bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of synthetic estrogens with human estrogen receptors
Authors: Nikov GN , Eshete M , Rajnarayanan RV , Alworth WL
Published: December 2004

Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors (ERs). Modeling studies suggest that 4-(1-adamantyl)phenol (AdP) and 4,4'-(1,3-adamantanediyl)diphenol (AdDP) can bind in the ligand binding site of ERalpha. We used fluorescence polarization (FP) to compare the binding affinities of AdP, AdDP and 2-(1-adamantyl)-4-methylphenol (AdMP) for human ERalpha and ERbeta with the binding affinities of the known ER ligands, diethylstilbestrol (DES) and 4hydroxytamoxifen (4OHT). Competition binding experiments show that AdDP has greater affinity for both ERs than does AdP, while AdMP does not bind the receptor proteins. The relative binding affinities of AdDP and AdP are weaker than the affinity of DES or 4OHT for both ERs with the exception of AdDP, which binds ERbeta with higher affinity than does 4OHT. We also found that AdDP and AdP cause differential conformational changes in ERalpha and ERbeta, which result in altered affinities of the ERs for fluorescein-labeled estrogen response elements (EREs) using a direct binding FP assay. The results show that ERbeta liganded with either AdDP or AdP has greater affinity for human pS2 ERE than the ERbeta-4OHT complex. The data suggest that synthetic molecules like adamantanes may function as biologically active ligands for human ERs. This demonstrates the importance of considering the potential of novel classes of synthetic compounds as selective ER modulators.
Hormonal regulation of tight junction closure in the mouse mammary epithelium during the transition from pregnancy to lactation
Authors: Nguyen DA , Parlow AF , Neville MC
Published: December 2004

Closure of the tight junctions of the mammary epithelium has been shown to accompany the onset of copious milk secretion or lactogenesis, stage 2, in both goats and humans. Here we use injection of [(14)C]sucrose and FITC-albumin (fluorescein isothiocyanate-albumin) into the mammary duct to follow the course of tight junction closure during lactogenesis in mice. To examine the hormonal changes responsible, we ovariectomized day 16 or 17 pregnant mice and found that closure followed ovariectomy with a mean delay of 13.6+/-1.5 (s.e.m. ) h. That progesterone withdrawal is the trigger for closure was shown by the finding that injection of progesterone within 4 h of ovariectomy delayed closure and that closure occurred after injection of the progesterone antagonist RU 486 in intact late pregnant mice. Endocrine ablation studies showed that low to moderate concentrations of corticosterone and either placental lactogen or prolactin are necessary for tight junction closure triggered by progesterone withdrawal. Thus the hormonal requirements for tight junction closure are similar to those shown by other investigators to promote lactogenesis, stage 2. Further, the tight temporal control of tight junction permeability suggests that ovariectomy of the late pregnant mouse may be a good model for molecular studies of the lactogenic switch.
Developmental changes in galanin in lumbosacral sympathetic ganglionic neurons innervating the avian uterine oviduct and galanin induction by sex steroids
Authors: Ubuka T , Sakamoto H , Li D , Ukena K , Tsutsui K
Published: December 2004

We recently found lumbosacral sympathetic ganglionic galanin neurons innervating the quail uterine oviduct. Galaninergic innervation of the uterine muscle may be essential for avian oviposition, as galanin evoked oviposition through a mechanism of induction of vigorous uterine contraction. The questions arising from these findings are: what changes occur in galanin expression in the sympathetic ganglionic galanin neuron during development, and what is the hormonal factor(s) that induces galanin expression in this neuron? Therefore, the present study examined the developmental changes in galanin of the quail sympathetic ganglionic neuron and uterus, and the effect of administration of ovarian sex steroids on galanin induction. Immature birds reared under long-day photoperiods from 4 weeks of age demonstrated progressive increases in galanin levels both per unit ganglionic protein (concentration) and per ganglia (content) concurrent with ganglionic development during weeks 4--13. The uterine galanin content and uterine weight also increased progressively during the same period, but the galanin concentration in the uterus at 4 weeks was high due to the much smaller tissue mass. Immunocytochemical analysis with anti-galanin serum showed that immunoreactive ganglionic cells were few and small at 4 weeks and increased progressively thereafter. Administration of oestradiol-17 beta to immature birds at 3 weeks of age for 1 week increased both the galanin concentration and content in the ganglia without ganglionic growth. A marked increase in galanin-immunoreactive ganglionic cells was detected following oestradiol treatment. In contrast, progesterone increased ganglionic galanin levels, but the effects were low. Expression of the mRNAs encoding oestrogen receptor-alpha and -beta (ER alpha and ER beta) in the ganglionic tissue was verified by RT-PCR/Southern blot analysis. Immunocytochemical staining with anti-ER serum further revealed an intense immunoreaction restricted to the nucleus of ganglionic neurons. These results suggest that ovarian sex steroids, in particular oestradiol-17 beta, contribute as hormonal factors to galanin induction, which takes place in the lumbosacral sympathetic ganglionic neurons innervating avian uterine oviduct during development. Oestradiol may act directly on this ganglionic neuron through intra-nuclear receptor-mediated mechanisms to induce galanin.
Large body size in the dog is associated with transient GH excess at a young age
Authors: Favier RP , Mol JA , Kooistra HS , Rijnberk A
Published: December 2004

The main determinants of body size are GH and IGFs. The aim of this study was to investigate whether differences in adult body size of medium-sized and giant dog breeds can be explained by differences in GH release and/or in plasma IGF-I and IGF-II concentrations at a young age. The basal plasma concentrations of GH, IGF-I and IGF-II were determined once weekly in six Great Danes and six beagles from the age of 6 weeks until the age of 24 weeks. In addition, the 6 h secretory profile of GH was determined every 2 weeks. Basal plasma GH concentrations as well as the total area under the curve (AUC) and the AUC above the baseline for GH were significantly higher in Great Danes than in beagles of the same age. In contrast, plasma IGF-I and IGF-II concentrations did not differ significantly between the two breeds. Compared with values in adults, the basal plasma GH concentrations were high until the age of 7 weeks in the beagles, whereas in the Great Danes the basal plasma GH levels remained high during the entire observation period, albeit with a gradual decline. The mean frequency and the mean amplitude of GH pulses tended to be higher in Great Danes than in beagles, although a significant difference was only reached at the age of 19 and 23 weeks for the frequency and at the ages of 9, 11 and 13 weeks for the amplitude. An age-dependent decrease in pulse frequency occurred in the Great Danes. The results of this study demonstrate that differences in adult body size of medium-sized and giant dog breeds are preceded by differences in GH release and not by differences in circulating IGF-I or IGF-II concentrations. Both young Great Danes and young beagles experience a period of high GH release, but this period persists much longer in Great Danes. It is discussed that this difference may be due to delayed maturation of the inhibitory influences of somatostatin on pituitary GH release in the latter dogs.
GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells
Authors: Aust G , Steinert M , Boltze C , Kiessling S , Simchen C
Published: December 2004

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.
Repeated in vivo determinations of bone mineral density during parathyroid hormone treatment in ovariectomized mice
Authors: Andersson N , Lindberg MK , Ohlsson C , Andersson K , Ryberg B
Published: December 2004

The recent development of different genetically modified mice with potentially interesting bone phenotypes has increased the demand for effective non-invasive methods to evaluate effects on bone of mice during growth and development, and for drug evaluation. In the present study, the skeleton was analyzed by repeated in vivo scans using dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Ovariectomized (ovx) mice treated with parathyroid hormone (PTH) were used as an animal model to evaluate these two techniques at different times after the onset of treatment. Female mice (6 weeks of age) were allocated randomly to four groups: (1) sham-operated+vehicle; (2) ovx+vehicle; (3) sham-operated+PTH(1-84) 150 microg/kg per day; (4) ovx+PTH. Six weeks after ovariectomy the drug treatment began and was continued for 8 weeks. The total body bone mineral content (BMC) and total body areal bone mineral density (BMD) were measured by DXA. Ovariectomy reduced total body BMC and total body areal BMD by 6.2+/-1.7% and 2.6+/-0.9% respectively. No effect of PTH on total body BMC was seen during the treatment period. The trabecular volumetric BMD was measured by pQCT. Ovariectomy reduced the trabecular volumetric BMD by 52+/-6.7%. The pQCT technique detected a clear effect on trabecular volumetric BMD after 2 weeks of PTH treatment (ovx 94+/-29% and sham-operated 46+/-10% more than vehicle-treated). The cortical bone was measured in a mid-diaphyseal pQCT scan of the tibia. Ovariectomy reduced the cortical BMC by 9+/-2%. PTH treatment for 8 weeks increased cortical BMC in ovx mice. In conclusion, the pQCT technique is more sensitive than the DXA technique in the detection of bone loss after ovariectomy and increased bone mass after PTH treatment in mice. Notably, the pQCT, but not the DXA, technique detected a dramatic effect as early as after 2 weeks of PTH treatment. Dynamic pQCT measurements will be useful for monitoring skeletal changes during growth and development, and for drug evaluation in mice.
Insulin-like growth factor-binding protein-1 in umbilical artery and vein of term fetuses with signs suggestive of distress during labor
Authors: Verhaeghe J , Billen J , Giudice LC
Published: December 2004

Insulin-like growth factor-binding protein-1 (IGFBP-1) is believed to be an inhibitory factor for fetal growth. The regulation of IGFBP-1 secretion in the fetus is uncertain, although insulin and oxygen tension (PO2) and saturation are thought to play a role. We studied IGFBP-1 levels in umbilical cord artery (UA) and vein (UV) of 98 singleton fetuses at term with clinical signs of distress during labor, i.e. meconium-stained liquor or/and an abnormal fetal heart rate tracing. Blood gas values and serum C-peptide and IGFBP-1 concentrations were measured in both UA and UV. Twenty-five fetuses had an UA pH<7.20. The concentrations of IGFBP-1 were similar in UA and UV and were highly correlated (r=0.98). IGFBP-1 levels were inversely correlated with birth weight, with increased concentrations in small-for-gestational age fetuses (< or =10th weight percentile). IGFBP-1 levels were negatively correlated with C-peptide concentrations, and remained so after correction for birth weight (r=-0.37 for both UA and UV; P<0.001); more specifically, IGFBP-1 levels were increased in the lowest C-peptide quartile (<0.23 nmol/l) compared with the other quartiles. In addition, IGFBP-1 levels were inversely correlated with PO(2) values (r=-0.39 in UA and r=-0.34 in UV; P<0.001); quartiles of UA and UV PO2 showed a gradual increase in IGFBP-1 concentrations with lower PO2 values. A regression model with C-peptide and PO2 values as independent variables predicted IGFBP-1 concentrations (R2 of model was 0.25 and 0.22 for UA and UV respectively; P<0.001). Other blood gas values (pH, PCO2, HCO3- and base deficit) did not correlate with IGFBP-1 levels. The data of this study indicate that serum IGFBP-1 levels in term fetuses are determined by both insulin and PO2 levels, and suggest that acute hypoxemia stimulates IGFBP-1 secretion in the fetus.
Early exposure of the rat mammary gland to estrogen and progesterone blocks co-localization of estrogen receptor expression and proliferation
Authors: Sivaraman L , Hilsenbeck SG , Zhong L , Gay J , Conneely OM , Medina D , O'Malley BW
Published: December 2004

An early single full-term pregnancy induces a long-lasting protective effect against mammary tumor development in humans and rodents. This protective effect can be mimicked in rats by short-term administration of estrogen and progesterone hormones prior to carcinogen administration. The hormones of pregnancy are able to induce a proliferative block upon carcinogen challenge that is not observed in the age-matched virgin. We wished to determine whether carcinogen is needed to induce a paracrine-to-autocrine shift of proliferation in steroid receptor positive cells or if such a cell population already exists in the age-matched virgin mammary gland. Here we show that estrogen receptor positive (ER+) proliferating cells are rare in the developing mammary gland of the virgin rat but represent the majority of the proliferating cells in the mature (96-day-old) mammary gland of the virgin rat. As the majority of the proliferating cells before carcinogen challenge were ER positive, the ER+ proliferating cells in the mature mammary gland may represent the target cells for carcinogen-induced transformation. Importantly, prior exposure of the mammary gland to pregnancy levels of estrogen/progesterone blocked this positive association. This ability to block the proliferation of the ER+ cells may be one factor by which pregnancy induces protection against breast cancer.
Administration of bovine, porcine and equine growth hormone to the horse: effect on insulin-like growth factor-I and selected IGF binding proteins
Authors: De Kock SS , Rodgers JP , Swanepoel BC , Guthrie AJ
Published: December 2004

This study investigated the biochemical effects of administration of three types of recombinant growth hormone (GH; somatotropin) to the Thoroughbred horse. Equine or bovine or porcine GH was administered at a recommended dosage to 3-5-year old Thoroughbred geldings, for up to 21 days. It was shown that, in addition to equine GH, bovine and porcine GH were active in the horse; however, porcine GH caused injection-site reactions that were so serious that administration had to be terminated. The concentrations of a range of GH-related serum protein markers were determined before, during and after the administration period. Because of the short half-life of GH itself, the objective was to identify GH-related markers that showed changes in concentration and which could be used as indicators of the abuse of these hormones. Among the possible markers identified, serum total insulin-like growth factor (IGF)-I was shown to be the most promising, increasing to 270% of the basal concentration for equine GH administration. After GH administration, IGF-I took longer to attain baseline concentrations than the time required for GH concentrations to recover to normal. The concentration obtained from the administration significantly exceeded natural concentrations for IGF-I, as was determined from a population of more than 2000 Thoroughbred horses in three continents. The concentrations of serum free IGF-I and IGF binding protein 3 (IGFBP-3) were also shown to be significantly affected by equine and bovine GH.
The metabolic effects of endotoxin are differentially affected by the pattern of GH administration in the rat
Authors: Roelfsema V , Thomas GB , Lin H , Breier BH , Maxwell L , Oliver MH , Heineman E , Clark RG , Gluckman PD
Published: December 2004

GH treatment can increase the mortality and morbidity of critically ill patients. The mechanisms of these harmful effects of GH are unknown but have been, in part, ascribed to interactions between GH and the immune system. Because GH has pattern-dependent actions we have now compared the dose-related effects of continuous and intermittent GH treatment given with or without an endotoxin (lipopolysaccharide; LPS) challenge. Male Wistar rats (n=6 per group) were treated for 5 days with recombinant human GH (0, 10, 100 or 1000 microg/kg per day) using either continuous s.c. infusion by osmotic minipump or intermittent twice daily s.c. injections. On day 4, endotoxin (5 mg/kg, i.p.) was injected and the animals monitored for a further 16 h. LPS administration alone led to neutrophilia and lymphopoenia, with increased plasma concentrations of urea, cholesterol, triglyceride, insulin and leptin, and decreased levels of IGF-I. High dose GH infusion (1000 microg/kg per day) followed by LPS caused greater increases in plasma urea, cholesterol, triglyceride, sodium and magnesium, but lower plasma glucose and insulin levels, than treatment with LPS alone. In contrast, twice daily injections of GH did not enhance these effects of endotoxin. In conclusion, the effects of endotoxin on plasma electrolytes, lipids, urea, glucose and insulin are differentially affected by the pattern of GH administration in the rat.
Characterization of uptake and compartmentalization of 3,5,3'-tri-iodothyronine in cultured neonatal rat cardiomyocytes
Authors: van der Putten HH , Joosten BJ , Klaren PH , Everts ME
Published: December 2004

The uptake of tri-iodothyronine (T(3)) in cultured neonatal rat cardiomyocytes was investigated and compared with the uptake of reverse T(3 )(rT(3)) and thyroxine (T(4)). Cellular compartmentalization of T(3) was studied by distinguishing T(3) activity associated with the plasma membrane from that in the cytosol or incorporated in the cell nucleus. T(3) and T(4) uptake displayed similar temperature dependencies which, in magnitude, differed from that of rT(3) uptake. T(3) uptake was Na(+ )independent, and sensitive to oligomycin and monodansylcadaverine (42-49% and 25% inhibition of 15-min cellular uptake respectively). Furthermore, T(3) uptake could be inhibited by tryptophan (20%) and tyrosine (12%), while 2-aminobicyclo[2,2,1]heptane-carboxylic acid had no effect. Co-incubation with tryptophan and oligomycin resulted in an additive inhibition of T(3) uptake (77%). We therefore conclude that (i) T(3) uptake is energy dependent, (ii) receptor-mediated endocytosis may be involved and (iii) the aromatic amino acid transport system T may play a role, while system L is not involved in T(3) transport in cardiomyocytes. Co-incubation with unlabeled iodothyronines showed that 3,3'-di-iodothyronine and T(3) itself were the most effective inhibitors of T(3) uptake (30% and 36% inhibition of 15-min cellular uptake respectively). At 15-min incubation time, 38% of the total cell-associated T(3) was present in the cytosol and nucleus, and 62% remained associated to the plasma membrane. Unidirectional uptake rates did not saturate over a free T(3) concentration range up to 3.9 microM. We have concluded that T(3) uptake in neonatal rat cardiomyocytes occurs by an energy- and temperature-dependent mechanism that may include endocytosis and amino acid transport system T, and is not sensitive to the Na(+) gradient. Elucidation of the molecular basis for the T(3) transporter is the subject of current investigation.
Effect of eicosapentaenoic acid ethyl ester on hypothyroid function
Authors: Makino M , Oda N , Miura N , Imamura S , Yamamoto K , Kato T , Fujiwara K , Sawai Y , Iwase K , Nagasaka A , Itoh M
Published: December 2004

Thyroid hormones affect reactions in almost all pathways of lipid metabolism. It has been reported that plasma free fatty acid (FFA) concentration in hypothyroidism is generally within the normal range. In this study, however, we show that plasma FFA concentration in some hypothyroid patients is higher than the normal range. Symptoms of thyroid dysfunction in these individuals were less severe than those of patients with lower plasma FFA concentrations. From these findings we hypothesized that the change in FFA concentration must correlate with thyroid function. Using an animal model, we then examined the effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E), a n-3 polyunsaturated fatty acid derived from fish oil, on thyroid function in 1-methyl-2-imidazolethiol (MMI)-induced hypothyroid rats. Oral administration of EPA-E inhibited reduction of thyroid hormone levels and the change of thyroid follicles in MMI-induced hypothyroid rats. These findings suggest that FFA may affect thyroid functions and EPA-E may prevent MMI-induced hypothyroidism.
Insulin resistance in obesity: body-weight or energy balance?
Authors: Assali AR , Ganor A , Beigel Y , Shafer Z , Hershcovici T , Fainaru M
Published: December 2004

Weight reduction is recommended for the treatment of subjects with insulin resistance (IR) syndrome; however, the relative importance of the decrease in body fat or the negative energy balance achieved during a hypo-energetic diet in the improvement of this metabolic syndrome is still debated. Therefore, we undertook to study their relative impact on amelioration of the metabolic abnormalities associated with IR in obese subjects. Twelve obese subjects (six males and six females, mean+/-s.d. body mass index 36.1+/-4.7 kg/m(2)) aged 38-57 years were investigated. During the first phase they were fed a hypo-energetic diet for 6 weeks (week 0-6). During the second phase, lasting 4 weeks (week 6-10) they consumed an iso-energetic diet. During the third phase (week 10-16) the subjects were put again on a hypo-energetic diet. Insulin sensitivity (SI) was assessed by an insulin-enhanced, frequently sampled i.v. glucose tolerance test with minimal model analysis. All subjects reduced weight during both hypo-energetic periods: 5.49+/-0.75 and 2.32+/-0.37%, means+/-s.e.m., P<0.005, week 0-6 and 10-16 respectively. One-third of this loss was achieved within the first week of each period. SI increased by 353+/-121 and 147+/-38% (P<0.005), means+/-s.e.m., at the end of both hypo-energetic periods (week 6 vs 0 and 16 vs 10 respectively). Two-thirds of this improvement were observed within the first week of each period (week 1 vs 0 and 11 vs 10 respectively). During the iso-energetic weight-maintaining period (week 10 vs 6), SI decreased by 43.5+/-7.9% (P<0.002). Serum levels of leptin and triglyceride followed a similar pattern, but to a lesser extent. It may be concluded that negative energy balance is more effective when compared with maintaining a stable lower weight in achieving an improvement in the metabolic parameters of the IR syndrome.
Factors mediating the transdifferentiation of islets of Langerhans to duct epithelial-like structures
Authors: Wang R , Li J , Rosenberg L
Published: December 2004

We have previously shown that isolated islets embedded in type 1 collagen gel in the presence of a defined medium undergo transdifferentiation within 96 h to duct epithelial structures. The aim of this study was to identify the factors implicated in this process. Freshly isolated canine islets were embedded in type 1 collagen gel, Matrigel or agarose for up to 120 h and cultured in (i) Dulbecco's modified Eagle's medium (DMEM)/F12 plus cholera toxin (CT), (ii) medium CMRL1066 plus CT, (iii) CMRL1066 plus forskolin and (iv) CMRL1066 alone. At 16 h, intracellular levels of cAMP (fmol/10(3) islets) were increased in groups i-iii (642+/-17, 338+/-48, 1128+/-221) compared with group iv (106+/-19, P<0.01). Epithelial differentiation correlated with the total amount of intracellular cAMP measured over 120 h. Islet-epithelial transformation during the initial 36 h was associated with a wave of apoptosis which was followed by a wave of cell proliferation. During epithelial differentiation there was a progressive loss of all islet hormones and the concomitant expression of cytoskeletal proteins characteristic of duct epithelial cells. Islets in collagen and Matrigel demonstrated high rates of epithelial differentiation (63+/-2% and 71+/-4% respectively) compared with those in agarose gel (0+/-0%, P<0.001). Islets suspended in DMEM/F12 plus CT supplemented with soluble laminin or fibronectin did not undergo transformation. Prior incubation of freshly isolated islets with an integrin-binding arginine-glycine-aspartate motif-presenting synthetic peptide also reduced islet transformation. These studies confirm the biological potential of islets of Langerhans to differentiate to duct epithelial structures. cAMP-mediated signal transduction and an appropriate integrin-matrix interaction are necessary for this process to proceed.
Adrenomedullin augments the neurohumoral response to haemorrhage in non-pregnant but not in pregnant sheep
Authors: Charles CJ , Rademaker MT , Richards AM , Nicholls MG
Published: December 2004

Adrenomedullin (ADM) is a novel peptide with actions which include reduction of arterial pressure and interaction with a number of hormone systems. In order to assess possible interactions with the renin-angiotensin system (RAS) and the hypothalamo-pituitary-adrenal (HPA) axis, we have examined neurohumoral responses to hypotensive haemorrhage (15 ml/kg over 15 min) with or without co-infusions of ADM (5.5 pmol/kg per min) in six non-pregnant and eight pregnant conscious sheep. Haemorrhage induced a greater decrease in arterial pressure, but a blunted increase in heart rate in pregnant sheep. There was no significant effect of ADM on haemodynamic responses to haemorrhage in either group. In non-pregnant sheep, haemorrhage-induced activation of both RAS and HPA was significantly augmented by ADM, as indicated by greater increases in plasma renin activity (P<0.01), angiotensin II (P<0.05) and arginine vasopressin (P<0.01). In contrast, ADM did not augment these responses to haemorrhage in pregnant sheep. Rather, plasma concentrations of aldosterone (P=0.039) and adrenocorticotrophic hormone (P=0.012) were decreased by ADM. In conclusion, ADM-induced augmentation of the RAS and HPA responses to hypotensive haemorrhage is abolished in the pregnant state.
Effect of dietary polyunsaturated fatty acids on uterine prostaglandin synthesis in the cow
Authors: Cheng Z , Robinson RS , Pushpakumara PG , Mansbridge RJ , Wathes DC
Published: December 2004

Dietary polyunsaturated fatty acid (PUFA) intake in humans can affect the incidence of a variety of diseases including coronary heart disease. Feeding high PUFA diets to cows can alter the PUFA content of milk for human consumption. PUFAs supply the precursors for prostaglandin (PG) synthesis and PGs in turn influence many aspects of reproduction. This study examined the effects of a control (CONT), a high n-6 PUFA diet (derived from protected soya) and a high n-3 diet (derived from protected linseed) on uterine PG synthesis in the lactating dairy cow. Endometrial explants obtained on days 15-17 of the oestrous cycle were cultured for an initial 42 h in vitro in fully defined medium (basal production) and then challenged with control medium, oxytocin (OT; 20 or 200 nM) or calcium ionophore A23187 (CaI; 10 microM). PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were measured in the spent medium. The experiments were repeated using tissue from two groups of cows, nine in Experiment 1 (three cows per diet) and seven in Experiment 2 (four CONT and three n-6). Results of the two experiments were consistent. The basal concentrations of all three PGs were significantly lower (>50% reduction) in the n-6-fed group in comparison with CONT and n-3 groups. The n-3 diet did not alter basal PGF(2 alpha) and PGE(2) but increased 6-keto-PGF(1 alpha). The n-6 diet also inhibited the ability of the tissue to respond to both OT and CaI, with significant reductions in the stimulated levels of all three PGs. In contrast, the n-3 diet only had minor effects; it did not alter the response to OT but did reduce the long-term response to CaI at 24 h post treatment. In conclusion, dietary PUFA intake can inhibit PG production in bovine endometrial explants, with a more pronounced effect following n-6 rather than n-3 supplementation. These data suggest that a high n-6 diet reduces the endometrial capacity to produce PGs and may therefore have implications for the control of luteolysis and other PG-mediated events such as ovulation.
Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters
Authors: Massa ML , Borelli MI , Del Zotto H , Gagliardino JJ
Published: December 2004

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, (14)CO(2) and (3)H(2)O production from D-[U-(14)C]-glucose and D-[5-(3)H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3+/-0.1 vs 0.6+/-0.03 ng/ml, P<0.001). Islets from S5 hamsters released significantly more insulin than C5 islets in the presence of low and high glucose (3.3 mM glucose: 0.77+/-0.04 vs 0.20+/-0.06 pg/ng DNA/min, P<0.001; 16.7 mM glucose: 2.77+/-0.12 vs 0.85+/-0.06 pg/ng DNA/min, P<0.001) and produced significantly higher amounts of (14)CO(2) and (3)H(2)O at both glucose concentrations ((14)CO(2): 3.3 mM glucose: 0.27+/-0.01 vs 0.18+/-0.01, P<0.001; 16.7 mM glucose: 1.44+/-0.15 vs 0.96+/-0.08, P<0.02; (3)H(2)O: 3.3 mM glucose: 0.31+/-0.02 vs 0.15+/-0.01, P<0.001; 16.7 mM glucose: 1.46+/-0.20 vs 0.76+/-0.05 pmol glucose/ng DNA/min, P<0.005). The hexokinase K(m) and V(max) values from S5 animals were significantly higher than those from C5 ones (K(m): 100.14+/-7.01 vs 59.90+/- 3.95 microM, P<0.001; V(max): 0.010+/-0.0005 vs 0.008+/- 0.0006 pmol glucose/ng DNA/min, P<0.02). Conversely, the glucokinase K(m) value from S5 animals was significantly lower than in C5 animals (K(m): 15.31+/-2.64 vs 35.01+/-1.65 mM, P<0.001), whereas V(max) figures were within a comparable range in both groups (V(max): 0.048+/-0.009 vs 0.094+/-0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26+/-0.02) than in C5 animals (0.11+/-0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by beta-cells, and in beta-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the beta-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate beta-cell function.
Stimulation of extracellular signal-regulated kinase by pituitary adenylate cyclase-activating polypeptide in alpha T3-1 gonadotrophs
Authors: Fowkes RC , Burch J , Burrin JM
Published: December 2004

The putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates glycoprotein hormone alpha-subunit (alpha GSU) gene transcription and secretion in the clonal gonadotroph alpha T3-1 cell line. The specific signalling pathways regulating these actions of PACAP have not been clearly defined. We have examined the possibility that mitogen activated protein kinases (MAPKs) may play a role in mediating the effects of PACAP on alpha T3-1 gonadotrophs. Treatment of alpha T3-1 cells with PACAP (100 nM) or epidermal growth factor (EGF, 10 nM) for 5 min significantly stimulated extracellular signal-regulated kinase activity (ERK, a component of the MAPK pathway) as determined by an immunocomplex assay. Pre-treatment of alpha T3-1 cells with the specific MAPK kinase (MEK) inhibitor, U0126, blocked PACAP and EGF-induced activation of ERK. Transcriptional stimulation of a human alpha GSU-luciferase reporter construct by PACAP was unaffected by U0126 treatment. However, pre-treatment with U0126 significantly inhibited PACAP stimulation of [(3)H]-thymidine incorporation in alpha T3-1 cells. Thus our results suggest that PACAP stimulates ERK activation in alpha T3-1 cells, and that the functional effect of this ERK activation is increased DNA synthesis and cell proliferation rather then transcriptional activation of the alpha GSU gene.
Development and validation of a new monoclonal antibody to mammalian aromatase
Authors: Turner KJ , Macpherson S , Millar MR , McNeilly AS , Williams K , Cranfield M , Groome NP , Sharpe RM , Fraser HM , Saunders PT
Published: December 2004

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.
Retinoic acids promote the action of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 on the biosynthesis of 17beta-estradiol in placental cells
Authors: Zhu SJ , Li Y , Li H , Wang YL , Xiao ZJ , Vihko P , Piao YS
Published: December 2004

The biosynthesis of 17beta-estradiol (E(2)) in human placenta involves the actions of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). Aromatase, an enzyme complex comprised of P450aromatase (P450arom) and NADH-cytochrome P450 reductase, converts androgens to estrogens, whereas 17HSD1 catalyzes the reduction of estrone to E(2). In the present study, the effects of retinoic acids (RAs) on P450arom and 17HSD1 expression in placental cells were investigated. Treatment with all-trans-RA (at-RA) or 9cis-RA increased E(2) production in JEG-3 choriocarcinoma cells and cytotrophoblast (CTB) cells isolated from normal early placentas. Meanwhile, the activity of aromatase and expression of P450arom mRNA were induced by at-RA in JEG-3 cells. Northern blot analysis showed that the effect on P450arom mRNA expression occurs in a dose- and time-dependent fashion. Similar to at-RA and 9cis-RA, Ro40-6055, the retinoic acid receptor alpha (RARalpha)-selective activator, increased the expression of P450arom and 17HSD1 mRNA in JEG-3 cells. On the other hand, Ro41-5253 (Ro41), the RARalpha-selective antagonist, blocked the stimulatory effect of RAs on P450arom expression. Surprisingly, Ro41 induced the activity and mRNA expression of 17HSD1 in JEG-3 cells, which is in contrast to the expected inhibitory effect and, moreover, remarkably potentiated the induction by at-RA and 9cis-RA. However, reporter gene analysis revealed that the influence of Ro41 on the transcription of the HSD17B1 gene, which encodes 17HSD1, is considerably milder in JEG-3 cells, and it only additively enhanced the effect of at-RA. Finally, it was found that at-RA and 9cis-RA increased the expression of P450arom and 17HSD1 mRNA in CTB cells, but to a lesser extent. The data suggest that RAs may play a role in promoting the biosynthesis of E(2 )in the placenta. In addition, Ro41 has divergent effects on gene expression in JEG-3 cells.
Impaired insulin signaling in the liver of transgenic rats with low circulating growth hormone levels
Authors: Furuhata Y , Yonezawa T , Takahashi M , Nishihara M
Published: December 2004

GH is known to regulate glucose and lipid metabolism as well as body growth. Controversy exists as to whether GH-deficient adults are indeed insulin sensitive or insulin resistant. In GH-deficient animal models, however, no clear observation indicating insulin resistance has been made, while increased insulin sensitivity has been reported in those animals. We have produced human GH (hGH) transgenic rats characterized by low circulating hGH levels and virtually no endogenous rat GH secretion. Although the body length of the transgenic rat is normal, they develop massive obesity and insulin resistance, indicating that the transgenic rat is a good model for the analysis of insulin resistance under GH deficiency. In this study, we have examined how GH deficiency affects the early steps of insulin signaling in the liver of the transgenic rat. Circulating glucose and insulin concentrations were significantly higher in the transgenic rats than in their littermates. In addition, impaired glucose tolerance was observed in the transgenic rat. The amount of insulin receptor was smaller in the liver of the transgenic rat, resulting in decreased tyrosine phosphorylation in response to insulin stimulation. The amounts of insulin receptor substrate-1 and -2 (IRS-1 and -2) and insulin-stimulated phosphorylation of IRSs were also smaller in the transgenic rat. Despite the decrease in tyrosine phosphorylation levels of IRSs being mild to moderate (45% for IRS-1 and 16% for IRS-2), associated phosphatidylinositol 3-kinase (PI3-kinase) activity was not increased by insulin stimulation at all in the transgenic rat. To elucidate whether this discrepancy resulted from the alteration in binding of the p85 subunit of PI3-kinase to phosphotyrosine residues of the IRSs, we determined the amount of p85 subunit in the immunocomplexes with anti-phosphotyrosine antibody. Insulin did not affect the amount of p85 subunit associated with phosphotyrosine in the transgenic rats, while it significantly increased in the controls, indicating that alteration may have occurred at the sites of phosphorylated tyrosine residues in IRSs. These results suggest that GH deficiency in the transgenic rat leads to impairment in at least the early steps of insulin signaling in the liver with a resultant defect in glucose metabolism.
Dual role of the tyrosine kinase GTK and the adaptor protein SHB in beta-cell growth: enhanced beta-cell replication after 60% pancreatectomy and increased sensitivity to streptozotocin
Authors: Anneren C
Published: December 2004

Transgenic CBA mice expressing either the tyrosine kinase GTK (gut tyrosine kinase) or the adaptor protein SHB (Src homology 2 protein of beta-cells) under the control of the rat insulin promoter exhibited an increased beta-cell mass, but also elevated cytokine-induced islet cell death compared with control mice. To further investigate the importance of GTK and SHB for beta-cell death and proliferation, these mice were subjected to a 60% partial pancreatectomy (Px) or a sham-operation and beta-cell replication was determined by autoradiographic detection of [(3)H]thymidine incorporation into islet cells positively stained for insulin. The Px-operated control mice exhibited a moderate and insignificant increase in beta-cell replication 4 days after Px compared with the sham-operated mice (0.27+/-0.08% vs 0.08+/-0.02%). In contrast, the Px-induced beta-cell proliferation was significantly increased in both the GTK- and SHB-transgenic mice compared with the corresponding sham-treated animals (0.64+/-0.12% vs 0.11+/-0.04% and 0.44+/-0.11% vs 0.09+/-0.04% respectively). This effect was dependent on intracellular signal transduction pathways activated or enhanced by GTK and SHB overexpression, since the proliferation of acinar cells, located in the vicinity of the islets, was equal in the transgenic and control mice. GTK- and SHB-transgenic mice, treated with a sub-diabetogenic dose of the beta-cell toxin streptozotocin (STZ) on day 0 and subjected to a glucose tolerance test on day 3, exhibited an impaired glucose tolerance in comparison with the STZ-treated control mice. Pretreatment with STZ blunted the regenerative response to Px in the transgenic mice. Furthermore, the SHB-transgenic islets were significantly more damaged with respect to beta-cell loss, compared with the islets of the control mice. Previous and present data suggest a dual role of GTK and SHB for beta-cell growth: whereas these proteins increase the beta-cell mass and induce beta-cell proliferation after 60% Px, SHB and GTK also enhance beta-cell death under certain stressful conditions.
The influence of maternal size on placental, fetal and postnatal growth in the horse. II. Endocrinology of pregnancy
Authors: Allen WR , Wilsher S , Stewart F , Stewart F , Ousey J , Ousey J , Fowden A
Published: December 2004

Within-breed artificial insemination and between-breed embryo transfer were carried out in small pony (P) and large Thoroughbred (Tb) mares to create 4 types of horse pregnancy in which the fetus experienced spatial and nutritional deprivation (Tb-in-P; n=8), luxury (P-in-Tb; n=7) or normality (Tb-in-Tb; n=7 and P-in-P; n=7) in utero. Measurement of equine chorionic gonadotrophin (eCG), total conjugated oestrogens and progestagen concentrations in serial peripheral serum samples recovered from all the mares throughout gestation showed that the amount of eCG produced during the first half of gestation was dependent upon the breed of the mare rather than the breed of the fetus being carried. In contrast, the mean total amounts of oestrogens produced, as measured by area under the curve, were significantly greater (P=0.003) in the two types of pregnancy in which a Thoroughbred fetus was being carried (Tb-in-Tb and Tb-in-P) than those in which a pony fetus was gestated (P-in-P and P-in-Tb); the evidence suggests that the Tb fetus may have larger gonads than the P fetus and thereby secrete more C-19 precursor steroids for aromatisation to oestrogens by the placenta. In the final weeks of pregnancy mean plasma progestagen concentrations rose much earlier, and to significantly higher levels (P<0.001), in the Tb-in-P than in the P-in-Tb pregnancies, thereby reflecting the increased fetal stress in the former causing premature maturation of the fetal adrenal gland. This, in turn, resulted in increased secretion of pregnenolone by the adrenal cortex for conversion to progestagens by the placenta.
Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner
Authors: Brar BK , Railson J , Stephanou A , Knight RA , Latchman DS
Published: December 2004

We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.
A detailed study of the L2beta long-loop region of human chorionic gonadotrophin suggests it to be spatially close to, but not part of, the receptor-binding site
Authors: Jagtap DD , Mahale SD , Mishra AK , Nandedkar TD , Iyer KS
Published: December 2004

An in-depth study of the L2beta long-loop region of human chorionic gonadotrophin (hCG), earlier identified to be a conformational bioneutralization epitope and receptor-binding site of the hormone, was carried out. The linear 38-57 hCGbeta peptide and the corresponding cyclic disulphide peptide were synthesized and antipeptide antibodies developed. Binding studies with antibodies to the linear peptide, and with hCGbeta, hCG and human LH suggest that part of the region is buried at the alpha/beta interface and part exposed in hCG. Observation of the surface exposure of residues 47-53 from the crystal structure of hCG was confirmed by epitope mapping studies of the region. The region is not unique to hCG as a majority of the antibodies to both the linear and cyclic peptides did not exhibit the required specificity. Competitive inhibition studies with the linear and cyclic peptides failed to show inhibition of radiolabelled hCG binding to its receptors. However, both the antipeptide antibodies were able to bioneutralize the hormone in an in vivo assay. Taken together, these results seem to indicate that the L2beta long-loop region is not a receptor-binding site of hCG but spatially close to it.