Saccharomyces cerevisiae produces a yeast substance that exhibits estrogenic activity in mammalian systems.

Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes.

Ketoconazole has recently been shown to interfere with steroidogenesis in patients and rat in vitro systems. In this study we attempted to elucidate the site of inhibition in the adrenal gland. Although ketoconazole impaired adrenocorticotropic hormone stimulated cyclic (c)AMP production, dibutyrl cAMP addition did not bypass the steroidogenic blockade indicating that the critical ketoconazole-inhibited step was distal to cAMP. Addition of radiolabeled substrates to isolated adrenal cells and analysis of products by high performance liquid chromatography demonstrated a ketoconazole block between deoxycorticosterone (DOC) and corticosterone. This 11-hydroxylase step is carried out by a P450-dependent mitochondrial enzyme. No restriction of progesterone or pregnenolone conversion to DOC was detected, steps carried out by non-P450-dependent microsomal enzymes. Inhibition of cholesterol conversion to pregnenolone by mitochondrial fractions indicated a second block at the side chain cleavage step, another mitochondrial P450-dependent enzyme. Adrenal malate dehydrogenase, a non-P450-dependent mitochondrial enzyme was not inhibited while renal 24-hydroxylase, a P450-dependent mitochondrial enzyme in another organ, was blocked by ketoconazole. We conclude that ketoconazole may be a general inhibitor of mitochondrial P450 enzymes. This finding suggests that patients receiving ketoconazole be monitored for side effects relevant to P450 enzyme inhibition. Further, we raise the possibility that this drug action may be beneficially exploited in situations where inhibition of steroidogenesis is a therapeutic goal.

High level expression of wild type and variant mouse glucocorticoid receptors in Chinese hamster ovary cells.

We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent doublet at 90/92 kilodaltons was observed by Western blotting or labeling with [3H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [3H]dexamethasone binding revealed the presence of approximately 10(6) receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells. Function of the amptified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endogenous activity of the CHO GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration by confocal microscopy that unliganded overexpressed glucocorticoid receptors are distributed in a nonrandom manner throughout all planes of the nucleus.

Mouse glucocorticoid receptors (GR) that are over-expressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no hormone-binding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells.

The pig kidney cell line (LLC-PK1) has been shown to possess 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and to exhibit functional responses to vitamin D metabolites. Here we report that these receptors appear to undergo homologous up-regulation by 1,25-(OH)2D3 and other vitamin D analogs. This phenomenon was also observed in other cell lines, including human skin fibroblasts and human mammary cancer cells (MCF-7). Treatment with active hormone or vitamin D analogs results in a substantial increase (200-400%) in the number of 1,25-(OH)2D3 receptors without altering the affinity of receptor for hormone. The up-regulated receptor, like the basal receptor, has an apparent Kd of about 0.04 nM and sediments at 3.3S on hypertonic sucrose gradients. In addition, approximately 50% of the total receptors from both control and treated cells bind to DNA-cellulose and elute at 0.18 m KCl. These results indicate that the up-regulated receptor is similar to the classical 1,25-(OH)2D3 receptor. While the time necessary to achieve the maximal receptor increment is 16-20 h, there is a rapid component in the rise observed within 5 min. The maximal effect persists for 4-6 h after hormone removal. The increased binding is not a result of differential receptor localization or extractability. 1,25-(OH)2D3, 1,24,25-trihydroxyvitamin D3, 24,25-(OH)2D3, and 25-hydroxyvitamin D3 all increase receptor binding to similar levels, and the dose required closely reflects the affinities of the various metabolites for the receptor. Treatment of cells with the RNA synthesis inhibitor actinomycin D indicates that the increase in receptors is partially dependent on RNA synthesis. Mutant skin fibroblasts from patients with vitamin D-dependent rickets type II, containing nonresponsive 1,25-(OH)2D3 receptors, failed to exhibit the characteristic up-regulation observed in normal cells. Taken together, these results indicate that vitamin D metabolites regulate the number of 1,25-(OH)2D3 receptors in part by receptor occupancy and, more importantly, by a receptor-mediated induction mechanism.

Vitamin D resistance and alopecia: a kindred with normal 1,25-dihydroxyvitamin D binding, but decreased receptor affinity for deoxyribonucleic acid.

A new kindred exhibiting vitamin D resistance and alopecia is described. Clinically, three of seven sisters demonstrated rickets, hypocalcemia, elevated serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels, and alopecia. Biochemical analysis of cultured fibroblasts from skin biopsy explants in two affected and one normal sister revealed normal [3H]1,25-(OH)2D3 binding to receptors (Kd = 0.05 nM; Nmax = 30-50 fmol/mg protein). Despite normal steroid binding, cells from the two affected sisters failed to respond to 1,25-(OH)2D3 in vitro, as measured by induction of the enzyme 25-hydroxyvitamin D-24-hydroxylase. The cells from the normal sister showed a response within the range of five normal cell lines. Sucrose gradient analysis yielded a typical 3.2S protein under high salt conditions in extracts from the three siblings, but with reduced capacity to aggregate to a 6S moiety in low salt gradients in the two affected cells. Whole cell [3H]1,25-(OH)2D3 binding studies revealed nearly normal localization of bound receptor to the nuclear compartment. Elution of bound receptors by KCl gradients from both DNA-cellulose or fibroblast nuclei demonstrated that the receptors from the affected sisters exhibited decreased affinity for DNA compared to those from normal subjects. We conclude that 1,25-(OH)2D3 receptors from these resistant fibroblasts have a normal steroid-binding domain, but a defective nuclear binding domain. We believe that this abnormality may be responsible for the vitamin D resistance observed both in vivo and in vitro.

1,25-dihydroxyvitamin D resistance, rickets, and alopecia: analysis of receptors and bioresponse in cultured fibroblasts from patients and parents.

To investigate further the cellular defects of vitamin D-dependent rickets type II with alopecia, we studied 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and the response to 1,25-(OH)2D3 in cultured skin fibroblasts from rachitic patients. Our studies included cells from four affected patients from three kindreds and their parents and cells from five normal subjects. We measured total 1,25-(OH)2D3 receptor binding in cell extracts and the capacity of 1,25-(OH)2D3 to induce the enzyme 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) as a marker of functional response. In normal fibroblasts, the 1,25-(OH)2D3 maximal binding capacity was 52 +/- 5 fmol/100 micrograms DNA (mean +/- SE), and the apparent dissociation constant (Kd) was 0.05 +/- 0.01 nM. The maximal induced 24-hydroxylase activity after 1,25-(OH)2D3 treatment was 11.5 +/- 1 fmol/10(6) cells X 30 min, and the dose of 1,25-(OH)2D3 that achieved half-maximal induction was 2.3 +/- 0.3 nM. Fibroblasts from all four rachitic patients had the same defect: no measurable 1,25-(OH)2D3 receptor binding and no detectable response above basal activity even after high doses of 1,25-(OH)2D3. Cells from all parents except one had normal 1,25-(OH)2D3 binding characteristics and normal 24-hydroxylase bioresponse to 1,25-(OH)2D3. One parent despite a normal phenotype had only half the normal level of binding sites and only half the normal bioresponse. In summary, the cultured fibroblasts from four affected children representing three different kindreds with 1,25-(OH)2D3 resistance failed to exhibit detectable 1,25-(OH)2D3 receptors. We postulate that this biochemical defect produced both the inability to respond to 1,25-(OH)2D3 in vitro and the 1,25-(OH)2D3 resistance in vivo. The obligate heterozygotic parents were normal, except for one who had both half the normal number of receptors and half the normal response to 1,25-(OH)2D3. The data confirm the critical role of the receptor in 1,25-(OH)2D3 action and the close coupling of receptor content and functional responsiveness.

Evaluating the Malaise Inventory: a comparison of measures of stress.

The Malaise Inventory developed by Rutter and his colleagues has been widely used to measure the level of stress experienced by mothers of severely disabled children. The results obtained using the Inventory in a survey of 210 mothers with a disabled child are compared with two alternative measures of stress: a scale of symptoms and the taking of medication related to mental health. Most of the Inventory items and the malaise score--the total number of items reported--are moderately correlated with the other measures of stress. The results obtained from two successive surveys of the sample are compared to check the consistency of the findings.

Disabilities, benefits, and disability benefits.

The purpose of this paper is to relate the patterns of disabilities in a sample of young adults to the amounts they received in social security benefits towards extra arising from disablement. Data on 248 young people aged 18 to 22 who lived in England were obtained by means of interviews with their parents. There was wide variation in weekly amounts received in benefits for the extra costs of disablement. The most severely disabled did not receive invariably the highest levels of support and some disabilities were not associated with amounts received in benefits. The findings indicate that the United Kingdom social security system is biased towards meeting the extra costs which arise from physical disablement; costs which arise because of mental disablement attract less recognition. It is concluded that help with extra costs should be based on a comprehensive assessment of disablement and its financial consequences.

Variations in take-up of the Family Fund.

This paper investigates geographical variations in successful and unsuccessful applications to the Family Fund Trust which provides grants to families with severely disabled children. A new measure of take-up is developed which takes account of demographic differences between local social services authorities and relates local take-up levels to national rates. The take-up of grants varies more than twofold across local authorities but is generally higher in deprived areas, suggesting that the help available from the fund is targeting those most in need. Nonetheless use of the fund is less than expected in metropolitan areas characterized by rented accommodation, high rise dwellings and minority ethnic groups. Ethnic monitoring and other measures to promote equal opportunities have recently been introduced. If take-up were uniformly high across England and Wales the current caseload and budget would increase by more than half. Local authorities where renewed efforts might be most effectively targeted to encourage families to apply for a grant can be identified but any publicity would need to ensure that inappropriate applications are kept to a minimum.

Young people with disabilities: what happens after 16?

This article presents some findings from a recent postal survey of young adults aged 16-21 years with severe disabilities. The study was funded by the Department of Health and Social Security and is based on a sample of over 1,000 drawn from the register of families helped by the Family Fund. Information was collected on the usual weekday activities of the young people. Parents were asked whether they were satisfied with the way in which their son/daughter was occupied and about any changes or difficulties since he/she left school. The research indicates that: there is a considerable gap in access to paid employment for young people with disabilities compared with young people in general; there is substantial variation in the occupational experience of young adults with different types of impairment; and the transition from school to further education, training, employment, unemployment or day care can be difficult. Implications of these findings are discussed.

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.

Inhibition of steroidogenic cytochrome P-450 enzymes in rat testis by ketoconazole and related imidazole anti-fungal drugs.

Ketoconazole, an imidazole antifungal drug, has previously been shown to diminish testosterone and cortisol production in patients as well as rat and mouse cells in vitro. Inhibition of adrenal mitochondrial cytochrome P-450 enzymes was demonstrated. In this study we tested several imidazole antifungal drugs and examined the individual steps in testicular steroidogenesis to determine which enzymes in the androgen pathway were blocked. In addition, we studied 25-hydroxyvitamin D 24-hydroxylase activity in cultured pig kidney cells (LLC-PK1) to assess a mitochondrial P-450 enzyme in another organ. All imidazoles tested inhibited both total testosterone production and 24-hydroxylase activity but the relative potencies differed. We next studied the individual testicular enzymatic steps between cholesterol and testosterone. Ketoconazole inhibited cholesterol-side-chain-cleavage enzyme (mitochondrial) and C-17,20 lyase (microsomal). The three inhibited enzymes (two testicular and one renal) are all P-450 cytochromes. Testicular 17-hydroxylase, also a P-450 cytochrome, was not inhibited even at high doses of ketoconazole. This is an interesting finding because the testicular hydroxylase and lyase have been shown to be a single protein. Non-cytochrome P-450 enzymes in the androgen pathway were not inhibited. The results demonstrate that several imidazole antifungal drugs all inhibit both microsomal and mitochondrial cytochrome P-450 enzymes in multiple organs.

Activation of cytochrome P450IA1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in wild-type and high-activity variant mouse hepatoma cells.

We analyzed the function of a DNA domain located upstream of the cytochrome P450IA1 gene in wild-type (Hepa 1c1c7) mouse hepatoma cells and in high-activity variant (HAV) cells that overtranscribe the gene in response to the inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transfection experiments indicated that both wild-type and HAV DNA confer responsiveness to TCDD upon the bacterial chloramphenicol acetyltransferase (CAT) gene. However, the level of CAT activity was four- to fivefold higher when the hybrid genes were expressed in the HAV cells. These findings imply that an alteration in a trans-acting function confers the HAV phenotype. Studies of mRNA accumulation imply that TCDD acts by enhancing the rate of mRNA initiation rather than by removing a block in mRNA elongation. We found that both wild-type and HAV cells used the same transcriptional promoter as that described previously for the cytochrome P450IA1 gene in C57BL/6 mouse liver. Both cell types exhibited superinduction of cytochrome P450IA1 gene expression in response to TCDD plus cycloheximide.

Discrimination of DNA response elements for thyroid hormone and estrogen is dependent on dimerization of receptor DNA binding domains.

We and others have previously shown that a two-amino acid substitution in the base of the first zinc finger of the glucocorticoid receptor DNA binding domain (DBD) is sufficient to alter the receptor's target DNA from a glucocorticoid response element (GRE) to an estrogen response element (ERE). Activation of a thyroid hormone response element (TRE) has been shown to require an additional five-amino acid change in the second zinc finger of the thyroid hormone receptor (TR). Using closely related TRE and ERE sequences, we report that a receptor containing the TR DBD activates the ERE poorly, and receptors containing essential amino acids of the estrogen receptor (ER) DBD activate the TRE poorly. The ER DBD (expressed in Escherichia coli) selectively bound to a 32P-labeled ERE (32P-ERE) as a dimer and a 32P-TRE as a monomer, whereas the TR DBD bound 32P-TRE as a dimer and 32P-ERE as a monomer. When hybrid receptor DBDs were examined, we found that the five amino acids in the second zinc finger of the TR necessary for TRE activation were also essential for dimer formation on a TRE. Dimer formation of ER on an ERE was localized to the second half of the second zinc finger. These results suggest that the ability of ER and TR to functionally discriminate between an ERE and a TRE is a result of dimerization of their DBDs.