bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxygen-dependence of ACTH-stimulated aldosterone and corticosterone synthesis in the rat adrenal cortex: developmental aspects
Authors: Bruder ED , Nagler AK , Raff H
Published: December 2004

The control of ACTH-stimulated steroidogenesis under decreasing levels of O(2) is not fully understood. The purpose of this study was to examine the effects of decreased O(2) in vitro on rat adrenocortical steroid synthesis at different stages of development. Of interest was the evaluation of the effect of low O(2) on steroidogenesis during the stress hyporesponsive period of the neonate. Rats were killed at 7, 14, or 42 days of age, adrenals collected and capsules (zona glomerulosa, ZG) separated from subcapsules (zona fasciculata/reticularis, ZFR). Cells were dispersed and placed into glass vials each gassed with a different level of O(2) (21, 5, 2, 1, or 0% O(2)). The entire steroidogenic pathway was analyzed by measuring ACTH-stimulated cAMP, corticosterone and aldosterone production during a 2 h incubation. In addition, the early (P450 scc) and late (P450c11 beta and P450 aldo) pathway activities were examined in the presence of cyanoketone. The PO(2) for half-maximal activity (P(50)) for aldosterone synthesis in ZG cells from 7- and 42-day-old rats was approximately 28 mmHg and 7 mmHg respectively, indicating that cells from older rats were more resistant to inhibition by low O(2). The P(50) for cAMP production from the ZG was approximately 14 mmHg for both age groups. The P(50) for corticosterone synthesis was approximately 28 mmHg and <7 mmHg in ZFR cells from 7- and 42-day-old cells respectively. The only enzyme activities affected by low O(2) (<35 mmHg) were P450 aldo and P450 scc. Moderate decreases in O(2) (from approximately 150 mmHg) decreased aldosteronogenesis, possibly due to observed decreases in cAMP generation, but not due to decreases in steroidogenic enzyme activity (7-day-old). Severe decreases in O(2) presumably inhibited P450 aldo through a direct effect on enzyme activity (both ages). P450 scc activity (including cholesterol transport) also seems to be decreased by very low O(2) (7-day-old). These findings illustrate a novel developmental alteration in O(2)-regulated steroid production, and may have implications for neonatal health and disease.
Gender- and age-related differences in osteoclast formation from circulating precursors
Authors: Jevon M , Sabokbar A , Fujikawa Y , Hirayama T , Neale SD , Wass J , Athanasou NA
Published: December 2004

A number of bone diseases characterised by excessive osteolysis (e.g. osteoporosis and Paget's disease) exhibit a marked gender difference in prevalence and are more common in the elderly population. Bone resorption is carried out by osteoclasts, which are formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. In this study, we have determined whether there are gender- and age-related differences in osteoclast formation from circulating precursors. Peripheral blood mononuclear cells (PBMCs) were co-cultured with UMR106 osteoblast-like cells in the presence of macrophage-colony stimulating factor (M-CSF) and 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) or cultured alone in the presence of sRANKL (soluble receptor activator of nuclear factor kappa B ligand) and M-CSF. As assessed by the formation of tartrate resistant acid phosphatase (TRAP)-positive (TRAP(+)) and vitronectin receptor-positive (VNR(+)) multinucleated cells (MNCs), there was no difference in the number of circulating osteoclast precursors in males and females. Lacunar resorption carried out by osteoclasts formed from these precursors was generally increased in males compared with females (P=0.03). An increase in the number of TRAP(+) and VNR(+) MNCs formed from male PBMCs was noted in response to 1,25(OH)(2)D(3) (P<0.005). An increase in lacunar resorption in cultures of PBMCs (10(5) per well) from males was also noted in response to 10(-9) M 1,25(OH)(2)D(3) (P<0.05) and sRANKL (P=0.05), but not M-CSF. The addition of dexamethasone resulted in a marked increase in osteoclast formation and lacunar resorption in both males and females. Post-menopausal females and males of comparable age showed similar levels of osteoclastogenesis. Pre-menopausal women showed similar levels of osteoclastogenesis but less resorption (P=0.01) compared with males of comparable age. These results show that there are specific gender/age-related differences in osteoclast formation and bone resorption and have implications for evaluating osteoclastogenesis in skeletal diseases such as primary osteoporosis and Paget's disease.
Glucocorticoid-induced glucose release is abolished in trout hepatocytes with elevated hsp70 content
Authors: Boone AN , Ducouret B , Vijayan MM
Published: December 2004

The metabolic potential of cells with elevated heat shock protein 70 (hsp70) content was examined by measuring unstimulated and glucocorticoid-stimulated glucose release in trout hepatocytes maintained in primary culture. Exposure of hepatocytes to either heat shock (HS;+15 degrees C) or sodium arsenite (50 microM) did not affect cell viability, but resulted in significantly higher hsp70 levels over a 24 h recovery period. Hsp70 accumulation had no significant impact on unstimulated glucose release, but completely abolished cortisol-induced glucose release in trout hepatocytes. This lack of glucocorticoid responsiveness corresponded with lower glucocorticoid receptor protein levels. Together, our results suggest that stressor-induced hsp70 accumulation, while important for maintaining cellular homeostasis, may impair metabolic adjustments to subsequent stressors in animals, especially those that are glucocorticoid-dependent.
Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines
Authors: Jeffery PL , Herington AC , Chopin LK
Published: December 2004

This study has examined the expression of two new facets of the growth hormone axis, the growth hormone secretagogue receptor (GHS-R) and its recently identified putative natural ligand ghrelin, in prostate cancer cells. GHS-R 1a and 1b isoforms and ghrelin mRNA expression were detected by RT-PCR in the ALVA-41, LNCaP, DU145 and PC3 prostate cancer cell lines. A normal prostate cDNA library expressed GHS-R1a, but not the 1b isoform or ghrelin. Immunohistochemical staining for the GHS-R 1a isoform and ghrelin was positive in the four cell lines studied. PC3 cells showed increased cell proliferation in vitro in response to ghrelin to levels 33% above untreated controls, implying a potential tumour-promoting role for ghrelin in this tissue. This study is the first to demonstrate the co-expression of the GHS-R and ghrelin in prostate cancer cells. It is also the first study to provide evidence that a previously unrecognised autocrine/paracrine pathway involving ghrelin, that is capable of stimulating growth, exists in prostate cancer.
Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors
Authors: McArdle CA , Franklin J , Green L , Hislop JN
Published: December 2004

Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca(2+)-mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of beta-arrestin, or both. The association with beta-arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca(2+) mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.
Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption
Authors: Elton CW , Pennington JS , Lynch SA , Carver FM , Pennington SN
Published: December 2004

Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.
The effect of maternal undernutrition on ovine fetal growth
Authors: Osgerby JC , Wathes DC , Howard D , Gadd TS
Published: December 2004

Modifications in maternal nutrition during pregnancy can significantly disrupt fetal growth and subsequent post-natal health and survival. This study investigated the effects of undernutrition on fetal growth and the potential mechanisms involved. Tissue from pregnant ewes (n=27) was investigated on days 45, 90 and 135 of gestation (term = approximately 150 days). The thoracic girth (P<0.05) was greater in fetuses from nutrient restricted ewes on day 45 and there was also a trend towards an increased gut weight (P<0.08). By day 90, the fetal brain and thymus weight were lighter in underfed than in well-fed animals whilst the weight of the fetal ovaries was heavier (P<0.05). On day 135 the fetal heart, pancreas, thymus, gut and kidney weights were lighter in undernourished ewes (P<0.05). When expressed as a percentage of fetal body weight, significance was retained in the heart, pancreas and thymus (P<0.05). Bone growth was also affected. At day 90 the fetal femur and metatarsal were longer in underfed mothers (P<0.05). In contrast, the fetal humerus and scapula were shorter in underfed than in well-fed animals on day 135 (P<0.05) when the weight of the semitendinosus muscle (P<0.05) was also reduced. The fall in fetal glucose (P<0.1), insulin (P<0.01) and IGF-I (P<0.01) levels in underfed ewes on day 135 may have compromised fetal growth. Fetal plasma IGF binding protein-2 also increased between days 90 and 135 in underfed ewes (P<0.03), whilst levels were unaltered in well-fed animals. Although maternal and fetal plasma IGF-I levels increased with gestation (P<0.01) and the placentome morphology altered in all ewes (P<0.05), the fall in placental mass (P<0.05), amniotic and allantoic glucose concentrations (P<0.05) and maternal plasma glucose and insulin levels (P<0.05) in underfed ewes in late gestation may have compromised fetal substrate delivery. These perturbations in fetal development may have significant implications on adult health and carcass conformation, raising important health and economic issues in medical and agricultural sectors.
Perinatal growth and plasma GH profiles in adolescent and adult sheep
Authors: Gatford KL , Clarke IJ , De Blasio MJ , McMillen IC , J S Robinson , Owens JA
Published: December 2004

Poor prenatal growth is associated with limited evidence of GH deficiency in adult humans, which may contribute to their increased risk of cardiovascular and metabolic disease. We therefore examined the effects of placental restriction of fetal growth (PR) on size at birth, neonatal fractional growth rate (FGR) and the circulating GH profile in adolescent and young adult sheep of both sexes. Moderate or severe PR decreased birth size and increased neonatal FGR of weight, crown-rump length and abdominal circumference. In adolescent males, mean and baseline GH concentrations correlated negatively and independently with birth weight and FGR of weight, and mean GH concentrations correlated negatively with current weight. In young adult males, mean GH concentrations correlated negatively and independently with birth shoulder height and FGR of shoulder height whilst, in young adult females, these correlations were positive. This suggests that restricted fetal growth and reduced neonatal growth rate in sheep are followed by elevated circulating GH in adolescent and adult males, but GH deficiency or increased GH clearance in adult females.
Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes
Authors: van den Hove MF , Stoenoiu MS , Croizet K , Couvreur M , Courtoy PJ , Devuyst O , Colin IM
Published: December 2004

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1alpha (100 U/ml), alone or in combination with interferon-gamma (IFN-gamma; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1alpha with IFN-gamma, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-gamma was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1alpha effect on LDH release. The NOS inhibitor, L-nitro-arginine-methyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1alpha+IFN-gamma and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier.
gamma-Linoleic acid and ascorbic acid ameliorate the effects of experimental diabetes on electrolyte and bone homeostasis in pregnant rats
Authors: Braddock R , Siman CM , Hamilton K , Devlin H , Garland H , Sibley CP
Published: December 2004

Experimental diabetes in rats is associated with excessive electrolyte loss in the urine, which is further accentuated by pregnancy, particularly of Ca. Supplementation with essential fatty acids and antioxidants has proven beneficial in treating several types of complications, including nephropathy. The present study investigated the effect of gamma-linoleic acid (GLA; 500 mg/kg per day; group DG) and ascorbate (290 mg/kg per day; group DA), alone and in combination (group DGA), as well as ascorbyl-GLA (790 mg/kg per day; group DASG), on urinary electrolyte output and skeletal composition in pregnant streptozotocin-diabetic rats. Urine was collected in metabolism cages before and throughout pregnancy. Diabetic rats (DP) increased their urine volume as compared with control (CP) throughout the experiment, reaching an output of more than 13 times that of the control group by the end of pregnancy (CP 24+/-4, DP 316+/-21, DG 223+/-21, DA 221+/-14, DASG 163+/-17, DGA 220+/-19 ml urine/24 h). Concomitant with increased urine volume was a reduction of urinary Na (CP 47+/-14, DP 22+/-5 mmol/l), K (CP 210+/-34, DP 31+/-1 mmol/l) and Mg (CP 14+/-1, DP 3.8+/-0.2 mmol/l) concentration, but not of Ca concentration (CP 5.4+/-1.5, DP 6.3+/-0.6 mmol/l), and hence total Ca loss was relatively most severe. All the treatments reduced urine volume with no effects on electrolyte concentration as compared with DP, with no significant difference between the treatments. A reduced bone size and bone Ca content was partially ameliorated by the diet supplementation. We have concluded that GLA and ascorbate, alone or in combination, prevent urinary electrolyte loss in pregnant rats and do so by reducing urine production.
Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages
Authors: Boiti C , Zampini D , Guelfi G , Paolocci F , Zerani M , Gobbetti A
Published: December 2004

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.
Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells
Authors: Riera MF , Meroni SB , Schteingart HF , Pellizzari EH , Cigorraga SB
Published: December 2004

By using cultured rat Sertoli cells as a model, both the action of basic fibroblast growth factor (bFGF) on lactate production and the site of this action were studied. bFGF stimulated Sertoli cell lactate production in a dose-dependent manner (basal: 7.3+/-0.5; 0.1 ng/ml bFGF: 7.5+/-0.5; 1 ng/ml bFGF: 7.5+/-0.6; 10 ng/ml bFGF: 10.3+/-1.0; 30 ng/ml bFGF: 15.2+/-1.5; 50 ng/ml bFGF: 15.4+/-1.6 microg/microg DNA). Two major sites for the action of this growth factor were identified. First, bFGF was shown to exert short- and long-term stimulatory effects on glucose transport (basal: 1170+/-102; 30 ng/ml bFGF for 120 min: 1718+/-152 and basal: 718+/-64; 30 ng/ml bFGF for 48 h: 1069+/-69 d.p.m./microg DNA respectively). Short-term bFGF stimulation of glucose transport was not inhibited by the protein synthesis inhibitor cycloheximide. These results indicate that short-term bFGF stimulation of glucose uptake does not involve an increase in the number of glucose transporters. On the other hand, stimulation with bFGF for periods of time longer than 12 h increased glucose transporter 1 (GLUT1) mRNA levels. These increased mRNA levels were probably ultimately responsible for the increments in glucose uptake that are observed in long-term treated cultures. Secondly, bFGF increased lactate dehydrogenase (LDH) activity (basal: 31.0+/-1.4; 30 ng/ml bFGF: 45.7+/- 2.4 mIU/microg DNA). The principal subunit component of those LDH isozymes that favors the transformation of pyruvate to lactate is subunit A. bFGF increased LDH A mRNA levels in a dose- and time-dependent manner. In summary, the results presented herein show that glucose transport, LDH activity and GLUT1 and LDH A mRNA levels are regulated by bFGF to achieve an increase in lactate production. These observed regulatory actions provide unequivocal evidence of the participation of bFGF in Sertoli cell lactate production which may be related to normal germ cell development.
Arthritis-induced increase in serum levels of IGF-binding protein-3 in rats is secondary to the decrease in its proteolytic activity
Authors: Ibanez De Caceres I , Holly JM , Priego T , Martin AI , Lopez-Calderon A , Villanua MA
Published: December 2004

Adjuvant-induced arthritis is a chronic inflammatory illness that induces a catabolic state, with a decrease in pituitary GH and hepatic IGF-I synthesis. We have previously observed an increase in serum IGF-binding protein-3 (IGFBP-3) in arthritic rats, and found that GH administration prevents the increase in circulating IGFBP-3 in arthritic rats. The aim of this work was therefore to study IGFBP-3 synthesis in the liver as well as its proteolysis in serum as the two possible causes of the increased circulating IGFBP-3 in arthritic rats. The effect of recombinant human GH (rhGH) administration was also analysed. Adult male Wistar rats were injected with complete Freund's adjuvant or vehicle, and 14 days later they were injected s.c. daily until day 22 after adjuvant injection with rhGH (3 IU/kg) or saline. Three hours after the last GH injection, all rats were killed by decapitation. Arthritis increased serum IGFBP-3 levels (P<0.01). The increase in serum IGFBP-3 levels in arthritic rats seems to be due to decreased proteolysis (P<0.01) rather than to an increased synthesis, since liver IGFBP-3 mRNA content was not modified by arthritis. GH administration to control rats resulted in an increase in both hepatic IGFBP-3 mRNA content and in serum IGFBP-3 levels in spite of the increase in IGFBP-3 proteolysis in serum. In arthritic rats, GH treatment did not modify liver IGFBP-3 synthesis, but it increased serum proteolysis of IGFBP-3, leading to a serum concentration of IGFBP-3 similar to that of control rats. Furthermore, there was a negative correlation between circulating IGFBP-3 and its proteolytic activity in the serum of adjuvant-induced arthritic rats. These data suggest that in chronic arthritis the increase in IGFBP-3 serum concentration is secondary to a decrease in proteolytic activity, rather than to an increase in hepatic IGFBP-3 gene expression.
Molecular cloning, identification and characterization of four distinct receptor subtypes for insulin and IGF-I in Japanese flounder, Paralichthys olivaceus
Authors: Nakao N , Tanaka M , Higashimoto Y , Nakashima K
Published: December 2004

Insulin receptor (IR) and IGF-I receptor (IGF-IR) are structurally and functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported, but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned and analysed four distinct cDNA clones for IR and IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (fIR-1 and fIR-2) belong to IR members and the other two (fIGF-IR-1 and fIGF-IR-2) are IGF-IRs. fIR-1 and fIR-2 comprised 1369 and 1368 amino acid residues respectively, and fIGF-IR-1 and fIGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their alpha-subunits, and conserved each transmembrane and tyrosine kinase domains in their beta-subunits. The amino acid sequences of fIRs and fIGF-IRs showed more than 90% sequence identity with turbot IR and IGF-IR respectively. When compared with their mammalian homologues, fIGF-IR-1 and fIGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of mRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues including heart, liver, ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.
Calcium balance in sea bream (Sparus aurata): the effect of oestradiol-17beta
Authors: Guerreiro PM , Fuentes J , Canario AV , Power DM
Published: December 2004

In all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17beta (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 microg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2.5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60-70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts.
Bone marrow and bone: a functional unit
Authors: Compston JE
Published: December 2004

Bone and bone marrow, although often regarded as separate systems, function as a single unit. Cells in the bone marrow are the precursors of bone remodelling cells and exert an important regulatory role both on their own development and the remodelling process, acting as mediators for the effects of systemic and local factors. Other cells, such as immune cells and megakaryocytes, also contribute to the regulation of bone cell development and activity. Many diseases that affect the bone marrow have profound effects on bone, involving interactions between abnormal and normal marrow cells and those of bone. Although recent advances in bone physiology have produced new insights into the relationship between bone marrow and bone cells, much remains to be learnt about the mechanisms by which marrow and bone act in synergy to regulate bone remodelling, both in health and disease.
Differential effects of interleukin-6 receptor activation on intracellular signaling and bone resorption by isolated rat osteoclasts
Authors: Moonga BS , Adebanjo OA , Wang HJ , Li S , Wu XB , Troen B , Inzerillo A , Abe E , Minkin C , Huang CL , Zaidi M
Published: December 2004

The effects of the related cytokines interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and oncostatin-M on bone resorption and cytosolic Ca(2+) signaling were compared in isolated rat osteoclasts. In the traditional disaggregated osteoclast (pit) assay, IL-6 and LIF, but not oncostatin-M, conserved the bone resorption otherwise inhibited by high extracellular [Ca(2+)] (15 mM). It produced a paradoxical, concentration-dependent stimulation of resorption by elevated extracellular Ca(2+). In the micro-isolated single osteoclast resorption assay, IL-6, high [Ca(2+)] or IL-6 plus high [Ca(2+)] all increased pit formation. In contrast, the IL-6 receptor (IL-6R)-specific agonist antibody MT-18 inhibited bone resorption in a concentration-dependent manner (1:500 to 1:500 000). MT-18 triggered cytosolic Ca(2+) signals in fura 2-loaded osteoclasts within approximately 10 min of application. Each cytosolic Ca(2+) transient began with a peak deflection that persisted in Ca(2+)-free, EGTA-containing extracellular medium, consistent with a release of intracellularly stored Ca(2+). This was followed by a sustained elevation of cytosolic [Ca(2+)] that was abolished in Ca(2+)-free medium, as expected from an entry of extracellular Ca(2+), and by the Ca(2+) channel antagonist Ni(2+). The inclusion of either IL-6 or soluble human (sh) IL-6R specifically reversed both the above effects of MT-18, confirming that both effects were specific for the IL-6R. The findings suggest that IL-6R activation by IL-6 stimulates osteoclastic bone resorption either by reversing the inhibitory effect of high extracellular Ca(2+) in stromal-containing systems or itself stimulating bone resorption along with Ca(2+) by micro-isolated osteoclasts. In contrast, activation of the IL-6R by an agonist antibody produces an inhibition of bone resorption and an associated triggering of the cytosolic Ca(2+) signals previously associated with regulation of bone resorptive function in other situations.
Structural analysis of the DNA-binding domain of alternatively spliced steroid receptors
Authors: Wickert L , Selbig J
Published: December 2004

We have generated 24 DNA-binding domain structure models of alternatively spliced or mutated steroid receptor variants by homology-based modeling. Members of the steroid receptor family dispose of a DNA-binding domain which is built from two zinc fingers with a preserved sequence homology of about 90%. Data from crystallographic analysis of the glucocorticoid receptor DNA-binding domain are therefore appropriate to serve as a template structure. We inserted or deleted amino acid residues in order to study the structural details of the glucocorticoid, mineralocorticoid, and androgen receptor splice variants. The receptor variants are created by QUANTA- and MODELLER-based modeling. Subsequently, the resulting energy-minimized models were compared with each other and with the wild-type receptor respectively. A prediction for the receptor function based mainly on the preservation or destruction of secondary structures has been carried out. The simulations showed that amino acid insertions of one, four or nine additional residues of existing steroid receptor splice variants were tolerated without destruction of the secondary structure. In contrast, a deletion of four amino acids at the splice site junction leads to modifications in the secondary structure of the DNA-recognition helix which apparently disturb the receptor-DNA interaction. Furthermore, an insertion of 23 amino acid residues between the zinc finger of the androgen receptor leads to a large loop with an additional alpha-helical structure which seems to disconnect a specific contact from its hormone response element. Thereafter, the prediction of receptor function based on the molecular models was compared with the available experimental results from the in vitro function tests. We obtained a close correspondence between the molecular modeling-based predictions and the conclusions of receptor function drawn from in vitro studies.
Maternal undernutrition alters triiodothyronine concentrations and pituitary response to GnRH in fetal sheep
Authors: Rae MT , Rhind SM , Kyle CE , Miller DW , Brooks AN
Published: December 2004

The aims of this study were to determine which hormones may have a role in the expression of maternal undernutrition effects on reproductive function, in both the developing fetus and the adult offspring. This was undertaken by measuring the effects of long-term maternal undernutrition on metabolic hormone profiles and pituitary responses to single doses of GnRH and GH-releasing factor (GRF) in fetal sheep. From mating, groups of ewes were fed rations providing either 100% (HIGH) or 50% (LOW) of estimated metabolisable energy requirements for pregnancy throughout the experiment until slaughter at approximately 119 days of gestation. Fetal and maternal blood samples were collected from 113 until 119 days of gestation, via carotid and jugular catheters respectively, and assayed for insulin, IGF-I, GH, thyroxine and triiodothyronine (T(3)). Undernutrition had no effects on fetal weight, fetal gonad weight of either sex, fetal insulin or IGF-I concentrations. Male LOW fetuses exhibited a significantly attenuated response (P<0.05) to a bolus challenge of GnRH compared with HIGH fetuses. Basal fetal GH concentrations and the response to exogenous GRF were similar in both treatment groups, although LOW fetuses exhibited more secretory episodes (P<0.01). Mean T(3) concentrations were significantly lower in both the maternal (P<0.01) and fetal (P<0.05) plasma of LOW animals compared with HIGH animals. It is concluded that pituitary function was altered in fetal males and could influence male reproductive development. On the other hand, in female sheep, fetal gonadal abnormalities and reductions in reproductive capacity in adult life which are associated with fetal undernutrition are unlikely to be attributable to altered pituitary function. Additionally, these studies raise the possibility that thyroid hormones may have a role in the expression of maternal undernutrition effects on fetal development.
Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human myocytes
Authors: Luque MA , Gonzalez N , Marquez L , Acitores A , Redondo A , Morales M , Valverde I , Villanueva-Penacarrillo ML
Published: December 2004

Glucagon-like peptide-1 (GLP-1) has been shown to have insulin-like effects upon the metabolism of glucose in rat liver, muscle and fat, and on that of lipids in rat and human adipocytes. These actions seem to be exerted through specific receptors which, unlike that of the pancreas, are not - at least in liver and muscle - cAMP-associated. Here we have investigated the effect, its characteristics, and possible second messengers of GLP-1 on the glucose metabolism of human skeletal muscle, in tissue strips and primary cultured myocytes. In muscle strips, GLP-1, like insulin, stimulated glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity, all of this at physiological concentrations of the peptide. In cultured myotubes, GLP-1 exerted, from 10(-13) mol/l, a dose-related increase of the D-[U-(14)C]glucose incorporation into glycogen, with the same potency as insulin, together with an activation of glycogen synthase a; the effect of 10(-11) mol/l GLP-1 on both parameters was additive to that induced by the equimolar amount of insulin. Synthase a was still activated in cells after 2 days of exposure to GLP-1, as compared with myotubes maintained in the absence of peptide. In human muscle cells, exendin-4 and its truncated form 9-39 amide (Ex-9) are both agonists of the GLP-1 effect on glycogen synthesis and synthase a activity; but while neither GLP-1 nor exendin-4 affected the cellular cAMP content after 5-min incubation in the absence of 3-isobutyl-1-methylxantine (IBMX), an increase was detected with Ex-9. GLP-1, exendin-4, Ex-9 and insulin all induced the prompt hydrolysis of glycosylphosphatidylinositols (GPIs). This work shows a potent stimulatory effect of GLP-1 on the glucose metabolism of human skeletal muscle, and supports the long-term therapeutic value of the peptide. Further evidence for a GLP-1 receptor in this tissue, different from that of the pancreas, is also illustrated, suggesting a role for an inositolphosphoglycan (IPG) as at least one of the possible second messengers of the GLP-1 action in human muscle.
Thyroid follicular cells secrete plasminogen activators and can form angiostatin from plasminogen
Authors: Ramsden JD , Yarram S , Mathews E , Watkinson JC , Eggo MC
Published: December 2004

Angiostatin, a 38 kDa fragment of plasminogen, potently inhibits the growth of blood vessels. Angiostatin is generated from plasminogen by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in the presence of free sulphydryl donors. Angiogenesis inhibitors may be important in regulating angiogenesis in developing goitre. We have examined angiostatin formation in human primary thyrocyte cultures and a rat thyrocyte cell line (FRTL-5). We found that human thyroid cells in culture secrete plasminogen activators (both tPA and uPA) as well as matrix metalloproteinase 2 into the medium. When human thyrocyte conditioned medium was incubated with plasminogen (10 microg/ml) and N-acetylcysteine (100 microM) for 24 h, a 38 kDa fragment of plasminogen, which is consistent with angiostatin, was generated. The appearance of the 38 kDa fragment was increased by agents that increase cAMP (forskolin and 8 BrcAMP). FRTL-5 cells, which do not secrete uPA or tPA, did not generate angiostatin. Thyroid cells produce several angiogenic growth factors, and human thyrocyte conditioned medium stimulated growth of endothelial cells. When the conditioned medium was incubated with plasminogen and N-acetylcysteine, this stimulatory effect was lost, consistent with the production of a growth inhibitory factor. We conclude that thyroid cells can produce angiostatin from plasminogen in vitro, and this may play a role in vivo in limiting goitre size.
Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula)
Authors: Saunders MC , Gemmell RT , Waters MJ , Curlewis JD
Published: December 2004

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8+/-1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximately 65 kDa, similar to that identified in other mammalian species, and binding of (125)I-labelled human GH (hGH) was displaced by excess hGH (20 microg). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with (125)I-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K(a)) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96+/-0.2 x 10(9)/M, n=6). Binding capacity (B(max)) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4+/-0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.
Vitamin D inhibits the activation of stress-activated protein kinases by physiological and environmental stresses in keratinocytes
Authors: Ravid A , Rubinstein E , Gamady A , Rotem C , Liberman UA , Koren R
Published: December 2004

In addition to its known effects on keratinocyte proliferation and differentiation, the hormonal form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to protect keratinocytes from UV- and chemotherapy-induced damage. Epidermal keratinocytes contain both the machinery needed to produce 1,25(OH)(2)D(3) and vitamin D receptors. The activation of the stress-activated protein kinases (SAPKs), such as c-Jun N-terminal kinase (JNK) and p38, is an early cellular response to stress signals and an important determinant of cell fate. This study examines whether modulation of these SAPKs is associated with the effects of 1,25(OH)(2)D(3) on keratinocytes under stress. HaCaT keratinocytes were exposed to heat shock, hyperosmotic concentrations of sorbitol, the epidermal growth factor receptor tyrosine kinase inhibitor AG1487, the pro-inflammatory cytokine tumor necrosis factor alpha, and H(2)O(2). These stresses activated both SAPKs. Pretreatment with 1,25(OH)(2)D(3) inhibited the activation of JNK by all stresses and the activation of p38 by heat shock, AG1478 and tumor necrosis factor alpha. Under the same conditions, treatment with 1,25(OH)(2)D(3) protected HaCaT keratinocytes from cytotoxicity induced by exposure to H(2)O(2) and hyperosmotic shock. The effect of 1,25(OH)(2)D(3) was dose-dependent, already apparent at nanomolar concentrations, and time-dependent, maximal after a 24-h pre-incubation. We suggest that inhibition of SAPK activation may account for some of the well-documented protective effects of 1,25(OH)(2)D(3) on epidermal cells during exposure to UV or chemotherapy and may also be related to the anti-inflammatory actions of the hormone in skin.
Diabetes causes marked changes in lymphocyte metabolism
Authors: Otton R ,   Mendonca, Curi R
Published: December 2004

An enhanced susceptibility to infections is well known to occur in a poorly controlled diabetic state. Since glucose and glutamine are essential for lymphocyte function, we investigated whether their metabolism is changed in lymphocytes obtained from mesenteric lymph nodes of alloxan-induced diabetic rats (40 mg/kg body weight). The activities of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase (G6PDH), citrate synthase and phosphate-dependent glutaminase were determined. Decarboxylation of metabolites [U-14C]-, [1-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvic acid, [U-14C]-palmitic acid and [U-14C]-glutamine was evaluated in incubated lymphocytes isolated from mesenteric lymph nodes. The measurements were carried out in cells following three experimental protocols: (1) lymphocytes freshly obtained from control and alloxan-induced diabetic rats, (2) lymphocytes from insulin-treated (2 U/rat per day) diabetic rats and (3) lymphocytes obtained from control and diabetic rats and cultured in the presence of insulin (1 mU/ml) for 6 h. The activities of hexokinase, G6PDH and citrate synthase were decreased by the diabetic state, whereas that of phosphofructokinase was raised. Decarboxylation of [U-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvate and [U-14C]-glutamine were also decreased in lymphocytes from diabetic rats, whereas [U-14C]-palmitic acid decarboxylation was increased. Insulin administration in vivo or added to the culture medium reversed the changes observed in freshly obtained lymphocytes. Alloxan-induced diabetes did change lymphocyte metabolism and this may be an important mechanism leading to impairment of lymphocyte function.