bluuebunny/crossref_metadata_2025 huggingface dataset:
A placebo-controlled study of three osteocalcin assays for assessment of prednisolone-induced suppression of bone turnover
Authors: Heuck C , Wolthers OD
Published: December 2004

Serum osteocalcin is a sensitive marker of suppressive effects of exogenous glucocorticoids on bone turnover. It has been suggested, however, that the degree of suppression detected by different assays may vary. Whether discrepancies between various assays influence conclusions from group studies of exogenous glucocorticoids has not been evaluated. The aim of the present study was to compare the CAP fluoroimmunoassay (FEIA), OSTK-PR and ELSA-OSTEO assays for assessment of prednisolone-induced effects on serum osteocalcin. Twelve men and eight premenopausal women aged 19-45 (mean 31) years were studied. All subjects were healthy. The design was a randomised double-blind, placebo-controlled parallel- group study with 2 days run-in, 3 days treatment and 4 days run-out. During run-in and run-out no medication was given. During the treatment period the subjects took either 20 mg prednisolone twice daily or placebo. Blood was collected on the last day of each period. Intra- and intergroup comparisons showed prednisolone treatment to be associated with a statistically significant suppression of osteocalcin which was detected by all assays (ANOVA;P<0.0001). In the individual subjects the response to prednisolone was the same for each assay. The CAP FEIA, OSTK-PR and ELSA-OSTEO assays seem equally sensitive for evaluation of osteocalcin in group studies of oral glucocorticoids.
Modulation of skin cell functions by transforming growth factor-beta1 and ACTH after ultraviolet irradiation
Authors: Dissanayake NS , Mason RS
Published: December 2004

The adaptive responses in skin to ultraviolet (UV) radiation include increased cornification of keratinocytes and increased synthesis and distribution of melanin by melanocytes. The possible involvement of paracrine factors in the generation of these responses was studied in a novel two-stage culture model. Human melanocytes or keratinocytes were first irradiated or sham-irradiated and then the conditioned media collected from these cells after 24 h were used to treat unirradiated skin cells. Immunofluorescent staining for transforming growth factor (TGF)-beta1 was increased in UV-irradiated keratinocytes compared with sham-irradiated cells. Increased TGF-beta1 was also detected in the culture media of irradiated keratinocytes. Treatment of unirradiated keratinocytes with conditioned media collected from UV-irradiated keratinocytes resulted in increased absolute numbers and percentages of cornified envelopes per well compared with treatment with conditioned media from sham-irradiated keratinocytes. The magnitude of this effect increased with increased dose of initial irradiation. The effects of conditioned media from UVR-treated cells were mimicked by authentic TGF-beta1. Treatment of conditioned media from irradiated cells with an antibody shown to neutralise the effects of TGF-beta1 but not with a non-immune antibody of similar isotype, abolished this bioactivity of the conditioned media from UV-irradiated cells. Immunofluorescent staining for ACTH was also increased in UV-irradiated keratinocytes. Conditioned media from UV-irradiated keratinocytes increased tyrosinase activity of unirradiated melanocytes, an effect which was mimicked by authentic ACTH. This bioactivity of conditioned media from irradiated keratinocytes was abolished in the presence of an antibody which neutralised the activity of ACTH but not MSH. These results provide evidence to support the involvement of TGF-beta1 and ACTH in the cornification and pigmentary responses respectively of skin cells after UV exposure.
Effects of endotoxin lipopolysaccharide administration on the somatotropic axis
Authors: Soto L , Martin AI , Millan S , Vara E , Lopez-Calderon A
Published: December 2004

The aim of this work was to study the effect of chronic activation of the immune system on the somatotropic axis. Accordingly, the changes in growth hormone (GH) secretion, circulating insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in response to endotoxin lipopolysaccharide (LPS) administration were examined in adult male Wistar rats. Acute LPS injection (2.5, 25 or 250 microg/kg) increased serum corticosterone in a dose-dependent manner and decreased serum levels of insulin and IGF-I, serum GH concentration declined linearly as the LPS dose increased. Western ligand blot showed an increase in the 33 kDa band (corresponding to IGFBP-1 and IGFBP-2) in the rats that received the highest dose of LPS (250 microg/kg). Chronic LPS administration (250 microg/kg daily for 8 days) significantly decreased body weight, serum levels of IGF-I and pituitary GH content, whereas it increased circulating IGFBP-3 (47 kDa band), IGFBP-1 and IGFBP-2 (33 kDa band) and the 24 kDa band (which possibly corresponds to IGFBP-4). Serum concentration of corticosterone and hypothalamic somatostatin content were also increased by chronic LPS treatment. These data suggest that the decrease in GH and IGF-I secretion and the increase in circulating IGFBPs are important mechanisms in body weight loss during chronic inflammation.
UCP2, UCP3 and leptin gene expression: modulation by food restriction and leptin
Authors: Scarpace PJ , Nicolson M , Matheny M
Published: December 2004

To determine the effects of food restriction and leptin administration on several transcripts involved in energy homeostasis, we examined leptin, uncoupling proteins (UCP) 1, 2 and 3, lipoprotein lipase (LPL), beta3-adrenergic receptors (beta3AR) and hormone-sensitive lipase (HSL) mRNA levels in brown adipose tissue (BAT) and epididymal (EWAT) and perirenal (PWAT) white adipose tissue in three groups of rats. The groups were administered leptin for 1 week, or had food restricted to the amount of food consumed by the leptin-treated animals, or had free access to food. Leptin administration increased serum leptin concentrations 50-fold and decreased food consumption by 43%, whereas serum insulin and corticosterone concentrations were unchanged. Leptin increased LPL mRNA by 80%, UCP1 mRNA twofold, and UCP3 mRNA levels by 62% in BAT, and increased UCP2 mRNA levels twofold in EWAT. In contrast, UCP2 mRNA levels were unchanged in PWAT and BAT. In WAT from food-restricted rats, leptin gene expression was diminished by 40% compared with those fed ad libitum. With leptin administration, there was a further 50% decrease in leptin expression. LPL mRNA levels were decreased by food restriction but not by leptin in WAT, whereas beta3AR and HSL mRNA levels were unchanged with either food restriction or leptin treatment. The present study indicates that leptin increases the gene expression of UCP2 in EWAT and that of UCP1, UCP3 and LPL in BAT, whereas reduced food consumption but not leptin, decreases LPL expression in WAT. In addition, with leptin administration there is a decrease in leptin gene expression in WAT, independent of food intake and serum insulin and corticosterone concentrations.
Rabbit ovarian production of interleukin-6 and its potential effects on gonadotropin-induced progesterone secretion in granulosa and theca cells
Authors: Breard E , Benhaim A , Feral C , Leymarie P
Published: December 2004

Recent studies suggest that non-steroid factors, such as cytokines, may play a role in ovarian processes. The purpose of this study was to explore cellular sites of interleukin (IL)-6 biosynthesis in rabbit follicles and to investigate IL-6 modulation in granulosa and theca cell functions. In this report development of rabbit preovulatory follicles was induced by 200 mIU equine chorionic gonadotropin (eCG) daily for 2 days. Seventy-two hours after the last injection ovaries were excised and granulosa and theca cells isolated. The two types of cells were preincubated for 24 h in Minimum Essential Medium (MEM) with 5% fetal calf serum (FCS), and then incubated for 24 h in MEM-2.5% FCS with appropriate stimulants. Results showed that rabbit granulosa and theca cell culture supernatants contained IL-6 bioactivity and that its production was inhibited by FSH and human CG and stimulated by IL-1. IL-6 inhibited gonadotropin-induced progesterone production, but not basal secretion, in both cell types, without a cytotoxic effect. IL-6 affected cAMP generation and steps distal to cAMP formation, but the mechanism of IL-6 action on progesterone differed in granulosa and theca cells. Taken together our results suggest that gonadotropins, by inhibiting IL-6 production, could control, in our model, IL-6 modulation of gonadotropin action on steroidogenesis.
The role of corticotropin-releasing factor in depression and anxiety disorders
Authors: Arborelius L , Owens MJ , Plotsky PM , Nemeroff CB
Published: December 2004

Corticotropin-releasing factor (CRF), a 41 amino acid-containing peptide, appears to mediate not only the endocrine but also the autonomic and behavioral responses to stress. Stress, in particular early-life stress such as childhood abuse and neglect, has been associated with a higher prevalence rate of affective and anxiety disorders in adulthood. In the present review, we describe the evidence suggesting that CRF is hypersecreted from hypothalamic as well as from extrahypothalamic neurons in depression, resulting in hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and elevations of cerebrospinal fluid (CSF) concentrations of CRF. This increase in CRF neuronal activity is also believed to mediate certain of the behavioral symptoms of depression involving sleep and appetite disturbances, reduced libido, and psychomotor changes. The hyperactivity of CRF neuronal systems appears to be a state marker for depression because HPA axis hyperactivity normalizes following successful antidepressant treatment. Similar biochemical and behavioral findings have been observed in adult rats and monkeys that have been subjected to early-life stress. In contrast, clinical studies have not revealed any consistent changes in CSF CRF concentrations in patients with anxiety disorders; however, preclinical findings strongly implicate a role for CRF in the pathophysiology of certain anxiety disorders, probably through its effects on central noradrenergic systems. The findings reviewed here support the hypothesis that CRF receptor antagonists may represent a novel class of antidepressants and/or anxiolytics.
Tri-iodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis
Authors: Varga F , Luegmayr E , Fratzl-Zelman N , Glantschnig H , Ellinger A , Prinz D , Rumpler M , Klaushofer K
Published: December 2004

Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine (T3), even though it enhances the expression of many osteoblastic features, attenuates the multilayer formation to about two layers. The aim of this study was to investigate how T3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that T3 did not inhibit DNA synthesis. Analysis of the cell cycle phases showed an increase in the number of cells in G0/G1 with increasing cell density, but no significant effect of T3 treatment was found. Morphological investigations showed apoptotic features in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and phagocytosed apoptotic bodies. T3 treatment significantly increased the number of apoptotic cells. We conclude from our data that T3 inhibits multilayer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulatory event during bone tissue differentiation, our findings emphasise the importance of thyroid hormones in bone maintenance and development.
Amino acid transformation of oscillatory Ca2+ signals in mouse pancreatic beta-cells
Authors: Ahmed M , Grapengiesser E , Hellman B
Published: December 2004

Glucose-induced increase of cytoplasmic Ca2+ in pancreatic beta-cells is usually manifested as slow oscillations from the basal level. The significance of this rhythmicity for maintaining normal beta-cell function with periodic variations of circulating insulin made it of interest to investigate how the oscillatory Ca2+ signal was affected by various amino acids. Individual mouse beta-cells were very sensitive to alanine, glycine and arginine, sometimes responding with a transformation of the oscillations into sustained elevation of cytoplasmic Ca2+ at amino acid concentrations as low as 0.1 mM. Stimulation of the entry of Ca2+, obtained either by raising the extracellular concentration or by prolonging the open state of the voltage-dependent Ca2+ channels with BAY K 8644, resulted in reappearance of the rhythmic activity in the presence of the amino acids. Oscillatory Ca2+ signals in intact islets were more resistant to transformation by amino acids than those of individual beta-cells. It is therefore suggested that signals from the adjacent cells make it possible for beta-cells situated in islets to overcome a suppression of the oscillatory activity otherwise seen in the presence of alanine, glycine or arginine.
GH but not IGF-I or insulin increases lipoprotein lipase activity in muscle tissues of hypophysectomised rats
Authors: Oscarsson J , Ottosson M , Vikman-Adolfsson K , Frick F , Enerback S , Lithell H , Eden S
Published: December 2004

Changes in GH secretion are associated with changes in serum lipoproteins, utilisation of fuels and body composition. Since lipoprotein lipase (LPL) is a key enzyme in the regulation of lipid and lipoprotein metabolism, changes in LPL activity may contribute to these effects of GH. The present study was undertaken to investigate the role of GH and the GH-dependent growth factor, IGF-I, in the regulation of LPL in heart, skeletal muscle and adipose tissue. Female rats were hypophysectomised at 50 days of age. One week later, hormonal therapy was commenced. All hypophysectomised rats received l-thyroxine and cortisol. Adipose tissue, the heart, soleus and gastrocnemius muscles were excised after 1 week of hormonal therapy. The effect of insulin injections on adipose tissue and heart LPL activity was also studied. In separate experiments, LPL activity in post-heparin plasma was measured.Hypophysectomy had no effect on adipose tissue LPL activity, whereas activity was reduced in heart, soleus and gastrocnemius muscle tissues. GH treatment had no significant effect on LPL activity in adipose tissue or soleus muscle, but increased the LPL activity in heart and gastrocnemius muscle. GH treatment increased post-heparin plasma LPL activity. Recombinant human IGF-I treatment (1.25 mg/kg per day) markedly reduced LPL activity in adipose tissue, but had no effect in muscle tissues. The effect of IGF-I treatment on adipose tissue LPL was not reflected by a decrease in post-heparin plasma LPL activity. Daily injections of insulin for 7 days increased LPL activity in adipose tissue but had no effect on heart LPL activity. In adipose tissue, LPL mRNA levels tended to decrease as a result of IGF-I treatment. In the muscle tissues, no significant effects of hypophysectomy, GH or IGF-I treatment on LPL mRNA levels were observed.%It is concluded that GH increases heart and skeletal muscle tissue LPL activity, which probably contributes to an increased post-heparin plasma LPL activity. The effect of GH on muscle LPL activity is probably not mediated by IGF-I or insulin. Insulin and IGF-I have opposite effects on LPL activity in adipose tissue.
Nitric oxide synthase acutely regulates progesterone production by in vitro cultured rabbit corpora lutea
Authors: Gobbetti A , Boiti C , Canali C , Zerani M
Published: December 2004

We examined the presence and the regulation of nitric oxide (NO) synthase (NOS) using in vitro cultured corpora lutea (CL) obtained from rabbits at days 4 and 9 of pseudopregnancy. The role of NO and NOS on steroidogenesis was also investigated using the same CL preparations after short-term incubations (30 min and 2 h) with the NO donor, sodium nitroprusside (NP), the NOS inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME) and prostaglandin (PG) F-2alpha. The basal NOS activity was greater in CL at day 4 than at day 9, and was also differently modulated by PGF-2alpha, depending on the age of the CL. The addition of PGF-2alpha to day 4 CL had no effect, but PGF-2alpha on day 9 caused a threefold increase in NOS activity. NP caused a two- to fivefold decrease in release of progesterone from CL of both ages, and this inhibitory effect on steroidogenesis was reversed by l-NAME. All treatments failed to modify basal androgens and 17beta-oestradiol was not detectable in either control or treated CL. These results suggest that NO is effectively involved in the regulation process of steroidogenesis, independently of 17beta-oestradiol. PGF-2alpha had no effect on day 4, but induced luteolysis on day 9, by reducing progesterone (P</=0. 01) to about 18% of control. The luteolytic action of PGF-2alpha was completely reversed by co-incubation with l-NAME, thus supporting the hypothesis that luteolysis is mediated by NO. The addition of NP or l-NAME did not modify the in vitro release of PGF-2alpha. We hypothesised that PGF-2alpha upregulates NOS activity and, consequently, the production of NO, which acutely inhibits progesterone release from day 9 CL of pseudopregnant rabbits.
Glucose transporter 2 concentrations in hyper- and hypothyroid rat livers
Authors: Mokuno T , Uchimura K , Hayashi R , Hayakawa N , Makino M , Nagata M , Kakizawa H , Sawai Y , Kotake M , Oda N , Nakai A , Nagasaka A , Itoh M
Published: December 2004

The deterioration of glucose metabolism frequently observed in hyperthyroidism may be due in part to increased gluconeogenesis in the liver and glucose efflux through hepatocyte plasma membranes. Glucose transporter 2 (GLUT 2), a facilitative glucose transporter localized to the liver and pancreas, may play a role in this distorted glucose metabolism. We examined changes in the levels of GLUT 2 in livers from rats with l-thyroxine-induced hyperthyroidism or methimazole-induced hypothyroidism by using Western blotting to detect GLUT 2. An oral glucose tolerance test revealed an oxyhyperglycemic curve (impaired glucose tolerance) in hyperthyroid rats (n=7) and a flattened curve in hypothyroid rats (n=7). GLUT 2 levels in hepatocyte plasma membranes were significantly increased in hyperthyroid rats and were not decreased in hypothyroid rats compared with euthyroid rats. The same results were obtained with a densitometric assay. These findings suggest that changes in the liver GLUT 2 concentration may contribute to abnormal glucose metabolism in thyroid disorders.
Mediation of humoral catecholamine secretion by the renin-angiotensin system in hypotensive rainbow trout (Oncorhynchus mykiss)
Authors: Bernier NJ , Kaiya H , Takei Y , Perry SF
Published: December 2004

The individual contributions of, and potential interactions between, the renin-angiotensin system (RAS) and the humoral adrenergic stress response to blood pressure regulation were examined in rainbow trout. Intravenous injection of the smooth muscle relaxant, papaverine (10 mg/kg), elicited a transient decrease in dorsal aortic blood pressure (PDA) and systemic vascular resistance (RS), and significant increases in plasma angiotensin II (Ang II) and catecholamine concentrations. Blockade of alpha-adrenoceptors before papaverine treatment prevented PDA and RS recovery, had no effect on the increase in plasma catecholamines, and resulted in greater plasma Ang II concentrations. Administration of the angiotensin-converting enzyme inhibitor, lisinopril (10(-4) mol/kg), before papaverine treatment attenuated the increases in the plasma concentrations of Ang II, adrenaline, and noradrenaline by 90, 79, and 40%, respectively and also prevented PDA and RS recovery. By itself, lisinopril treatment caused a gradual and sustained decrease in PDA and RS, and reductions in basal plasma Ang II and adrenaline concentrations. Bolus injection of a catecholamine cocktail (4 nmol/kg noradrenaline plus 40 nmol/kg adrenaline) in the lisinopril+papaverine-treated trout, to supplement their circulating catecholamine concentrations and mimic those observed in fish treated only with papaverine, resulted in a temporary recovery in PDA and RS. These results indicate that the RAS and the acute humoral adrenergic response are both recruited during an acute hypotensive stress, and have important roles in the compensatory response to hypotension in rainbow trout. However, whereas the contribution of the RAS to PDA recovery is largely indirect and relies on an Ang II-mediated secretion of catecholamines, the contribution from the adrenergic system is direct and relies at least in part on plasma catecholamines.
High concentration of glucose decreases glucose transporter-1 expression in mouse placenta in vitro and in vivo
Authors: Ogura K , Sakata M , Yamaguchi M , Kurachi H , Murata Y
Published: December 2004

Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1.
Spatiotemporal expression of alternatively spliced IGF-I mRNA in the rat costochondral growth plate
Authors: Lin WW , Oberbauer AM
Published: December 2004

IGF-I acts as a local proliferation and maturation factor for chondrocytes in the growth plate. However, the expression of different alternative IGF-I mRNA classes in the growth plate has not been characterized. Using quantitative reverse transcription PCR, the abundance of each alternative IGF-I mRNA class in resting, proliferative and hypertrophic chondrocytes was measured in rat costochondral growth plates. Class 1Ea mRNA was the most abundant IGF-I transcript overall and was highly expressed in proliferative chondrocytes at 2 and 4 weeks of age; by 6 weeks, the majority of 1Ea mRNA expression had shifted to hypertrophic chondrocytes. Class 1Eb mRNA was the second most abundant transcript and its distribution was uniform across all the cell types at 2 weeks of age. The expression pattern changed with increasing age such that at 6 weeks a gradient existed with hypertrophic chondrocytes expressing higher levels of 1Eb than resting chondrocytes. Class 2Ea mRNA was constitutively expressed at low levels across the growth plate at all ages, while class 2Eb mRNA expression was negligible. The distribution of total IGF-I mRNA also shifted across growth plate cell types as the animals aged from 2 to 6 weeks. These findings suggest that IGF-I class 1 mRNA plays the predominant role in the maturation of the growth plate.
Augmentation of diabetes-associated renal hyperfiltration and nitric oxide production by pregnancy in rats
Authors: Omer S , Shan J , Varma DR , Mulay S
Published: December 2004

We tested the hypothesis that pregnancy might increase diabetes-associated nitric oxide (NO) production and renal hyperfiltration. Two weeks following i.v. streptozotocin (40 mg/kg), mean arterial pressure (MAP) was not modified by diabetes; glomerular filtration rate (GFR), renal plasma flow (RPF) and filtration fraction (FF) were higher in pregnant than in virgin controls and increased by diabetes to a greater extent in pregnant than in virgin rats. Urinary volume (UV), creatinine, albumin and sodium (UNaV) were significantly increased by diabetes. Diabetes led to an increase in renal, cardiac, aortic and uterine but not in placental NO synthase activities. Infusion of NG-nitro-l-arginine (l-NA) caused a dose-dependent reduction in GFR, RPF, plasma NO2-/NO3-, UV and UNaV; in general, diabetes increased these effects to a greater extent in pregnant than in virgin rats. l-NA increased MAP in all groups of rats but did not alter FF. Diabetes did not alter responses of thoracic aorta rings to vasoconstrictor effects of phenylephrine and the vasorelaxant effects of sodium nitroprusside but increased endothelium-dependent relaxant effects of acetylcholine. In general the effects of diabetes of 7 days duration were similar to those described above for diabetes of 14 days duration. These data suggest that diabetes-associated renal hyperfiltration and NO production are augmented by pregnancy.
Functional inactivation of the oestrogen receptor by the antioestrogen, ZM 182780, sensitises tumour cells to reactive oxygen species
Authors: Newton CJ , Drummond N , Burgoyne CH , Speirs V , Stalla GK , Atkin SL
Published: December 2004

Reactive oxygen species (ROS) play a fundamental role in both apoptotic and necrotic cell death. Their importance is highlighted by studies showing that they mediate cell death in response to radiotherapy and to some forms of chemotherapy. Here we provide the first evidence for a role of ROS in response to an antiendocrine agent currently undergoing clinical trials. Using the oestrogen receptor (ER) containing rat pituitary GH3 cell line, we show that cell death is induced by the pure steroidal antioestrogen, ZM 182780, and that this is blocked by the antioxidant, N-acetyl cysteine (NAC). By flow cytometry, we show that, prior to the onset of DNA breakdown measured by ELISA, ZM 182780 exposure has no significant effect on intracellular oxidant concentrations. In contrast, ZM 182780 exposure greatly increases sensitivity to oxidants generated by blocking cellular antioxidant pathways and from exogenous administration of hydrogen peroxide (H2O2). As both necrosis and apoptosis are controlled by mitochondrial function, further experiments conducted to determine mitochondrial membrane potential (Delta|gWm) have indicated that the ZM 182780-induced loss of ER function increases the ease with which oxidants collapse mitochondrial activity and, as a consequence, cell death.
Changes in the suppressive effects of recombinant inhibin A on FSH secretion in ram lambs during sexual maturation: evidence for alterations in the clearance rate of inhibin
Authors: Tilbrook AJ , de Kretser DM , Clarke IJ
Published: December 2004

We have investigated the effectiveness of human recombinant inhibin A (hr-inhibin A) to suppress the secretion of follicle-stimulating hormone (FSH) in ram lambs from 1 to 18 months of age. Seventeen rams (nine castrated, eight intact) were used. At 1, 3, 6, 9, 12 and 18 months of age the rams were given an i.v. injection of either vehicle or hr-inhibin A (0.64 microgram/kg). Blood samples were taken over 24 h. Plasma concentrations of FSH were suppressed in castrated and intact rams following injection of hr-inhibin A with maximal suppression occurring 6 h after injection. Vehicle injection had no effect. At 12 months of age the suppression in FSH was most rapid in castrated rams and was maximal in intact rams. The clearance rate of inhibin was greatest at older ages but during the period of seasonally induced testicular activity, there was a significant decrease in the inhibin clearance. The testicular weight was reduced in rams treated with hr-inhibin A, indicating the importance of FSH for testicular development during the pubertal period. There was no effect of hr-inhibin A on plasma concentrations of luteinizing hormone. We conclude that inhibin is capable of suppressing the secretion of FSH in rams from 1 month of age onwards and that the pituitary gland becomes maximally responsive to the actions of inhibin by the age of puberty.
Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins
Authors: Zhang Y , Marchant TA
Published: December 2004

The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (Mr) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and low-capacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (Ka) of 20.1x10(9) M-1 and a maximum binding capacity (Bmax) of 161 fmol ml-1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with Mr ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with Mr of 66 kDa and a minor band with Mr of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with Mr of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.
Maternal hypothyroxinemia disrupts neurotransmitter metabolic enzymes in developing brain
Authors: Evans IM , Sinha AK , Pickard MR , Edwards PR , Leonard AJ , Ekins RP
Published: December 2004

Maternal thyroid status influences early brain development and, consequently, cognitive and motor function in humans and rats. The biochemical targets of maternal thyroid hormone (TH) action in fetal brain remain poorly defined. A partially thyroidectomized rat dam model was therefore used to investigate the influence of maternal hypothyroxinemia on the specific activities of cholinergic and monoaminergic neurotransmitter metabolic enzymes in the developing brain. Maternal hypothyroxinemia was associated with reduced monoamine oxidase (MAO) activity in fetal whole brain at 16 and 19 days gestation (dg). A similar trend was observed for choline acetyltransferase (ChAT) activity. In contrast, DOPA decarboxylase (DDC) activity was markedly elevated at 21 dg. Further study of these enzymes at 14 dg showed no differences between normal and experimental progeny - suggesting they become TH sensitive after this age. Tyrosine hydroxylase (TyrH) and acetylcholinesterase (AChE) activities were unaffected prenatally. During postnatal development, the activities of TyrH, MAO, DDC and, to a lesser extent, AChE were increased in a brain region- and age-specific manner in experimental progeny. The prenatal disturbances noted in this study may have wide-ranging consequences since they occur when neurotransmitters have putative neurotropic roles in brain development. Furthermore, the chronic disturbances in enzyme activity observed during postnatal life may affect neurotransmission, thereby contributing to the behavioural dysfunction seen in adult progeny of hypothyroxinemic dams.
The expression of inhibin/activin subunits in the human adrenal cortex and its tumours
Authors: Munro LM , Kennedy A , McNicol AM
Published: December 2004

Inhibins and activins are dimeric proteins of the transforming growth factor-beta superfamily which have been shown to be expressed in the adrenal cortex. Recent studies have suggested a role for these peptides in the pathogenesis and/or function of adrenal tumours. To investigate further their physiological and pathological roles, we have documented immunoreactivity for inhibin alpha, betaA and betaB subunits in normal adult and fetal human adrenals, in hyperplastic adrenals and in adrenal tumours. In the normal and hyperplastic adult gland, diffuse immunopositivity was demonstrated for beta subunits, suggesting that activins (beta beta dimers) can be expressed in all zones. Inhibin alpha was limited to the zona reticularis and the innermost zona fasciculata in the normal gland, extending centripetally into the zona fasciculata in hyperplasia, supporting a role for ACTH in the regulation of expression, and suggesting that expression of inhibins (alpha beta dimers) is restricted. Immunopositivity for all three subunits was seen in both fetal and definitive zones of the fetal cortex, indicating that both inhibins and activins could be expressed in both. Immunopositivity for all three subunits was seen in most adrenocortical tumours. Loss of immunopositivity for inhibin alpha in a subgroup of carcinomas might indicate a role in tumour progression. The greater intensity of staining for inhibin alpha in tumours associated with Cushing's syndrome again suggests a link with cortisol production.
Changes in the expression of neurohypophyseal prolactins during the estrous cycle and after estrogen treatment
Authors: Torner L , Nava G , Duenas Z , Corbacho A , Mejia S , Lopez F , Cajero M , Martinez de la Escalera G , Clapp C
Published: December 2004

Estrogens are recognized regulators of the expression of neurohypophyseal hormones and of anterior pituitary prolactin (PRL). Here we have investigated whether the levels of PRL mRNA and of 23 and 14 kDa PRL variants present in the hypothalamo-neurohypophyseal system change during the estrous cycle or in response to estrogen treatment. The reverse transcription polymerase chain reaction (RT-PCR) was performed to examine PRL mRNA expression in isolated paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei. In both nuclei PRL mRNA levels appeared higher in cycling females than in male rats, with the highest level occurring at estrus. This increase may involve estrogen action, since estrogen administration to ovariectomized rats was associated with apparently higher PRL mRNA levels in both the PVN and SON. Expression of the PRL gene at these sites may occur via both transcriptional factor Pit-1-dependent and -independent mechanisms. RT-PCR detected the mRNA for Pit-1 in the PVN but only at estrus. The concentration of the 23 kDa immunoreactive PRL determined in the neurohypophysis was significantly higher during estrus and after estrogen treatment. However, no difference was detected in the levels of the neurohypophyseal 14 kDa PRL-like fragment along the estrous cycle nor after estrogen administration. This lack of parallelism between neurohypophyseal PRLs could relate to an estrogen-induced inhibition of the proteolysis of 23 kDa PRL at this site, since estrogen treatment reduced the activity of neurohypophyseal proteolytic enzymes able to cleave PRL. Altogether our results are consistent with estrogens having a stimulatory effect on PRL gene expression in the hypothalamo-neurohypophyseal system and a concomitant inhibitory action on PRL proteolysis at this site.
Direct effects of the prostaglandins E2 and f2alpha on progesterone release by the corpus luteum of the marmoset monkey (Callithrix jacchus) studied by in vitro microdialysis
Authors: Fehrenbach A , Hodges JK , Einspanier A
Published: December 2004

The effects of the prostaglandins (PG) E2 and F2alpha on progesterone secretion in luteal tissue (32 corpora lutea) explanted from the mid-luteal ovary of the marmoset monkey (n=13) were investigated using an in vitro microdialysis system. Consecutive applications of 1, 10 and 100 microg/ml PGE2 resulted in a significant increase in secretion of progesterone at the maximum dose of 100 microg/ml, which was shown to be the stimulatory dose in both long-period and 20-min pulse (time to collect one fraction) applications. The response varied individually between 1.4- and 3. 4-fold above the baseline concentrations. Application of 500 microg/ml PGF2alpha led to similar hormone responses. In contrast, lower doses of PGF2alpha (0.5, 5 and 50 microg/ml) resulted in significantly increased levels of secretion of progesterone, to approximately 1.4-fold baseline values, only after the application was terminated (echo effect). Responses were less variable when a short pulse of 20 min duration was applied, instead of long applications of 1-2 h. On the basis of the passage rates measured for tritiated PGF2alpha, transfer through the dialysis membrane was assumed to be in the range of 1% for both PGs. Ultrastructurally, luteal cells lying in a sheath of five to seven cell layers around the dialysis tubing appeared intact and were interconnected by gap junctions. Vesiculation of the smooth endoplasmic reticulum was more prominent after PG treatment, indicating a stimulation of cellular synthesis/secretory activities that was in accordance with the stimulatory action of both PGs on progesterone release under these in vitro conditions.
A missense mutation in the GHR gene of Cornell sex-linked dwarf chickens does not abolish serum GH binding
Authors: Hull KL , Marsh JA , Harvey S
Published: December 2004

Sex-linked dwarfism (SLD) in chickens is characterized by impaired growth despite normal or supranormal plasma growth hormone (GH) levels. This resistance to GH action is thought to be due to mutations of the GH receptor (GHR) gene that reduce or prevent GH binding to target sites. The genetic lesion causing GH resistance in Cornell SLD chickens is, however, not known. Previous studies have shown that hepatic GH-binding activity is abnormally low in these birds, yet the GHR gene is transcribed into a transcript of appropriate size and abundance. Point mutations or defects in translation could therefore account for the impaired GHR activity in this strain. These possibilities were addressed in the present study. A missense mutation resulting in the substitution of serine for the conserved phenylalanine was identified in the region of the GHR cDNA encoding the extracellular domain. Translation of this mutant transcript was indicated by the presence of GHR/GH-binding protein (GHBP)-immunoreactive proteins in liver (55, 70 and 100 kDa) and serum (70 kDa) of normal (K) and SLD birds. Radiolabelled GH did not, however, bind to the hepatic membranes of most SLD chickens. Serum GH-binding activity, in contrast, was readily detectable, although at significantly lower levels than in normal birds. The missense mutation in the SLD GHR gene may thus affect targeting of GHRs to hepatic plasma membranes.
Effects of prolonged infusion of basic fibroblast growth factor and IGF-I on adrenocortical differentiation in the autotransplanted adrenal: an immunohistochemical study
Authors: Vendeira P , Pignatelli D , Neves D , Magalhaes MM , Magalhaes MC , Vinson GP
Published: December 2004

Adrenocortical regeneration after adrenal autotransplantation provides a model for the study of local autocrine/paracrine mechanisms involved in the growth and differentiation of the adrenal cortex. To study the possible involvement of some growth factors, namely basic fibroblast growth factor (bFGF, FGF-2) and insulin-like growth factor I (IGF-I), in cell differentiation, immunohistochemical and ultrastructural studies were carried out on adrenal autotransplants in adult male rats. To distinguish between fasciculata and glomerulosa-like cells with accuracy, tissue sections were immunostained with IZAb, which recognizes the inner zone antigen (IZAg) present in fasciculata and reticularis cells but absent from the glomerulosa, and by electron microscopy. IGF-I-treated animals exhibited a clear glomerulosa-like zone that was devoid of IZAb immunostaining. In this outer subcapsular area, ultrastructural examination showed cells containing mitochondria with irregular cristae resembling those of the fetal or immature glomerulosa cells. In contrast, no significant morphological differences were observed in bFGF-treated animals when compared with those from saline-treated controls, in both of which, IZAb immunostaining occurred in almost all adrenocortical cells, with no clear zonation or glomerulosa, as seen in the intact animal. Plasma aldosterone and corticosterone concentrations were lower in autotransplanted control animals than in intact controls, although plasma renin activities were similar. IGF-I treatment significantly increased aldosterone concentrations, whereas corticosterone and plasma renin activity were reduced. bFGF infusion further reduced plasma aldosterone, although plasma renin activity and corticosterone were unaffected. These results suggest that the two growth factors have different effects on zonal differentiation and function in the autotransplanted gland. In particular, bFGF, by reducing glomerulosa function, appears partly to replicate the actions of ACTH in normal animals. In contrast, IGF-I enhances the glomerulosa secreting phenotype and diminishes that of the fasciculata/reticularis, possibly replicating the actions of angiotensin II or a low sodium diet.
The de novo synthesis of numerous proteins is decreased during vitamin D3 deficiency and is gradually restored by 1, 25-dihydroxyvitamin D3 repletion in the islets of langerhans of rats
Authors: Bourlon PM , Faure-Dussert A , Billaudel B
Published: December 2004

Since both the release and de novo biosynthesis of insulin are severely decreased by vitamin D3 deficiency and improved by 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) repletion following a 6-h delay in the rat, the present experiments investigated the effects of vitamin D3 deficiency on the biosynthesis of heavier molecular weight proteins using electrophoretic separation. Gel protein staining by Coomassie blue showed very different profiles for islets protein production from 4-week vitamin D3-deficient rats compared with normal islets. The pattern was characterised by a decrease in high molecular weight proteins, concomitantly accompanied by an increase in low molecular weight proteins. This tendency was partially reversed in vivo by 1,25(OH)2D3 repletion treatment for 7 days and was evident after only 16 h of treatment. In parallel with these in vivo observations, which represent a static index of islets protein production, a kinetic study was performed in vitro by a double-labelling method allowing us to measure the de novo synthesis of proteins in islets during a strong 16.7 mM glucose stimulation. Comparison of 3H and 14C labelled samples was achieved via coelectrophoresis to avoid experimental artefacts. The study of the ratio of d.p.m. 3H/d.p.m. 14C for each molecular weight protein in islets stimulated by 16.7 mM glucose (versus basal 4.2 mM glucose) showed an increase in the height of certain peaks: 150, 130 and 8.5 kDa. Under the same conditions, islets from 4-week vitamin D3-deficient rats (versus normal islets) presented a large deficit of numerous newly synthesised proteins and particularly those implicated in the response to glucose stimulation. In vitro repletion of 1,25(OH)2D3 tended to reverse, at least in part, the deleterious effect of vitamin D3 deficiency on the de novo protein synthesis of islets but these effects were gradual. Indeed, there was no detectable effect at 2 h incubation, but 1,25(OH)2D3 increased the 60 to 65 kDa, 55 kDa, and 9 to 8 kDa molecular mass proteins at 4 h, and increased the level of most newly synthesised proteins at 6 h. These data support the hypothesis of a beneficial genomic influence of 1,25(OH)2D3 that occurs progressively within the islets of Langerhans and which may prepare the beta cells for an enhanced response to glucose stimulation.
A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11
Authors: Phillips DJ , Brauman JN , Mason AJ , de Kretser DM , Hedger MP
Published: December 2004

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.