N-glycans are not the signal for apical sorting of corticosteroid binding globulin in MDCK cells.
(1/134)
It has been suggested that N-glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96-98]. Human corticosteroid binding globulin contains six consensus sites for N-glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild-type corticosteroid binding globulin is N-glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N-glycosylation sites, and a mutant lacking all six N-glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N-glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells. (+info)
Abnormalities of hypothalamic-pituitary-adrenal and hypothalamic-somatotrophic axes in Fawn-Hooded rats.
(2/134)
Fawn-Hooded (FH) rats show central and peripheral abnormalities in serotoninergic functions and have attracted attention as an animal model of some pathologies, including depression and hypertension. In addition, these rats show a reduced growth rate. As the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in both depression and hypertension, and the hypothalamic-somatotrophic (HSM) axis has a major role in growth, these two endocrine axes were characterised in FH rats as compared with outbred Sprague-Dawley (SD) rats in basal conditions. FH rats showed normal serum ACTH and corticosterone concentrations, but reduced serum corticosterone binding capacity. At a central level, normal expression of mRNA for glucocorticoid type II receptors in the hippocampal formation and mRNA for corticotrophin-releasing factor (CRF) in the paraventricular nucleus of the hypothalamus were observed in FH rats, whereas expression of mRNA for CRF in the central nucleus of the amygdala was enhanced compared with the expression in SD rats. Serum GH concentrations were normal in FH rats, IGF-I tended to be lower, and mRNA for somatostatin (SRIF) in the periventricular nucleus of the hypothalamus was significantly lower in FH rats than in SD rats. The reduced SRIF gene expression in rats with normal or slightly reduced GH and IGF-I, respectively, might be secondary to a defective central and peripheral response to IGF-I, compatible with the reduced growth of FH rats. The present results suggest that FH rats have abnormalities in both HPA and HSM axes that might be related to some of their physiopathological characteristics. (+info)
Identification and characterization of nuclear matrix-attachment regions in the human serpin gene cluster at 14q32.1.
(3/134)
Matrix-attachment regions (MARs) are DNA elements that are defined by their abilities to bind to isolated nuclear matrices in vitro. The DNA sequences of different matrix-binding elements vary widely. The locations of some MARs at the ends of chromatin loops suggest that they may represent boundaries of individual chromatin domains. As such, MARs may play important roles in regulating transcription and chromatin structure. As a first step towards assessing the roles of MARs in these processes, we assayed DNA sequences from the human serine protease inhibitor (serpin) gene cluster at 14q32.1 for matrix-binding activity in vitro. This approximately 150 kb region contains the cell-specific genes encoding alpha1-anti-trypsin (alpha1AT) and corticosteroid-binding globulin (CBG), as well as an antitrypsin-related sequence termed ATR. A DNase I-hypersensitive site (DHS) map of the locus has recently been described. We report here that the alpha1AT-ATR-CBG region contains five distinct MARs. There is a strong matrix-binding element approximately 16 kb upstream of alpha1AT; three MARs are between ATR and CBG and one MAR is within the CBG gene itself. These MARs were matrix-associated in all cell types examined. DNA sequencing indicated that the serpin MARs contained predominantly repetitive DNA, although the types of DNA repeats differed among the MARs. (+info)
Few differences found between early- and late-weaned pigs raised in the same environment.
(4/134)
Segregation and medicated early weaning are technologies used to optimize the productivity and health of pigs, but these practices may also cause aberrant behaviors indicative of stress. Thus, differences in early- (=10 d of age) and late- (=30 d of age) weaned pigs were investigated. At weaning, pigs were housed in groups of four in 16 pens (eight pens per treatment) in the same facility, and, thus, they were not segregated. Body weights were recorded at birth, weaning, and at approximately 42, 65, 102, 137, and 165 d of age (at slaughter). One-minute, instantaneous scan samples during a 10-min period (at 0600, 1000, 1400, and 1800) were used to record the frequency of lying, standing, and sitting, total number of drinks, feeder investigations, and time spent playing/fighting on 2, 3, and 4 d after weaning. Five-minute, direct observations of each pig were conducted at approximately 40, 60, 80, and 150 d of age. Direct observations were also made of the entire pen for 10 min at approximately 50, 95, 123, and 160 d of age to record aberrant behaviors. At 62 d of age, a handling and blood collection stress was imposed. At 165 d of age, a second stress test was conducted in response to rough handling and transport. Early-weaned pigs spent more time playing/ fighting (P < .006) than late-weaned pigs during the 4 d after weaning, manipulated conspecifics more often at 40 d of age (P < .002), had greater percentage of hemoglobin (P < .03) during Stress Test 1, had greater ADG at 42 d of age (P < .03), and had greater hypothalamic growth hormone-releasing hormone receptor mRNA at slaughter (P < .06). Late-weaned pigs had greater ADG between 137 and 165 d of age (P < .03) and greater pro-opiomelanocortin at slaughter (P < .04). Overall, most differences found between early-weaned and late-weaned pigs were evident soon after weaning, but they disappeared before slaughter. (+info)
Neonatal handling permanently alters hypothalamic-pituitary- adrenal axis function, behaviour, and body weight in boars.
(5/134)
Neonatal handling permanently alters hypothalamic- pituitary-adrenal axis (HPA) function in rats. In the rat, this treatment increases hippocampal glucocorticoid receptors (GR) and dampens plasma ACTH and corticosterone responses to stressors. The objectives of this study were to determine whether neonatal handling of pigs would effect permanent changes in plasma corticosteroid binding capacity (CBG), basal or stressor-induced plasma cortisol and ACTH concentrations, brain or pituitary GR levels, dexamethasone suppression of plasma cortisol and ACTH concentrations, behaviour in an open field-test pen, and body weights. Twelve litters of pigs were randomly assigned to either neonatal handling or no disturbance. Handled litters were removed from the farrowing crate for 10 min per day for the first 14 days of life. Male pigs were kept for the study and the boars were weighed monthly. At 7 months of age, boars were tested for locomotory behaviour in an open field-test pen. The boars were implanted with indwelling ear-vein catheters and blood samples were obtained basally, during and after application of a nose snare, and after 0.04 mg/kg dexamethasone. Boars were killed and blood samples were obtained and the brain and pituitary glands collected. Handled boars had greater (P<0.05) plasma CBG binding and lower basal total (P<0.05) and calculated free (P<0.03) plasma cortisol concentrations. No significant differences between treatments were found in plasma ACTH or cortisol responses to a nose-snare stressor; however, when killed, handled boars had greater (P<0.02) plasma ACTH concentrations. Handled and non-handled boars did not differ in plasma ACTH or cortisol responses to dexamethasone. There was no treatment effect on GR expression in the pituitary gland, frontal cortex, hippocampus, or hypothalamus. Behaviourally, the handled boars had higher (P<0.03) locomotor scores over inner squares and a lower (P<0.05) ratio of outer:inner squares entered in open field-tests. During the first 7 months of life, body weights were lower (P<0.004) for handled boars. In conclusion, neonatal handling permanently altered HPA function in pigs, but in a manner dissimilar to that found in the rat. These changes induced in the pig were not beneficial for commercial production with respect to body weight. (+info)
Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.
(6/134)
A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan. (+info)
Identification of selective estrogen receptor modulators by their gene expression fingerprints.
(7/134)
Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile. (+info)
Circulating concentrations of the antiprogestins CDB-2914 and mifepristone in the female rhesus monkey following various routes of administration.
(8/134)
The overall aim of these studies was to investigate the oral and i.m. bioavailability of CDB-2914 in intact female rhesus monkeys, and to compare the serum concentrations of CDB-2914 with that of mifepristone following oral administration. In the first study, a 50 mg bolus of CDB-2914 per monkey was administered intravenously, orally or intramuscularly. The area under the serum concentration-time curve for 72 h (AUC(0-72)) following i.v. injection was 18 320 +/- 2718 ng/ml*h, and that for oral administration was 10 464 +/- 3248 ng/ml*h. Thus, the oral bioavailability of CDB-2914 equivalents was 56%. The AUC(0-168 h) following i.m. injection was 11 226 +/- 1130 ng/ml*h. Therefore, the i.m. bioavailability of CDB-2914 equivalents was 62%. In the second study, the serum concentrations of CDB-2914 and mifepristone equivalents were compared following an oral bolus dose in two different formulations. When administered at 5 mg/kg in aqueous suspending vehicle (ASV), the mean peak serum concentration (C(max)) of CDB-2914 equivalents (192 +/- 64 ng/ml) occurred at 5 +/- 1 h, whereas the C(max) of mifepristone equivalents (82 +/- 25 ng/ml) occurred at 3 +/- 1 h. Following administration in gelatin capsules (35 mg/monkey), the C(max) of CDB-2914 equivalents (129 +/- 24 ng/ml) occurred at 5 +/- 1 h, while the C(max) of mifepristone equivalents (31 +/- 8 ng/ml) occurred at 3 +/- 1 h. The serum concentration (AUC(0-120 h)) of CDB-2914 equivalents was 4.7- or 5. 3-fold greater than that of mifepristone equivalents when administered orally in ASV or gelatin capsules respectively. The serum protein binding characteristics of CDB-2914 were also studied. CDB-2914 bound to human alpha(1)-acid glycoprotein (AAG), but not with as high an affinity as mifepristone. In contrast, neither CDB-2914 nor mifepristone bound with high affinity to AAG, corticosteroid binding globulin or sex hormone binding globulin in monkey serum. Collectively, these results indicated that CDB-2914 was more efficiently absorbed than mifepristone following oral administration to female rhesus monkeys. (+info)