Age-dependent induction and repression of rat liver microsomal hydroxylase systems by estradiol

The activities of the hepatic microsomal 2α-, 2β-, 18- and 7α-hydroxylase systems active on 5α-[4-^14C] androstane-3α,17β-diol were studied in male and female rats which had been castrated and spayed at 14, 24, 35 and 45 days of age, treated for 5 days with 500 μg of estradiol benzoate per kg body weight and killed 6 days after gonadectomy. The hepatic microsomal 15β-hydroxylase enzyme system active on 5α-[1,2-³H] androstane-3α, 17β-diol 3,17-disulphate was also measured in these animals as well as in an additional group of animals that were gonadectomized at 56 days of age, treated with estradiol benzoate and killed at 62 days of age. The hydroxylase systems active on the free steroid substrate were all relatively unresponsive towards enstradiol in the developing rat, in contrast to the strong effects on hepatic hydroxylase activities previously noted following treatment of adult male rats with estradiol. On the other hand, the 15β-hydroxylase system active on disulphurylated 5α-androstane-3α, 17β-diol was inducible in both male and female rats of 41 and 51 days of age and in male rats of 61 days of age.     

Measurement and characterization of swine uterine estradiol receptors: the effects of puberty induction on estradiol receptors and corpus luteum function

Two types of cytoplasmic 17 beta-estradiol (E2) binding activity were identified and characterized in the uteri of pregnant, cycling and prepubertal, cycle-induced (400 IU pregnant mare's serum gonadotrophin (PMS) + 200 IU human chorionic gonadotrophin (hCG)) gilts. Overall, type I affinity and capacity were Kd 1.94 +/- 0.51 nM and 5.410 +/- 1.09 pmol/mg protein, respectively; type II apparent dissociation constant and capacity were Kd 21.34 +/- 6.83 nM and 62.58 +/- 15.96 pmol/mg protein, respectively. Cytoplasmic luteal E2 receptors were undetectable in all groups. Uterine E2 receptor activity was eluted from diethylaminoethyl columns by a 0.05-0.15 M KCl gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated a molecular weight of 70 400-79 000. Excluding gilts with cystic ovarian follicles (16.67%), prepubertal gilts treated with PMS + hCG versus cycling sows had lower serum progesterone on days 6 and 9-13 of the estrous cycle and lower 13,14-dihydro-15-keto prostaglandin F2 alpha levels on days 0-9 and 13-17 of the cycle. Implants, containing 200 mg estrone inserted subcutaneously on days 12-19 after PMS + hCG treatment in gilts, had no discernible effects on these parameters. These results indicate that the diminished reproductive capacity of the gilt, in which cycle activity is induced by PMS + hCG, is likely due to decreased luteal progesterone secretion. Preliminary data also suggest that the lack of E2 receptors may contribute to the low reproductive performance in gilts with cystic ovarian follicles.     

Structural properties of the microsomal triglyceride-transfer protein complex

The microsomal triglyceride-transfer protein (MTP), which catalyzes the transport of triglyceride and cholesteryl ester between membranes, is a complex composed of two proteins having apparent molecular weights of 58,000 and 88,000. The 58,000 molecular weight component of MTP has been identified as the multifunctional protein, protein disulfide isomerase (PDI). The multisubunit nature of MTP as well as the presence of PDI as one of the subunits distinguishes this protein from previously characterized lipid-transfer proteins. In this study, we have more clearly defined structural elements of MTP that may play important functional roles. The molecular weight of the transfer protein complex was determined to be 150,000 by sedimentation equilibrium experiments performed at three different speeds, suggesting that MTP is a complex of one PDI and one 88,000 molecular weight polypeptide (88K). Following SDS-polyacrylamide gel electrophoresis, the Coomassie Blue staining intensity of PDI in a known amount of MTP was compared to that of known amounts of a PDI standard. A 1 to 0.98-1.30 ratio of PDI to 88K was determined, confirming the 1:1 stoichiometry of MTP. The sedimentation coefficient (5.85) determined by analytical ultracentrifugation and the Stokes radius (47 A) determined by polyacrylamide gradient gel electrophoresis indicate that the 150,000 molecular weight MTP complex is asymmetric and/or has an unusually high water of hydration. PDI and 88K form a stable protein complex; there was no evidence of a dissociation-reassociation reaction occurring between the two components. Analysis of far-ultraviolet circular dichroism spectra revealed MTP has about 28% alpha-helical and 28% beta-structural content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential up-regulation of microsomal and synaptic membrane mu opioid receptors

Naltrexone was administered to rats for 7 days by osmotic minipump (5 mg/kg/day) and thereupon, forebrain mu opioid receptor levels in subcellular fractions were monitored by homologous displacement of [3H]D-ala2-mePhe4-gly-ol5 enkephalin binding. Microsomes displayed increases in mu receptor concentrations that were twofold greater than those associated with synaptic plasma membrane fractions (92 vs. 51%). Levels in crude membranes rose 77%. Binding affinities were unchanged.     

Effect of prolactin and estradiol on rat striatal dopamine receptors

Chronic estrogen treatment has been found to increase the level of rat striatal dopamine receptors. Since it is well known that estrogen treatment increases circulating prolactin levels, we have investigated the possibility that the stimulatory effect of estrogens on dopamine receptors is exerted via prolactin. Ovariectomized female or intact male rats were implanted with three adenohypophyses under the kidney capsule or treated with 17 β-estradiol (10 μg, twice daily) for 2 weeks. In animals of both sexes, the pituitary-implanted and estradiol-treated rats showed higher levels of [³H]spiperone binding to striatal dopamine receptors. This effect of estradiol or pituitary implants on dopamine receptors was further investigated in ovariectomized rats. The pituitary-implanted and estradiol-treated rats had elevated plasma prolactin levels and an increased density of striatal dopamine receptors without alteration of their affinity. The role of the pituitary in the effect of estradiol was next investigated using hypophysectomized female rats treated with 17 β-estradiol (10 μg, twice daily), o-prolactin (500 μg, twice daily) or bearing three anterior pituitary implants. The implants as well as the treatment with estradiol or prolactin increased the level of striatal dopamine receptors in hypophysectomized rats while, as expected, the estradiol-treated animals did not have elevated plasma prolactin levels. The present data indicate that high prolactin levels lead, as observed with chronic estradiol treatment, to an increased density of striatal dopamine receptors. However, the effect of estradiol may not be explained exclusively by increased prolactin levels since a similar stimulatory effect is observed in hypophysectomized animals.     

Structural organization and chromosomal assignment of the gene encoding endothelin

Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.     

Structural biology of chemokine receptors

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involve interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective immunomodulatory and anti-HIV-1 therapeutic agents.     

Structural conservation of Notch receptors and ligands

The Notch signalling pathway is an evolutionarily conserved cell-to-cell communication system utilized multiple times and in many tissues during development. The outcome of an interaction between Notch and its ligands is highly influenced by factors both extrinsic and intrinsic to Notch expressing cells, suggesting that Notch functions either directly or in parallel with other signalling systems to regulate cellular differentiation events. Protein domains common to all ligands and receptors of this system suggest conserved functional properties that likely relate to regulatory mechanisms for Notch signalling. Within this review, the known functional properties of these domains are analyzed with respect to their contributions to ligand/receptor interactions and Notch signalling.     

Tamoxifen counteracts estradiol induced effects on striatal and hypophyseal dopamine receptors

We investigated the ability of Tamoxifen (TAM), an antiestrogen drug, to counteract the modifications induced by estrogens on dopamine (DA) receptors on striatum and on adenohypophysis of ovex female rats. Subacute treatment with 17 beta-estradiol (E2) at both low (0.1 micrograms/kg) and high (20 micrograms/kg) doses confirmed its ability to increase the number of striatal 3H-Spiperone (3H-SPI) binding sites in a dose dependent manner. By contrast in the pituitary, only high doses of estrogen were effective in reducing the number of DA receptors. We treated ovex female rats for 15 days with TAM alone or associated with E2, to see if these estrogenic effects could be suppressed by an antiestrogenic drug. TAM did not affect the number of striatal DA receptors, but significantly increased the adenohypophyseal DA binding sites, without varying their affinity. No changes were observed in pituitary and striatal DA receptor density, even when TAM was injected in association with estradiol. In conclusion: TAM is able to counteract the effects estrogens have on DA receptors. However there is some evidence that it could influence the pituitary DA systems independently of its antiestrogenic activity.     

Nuclear estradiol receptors in several non-target organs of rats]

Some properties of the macromolecules of the KCl-extracts of the nuclei of the uterus, kidney, liver, testis and prostate, specifically binding estradiol (E2), were studied. These macromolecules of the uterus and the liver were found to be maximally extracted from chromatin by the 0.6 M KCl concentration. The capacity of the macromolecules of the uterine, kidney and liver nuclear extracts to bind E2 specifically is destroyed completely by pronase, but not by RNA-ASe and DNA-ase, pointing to the protein nature of these macromolecules. Only estrogenic compounds, but not testosterone, 5alpha-dihydrotestosterone, progesterone or corticosterone were capable to compete with H3--E2 for the E2--binding sites of the macromolecules of the nuclear extracts of all the organs investigaeted. It is assumed that macromolecules of the nuclei of the investigated nontarget organs specifically binding E2 are estrogen receptors.     

Rat liver microsomal palmitoyl-coenzyme A synthetase. Structural properties

The structural properties of purified palmitoyl-CoA synthetase from rat liver microsomal material were investigated. The active enzyme has a molecular weight of 168000 and contains about 8.2mol of phospholipid bound/mol of enzyme protein, as well as bound fatty acids. Association of the active enzyme was shown to occur, without impairment of catalytic activity. The lowest-molecular-weight species obtained under denaturing conditions was 27000 daltons.     

ATP-dependent stabilization against microsomal factor-induced loss of unoccupied glucocorticoid receptors

ATP stabilizes the unoccupied glucocorticoid receptor from brain at 12 degrees C, but only in the presence of a destabilizing microsomal factor. This stabilization is optimal at an ATP concentration of about 1 mM, higher concentrations resulting in an increase in the rate of heat inactivation. Other nucleotides, including CTP, GTP, UTP, ADP, cAMP and cGMP were ineffective in stabilizing receptors, although additions of some of these nucleotides actually resulted in further destabilization of the unoccupied glucocorticoid receptor.