens such as BP is best achieved by choosing vehicles with a delta(v) substantially different to the solubility parameter of the membrane.

Synchronization of estrus and fertility in zebu beef heifers treated with three estrus synchronization protocols

The effects on estrus and fertility of 3 estrus synchronization protocols were studied in Brahman beef heifers. In Treatment 1 (PGF protocol; n=234), heifers received 7.5 mg, im prostianol on Day 0 and were inseminated after observed estrus until Day 5. Treatment 2 (10-d NOR protocol; n=220) consisted of norgestomet (NOR; 3 mg, sc implant and 3 mg, im) and estradiol valerate (5 mg, im) treatment on Day -10, NOR implant removal and 400 IU, im PMSG on Day 0, and AI after observed estrus through to Day 5. Treatment 3 (14-d NOR+PGF protocol; n=168) constituted a NOR implant (3 mg, sc) on Day -14, NOR implant removal on Day 0, PGF on Day 16, and AI after observed estrus through to Day 21. All heifers were examined for return to estrus at the next cycle and inseminated after observed estrus. The heifers were then exposed to bulls for at least 21 d. During the period of estrus observation (5 d) after treatment, those heifers treated with the PGF protocol had a lower (P

Synchronization of estrus and ovulation and associated endocrine changes in Bos indicus cows

The effects of 4 estrus synchronization treatments on intervals to and synchrony of estrus and ovulation, on timing of the preovulatory LH surge and associated changes in plasma progesterone, LH, FSH, and 17 beta-estradiol (E(2)) were investigated in 48 Bos indicus cows. Treatment 1 consisted of 2 injections of PGF(2 alpha) 14 d apart (n = 12); Treatment 2 of a subcutaneous 3-mg norgestomet implant and an intramuscular injection of 3 mg of norgestomet and 5 mg estradiol valerate, with the implant removed 10 d later (n = 12; norgestomet-estradiol); Treatment 3 of norgestomet-estradiol, with a subcutaneous injection of PMSG given at time of implant removal (Day 10; n = 12); and Treatment 4 of norgestomet implant (as for Treatments 2 and 3) inserted for 10 d, with an intramuscular injection of PGF(2 alpha) given at the time of implant removal (n = 12). The experiment was conducted in 2 replicates (24 cows/replicate, 6 cows/group). Estrus, ovulation and timing of the preovulatory surge of LH varied less in cows treated with norgestomet-estradiol and PMSG than in cows in Treatments 1 and 4 (P < 0.008). Treatment with PMSG;educed variation in ovulation times and timing of the LH surge in cows treated with norgestomet-estradiol (P < 0.02). Concentrations of E(2) were higher in cows in Treatments 2 and 3 on the final day of treatment and at about 6 h post ovulation compared with cows in Treatments 1 and 4 (P < 0.05). Different methods for synchronizing estrus did not alter sequential endocrine and behavioral changes in relation to the timing of the LH peak, and the results were consistent with current recommendations for insemination times in Bos taurus cattle. (C) 1997 by Elsevier Science Inc.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Ni(II)-doped CsCdBrCl2: Variation of spectral and structural properties via mixed-halide coordination

A new addition to the family of single-molecule magnets is reported: an Fete cage stabilized with benzoate and pyridonate ligands. Monte Carlo methods have been used to derive exchange parameters within the cage, and hence model susceptibility behavior.

A ferrocene functionalised macrocyclic receptor for cations and anions

The isolation and characterisation of a new macrocyclic hexaamine trans-6,13-bis(ferrocenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane (L-2) bearing two ferrocenyl groups appended to its exocyclic amines is reported. The crystal structures of L-2 and its dihydrochloride salt L-2. 2HCl . 2H(2)O have been determined. In the latter case cation-anion hydrogen bonding is observed in the solid state. Substrate binding by the electroactive L-2 in MeCN-CH2Cl2 solution has been examined by cyclic voltammetry and reveals the receptor electrochemically to recognise benzoate and chloride anions. The macrocyclic N-donors may also bind transition metal cations such as Cu-II and Zn-II.

Conversion of glucosamine to galactosamine and allosamine derivatives: Control of inversions of stereochemistry at C-3 and C-4

The reactions of sodium benzoate with a series of trimesylates derived from glucosamine have been examined in an attempt to gain facile access to galactosamine analogues. Trimesylate 17, in which the amino group was protected as a phthalimide, underwent double displacement at positions 4 and 6 to give the dibenzoate 18 with the desired galactosamine configuration. In contrast, trimesylates 21 and 27, in which the amino groups were protected as acetamides, unexpectedly underwent double displacement at positions 3 and 6, giving products 22 and 28, respectively, with allosamine configurations.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

Transition to raloxifene with and without low-dose estrogen therapy in postmenopausal women: effects on serum lipids and fibrinogen - a pilot study

Objective To compare the effects of transferring from low-dose transdermal estrogen to raloxifene (RLX), with a phase of alternate-day RLX therapy with or without low-dose transdermal estrogen, on serum lipids and fibrinogen in postmenopausal women previously administered estrogen plus progestogen therapy. Methods Sixty postmenopausal women (mean age 55 years) were randomized to one of two treatment groups: RLX + low-dose transdermal estrogen (RLX + E) or RLX + placebo. The study consisted of four 8-week phases: phase I (all subjects low-dose transdermal estrogen 25 mug/day), phase II (double-blind RLX 60 mg every 2nd day in combination with either low-dose transdermal estrogen or placebo), phase III (all subjects RLX 60 mg every 2nd day + placebo) and phase IV (all subjects RLX 60 mg/day + placebo). Results No significant differences existed between groups for baseline measurements prior to phase I. In phase I, for all subjects combined, total cholesterol and low-density lipoprotem cholesterol both showed a significant increase (median increase of 0.2 mmol/l, p = 0.008 and 0.4 mmol/l, p < 0.001, respectively), while triglycerides decreased significantly (median decrease of 0.2 mmol/l, p < 0.001). For the primary analysis (phase II to phase IV), the mean change from baseline observations showed no significant differences between the therapy groups for serum lipids, fibrinogen, vital signs or weight. In the comparison phase (phase II), changes in serum lipids, fibrinogen, vital signs and weight were not significantly different between groups. Conclusion Gradual conversion to RLX from low-dose transdermal estrogen, with a phase of alternate-day RLX therapy with or without low-dose transdermal estrogen, does not have any effect on the serum lipid profile or fibrinogen level.