Identification of a novel thyroid hormone receptor expressed in the mammalian central nervous system.

A complementary DNA clone derived from rat brain messenger RNA has been isolated on the basis of homology to the human thyroid hormone receptor gene. Expression of this complementary DNA produces a high-affinity binding protein for thyroid hormones. Sequence analysis and the mapping of this gene to a distinct human genetic locus indicate the existence of multiple human thyroid hormone receptors. Messenger RNA from this gene is expressed in a tissue-specific fashion with highest levels in the central nervous system.

Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor.

Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter. Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions, and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.

Identification of human glucocorticoid receptor complementary DNA clones by epitope selection.

Steroid hormones regulate cellular differentiation and physiologic functions predominantly through gene transcription. Regulation is achieved by the interaction of specific steroid receptor proteins and target genes. Expression cloning techniques were used to select human glucocorticoid receptor complementary DNA clones in order to define the mechanism by which the receptor exerts its transcriptional control. Immobilized fusion proteins from individual clones were used to select epitope-specific antibody which was subsequently eluted and identified by binding to protein blots of cellular extracts. Three cross-hybridizing clones containing inserts expressing antigenic determinants of the human glucocorticoid receptor were isolated.

Carbide and nitride phase characterization in a transition metal carbo-nitride using x-ray spectroscopy and atom probe tomography.

A multi-phase hafnium carbo-nitride was investigated by various analytical methods. Incomplete homogenization between mixed HfC-HfN starting powders subjected to hot isostatic pressing resulted in both carbon-rich and nitrogen-rich phases. The compositions of these two phases were quantified in detail by wavelength dispersive spectroscopy and atom probe tomography, with the atom probe tips having either a small or a large shank angle geometry. For each of the two phases, an agreement of the compositions obtained by wavelength dispersive spectroscopy and atom probe tomography was found. However, the quality of the mass spectrum and hit multiplicity (single hits) were generally higher for the carbon-rich as compared to the nitrogen-rich carbo-nitride. Though the atom probe tip geometry does not appear to influence the composition, the mass resolving power did improve with the larger shank angle geometry while the hit multiplicity deteriorated slightly. Finally, our results demonstrate that hafnium carbide requires less thermal assistance to field evaporate than hafnium nitride.

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

A model for farnesoid feedback control in the mevalonate pathway.

Mevalonate is the rate-limiting substrate leading to farnesyl pyrophosphate (FPP), the central intermediate for isoprenoids such as cholesterol, dolichols, ubiquinone, and carotenoids. One major challenge has been to identify the isoprenoid effector molecules and transcription factors mediating negative regulation in this metabolic pathway. A nuclear receptor called FXR has recently been characterized that is activated by farnesyl pyrophosphate metabolites such as farnesol, farnesal, farnesoic acid, and methyl farnesoate. FXR expression in isoprenoidogenic tissues suggests a hypothesis that these intracellular "farnesoids" may be signals for transcriptional feedback control of cholesterol biosynthesis.

An adenoviral vector system for functional identification of nuclear receptor ligands.

A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element. Corticosteroid- or thyroid hormone-induced transcription can be efficiently and accurately quantitated from cells coinfected with the appropriate complementary virus pair 20 h after infection in 96-well microtiter plates. This coinfection assay offers a convenient way to measure transcriptional activation by nuclear receptors and has certain key advantages over the commonly used cotransfection method. Its sensitivity and precision make it a practical approach to rapidly identify substances extracted from complex biological samples activating candidate "orphan" nuclear receptor molecules.

Tight linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA beta gene.

Multiple cDNAs belonging to the c-erbA gene family encode proteins that bind T3 with high affinity. However, the biological functions of these multiple thyroid hormone receptors have not yet been clarified. Generalized thyroid hormone resistance (GTHR) refers to a human syndrome characterized by tissue refractoriness to the action of thyroid hormones; several studies have suggested quantitative or qualitative defects in T3 binding to nuclear receptors in certain kindreds. To investigate the biological functions of the c-erbA genes, c-erbA alpha and c-erbA beta, we tested the hypothesis that an abnormal c-erbA gene product is present in GTHR by examining these genes in members of one kindred. Restriction enzyme analysis failed to identify an abnormal pattern in affected individuals suggesting no rearrangements or large deletions. However, we demonstrated that the gene conferring the GTHR phenotype is tightly linked to the c-erbA beta locus on chromosome 3. This linkage strongly suggests that the c-erbA beta gene is important in man as a thyroid hormone receptor and identifies a putative c-erbA beta mutant phenotype with central nervous system, pituitary, liver, metabolic, and growth abnormalities.

Estimation of dislocation density from precession electron diffraction data using the Nye tensor.

The Nye tensor offers a means to estimate the geometrically necessary dislocation density of a crystalline sample based on measurements of the orientation changes within individual crystal grains. In this paper, the Nye tensor theory is applied to precession electron diffraction automated crystallographic orientation mapping (PED-ACOM) data acquired using a transmission electron microscope (TEM). The resulting dislocation density values are mapped in order to visualize the dislocation structures present in a quantitative manner. These density maps are compared with other related methods of approximating local strain dependencies in dislocation-based microstructural transitions from orientation data. The effect of acquisition parameters on density measurements is examined. By decreasing the step size and spot size during data acquisition, an increasing fraction of the dislocation content becomes accessible. Finally, the method described herein is applied to the measurement of dislocation emission during in situ annealing of Cu in TEM in order to demonstrate the utility of the technique for characterizing microstructural dynamics.

Population and family planning.

PIP: The author agrees with the United Teheran Declaration of Human Rights which declared the right of each couple to choose the size of their own family. Government policies must preserve and increase informed choice for all couples. The Supreme Court recognized the right of all married couples to practice birth control in a 1965 decision; the Court extended this right to all persons in 1972. The 1970 Family Planning Services and Population Research Act involved the United States government in family planning support here and abroad. By 1973 organized family planning programs were serving more than 3.2 million women. They had been extended geographically too. Fertility research is being conducted. Before 1980 family planning programs will be integrated into general health care systems with comprehensive health care insurance covering the bills.^ieng

Neuroectodermal tumor and monoclonal antibodies.

Antibody-producing clones were obtained by hybridization of spleen cells from mice immunized with whole cultured human tumor cells and mouse myeloma cells, P3UX59AG8. The tumor cells were derived from a peripheral primitive neuroectodermal tumor. One of the antibodies produced by these clones reacts with the original cell line, SK-PN-DW, and other more differentiated neuroectodermal tumors such as neuroblastomas and melanomas. The cell line SK-PN-DW contains antigenic determinants recognized by monoclonal antibodies raised against melanoma, neuroblastoma, and human fetal brain. These data indicate that this primitive neuroectodermal tumor is derived from the neuroectoderm.

Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription.

Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

Adenovirus VAI RNA facilitates the initiation of translation in virus-infected cells.

The adenovirus VAI RNA is a small polymerase III-transcribed species that is required for optimal translation of mRNAs late after infection. Mutant dl331 fails to produce this RNA species and, as a result, grows poorly. Mutant-infected cells contain normal levels of late mRNAs, but reduced levels of polypeptides are synthesized late after infection. Translational elongation occurs at normal rates in mutant, as compared to wild-type, virus-infected cells. Initiation of translation occurs with reduced efficiency in dl331 -infected cells. VAI RNA is required for formation of a stable 48S preinitiation complex and very likely functions to facilitate the interaction between 43S preinitiation complex and mRNA to form the 48S species.

Adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection.

Two adenovirus type 5 mutants were constructed to probe the function of the virus-encoded RNA polymerase III transcripts (VA RNAs). Each mutant fails to synthesize one of the two VA RNA species. The variant that does not produce the minor VAII species grows normally. The mutant that cannot synthesize the major VAI species grows more poorly than its parent. Analysis of the mutant's growth defect indicates that the adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection.

Effect of mutant host RNA polymerase on the bifunctional activities of P22 gene c1.

The product of phage P22 gene c1 has two functions: (1) it promotes synthesis of repressor and (2) during the first minutes of infection it retards expression of some lytic genes. We call the second, negative function "c1 retardation". We investigated c1 retardation in a mutant host of Salmonella typhimurium that is resistant to rifampicin and carries an altered RNA polymerase. No c1 retardation of DNA synthesis was detectable in this host after infection with wild-type phages. This elimination of the normally detectable c1 function leads to the conclusion that the mutant RNA polymerase interferes with the expression of c1 gene activity. Wild-type genes form clear plaques on the mutant host. Mutants of P22 called cly were isolated by others. These mutants form turbid plaques on the altered RNA polymerase host. Infections with P22 cly in the mutant host resulted in detectable c1 retardation. The cly mutation therefore restores c1 activity in a host which wild-type c1 is not expressed. Two spontaneous mutants were isolated from the mutant host. These two strains allowed partial expression of c1 retardation, although they remained rifampicin resistant. We interpret our data to indicate that expression of the normal functions of the gene c1 product requires an interaction of that product with the host RNA polymerase.

Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary.

Multiple thyroid hormone receptor cDNAs have previously been identified in rat and are classified into alpha and beta subtypes. Alternative splicing of the alpha gene gives rise to the functional receptor, rTR alpha 1, and the non-thyroid hormone-binding isotype, rTR alpha 2. Recent evidence suggests the beta gene encodes two functional receptors, rTR beta 1, and the pituitary-specific receptor, rTR beta 2. By using synthetic DNA probes common to rTR beta transcripts and specific for rTR alpha 1 and rTR alpha 2 mRNAs, we mapped the expression of these transcripts in adult rat brain and pituitary by hybridization histochemistry. We also localized mRNAs encoding the putative nuclear receptor REV-ErbA alpha, a portion of which is derived from the opposite strand of the rTR alpha gene. rTR alpha 1 and rTR alpha 2 transcripts were widely distributed in a similar, if not identical, pattern. Highest levels of rTR alpha 1 and rTR alpha 2 transcripts were found in the olfactory bulb, hippocampus, and granular layer of the cerebellar cortex. REV-ErbA alpha and rTR beta mRNAs were found in more restricted patterns of expression distinct from those of rTR alpha 1 and rTR alpha 2. REV-ErbA alpha mRNA was highest in the neocortex. High levels of rTR beta transcripts in the anterior pituitary and the parvocellular part of the paraventricular hypothalamic nucleus suggest rTR beta gene products may mediate thyroid hormone feedback regulation of thyroid-stimulating hormone and thyrotropin-releasing hormone. Our results identify nuclei and structures in the mammalian central nervous system in which regulation of gene expression by specific thyroid hormone receptor subtypes may occur.