bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulsatile gonadotrophin secretion in hypothyroid women of reproductive age
Authors: Tomasi Paolo A , Fanciulli Giuseppe , Zini Michele , Demontis Maria A , Dettori Alessandra , Delitala Giuseppe
Published: December 2008

Hypothyroid women may have various disturbances of the reproductive system. Although menstrual cycle disturbances and infertility have been reported in hypothyroidism, gonadotrophin levels have usually been found in the normal range. We have investigated whether female hypothyroid patients of reproductive age have any alteration in the pulsatile secretory pattern of gonadotrophin secretion. LH and FSH were assayed on days 2–5 of the menstrual cycle in blood samples taken every 10 min for 8 h from six hypothyroid women and six age-matched control subjects. Pulsatility was analysed using the Cluster and Detect programs. There was no significant difference in the number of peaks identified (3·7±0·8 vs 3·7±0·8 for LH, and 3·7±0·8 vs 4·2±0·5 for FSH), the mean duration of peaks (LH: 68·0±6·9 vs 72·±5·1 min; FSH: 81·9±8·1 vs 71·2±10·3 min), the area under the peaks (LH: 91·5±20·4 vs 148·2±55·1IU/l per min; FSH: 71·5±4·5 vs 62·7±15·0IU/l per min), and the incremental amplitude from baseline (LH: 2·2±0·4 vs 3·0±0·8 IU/l; FSH: 1·4±0·2 vs 2·1±0·5IU/l). However, the absolute pulse amplitude was greater in hypothyroid patients (LH: 14·5±1·4 vs 8·3±1·3 IU/l, FSH: 9·0±1·5 vs 5·8±1·2 IU/l, P=0·04), as were the integrated concentrations (LH: 6·6±0·7 vs 3·3±0·4OU/l per min, P<0·01; FSH: 4·3±0·4 vs 2·1±0·5IU/l per min, Oestradiol values were comparable in the two groups 42·7±0·4 vs 43·±9·7 pg/ml). These results indicate that in hypothyroid women there is an increased baseline level with a normal pulsatility of the gonadotrophin secretion. Similar oestrogen levels in both groups, and normal or nearnormal cycles in our patients suggest either a decreased biological potency of the gonadotrophins or a mild ovarian resistance.

European Journal of Endocrinology 136 406–409
Lack of relationship between 17-hydroxyprogesterone response to buserelin testing and hyperinsulinemia in polycystic ovary syndrome
Authors: Şahin Yilmaz , Ayata Demet , Keleştimur Fahrettin
Published: December 2008

Objective: To determine whether hyperinsulinism affects cytochrome P450c 17α activity by investigating the correlation between 17–hydroxyprogesterone (17–OHP) hyper-responsiveness to the gonadotropin-releasing hormone (GnRH) agonist, buserelin, and the insulin response to oral glucose in polycystic ovary syndrome (PCOS).

Design: Ultrasound, clinical and hormonal parameters were used to define PCOS in this prospective clinical study. We investigated the correlation between the 17–OHP response to buserelin testing and the insulin response to oral glucose in PCOS.

Methods: Twenty-eight women with PCOS and eighteen normal women were included in the study. 17–OHP response to buserelin, and insulin and C–peptide responses to oral glucose were measured. Results: Twenty–live women with PCOS had an increased 17–OHP response. The PCOS patients showed significantly higher mean post–glucose load insulin and C–peptide levels than controls (P<0·05). No significant correlations were found between basal 17–OHP and fasting insulin or fasting C–peptide, between peak 17–OHP and fasting insulin, peak insulin or peak C–peptide, between 17–OHP area under the curve (AUC) and insulin AUC or C–peptide AUC, and between percent increment in 17–OHP and insulin AUC or C–peptide AUC (P >0·05).

Conclusions: Lack of a relationship between the 17–OHP response to the GnRH agonist buserelin and hyperinsulinism suggests that hyperinsulinism may not play a role in the dysregulation of the cytochrome P450c17α enzyme seen in PCOS.

European Journal of Endocrinology 136 410–415
Oestrogen replacement does not restore the reduced GH-releasing activity of Hexarelin, a synthetic hexapeptide, in post-menopausal women
Authors: Arvat E , Gianotti L , Broglio F , Maccagno B , Bertagna A , Deghenghi R , Camanni F , Ghigo E
Published: December 2008

Hexarelin (HEX), a synthetic hexapeptide, has a strong and reproducible GH-releasing activity in man after intravenous, subcutaneous, intranasal and oral administration. Its effect undergoes age-related variations, being reduced in elderly subjects. In spite of evidence in animals showing that the activity of GH-releasing peptides (GHRPs) is positively influenced by oestrogens, in young adults no sex-related difference has been found in the GH response to HEX or to other GHRPs. We aimed to clarify the influence of the menopause and oestrogens on the GH-releasing activity of HEX. We studied the GH response to the acute administration of the maximal effective dose of HEX (2 μg/kgi.v.) in 24 young women (YW, age: 27·3±0·5 years; body mass index (BMI): 20·7±0·3 kg/m2). 14 post-menopausal women (PW, age: 52·9±1·2 years; BMI: 23·2±0·9 kg/m2) and 14 aged women (AW, age: 68·9±1·5 years; BMI: 21·7±0·7 kg/m2). In 10 post-menopausal women the GH response to HEX was also studied after 3 months of transdermal oestradiol treatment (delivery 50 μg/die). Basal oestrogen and GH levels in PW were lower than those in YW (oestrogen: 4·8±3·6 vs 42·0±3·4 pg/ml (mean±s.e.m.), P< 0·001; GH: 1·5±0·5 vs 2·9±0·6 μg/l, P< 0·02) and similar to those in AW (oestrogen: 1·3±0·4 pg/ml; GH: 0·9±0·2 μg/l). IGF-I levels in PW were not different from those in YW (174·4±11·9 vs 195·5±14·9 μg/l) and higher than those in AW (109·8±15·8 μg/l, P<0·01). The GH response to HEX in PW (areas under the curve±s.e.m.: 453·6±56·0 μg.min/l) was lower (P<0·002) than that in YW (1630·4±259·7 μg.min/l) while it did not differ from that in AW (781·8±189·3 μg.min/l). In PW 3-month oestrogen administration increased oestradiol levels (38·3±5·9 vs 0·8±0·4 pg/ml, p<0·001) making them similar to those recorded in YW, while it failed to modify both basal GH and IGF-I levels (GH: 1·8±0·6 vs 1·5±0·7 μg/l; IGF-I: 164·6±14·3 vs 175·0±12·3 μg/l). Also the GH response to HEX was not modified by oestradiol treatment (518·4±125·6 vs 425·4±69·3 μg.min/l). In conclusion, present data confirm the strong GH-releasing effect of Hexarelin in humans and demonstrate that its activity is already reduced in post-menopausal women to an extent overlapping that in elderly women. Moreover, oestrogen treatment is not able to restore it. Thus, the lack of oestrogens does not seem to account for the reduced somatotroph responsiveness to GHRPs in the post-menopausal period.

European Journal of Endocrinology 136 483–487
Assessment of thyroid growth stimulating activity of immunoglobulins from patients with autoimmune thyroid diseases by cytokinesis arrest assay
Authors: Miyamoto Shinichi , Kasagi Kanji , Alam Mohammad Sayeedul , Misaki Takashi , Iida Yasuhiro , Konishi Junji
Published: December 2008

Objective: To develop a novel bioassay for the assessment of thyroid cell growth stimulating activity using cytochalasin B (CB) and to test immunoglobulins (IgGs) from patients with autoimmune thyroid diseases.

Design: The assay is based on the principle that growing cells during incubation with CB show an increased number of nuclei in a cell (N/C index), since CB. at appropriate concentrations, is known to inhibit cytoplasmic cleavage without affecting nuclear mitosis. The N/C index represents potential DNA production while cells are incubated with CB.

Methods: FRTL-5 thyroid cells were incubated with various thyroid stimulators in TSH-free medium containing 2 mg/l CB for 3 days. After the incubation, the cells were harvested in trypsin/EDTA to obtain single cell suspension, fixed, dropped onto a glass slide, stained and observed under a microscope to determine the N/C index.

Results: Bovine TSH at 10−3–1·0 U/l, forskolin at 1×10−7–10−5 mol/l, cholera toxin at 10×10−5–10−3 mg/l, or (Bu)2cAMP at 1× 10−5–10−3 mol/l increased the N/C index up to approximately 2·0 in a dose-dependent manner. IgGs not only from 27 patients with untreated goitrous Graves' disease but also from 14 patients with goitrous Hashimoto's thyroiditis elicited an increase in the N/C index, which exceeded the mean+2s.d. of the values for 17 normal subjects (mean ± s.d., 1·063 ±0.014). Four patients with primary myxedema displayed a normal N/C index. In Graves' disease, the N/C index did not correlate significantly with thyroid stimulating antibodies (TSAb) activities but did correlate significantly with estimated goiter size (P<0·05). IgGs containing blocking-type TSH-receptor antibodies inhibited the TSH- or Graves' IgG-stimulated increase in N/C index almost completely, but did not influence the stimulatory effect of IgG from two patients with Hashimoto's thyroiditis.

Conclusions: We have developed a sensitive and simple assay for thyroid growth stimulating activity by using CB, and found that all tested patients with goitrous Graves' disease and goitrous Hashimoto's thyroiditis have thyroid growth stimulating immunoglobulins whose activity does not correlate with TSAb.

European Journal of Endocrinology 136 499–507
Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism
Authors: Colin Ides M , Kopp Peter , Zbären Jakob , Häberli André , Grizzle William E , Jameson J Larry
Published: December 2008

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands.

In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients).

NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable.

In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

European Journal of Endocrinology 136 649–655
Effects of Benthic Prey Composition and Abundance on Diet and Growth of Black Crappies in Three Florida Lakes
Authors: Tuten Travis , Allen Micheal , Cichra Chuck
Published: December 2008

The influence of benthic prey availability on growth of black crappies Pomoxis nigromaculatus is poorly understood due to scant research on the relationships between benthic prey abundance and composition and black crappie diet. However, benthic taxa play an important role as prey items of black crappies during intermediate ontogenetic diet phases. We evaluated diet, growth, and abundance of black crappies in three large Florida lakes (Lochloosa, Marian, and Monroe), and we related diets to benthic prey availability at these lakes. Black crappies in Lake Monroe obtained the largest size at age, whereas those in Lake Marian had the smallest size at age. Black crappies in Lakes Lochloosa and Marian consistently exhibited positive selection of dipteran pupae; fish in Lake Monroe showed consistently positive selection of a higher‐quality prey item (the mysid shrimp Americamysis almyra) that was not available in Lakes Lochloosa and Marian. A similarity index indicated that benthic prey availability influenced consumption of benthic prey items by black crappies in all three lakes. Differences in prey availability (i.e., density and presence‐absence) and prey selection influenced diet composition and ontogenetic diet shifts of black crappies. Diet and prey availability differences among lakes probably contributed to the variation in black crappie growth rates; the highest growth was observed at Lake Monroe, where the additional prey resource (A. almyra) was available.
Larval Metamorphosis of Individual Pacific Lampreys Reared in Captivity
Authors: McGree Michelle , Whitesel Timothy A. , Stone Jen
Published: December 2008

This study is one of the first to follow individual Pacific lampreys Lampetra tridentata through the process of metamorphosis. Readily observable external changes were described for 13 individual Pacific lampreys undergoing metamorphosis. Changes occurred to the mouth, eyes, and branchial region from July to at least November. During metamorphosis, Pacific lampreys also exhibited asymmetric growth, including an increase in snout depth, that had not previously been reported in the literature. The order of the morphological changes and the patterns of asymmetric growth in the Pacific lamprey closely matched those reported for another Lampetra species, the American brook lamprey L. appendix, but exhibited unexpected variations from those reported in other species of lampreys. Excepting one catastrophic event, under captive rearing conditions 96.4% of the ammocoetes survived and the maximum growth rates of 0.040‐0.071 mm/d were within the range of those estimated for ammocoetes rearing naturally in stream environments. Supplemental feeding improved larval growth but did not influence the incidence of metamorphosis in captively reared animals. To develop effective conservation strategies for Pacific lampreys in the Columbia River basin, it would be prudent to consider that metamorphosis, a time when the animals are relatively vulnerable, may last from July to December.
Use of Microsatellites to Determine Population Structure and Migration of Pacific Herring in British Columbia and Adjacent Regions
Authors: Beacham T. D. , Schweigert J. F. , MacConnachie C. , Le K. D. , Flostrand L.
Published: December 2008

This study assessed genetic population structure of Pacific herring Clupea pallasii in British Columbia and adjacent regions by analyzing microsatellite variation; the utility of microsatellites for stock identification applications was also evaluated. Variation at 14 loci was surveyed in approximately 26,000 Pacific herring from 90 sampling locations. Estimates of the genetic differentiation index FST by locus varied between 0.000 and 0.014, and the mean across all loci was 0.003. Four stocks of Pacific herring were defined in British Columbia, and stocks were also identified in southeast Alaska, Washington, and California. In British Columbia, differences in timing of spawning are the main isolating mechanisms among stocks, although geographic isolation of the spawning populations may also have some effect in maintaining the genetic distinctiveness of each stock. The limited genetic differentiation among Pacific herring populations in British Columbia is consistent with among‐population straying rates that are sufficient to homogenize allele frequencies over broad areas. Analysis of simulated mixtures from fishery sampling suggested that acceptable estimates of stock composition were achieved by use of the microsatellites. Mixed‐stock samples (∼1,700 individuals) were collected during research vessel surveys from 14 locations in British Columbia. Analysis of summer mixed‐stock samples from the Strait of Georgia and adjacent waters indicated that resident fish on the west side of the strait were mainly derived from primary‐spawning populations (i.e., those that spawned during the primary period in March) that failed to migrate to traditional summer feeding grounds. On the east side of the strait, Pacific herring of mainland inlet origin were found in higher proportions and presumably did not move in appreciable numbers to feeding areas off the west coast of Vancouver Island. In northern British Columbia, fish from northern late‐spawning populations dominated fishery samples collected in waters adjacent to the mainland until the end of June; however, by late July, this group had been replaced almost entirely by primary‐spawning Pacific herring of British Columbia origin.
Body mass index for predicting hyperglycemia and serum lipid changes in Brazilian adolescents
Authors: Vieira Ana Carolina R , Alvarez Marlene M , Kanaan Salim , Sichieri Rosely , Veiga Gloria V
Published: December 2008

OBJECTIVE: To determine the best cut-offs of body mass index for identifying alterations of blood lipids and glucose in adolescents. METHODS: A probabilistic sample including 577 adolescent students aged 12-19 years in 2003 (210 males and 367 females) from state public schools in the city of Niterói, Southeastern Brazil, was studied. The Receiver Operating Characteristic curve was used to identify the best age-adjusted BMI cut-off for predicting high levels of serum total cholesterol (>150mg/dL), LDL-C (>100mg/dL), serum triglycerides (>100mg/dL), plasma glucose (>100mg/dL) and low levels of HDL-C (< 45mg/dL). Four references were used to calculate sensitivity and specificity of BMI cut-offs: one Brazilian, one international and two American. RESULTS: The most prevalent metabolic alterations (>50%) were: high total cholesterol and low HDL-C. BMI predicted high levels of triglycerides in males, high LDL-C in females, and high total cholesterol and the occurrence of three or more metabolic alterations in both males and females (areas under the curve range: 0.59 to 0.67), with low sensitivity (57%-66%) and low specificity (58%-66%). The best BMI cut-offs for this sample (20.3 kg/m² to 21.0 kg/m²) were lower than those proposed in the references studied. CONCLUSIONS: Although BMI values lower than the International cut-offs were better predictor of some metabolic abnormalities in Brazilian adolescents, overall BMI is not a good predictor of these abnormalities in this population.
O impacto da publicidade de bebidas alcoólicas sobre o consumo entre jovens: revisão da literatura internacional
Authors: Pinsky Ilana , Jundi Sami A R J El
Published: December 2008

OBJETIVO: Problemas relacionados ao consumo de álcool são freqüentes, especialmente entre a população jovem. A publicidade de bebidas alcoólicas é um dos fatores passíveis de modificação com impacto no aumento do consumo de álcool. MÉTODO: Foi realizada uma revisão bibliográfica de trabalhos que investigaram de diversos pontos de vista o impacto da publicidade do álcool sobre o consumo. A busca foi feita nas bases de dados Medline, SciELO, PsychoInfo e Google Scholar no período entre 1990 e 2008 e utilizou-se a técnica de " bola de neve" para a indicação de autores mais profícuos na área. Obtiveram-se mais de uma centena de artigos. RESULTADOS: O conjunto de trabalhos aponta que fatores como exposição à publicidade e atratividade da publicidade de bebidas alcoólicas estão relacionados com uma maior expectativa de consumo futuro e com um consumo maior e mais precoce, principalmente entre adolescentes e adultos jovens. Apesar das dificuldades metodológicas, estudos econométricos mais recentes consideram que a redução e/ou banimento de publicidade teria efeito de redução do consumo de álcool. Questões referentes às bases neurofisiológicas do processo de tomada de decisões e da liberdade de escolha no contexto da exposição à publicidade também são discutidas. CONCLUSÕES: O conhecimento atual sobre o tema indica que a redução da exposição à publicidade tem impacto sobre o consumo de álcool, principalmente entre os mais jovens.
Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal β-cell line HIT-T15
Authors: Hughes S. J. , Ashcroft S. J. H.
Published: December 2008

We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal β-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mm increased Cai by 36%, from 89 ± 4 to 121 ± 6 nm (mean ± s.e.m., n = 23). Further raising the medium glucose concentration to 10 mm increased Cai to 139 ± 6 nm. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mm K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 ± 48 nm. The increases in Cai in response to glucose or K+ were blocked by the simultaneous presence of verapamil (50μm). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.
Is intracellular pH a coupling factor in nutrient-stimulated pancreatic islets?
Authors: Best L. , Bone E. A. , Meats J. E. , Tomlinson S.
Published: December 2008

Intracellular pH (pHi) was monitored in dispersed pancreatic islet cells from rats using the fluorescent dye 2′7′bis-carboxyethyl-5′(6′)-carboxyfluorescein. The addition of a weak acid (acetate, propionate or formate) provoked a rapid fall in pHi, corresponding to approximately 0·2 units, followed by a slower return to the basal value. Amiloride also caused a rapid fall in pHi, but no recovery occurred in this case. Addition of NH4Cl induced a rise in pHi. Of the nutrients tested, only glyceraldehyde produced a fall in pHi, both glucose and α-ketoisocaproate causing a gradual and sustained rise in pHi.

Insulin secretion and inositol lipid metabolism in response to nutrient stimuli were markedly inhibited by NH4Cl. The responses to non-nutrient stimuli were unaffected. Glucose-induced insulin secretion and inositol lipid metabolism were potentiated in the presence of amiloride. No such potentiation, however, was observed in the presence of weak acids.

Amiloride and weak acids shared the ability to reduce the fractional outflow rate of 45Ca2+.

It is concluded that pharmacological manipulations of pHi can influence certain aspects of islet cell function, such as calcium handling, though it seems unlikely that the stimulation of islets by nutrient secretagogues occurs as a result of changes in pHi.
Pro-opiomelanocortin mRNA size heterogeneity in ACTH-dependent Cushing's syndrome
Authors: Clark A. J. L. , Lavender P. M. , Besser G. M. , Rees L. H.
Published: December 2008

As an approach to understanding the abnormalities of pro-opiomelanocortin (POMC) gene regulation in human ACTH-secreting tumours, we have analysed the POMC mRNA content of nine such tumours using the Northern blot technique. Most of the tumours and normal human pituitary contained easily detectable quantities of POMC mRNA. The length of this message in most tumours was similar to, or slightly larger than, that in the normal pituitary (1150–1200 bases). Ribonuclease H studies suggested that the origin of any size heterogeneity was a longer poly(A) tail in the tumour RNA. Some tumours, however, expressed a short POMC mRNA (800 bases) which may lack the first two exons of the POMC gene as has been described. A third POMC mRNA size variant (1500 bases) was also seen in low levels in two cases, and as the principal mRNA species in one case. Primer extension and S1 nuclease protection studies suggested that most transcripts in the tumours analysed originated from the conventional promoter, and thus the use of an alternative promoter is not an adequate explanation for the expression of this gene in ectopic ACTH-secreting tumours.
Molecular cloning and sequence analysis of putative chicken prolactin cDNA
Authors: Hanks M.C. , Alonzi J.A. , Sharp P.J. , Sang H.M.
Published: December 2008

A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).
Heterogeneity of responses to LH-releasing hormone and phorbol ester among rat gonadotrophs: a study using a reverse haemolytic plaque assay for LH
Authors: Lewis C. E. , Richards P. S. M. , Morris J. F.
Published: December 2008

Protein kinase C (PK-C) has been implicated in the action of LHRH because LHRH-induced release of LH is partially mimicked by phorbol esters which activate PK-C, and reduced in magnitude by inhibitors of PK-C. We have used a reverse haemolytic plaque assay for LH to visualize and compare the direct effects on individual rat gonadotrophs of (1) a 2-h exposure to a range of concentrations of LHRH and phorbol 12-myristate 13-acetate (PMA) and (2) a single maximally stimulatory dose (10 nm) of LHRH or PMA, or LHRH in the presence of inhibitors of PK-C (retinal and isoquinolone sulphonamide; H7) over six consecutive 30-min intervals.

Quantitative analysis of the size and number of haemolytic plaques indicated that LHRH induced a dose- and time-dependent increase in the amount of LH released by individual gonadotrophs, with no evidence of the priming effect of LHRH. Stimulation by 10 nm LHRH induced recruitment of actively secreting gonadotrophs which reached maximum levels by 90 min. There was a delay of 90–120 min before 10 nm PMA caused a significant release of LH, as assessed by both the size and number of plaques. During the first 30 min of exposure, the presence of 10 μm retinal or H7 augmented LHRH-induced secretion of LH, with the absence of any inhibition of the effects of LHRH until 90–120 min, when both the size and number of plaques were reduced compared with those formed in the presence of LHRH alone.

Heterogeneity of responsiveness to LHRH among gonadotrophs was evident from the bimodal distribution of plaque sizes formed after 60–180 min of exposure to LHRH. The size of gonadotrophs was correlated with that of plaques by 60 min of exposure to LHRH, with the larger cells secreting more LH. By contrast, not until 180 min did PMA induce such heterogeneity in secretion. PMA or LHRH in the presence of retinal or H7 resulted in only a unimodal distribution of plaque sizes, with the absence of a discrete population of cells producing the larger plaques.

These findings indicate that PK-C is not crucial for the immediate LH-releasing action of LHRH but could well be involved in other, slower LHRH-induced effects such as post-translational modification of LH, up-regulation of LHRH receptors and/or gonadotroph multiplication.
Specific effects of agonists of the calcium messenger system on secretion of 'late-pathway' steroid products by intact tissue and dispersed cells of the rat adrenal zona glomerulosa
Authors: Vinson G. P. , Laird S. M. , Whitehouse B. J. , Hinson J. P.
Published: December 2008

Recent data have implicated the phosphatidylinositol/calcium second-messenger system in the control of aldosterone secretion by the adrenal zona glomerulosa. However, in the rat adrenal there are few reports of a direct effect of protein kinase C activation on steroid secretion, while the effects of calcium mobilization may be variable. Since the rat adrenal zona glomerulosa is sensitive to the mode of tissue preparation, these mechanisms were reinvestigated in intact (non-dispersed) capsular tissue and collagenase-dispersed zona glomerulosa cells.

Steroidogenesis in the intact zona glomerulosa was markedly affected by agonists of the calcium messenger system. Most notably, aldosterone and 18-hydroxycorticosterone (18-OH-B) secretion were stimulated by A23187 (100 nmol to 10 μmol/l) and BAY K 8644 (500 nmol/l). Phorbol 12-myristate 13-acetate (TPA; 1 pmol to 1 μmol/l) stimulated aldosterone secretion at all doses and caused a dose-dependent increase in 18-OH-B and 18-hydroxydeoxycorticosterone (18-OH-DOC) secretion. Corticosterone secretion was slightly increased in the presence of A23187 but not by TPA or BAY K 8644. Production of 18-OH-DOC was unaffected by A23187 and BAY K 8644. The calcium channel antagonist verapamil (10 μmol/l) inhibited ACTH-stimulated aldosterone secretion by the intact zona glomerulosa but had no effect on corticosterone secretion.

Verapamil (10 μmol/l) also inhibited the increase in aldosterone secretion by collagenase-dispersed zona glomerulosa cells stimulated by ACTH (100 fmol to 100 nmol/l), angiotensin II (100 pmol to 10 nmol/l) and potassium (5·9 and 8·4 mmol/l); stimulated corticosterone secretion was unaffected. Aldosterone secretion by dispersed zona glomerulosa cells was unaffected by the calcium ionophore A23187 (10 nmol to 100 μmol/l) or by TPA (1 nmol to 10 μmol/l). Corticosterone secretion was also unaffected by A23187 over the same dose range, but was increased slightly by high doses of TPA (10 and 1 μmol/l), while the calcium channel agonist BAY K 8644 had no effect on either steroid.

The results show that calcium mobilization and protein kinase C activation can stimulate steroid secretion in the rat adrenal zona glomerulosa and that large and reproducible effects are seen when intact tissue is used. In general only 18-OH-B and aldosterone secretion were affected; effects on corticosterone and 18-OH-DOC were much less marked. Together with the effects of calcium blockers, the data strongly support the view that protein kinase C activation and calcium mobilization are primarily involved in the control of the 'late pathway' of aldosterone biosynthesis.
STRUCTURAL ORGANIZATION OF THE HUMAN ANDROGEN RECEPTOR GENE
Authors: Kuiper G.G.J.M. , Faber P.W. , van Rooij H.C.J. , van der Korput J.A.G.M. , Ris-Stalpers C. , Klaassen P. , Trapman J. , Brinkmann A.O.
Published: December 2008

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.
Activation of the thyroid peroxidase gene in human thyroid cells: effect of thyrotrophin, forskolin and phorbol ester
Authors: Collison K. S. , Banga J. P. , Barnett P. S. , Kung A. W. C. , McGregor A. M.
Published: December 2008

The enzyme thyroid peroxidase (TPO) plays a central role in thyroid hormone synthesis and is the target for the autoimmune attack in lymphocytic thyroiditis. We have examined the activation of the TPO gene in cultured human thyrocytes using slotblot hybridization with a synthetic 40 mer oligonucleotide probe derived from the nucleotide sequence of the human TPO gene. The oligonucleotide probe was shown by Northern blotting to hybridize specifically to an approximately 3 kb RNA species from thyroid tissue of patients with Graves' disease, but not to RNA preparations from human or bovine retinal tissue, providing compelling evidence for the specificity of the probe for TPO mRNA. Addition of TSH (10 mU/ml) to primary thyroid cultures for 4 h led to increased TPO mRNA levels which were maximal after 48 h and significantly higher than basal even after 7 days of co-culture. Activation of TPO mRNA by TSH showed dose dependency over a wide range (0·01–100 mU/ml), with a maximal effect at 10 mU TSH/ml in cells cultured for a period of 72 h.

Comparison of TPO mRNA levels with the accumulation of thyroglobulin mRNA levels following stimulation by TSH indicated that the induction of the gene encoding thyroglobulin precedes transcription of the TPO gene. The adenylate cyclase activator forskolin (1–100 μm) mimicked TSH in increasing TPO mRNA levels whilst, in contrast, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 0·01–1 μm) led to levels of TPO mRNA that were lower than basal. Thus TSH induces a specific dose-dependent activation of TPO mRNA which is mimicked by agents which increase cyclic AMP. In contrast, TPA-induced activation of protein kinase C inhibits this response.
Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli
Authors: Hanks M. C. , Talbot R. T. , Sang H. M.
Published: December 2008

The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1·5% of total cell protein.
Retinoic acid and tumour necrosis factor-α act in concert to control the level of alkaline phosphatase mRNA
Authors: Ng K. W. , Hudson P. J. , Power B. E. , Manji S. S. , Gummer P. R. , Martin T.J.
Published: December 2008

Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 μm retinoic acid for 24 h. Recombinant human tumour necrosis factor-α (recombinant TNF-α) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-α alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-α modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-α may serve as a model for other paracrine regulators of cell function.
Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-β subunit
Authors: Noce T. , Ando H. , Ueda T. , Kubokawa K. , Higashinakagawa T. , Ishii S.
Published: December 2008

A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using λ gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-β subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0·8 and 1·0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 °C. In-situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-β cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-β subunit supports the hypothesis that the number of proline residues increases in the LH-β subunit the closer phylogenetically the vertebrate is to mammals.
Gonadal and sex steroid feedback regulation of gonadotrophin mRNA levels and secretion in neonatal male and female rats
Authors: Pakarinen P. , Huhtaniemi I.
Published: December 2008

Serum and pituitary LH and FSH, and their pituitary mRNA levels, were measured in neonatal male and female rats after gonadectomy and after gonadectomy with sex steroid replacement. The animals were gonadectomized on day 3 of life, and those given sex steroid replacement were implanted with silicone elastomer capsules containing testosterone for males and diethylstilboestrol for females. Shamoperated rats served as controls. The animals were killed 4 or 8 days later and the sera and pituitaries collected. Pituitary contents of mRNAs for the α subunit, FSH-β and LH-β were determined by blot hybridization using corresponding cDNAs. Distinct sex differences were found in the mRNA responses to gonadectomy and steroid replacement. In the males, gonadectomy increased all mRNA levels at 7 days of age. In the females, a rise on day 7 was detected only for FSH-β; the other mRNAs were increased on day 11 of age. The steroid replacements reversed all the post-gonadectomy increases of mRNAs in both sexes. Moreover, the common α and LH-β mRNAs of the male animals were consistently suppressed below control levels. The serum concentrations of gonadotrophins increased after gonadectomy on day 7 in the males but only on day 11 in the females. The steroid replacements also suppressed the post-gonadectomy increases in serum gonadotrophins, but only the serum concentration of FSH in the females was reduced below controls. Pituitary gonadotrophin concentrations were not affected by gonadectomy, but the steroids suppressed LH in the males and FSH in the females.

It is concluded that the onset of negative-feedback regulation of gonadotrophin synthesis by gonads and/or gonadal steroids starts earlier in male rats, before 7 days of age. In female rats these responses appear between 7 and 11 days of age. Clear sex differences were observed in how gonadotrophin mRNAs and pituitary and serum hormone levels responded to gonadectomy and steroid replacement in the neonatal period. Some of the responses differed from those previously reported in adult animals.
Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by Ca2+
Authors: Takasu N. , Komiya I. , Nagasawa Y. , Asawa T. , Shimizu Y. , Yamada T.
Published: December 2008

The role of calcium in cytoplasmic pH (pHi) changes was studied using 2′,7′-bis(2-carboxyethyl)-5-(and 6-)carboxyfluorescein, an internalized fluorescent pH indicator, in cultured porcine thyroid cells. The Ca2+ ionophores A23187 and ionomycin stimulated thyroid cell alkalinization. An increase in cytoplasmic free calcium resulted in activation of Na+/H+ exchange which alkalinized the cells.
Insulin-like growth factor-I and its autocrine role in growth of MCF-7 human breast cancer cells in culture
Authors: Freed K. A. , Herington A. C.
Published: December 2008

Human MCF-7 breast cancer cells have been studied to determine their suitability as an autocrine model for the synthesis, secretion and action of insulin-like growth factor-I (IGF-I). Secretion of immunoreactive (ir-) IGF-I into serum-free medium was very low (<500 pg/106 cells per day). Northern blot hybridization detected at least two IGF-I messenger RNA transcripts (∼4·6 and ∼1·8 kb) which were similar in size to those reported in other human and rat tissues. IGF-II mRNA was also detected but at low abundance. Cell proliferation was stimulated in a dose-responsive manner by exogenous IGF-I (10–30 ng/ml). Addition of a monoclonal antibody against IGF-I to MCF-7 cells in serum-free medium caused an inhibition of cell proliferation, suggesting that endogenous locally produced IGF-I does play an autocrine/paracrine role in MCF-7 cell growth. Proliferation of MCF-7 cells was sensitive to oestradiol (10 nm) in the absence but not in the presence of the weakly oestrogenic pH indicator phenol red. Neither IGF-I secretion nor IGF-I mRNA synthesis, however, was affected by addition of oestradiol. Similarly, GH, dexamethasone or dexamethasone plus oestradiol had no effect on either parameter.

These data indicate that MCF-7 cells synthesize, secrete and respond to IGF-I. The very low levels of ir-IGF-I produced and their apparent lack of hormonal modulation suggest, however, that further studies are required to establish whether IGF-I plays a major physiological role in growth and development of MCF-7 cells.
Sex-steroid receptors in the diethylnitrosamine model of hepatocarcinogenesis: modifications by gonadal ablation and steroid replacement therapy
Authors: Tejura S. , Rodgers G. R. , Dunion M. H. , Parsons M. A. , Underwood J. C. E. , Ingleton P. M.
Published: December 2008

The results of this study confirm our previous report of increased androgen receptor expression in livers of female SUAH Wistar rats during development of liver tumours induced by diethylnitrosamine (DENA). In adult female rats not treated with DENA, removal of the ovary increased liver androgen receptor levels but testosterone did not further enhance the androgen receptor status of ovariectomized rats. In normal adult males the testis and/or testosterone maintained high levels of androgen receptors but oestrogen reduced them in castrated rats. Oestrogen receptor levels were not significantly changed in either males or females by gonadectomy. Treatment of female rats with DENA for 10 and 16 weeks increased liver androgen receptors but oestrogen receptors were only reduced by 16 weeks of DENA treatment, whether the rats were intact or ovariectomized. Concentrations of liver androgen receptors were increased in intact and castrated male rats by 10 and 16 weeks of DENA treatment, an increase not seen in the previous experiments. Oestrogen appeared to inhibit both the increases in liver androgen receptor expression and liver tumour development in rats treated with the weakly carcinogenic dose of 10 weeks of DENA. However, the full carcinogenic dose of 16 weeks of DENA increased liver androgen receptors and decreased oestrogen receptors in female rats regardless of sex-steroid status. Development of malignant hepatocellular carcinoma (HCC) was associated with both an increase in liver androgen receptors and a decrease in oestrogen receptors. Maintenance of relatively high levels of liver oestrogen receptors appeared to protect the liver against development of HCC.
EXPRESSION OF THE RAT AMYLIN (IAPP / DAP) GENE.
Authors: Ferrier G J M , Pierson A M , Jones P M , Bloom S R , Girgis S I , Legon S
Published: December 2008

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.
MELATONIN INHIBITS CYCLIC AMP PRODUCTION IN CULTURED OVINE PARS TUBERALIS CELLS
Authors: Morgan P.J. , Lawson W. , Davidson G. , Howell H.E.
Published: December 2008

Primary cultures of ovine pars tuberalis (PT) cells of the pituitary were established to investigate the mode of action of melatonin. The heterogeneous population of cells was shown to bind the radioligand 2-[125I]-melatonin over 72 h in culture, although there was a progressive decline in specific binding with time. In cells cultured for 24 h, forskolin (1 μmol/l) was found to stimulate a 12-fold increase in cyclic AMP accumulation. This response could be inhibited by melatonin in a dose-dependent manner, with an IC50 of approx. 6 pmol/l. However, melatonin did not inhibit basal levels of cyclic AMP.

In homogenates of ovine PT, forskolin stimulated a dose-dependent increase in cyclic AMP, although the magnitude of this response was found to be lower than that observed in cells. This response was not inhibited by either 10 nmol or 1 μmol melatonin/l, and was also unaffected by GTP.

These results provide the first evidence that the melatonin-binding site on ovine PT, recently characterized using the radioligand 2-[125I]-melatonin, functions as a physiological receptor.
Feedback-inhibition of growth hormone (GH) secretion in fowl: GH-induced down-regulation of thyrotrophin-releasing hormone binding to pituitary membranes
Authors: Harvey S. , Baidwan J. S.
Published: December 2008

Administration of ovine GH to immature domestic fowl blunted their subsequent GH response to thyrotrophin-releasing hormone (TRH), a GH secretagogue in birds. The in-vivo administration of GH also reduced the ability of radiolabelled TRH to bind to plasma membranes of the pituitary caudal lobe, in which GH cells predominate. These inhibitory effects of GH were mediated by extrapituitary actions, since GH had no direct inhibitory effects on TRH-induced GH release or on pituitary TRH binding in vitro. GH inhibition of GH secretion and TRH binding would not appear to be mediated by hypothalamic somatostatin (SRIF) or peripheral somatomedin (IGF-I), since SRIF and IGF-I had no direct effects in vitro.