cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues (M(r) = 33,258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M(r) of the receptor is smaller than the 40-45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase. A phosphorylation motif located in the third intracellular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3'-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.     

Interferon gamma upregulates its own gene expression in mouse peritoneal macrophages

Interferon gamma (IFN-gamma) exerts a variety of immunoregulatory effects on several cell targets. It is generally assumed that IFN-gamma is specifically produced by T and large granular lymphocytes. In this study, we show that IFN-gamma is constitutively expressed in resting mouse peritoneal macrophages (PM). Treatment of PM with cycloheximide results in a significant accumulation of IFN-gamma mRNA, suggesting that a short-lived IFN-gamma mRNA accumulates when protein synthesis is inhibited. Moreover, treatment of PM with IFN-gamma also results in a clear-cut accumulation of this mRNA. This effect is not observed in murine lymphocytes from mesenteric lymph nodes (which instead produce IFN-gamma after phytohemagglutinin treatment) and in mouse cell lines. The treatment of PM with IFN-gamma also results in secretion of IFN-gamma after 24-48 h. The upregulation of IFN-gamma expression is also found in PM from anti-asialo GM1-treated nude mice. We suggest that the ability of PM to produce this IFN-gamma is indicative of an autocrine mechanism. The macrophage IFN-gamma may play a role in the regulation of cell differentiation and immune response.     

Regulation of gonadotropin-releasing hormone (GnRH) receptor gene expression in sheep: interaction of GnRH and estradiol

GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 microg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 microg i.m.) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone. Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHbeta and FSHbeta subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHbeta subunit (P < 0.005) and FSHbeta subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHbeta subunit mRNA (P < 0.01) but did not affect amounts of LHbeta subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHbeta subunit (P < 0.01) and FSHbeta subunit (P < 0.005). From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell's ability to respond to estradiol.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Genetic heterogeneity of adrenocorticotropin (ACTH) resistance syndromes: identification of a novel mutation of the ACTH receptor gene in hereditary glucocorticoid deficiency

Hereditary primary adrenal insufficiency syndromes due to ACTH resistance include hereditary glucocorticoid deficiency (HGD) and Allgrove's syndrome (AS). Patients with both conditions present in childhood with failure to thrive, weakness, and fatigue or adrenal crisis; patients with AS in addition have alacrima and achalasia (triple A syndrome). We studied four kindreds with HGD and four kindreds with AS for abnormalities of the ACTH receptor (ACTHR) gene. The ACTHR coding sequence in all AS kindreds and two HGD kindreds was normal. Analysis of the ACTHR gene of the proband in one of the HGD kindreds showed him to be homozygous for the previously described G221T transition causing a Ser74Ile substitution of the protein, which has been shown to inactivate the ACTHR in signal transduction. The proband in another HGD kindred was found to be a compound heterozygote with the G221T transition in one allele and a novel C818A transition in the other allele of ACTHR. The C818A transition caused the substitution of the highly conserved Pro273 by His in the receptor protein. In vitro expression of the mutated ACTHR in mouse melanoma M3 cells showed that at a medium ACTH concentration of 3 nM, cells transfected with the wild-type ACTHR produced twofold and threefold, respectively, of the amount of intracellular cAMP when compared to cells transfected with the ACTHR carrying the Pro273His and the Ser74Ile mutation, respectively, confirming that HGD in this kindred is caused by loss-of-function mutations of the ACTHR. These results showed that the genetic cause of the ACTH-resistant syndromes is heterogeneous.     

PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus)

The authors analyze problems inherent to the treatment of spine tumors (in particular surgical indications) especially with regard to metastases. The purpose of treatment must be that of improving the quality of life and, perhaps, prognosis. In this sense it is hoped that when indications exist surgical treatment will be as timely and radical as possible.     

Mutation in the thyroid hormone receptor beta gene (A317T) in a Thai subject with resistance to thyroid hormone

Analysis of the thyroid hormone receptor beta (TRbeta) gene of a Thai female with the syndrome of resistance to thyroid hormone (RTH) revealed a missense mutation at codon 317, changing the guanine in nucleotide 1234 to an adenine that results in the replacement of the normal alanine (GCT) with a threonine (ACT). The proposita was heterozygous, and this mutation was not present in her parents and her sister, compatible with a neomutation. This is the first report of TRbeta gene mutation causing RTH in an individual of Thai origin.     

Insulin treatment (hormonal imprinting) increases the insulin production of the unicellular tetrahymena long term. Is there a simultaneous formation of hormone receptor and hormone?

Insulin treatment of Tetrahymena pyriformis resulted in a long-lasting increase in the insulin content of the cells. After about 200 generations, insulin levels significantly higher than in the control were demonstrated by using a quantitative immunocytochemical method. Although the insulin content fluctuated from day to day, it was always higher in the insulin pre-treated (imprinted) cells than in the controls. The results emphasise the simultaneous stimulation of hormone and receptor formation by hormonal imprinting.     

Hepatic apolipoprotein and LDL receptor gene expression in the genetically hypercholesterolemic (RICO) rat

Up and down-regulation of calcium and potassium conductances are associated with several forms of short-term synaptic modulation. Detailed investigation of synaptic plasticity in the marine gastropod Aplysia, and in other mollusks, indicates that synaptic transmission can be influenced in a number of ways by modulatory neurotransmitters acting through several second-messenger cascades. Modulation at the synapse itself occurs by means of the regulation of calcium current as well as through effects on processes directly involved in transmitter mobilization and exocytosis. Modulation of potassium current plays a major role in controlling neuronal excitability and may contribute to a lesser extent to the regulation of transmitter release through actions on the resting potential and on action potential configuration.