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Journal Article 2005; 26(5): 526-532 PubMed PMID: 16264406 Citation  Keywords:  Animals, Astrocytes:drug effects, Blotting, Western, Cell Line, Estradiol:pharmacology, Estrogen Antagonists:pharmacology, Estrogen Receptor alpha:drug effects, Estrogen Receptor beta:drug effects, Gene Expression Regulation:drug effects, Ligands, Lucifer.   

OBJECTIVE: The mechanism through which estrogen exerts its neuroprotective and anti-neurodegenerative effects in the central nervous system is poorly understood. Human glial cells are implicated in the pathogenesis of Alzheimer's disease and have both alpha and beta estrogen receptors (ER). We developed a glial cell model for ER function using the N20.1 mouse oligodendroglial cell line to evaluate the response of ERalpha and ERbeta to estradiol (E2), a raloxifene analog LY117018 (LY) and 4-hydroxytamoxifen (4OHT).

DESIGN: We tested the ability of exogenous ER to activate transcription in response to ligands (100 nM) using the glial cell line N20.1 in a transient cotransfection assay with an ERalpha or ERbeta expression vector, an ERE-driven reporter and a Renilla luciferase transfection control.

RESULTS: Endogenous ER was not detected in the N20.1 cells by Western immunoblotting. E2 stimulated both ERalpha and ERbeta on both ERE- and AP-1 driven promoters. The transcription stimulation by E2 in the ERalpha and ERbeta through the AP-1driven promoter, though significant, was not of the same magnitude as the stimulation of the ERalpha through the ERE-driven promoter. 4OHT and LY did not show significant transcriptional activation of either the ERalpha or ERbeta, through either the ERE or AP-1 driven promoters. LY, at a 10-fold higher concentration than E2, showed a difference in its antagonist activity on the ERbeta through the AP-1 pathway when compared with the ERE- driven promoter, demonstrating not only promoter specificity, but also receptor specificity.

CONCLUSIONS: This is the first description of the activity of 4OHT and LY on estrogen receptors in glia....

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Journal Article 2005; 26(3): 197-203 PubMed PMID: 15990721 Citation  Keywords:  Aging:physiology, Animals, Antibodies, Blood Pressure:physiology, Body Temperature:physiology, Caloric Restriction, Cell Survival:physiology, Estrogen Receptor alpha:immunology, Female, Immunohistochemistry, Luteinizing Hormone:metabolism, Mice, Mice, Inb.   

: Life-long calorie restriction (CR) remains the most robust and reliable means of extending life span in mammals. Among the several theories to explain CR actions, one variant of the neuroendocrine theories of aging postulates that changing hypothalamic sensitivity to endocrine feedback is the clock that times phenotypic change over the life span. If the feedback sensitivity hypothesis is correct, CR animals should display a significantly different pattern of hormone-sensitive cell density and distribution in the hypothalamus. Of the many endocrine signal receptors that may be involved in maintaining hypothalamic feedback sensitivity, our study has selected to begin mapping those for estrogen (E). Altered hypothalamic sensitivity to E is known to schedule reproductive maturation and influence reproductive senescence. Taking estrogen receptor-alpha (ERalpha) immunoreactivity as an index of sensitivity to E, we counted ERalpha immunoreactive and non-immunoreactive cells in the pre-optic hypothalamus of young (6 weeks), ad-libitum (Old-AL) fed old (22 months), and calorie restricted (Old-CR) old (22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each sampled section of pre-optic hypothalamus. Results show a 38% reduction in ERalpha immunoreactive cells and an 18% reduction in total cell numbers in AL-old mice in comparison to young mice. However, CR mice only show a 19% reduction in ERalpha immunoreactive cells and a 13% reduction in total cell numbers in comparison to young mice. This indicates CR prevents age-related cell loss and maintains estrogen sensitivity in the pre-optic hypothalamus of old female B6D2F1 mice....

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Case Reports 2005; 26(2): 109-112 PubMed PMID: 15855880 Citation  Keywords:  Adult, Aged, Autoimmune Diseases:complications, Female, Gene Frequency, Genetic Markers, Genetic Predisposition to Disease, HLA-DR Antigens:drug effects, Hepatitis C, Chronic:complications, Humans, Hypothyroidism:complications, Interferon-alpha:adverse ef.   

OBJECTIVES: The interferon-alpha (IFN-alpha) therapy for HCV hepatitis may exacerbate or induce underlying thyroid disorders. Besides viral factors, the human leukocyte antigen (HLA) may be an independent risk factor.

METHODS: We evaluated fifteen patients with HCV chronic hepatitis during a period of 40 months. At the enrollment, all the patients were negative for thyroid disorders, excluding one patient with subclinical hypothyroidism. Eleven patients received IFN-alpha therapy. The HLA system was examined in every patient, evaluating antigens (n=40) of locus A, B and Cw and alleles (n=19) of locus DRB1* and DQB1*. The HLA system was also examined in healthy subjects (n=107) as a control group.

RESULTS: The HCV genotype distribution in patients was: 1b=20%, 2a=60%, 3a=20%. Four IFN-treated patients presented clinical thyroid disorders, including autoimmune hypothyroidism (n=2), transient thyrotoxicosis (n=1) and subacute thyroiditis (n=1). The HLA susceptibility to thyroid disorders (antigen/allele frequency) in the whole group of patients was not different in respect to controls and normal Italian population. The patients with HCV chronic hepatitis that developed thyroid diseases after IFN- treatment had a double and specific association with the HLA system (Mantel-Haenszel X(c)(2)=4.706, p<0.05).

CONCLUSIONS: This case report suggests that HLA system examination is an important and promising diagnostic aspect that may be considered in order to evaluate the appearance of thyroid disorders during the IFN-alpha treatment for HCV-related chronic hepatitis....

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Journal Article 2005; 26(1): 43-49 PubMed PMID: 15726019 Citation  Keywords:  Adrenergic alpha-Agonists:pharmacology, Adrenergic alpha-Antagonists:administration & dosage, Animals, Atmosphere Exposure Chambers, Atmospheric Pressure, Hypoxia:drug therapy, Injections, Intraventricular, Male, Median Eminence:drug effects, Norepinephri.   

OBJECTIVE: We have previously found that chronic hypoxia inhibited thyrotropin-releasing hormone (TRH) mRNA expression in rat paraventricular nucleus (PVN). This study presented the effects of hypoxia on TRH secretion in rat hypothalamus, and the norepinephrine (NE) involvement in the modulation of TRH secretion during acute hypoxia exposure.

SETTING AND DESIGN: Hypoxia was simulated at altitudes of 5 km (10.8% O2) or 7 km (8.2% O2) in a ventilated hypobaric chamber, and control group was set at local altitude of 2.3 km (15.8% O2). The duration of hypoxia exposure was designed acutely and chronically for 0.5, 2, 24 h, 5, 10, and 30 d, respectively. TRH levels were measured by specific radioimmunoassay.

RESULTS: The results showed that hypoxia of 5 km or 7 km significantly enhanced TRH levels of the ME and PVN, and reduced serum T3 levels in most hypoxia-exposed groups. Intraventricular injection (icv) of NE (4 nmol/L) induced a decrease in TRH levels in the median eminence (ME) and PVN, and an increased serum T3 levels following hypoxia of 7 km exposure for 2 h, compared with icv saline control, indicating TRH release increased. The stimulating effect of NE on the TRH secretion was abolished by icv antagonist of adrenergic alpha2-receptor, yohimbine (40 nmol/L).

CONCLUSIONS: We conclude that acute and chronic hypoxia exposure produces an inhibition of hypothalamic TRH secretion from the ME and PVN. Central adrenergic system may play a stimulating role through alpha2-receptor in the acute hypoxia-modulating TRH release from rat hypothalamus....

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Journal Article 2005; 26(1): 35-38 PubMed PMID: 15726017 Citation  Keywords:  3-Oxo-5-alpha-Steroid 4-Dehydrogenase:metabolism, Animals, Aromatase:metabolism, Brain Chemistry:physiology, Catecholamines:metabolism, Chromatography, Thin Layer, Dopamine:metabolism, Female, Feminization:physiopathology, Hypothalamus:metabolism, Male, N.   

OBJECTIVES: To study the role of opioids in early postnatal changes of the hypothalamic testosterone metabolism and catecholamine content underlying feminizing effect of prenatal stress on male sexual behavior in rats.

MATERIAL AND METHODS: 10 day-old male and female offspring from mothers given naltrexone prior to daily 1-hour restraint during the last gestational week were used in the study. Aromatase and 5 alpha-reductase activities, noradrenaline and dopamine contents in the brain preoptic area and medial basal hypothalamus were studied by thin layer chromatography, radiometric and spectrofluorimetric techniques.

RESULTS: Sexual dimorphism of testosterone metabolism enzymes activity and catecholamine content in discrete brain regions of 10 day-old rat pups was found. Prenatal stress attenuated these gender-related differences. Naltrexone pre-treatment of stressed dams prevented modifying effect of prenatal stress on aromatase activity and noradrenaline content in the male preoptic area.

CONCLUSIONS: The results of the study demonstrate preventive effect of naltrexone on stress-induced alterations of testosterone aromatization and noradrenaline concentration in the developing brain preoptic area associated with neuroendocrine control of the male sexual behavior in adult rats. These findings indicate that endogenic opioids mediate detrimental effect of prenatal stress on neurochemical determinants of the brain sexual differentiation that may underlie feminization of the male sexual behavior....

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Journal Article 2004; 25(6): 403-406 PubMed PMID: 15665799 Citation  Keywords:  Adrenergic alpha-Antagonists:pharmacology, Adrenergic beta-Antagonists:pharmacology, Animals, Diabetes Mellitus, Experimental:drug therapy, Dose-Response Relationship, Drug, Insulin:blood, Islets of Langerhans:drug effects, Male, Oligopeptides:pharmacolog.   

OBJECTIVE: To examine the effect of ipamorelin (IPA), a novel pentapeptide with a strong growth hormone releasing potency, on insulin secretion from pancreatic tissue fragments of normal and diabetic rats.

MATERIALS AND METHODS: Diabetes mellitus was induced by streptozotocin (60 mg kg(-1)). Four weeks after the induction of diabetes, pancreatic tissue fragments of normal and diabetic rats were removed and incubated with different concentrations (10(-12) - 10(-6) M) of IPA. Insulin release from the pancreas was measured by radioimmunoassay.

RESULTS: Ipamorelin evoked significant (p<0.04) increases in insulin secretion from the pancreas of normal and diabetic rats. Either diltiazem or yohimbine or propranolol or a combination of atropine, propranolol and yohimbine inhibited IPA-evoked insulin secretion significantly (p<0.03) from the pancreas of normal and diabetic rats. Atropine caused a significant (p<0.007) reduction in the IPA-induced insulin secretion in diabetic but not in normal rats.

CONCLUSION: IPA stimulates insulin release through the calcium channel and the adrenergic receptor pathways. This is the first study to examine the effect of ipamorelin on insulin secretion in the pancreas....

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Journal Article 2003; 24(6): 440-444 PubMed PMID: 15073572 Citation  Keywords:  Adjuvants, Immunologic:pharmacology, Breast Neoplasms, Cell Division:drug effects, Cell Line, Tumor:cytology, Drug Interactions, Estradiol:pharmacology, Estrogen Receptor alpha, Estrogen Receptor beta, Female, Gene Expression Regulation, Neoplastic:drug e.   

OBJECTIVES: Detection of the antiestrogenic effect of melatonin on various breast cancer cell lines and its dependence of the differential expression of estrogen receptors (ERalpha and ERbeta) and melatonin receptors (mt1 and RZRalpha).

SETTING AND DESIGN: Dose-response curves of estradiol were determined in 6 different breast cancer cell lines using a colorimetric proliferation assay in the absence or presence of various melatonin concentrations.

METHODS: In order to detect the minor growth inhibitory effect of melatonin, a simple yet novel approach was employed: instead of incubating cells at single estradiol-concentrations at increasing melatonin levels, breast cancer cells were grown in microwell-plates for 4 days at increasing concentrations of estradiol (10(-12)M - 10(-10)M) in the absence or presence of melatonin (10(-9)M - 10(-8)M). Cell number was determined using Alamar blue and colorimetry. RT-PCR was performed for the expression of ERalpha, ERbeta, RZRalpha and mt1.

RESULTS: Melatonin at concentrations of 10(-9)M and 5 x 10(-9)M shifted the dose-response curves of estradiol to higher concentrations. Responsiveness to melatonin depended on expression of ERalpha but not on ERbeta. mRNA of ERbeta was not detectable in the breast cancer cell lines used. Only small amounts of mt1 transcripts were detectable in MCF-7 cells of one source. In MCF-7 cells transfected with the mt1 gene and in an ovarian cancer cell line mt1 was expressed at significant levels. RZRalpha was expressed in all tested cell lines at different amounts.

CONCLUSION: The growth of all ERalpha-positive breast cancer cell lines can be inhibited by melatonin. The effect in most cell lines is weak yet clearly reproducible. RZRalpha clearly contributes to the growth inhibitory effect of melatonin....

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Journal Article 2003; 24(3-4): 249-254 PubMed PMID: 14523365 Citation  Keywords:  17-alpha-Hydroxyprogesterone:blood, Adolescent, Adrenocorticotropic Hormone:blood, Adult, Female, Hirsutism:blood, Humans, Hydrocortisone:blood, Hyperandrogenism:genetics, Pituitary-Adrenal System:physiopathology, Polycystic Ovary Syndrome:genetics, Stero.   

OBJECTIVES: Impaired adrenal function is common in patients with polycystic ovary syndrome (PCOS). Abnormal regulation of cytochrome P450 17 alpha is believed to cause the exaggerated 17-hydroxyprogesterone (17OHP) response to ACTH stimulation. The aim of the study was to evaluate cortisol and 17OHP response to low dose (1 microg) ACTH test and to compare it with the standard ACTH (250 microg) test in hyperandrogenic women with PCOS.

DESIGN AND MEASUREMENTS: We studied 27 PCOS and 22 control women. All participants were examined for mutations of the CYP21 gene, Cortisol and 17OHP levels before, 30 and 60 minutes after the IV injection of 250 microg ACTH (SDT) and after 1 microg ACTH (LDT). Fasting serum levels of LH, FSH, testosterone, DHEAS were determined in all participants.

RESULTS: Basal and ACTH stimulated Cortisol during the SDT (470+/-138 nmol/L and 761+/-143, respectively) were significantly higher in PCOS vs. controls (232+/-124 and 670+/-130, respectively) (p<0.03, p<0.02, respectively). Basal 17OHP (6.1+/-2.1 nmol/L) and the peak response to SDT (14.2+/-3.6 nmol/L) were significantly higher in PCOS vs. controls (4.2+/-2.1, 10.9+/-3.0, respectively) (p<0.003, p<0.004, respectively). Abnormally elevated 17OHP response to SDT was detected in 6/27 PCOS women (22%). No statistically significant difference between the PCOS and control groups were noted during the LDT in both cortisol and 17OHP levels.

CONCLUSIONS: These data suggest that the exaggerated 17-hydroxyprogesterone (17OHP) response to ACTH stimulation in PCOS is revealed by stimulation at a pharmacological dose (250 microg) but not by a physiological dose (1 microg)....

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