bluuebunny/crossref_metadata_2025 huggingface dataset:
Sclerotome-related helix-loop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its level is enhanced by type-β transforming growth factor
Authors: Liu Y , Cserjesi P , Nifuji A , Olson E N , Noda M
Published: December 2004

Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ.

Journal of Endocrinology (1996) 151, 491–499
Diet-induced obesity and pancreatic islet blood flow in the rat: a preferential increase in islet blood perfusion persists after withdrawal of the diet and normalization of body weight
Authors: Svensson A M , Hellerström C , Jansson L
Published: December 2004

The aim of the present study was to evaluate the effects of diet-induced obesity on pancreatic islet blood perfusion in normal Wistar rats. Furthermore, we investigated to what extent any obesity-associated changes in islet blood flow could be reversed after reversion to a normal diet with normalization of body weight. Young adult female Wistar rats were offered a palatable mixed high-caloric diet (cafeteria diet) in addition to standard pelleted chow. Age-matched control rats received standard pelleted chow only. After 4 weeks the diet-treated rats had a body weight of approximately 15% more than that of the controls. All diet-treated rats had decreased glucose tolerance and increased serum insulin concentrations, but basal blood glucose concentrations were similar in anesthetized diet-treated and control rats. Whole pancreatic and islet blood flow rates were measured with a microsphere technique. The islet blood flow as well as fractional islet blood flow were increased (P<0·01) in rats fed the cafeteria diet, while blood perfusion of the whole pancreas was similar to that of the control rats. In a second experiment, rats received the cafeteria diet for 4 weeks and were then fed standard pelleted food alone for another 3 weeks, while controls received standard diet for 7 weeks. After this period total body weight, retroperitoneal fat pad weight and glucose tolerance were similar to those of the controls. Whole pancreatic blood flow was unchanged as compared with that of control rats. However, both islet blood flow (P<0·01) and fractional blood flow (P<0·01) were increased. We conclude that diet-induced obesity in rats is associated with decreased glucose tolerance, hyperinsulinemia and a specific increase in absolute and fractional islet blood perfusion. This increase persists for at least 3 weeks after the diet is withdrawn despite normalization of body weight and glucose tolerance.

Journal of Endocrinology (1996) 151, 507–511
Angiotensin II is generated from angiotensin I by bone cells and stimulates osteoclastic bone resorption in vitro
Authors: Hatton R , Stimpel M , Chambers T J
Published: December 2004

During bone resorption, osteoclasts are closely associated with endothelial cells. The latter are able to produce several agents that regulate bone resorption. In view of the increasing evidence that angiotensin II, which can be generated by endothelial cells, has actions outside the traditional renin-angiotensin system, we tested the effect of angiotensin II on bone resorption. Angiotensin II showed no effect either on osteoclast formation or on bone resorption by isolated osteoclasts. However, in co-cultures of osteoclasts with calvarial or MC3T3-E1 osteoblastic cells, and in osteoclastic cultures co-cultured with other bone cells obtained by prolonged sedimentation, angiotensin II stimulated bone resorption to a similar degree to that observed with 1,25(OH)2 vitamin D3. Stimulation of resorption was noted at concentrations of 10−7 m and above. We found that angiotensin I also stimulated bone resorption in co-cultures of osteoclasts with osteoblastic cells, and that this action was inhibited by inhibitors of angiotensin-converting enzyme. These results identify angiotensin I and II as potent stimulators of osteoclastic bone resorption, and raise the possibility that bone might contain a tissue-renin-angiotensin system that might play a role in the regulation of bone resorption.

Journal of Endocrinology (1997) 152, 5–10
Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium
Authors: Olson G E , Winfrey V P , Matrisian P E , Melner M H , Hoffman L H
Published: December 2004

A glycoprotein, termed GP42, was previously identified in uterine fluid obtained from peri-implantation-stage rabbits. N-terminus amino acid sequencing of purified GP42 demonstrated identity through the first 13 amino acids with the β subunit of liver haptoglobin. The present study was undertaken to determine if GP42 is indeed identical to haptoglobin and, if so, to determine whether it is expressed in the uterus as opposed to being present as a transudate from plasma. Reverse transcription-PCR amplification of poly(A)+ RNA prepared from implantation-stage rabbit endometrium with GP42- and haptoglobin-specific primers yielded a predicted 667 bp cDNA product. Sequence analysis of the cloned cDNA confirmed the identity of GP42 with β-haptoglobin. Northern blot analysis demonstrated the specific expression of haptoglobin mRNA in the peri-implantation-stage endometrium and the absence of its expression in the estrous or day 4 pseudopregnant endometrium. Non-isotopic in situ hybridization revealed that the haptoglobin mRNA was restricted to the epithelium lining the luminal surface and mucosal folds of day 6¾ pregnant or pseudopregnant uteri and that no haptoglobin mRNA was detectable in the epithelium of the deep glands or cells of the stroma or myometrium. Similarly, in situ hybridization revealed no expression of haptoglobin mRNA in any cell types of the estrous uterus. These data establish the identity of GP42 with β-haptoglobin and demonstrate that it is expressed in a stage-specific manner just prior to implantation, correlating with uterine receptivity to blastocyst implantation. Endometrial GP42 mRNA expression is not dependent on the presence of blastocysts.

Journal of Endocrinology (1997) 152, 69–80
Amniotic fluid inhibin-A in chromosomally normal and Down's syndrome pregnancies
Authors: Wallace E M , Crossley J A , Groome N P , Aitken D A
Published: December 2004

Recently, inhibin-A has been shown to be a useful new prenatal marker of Down's syndrome, significantly increasing detection rates. While the placenta is believed to be the major source of inhibin in pregnancy, there are actually very limited data available on specific inhibin dimers in pregnancy. Using a sensitive and specific ELISA we have measured the inhibin-A content of amniotic fluid (AF) to investigate further the biology of inhibin-A in chromosomally normal and abnormal pregnancies. AF from 51 Down's syndrome and 161 chromosomally normal pregnancies between 16 and 19 weeks of gestation were analysed, blinded as to whether the sample was from a Down's syndrome or normal pregnancy. There were no sex differences in inhibin-A content in either the control or Down's syndrome pregnancies. The median (10th–90th percentiles) inhibin-A level in the control pregnancies increased from 339·6 (175·2–649·1) pg/ml at 16 weeks to 592·9 (256·4–1027·3) pg/ml at 19 weeks of gestation. The median (95% confidence interval) inhibin-A in the Down's syndrome pregnancies, expressed as multiples of the median (MoM) to correct for gestation, was 0·77 (0·68–0·89) MoM, significantly lower than the controls (P<0·001, Mann–Whitney U test).

We believe that these data are compatible with more than one source of inhibin-A in pregnancy and suggest that the fetal membranes may be contributing significantly to AF inhibin-A content. Further, our data would suggest that the endocrine function of the placenta and the other inhibin source(s) are differentially regulated.

Journal of Endocrinology (1997) 152, 109–112
Presence of pituitary adenylate cyclase-activating polypeptide 38-immuno-like material in the brain and ovary of the female crested newt, Triturus carnifex: its involvement in the ovarian synthesis of prostaglandins and steroids
Authors: Gobbetti A , Zerani M , Miano A , Bramucci M , Murri O , Amici D
Published: December 2004

The presence of pituitary adenylate cyclase-activating peptide (PACAP) 38-immuno-like material (PACAP 38-IL) in the brain and ovary of the crested newt, Triturus carnifex, and its action on ovarian steroidogenesis and prostaglandin synthesis were evaluated. The HPLC brain and ovary extract peaks that eluted like PACAP 38 were considered PACAP 38-like material. The concentrations of PACAP 38-IL in the HPLC extracts were measured by RIA. T. carnifex ovary was incubated with PACAP 38, brain and ovary PACAP 38-IL, and inhibitors of cyclooxygenase (COX), adenylate cyclase (AC) and phospholipase C (PLC) for 30 and 60 min. PACAP 38, and brain and ovary PACAP 38-IL increased prostaglandin E2 (PGE2) (30 and 60 min), and progesterone and corticosterone (60 min), but decreased oestradiol-17β (60 min). COX and PLC inhibitors counteracted the increases in PGE2, progesterone and corticosterone and the decrease in oestradiol-17β, and the AC inhibitor also counteracted them except for PGE2. These results suggest that PACAP 38-IL, present in T. carnifex brain and ovary, acts on PLC, inducing the increase of PGE2 which, in turn, acting on AC, induces increases in progesterone and corticosterone and a decrease in oestradiol-17β.

Journal of Endocrinology (1997) 152, 141–146
Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells
Authors: Ogiwara T , Chik C L , Ho A K
Published: December 2004

In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release.

Journal of Endocrinology (1997) 152, 193–199
Growth hormone as an amplifier of insulin-like growth factor-I action: potentiated oestradiol accumulation
Authors: Xu Y P , Chedrese P J , Thacker P A
Published: December 2004

The interaction between GH and IGF-I in modulating oestradiol biosynthetic capacity was examined in cultured porcine granulosa cells. Granulosa cells (2 × 106 viable cells/culture) were initially cultured for 96 h (treatment period) in an androstenedione-free medium in the absence or presence of IGF-I (0 or 50 ng/ml), with or without GH at 100 ng/ml. At the conclusion of this period, culture media were discarded and the cells were washed twice with the androstenedione-free medium and reincubated for an additional 24 h (test interval) in an androstenedione (10−7 m)-supplemented medium for oestradiol accumulation. GH alone induced no stimulation (P>0·05) of basal oestradiol accumulation. In contrast, concurrent treatment with IGF-I produced a 4·3-fold increase (26 vs 112 ng/2 × 106 cells per 24 h, P<0·001) in oestradiol accumulation. GH amplified IGF-I-induced oestradiol production in a dose (minimal dose requirement of 0·3 ng/ml)- and time (minimal time requirement of 24–48 h)-dependent manner. Studies on the site(s) of action indicated that GH exerts its amplifying effects on IGF-I-induced oestradiol production both proximal and distal to cAMP generation. As the specificity study and the inhibitory study indicated, GH amplification of IGF-I-induced oestradiol production is a process involving gene transcription and/or translation and the synergism is not solely specific to IGF-I as IGF-II-induced oestradiol production was also amplified in the presence of GH.

Journal of Endocrinology (1997) 152, 201–209
Insulin and glucagon release of human islets in vitro: effects of chronic exposure to glucagon
Authors: Bertuzzi F , Berra C , Socci C , Davalli A M , Pozza G , Pontiroli A E
Published: December 2004

Hyperglucagonemia is commonly found in insulin-dependent as well as in non-insulin-dependent diabetes mellitus, and is likely to be caused by absolute or relative insulin deficiency. The aim of the present study was to evaluate whether a chronic glucagon exposure (1·0 μm for 4 h) modifies the insulin response to acute stimuli with glucagon (1·0 μm), arginine (10·0 mm) and glucose (16·7 mm), or the glucagon response to arginine and glucose, in human islets. Chronic exposure to glucagon did not affect the insulin response to glucose and arginine, but inhibited its response to glucagon (44·6 ± 9·3 vs 168·6 ± 52·3 pg/islet per 20 min, P<0·05); the latter effect was not observed when exposure to glucagon was discontinuous (2·0 μm glucagon alternated with control medium for 30 min periods). The chronic exposure to glucagon also reduced the glucagon response to arginine (−4·9 ± 5·7 vs 19·9 ± 7·9 pg/islet per 20 min, P<0·05) without affecting the inhibition of glucagon release exerted by glucose. These data indicate that chronic exposure to glucagon desensitizes pancreatic α and β cells in response to selected stimuli.

Journal of Endocrinology (1997) 152, 239–243
Distinct expression patterns and biological activities of two isoforms of the mouse orphan receptor TR2
Authors: Lee C-H , Chang L , Wei L-N
Published: December 2004

An alternatively spliced variant of a testis-specific nuclear orphan receptor TR2-11 was identified and designated as TR2-11-t. As a result of retaining intron 5 of this gene, TR2-11-t mRNA encoded a truncated receptor with the complete ligand-binding domain deleted. Protein expression of both isoforms was confirmed using a prokaryotic expression system. In the mouse, the expression of the two TR2 isoforms was elevated in the testis with distinct profiles beginning at puberty. TR2-11 expression increased at postnatal day 18, peaked between day 20 and day 24 and remained at high levels throughout adulthood, whereas TR2-11-t expression was elevated transiently at postnatal day 24. Among separated primary germ cells and established testicular cell lines, TR2-11 was expressed highly in meiotic and postmeiotic germ cells and weakly in a Leydig cell line and a germ cell line, but not expressed in a Sertoli cell line. In contrast, TR2-11-t was expressed at a much lower level in all the testicular cell types examined. In adult testes blocked at germ cell development by vitamin A depletion or hypophysectomy, TR2-11 expression was dramatically reduced whereas TR2-11-t was highly elevated. Based upon the RNA expression patterns of these isoforms, it was suggested that TR2-11 was specific to meiotic and postmeiotic germ cells whereas TR2-11-t was enriched in early germ cell populations such as premeiotic cells. The biological activities of TR2-11 and TR2-11-t on a direct repeat 5-type retinoic acid (RA) response element (RARE)-containing reporter gene was examined in Cos cells. TR2-11 repressed RA induction of this reporter whereas TR2-11-t enhanced RA induction of the same reporter, and the opposite biological effects of these isoforms were dose-dependent. Gel-shift experiments provided evidence for a direct interaction of TR2-11, but not TR2-11-t, with DNA fragments containing this RARE. Opposite roles of TR2-11 and TR2-11-t on RA induction of promoters containing this particular RARE are suggested.

Journal of Endocrinology (1997) 152, 245–255
Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells
Authors: Wang J F , Becks G P , Hill D J
Published: December 2004

In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44–46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44–46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44–46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5.

To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A–Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A–Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells.

Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 °C for 16 h. Both 44–46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells, and no IGFBP could be found in serum-free conditioned medium from agarose-covered FRTL-5 cells. This suggests that the 44–46 kDa IGFBP-3 in serum was degraded to yield a 31 kDa fragment, while any endogenous IGFBP-5 could not pass out of the agarose. The degradation of 44–46 kDa IGFBP-3 in the modified zymographic assay was inhibited by phenylmethylsulfonyl fluoride, EDTA, and aprotinin, but not by leupeptin. In summary, these results indicated that IGFBP-3 in calf serum added to culture medium could be degraded by FRTL-5 cells and that this may involve calcium-dependent serine proteases.

Journal of Endocrinology (1997) 152, 265–274
Effect of chronic food restriction on pulsatile luteinizing hormone secretion and hypothalamic neuropeptide Y gene expression in castrate male sheep
Authors: Adam C L , Findlay P A , Kyle C E , Young P , Mercer J G
Published: December 2004

Castrate male sheep (wethers, average liveweight 38 ± 0·6 kg) were given one of the following diets for 10 weeks followed by euthanasia (n=8/group): high-energy high-protein providing 1·5 times the energy required to maintain liveweight (maintenance) (group 1·5M), low-energy low-protein at 0·5 maintenance (0·5M), or low-energy high-protein at 0·5 maintenance (0·5M+P). 1·5M wethers gained 22% liveweight whereas 0·5M and 0·5M+P wethers lost 18 and 13% liveweight respectively. Relative to the 1·5M group, the 0·5M and 0·5M+P groups had similar plasma concentrations of glucose and cortisol throughout, but elevated non-esterified fatty acids (P<0·001) and reduced IGF-I and insulin (P<0·05, 0·01 or 0·001) from 1 week onwards. Each week blood samples were taken every 12 min for 4 h and plasma assayed for LH. Mean concentration over 4 h, LH pulse frequency and LH pulse amplitude showed no progressive change in 1·5M sheep. However, in both 0·5M and 0·5M+P groups mean LH increased (P<0·001 and P<0·01 respectively), pulse frequency decreased (P<0·01 and P<0·01) and pulse amplitude increased (P<0·001 and P<0·01) over the 10-week period. Anterior pituitary LH content was greater in 0·5M (P<0·01) and 0·5M+P (P<0·05) than in 1·5M sheep. Coronal sections (20 μm) of hypothalamic brain tissue were subjected to in situ hybridisation to determine gene expression for neuropeptide Y (NPY). NPY mRNA was concentrated in the arcuate nucleus and median eminence, with total amounts greater in both 0·5M (310%, P<0·001) and 0·5M+P (333%, P<0·01) groups than in 1·5M sheep (100%). These data reveal that chronic low dietary energy intake by long-term castrates, with high or low protein intake, reduces LH pulse frequency but increases the circulating levels of LH by virtue of an increase in pulse amplitude, and concomitantly increases hypothalamic NPY gene expression.

Journal of Endocrinology (1997) 152, 329–337
Role of the IGF-I receptor in mutagenesis and tumor promotion
Authors: Blakesley V A , Stannard B S , Kalebic T , Helman L J , LeRoith D
Published: December 2004

Introduction

The insulin-like growth factor-I (IGF-I) receptor is a transmembrane protein tyrosine kinase which mediates the biological effects of IGF-I (LeRoith et al. 1995, Nissley & Lopaczynski 1991) and most of the actions of insulinlike growth factor-II (IGF-II). Binding of IGF-I to the IGF-I receptor results in receptor autophosphorylation, phosphorylation of intracellular substrates and activation of specific signaling processes involved in growth regulation and differentiation (Lowe 1991).

The role of the IGF-I receptor in normal mammalian development is exemplified by the work of Efstratiadis and co–workers on mice that carried a null mutation for the IGF-I receptor gene (Baker et al. 1993, Liu et al. 1993c). The growth in utero of these mice is severely inhibited and the mice die immediately after birth. Fibroblasts derived from these mice cannot grow in IGF-I-supplemented serum-free medium whereas mouse fibroblasts with functional IGF–I receptors are capable of growth (Sell et al. 1994), suggesting
Vasopressin and oxytocin levels during normal pregnancy: effects of chronic dietary sodium restriction
Authors: van der Post J A M , van Buul B J A , Hart A A M , van Heerikhuize J J , Pesman G , Legros J J , Steegers E A P , Swaab D F , Boer K
Published: December 2004

Neurohypophysial hormones are thought to be involved in alterations in fluid balance during pregnancy and delivery. In the course of normal pregnancy intravascular volume is increased whereas sodium restriction is thought to reduce plasma volume and cardiac output. In the present study, we measured the effect of long-term severe sodium restriction on vasopressin (AVP) and oxytocin (OT) levels during normal pregnancy and after delivery.

Fifty-nine healthy nulliparous women were randomized either for a low sodium diet (20 mmol sodium daily) or for a normal diet from week 12 of pregnancy onwards. Circulating plasma levels and urinary excretion of AVP and OT, their neurophysins (Np-AVP and Np-OT) and AVP bound to platelets were determined at regular intervals during pregnancy and after delivery. After completion of the study, women on a sodium-restricted diet were compared with control women on a normal diet using repeated measurement ANOVA with adjustment for potentially confounding variables.

After randomization, a reduction in urinary sodium excretion of, on average, 40–82% was found. In general, no effect of sodium restriction could be demonstrated on the various parameters (0·53<P<0·98) with the exception of a significantly lower 24-h urinary AVP excretion by non-smokers with sodium restriction compared with non-smokers having a normal diet (P=0·018) For all parameters, clear changes were found in the course of pregnancy and puerperium (P<0·0001 to P<0·005). Platelet-bound AVP decreased and Np-OT increased during pregnancy. After birth, free plasma AVP, plateletbound AVP, OT, osmolality, sodium and potassium increased, while Np-AVP and Np-OT decreased.

Although elevated Np-AVP and Np-OT levels during pregnancy seem to indicate increased release of neurohypophysial hormones, pregnancy up to 36 weeks of gestation is accompanied by low circulating AVP and OT levels.

Long-term severe sodium restriction diminishes urinary AVP excretion in (non-smoking) pregnant women, without changing circulating levels of AVP and OT, despite the known reduction in circulating volume. The reduced circulating (platelet-bound) AVP levels during pregnancy, whether or not in combination with severe sodium restriction, support the absence of significant non-osmotic stimulation of AVP during pregnancy.

Journal of Endocrinology (1997) 152, 345–354
Expression of horse and donkey LH in COS-7 cells: evidence for low FSH activity in donkey LH compared with horse LH
Authors: Chopineau M , Martinat N , Troispoux C , Marichatou H , Combarnous Y , Stewart F , Guillou F
Published: December 2004

Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs in heterologous species, recombinant eLH (r.eLH/CG) and recombinant dkLH (r.dkLH/CG) were expressed, for the first time, in COS-7 cells. Their LH activities were assessed in a rat Leydig cell bioassay, and their FSH activities were estimated in a bioassay using Y1 cells stably expressing the human FSH receptor. Human CG (hCG) was expressed (r.hCG) and analysed in the same system. The results showed that, whereas r.dkLH/CG was about twice as active as r.eLH/CG in the LH bioassay, it was five times less active than r.eLH/CG in the FSH bioassay; r.hCG was about three times less active than r.eLH/CG in the LH bioassay but was completely inactive in the FSH bioassay. These results confirm that dkLH/CG possesses significant FSH activity in heterologous species that is not attributable to contamination with FSH.

Journal of Endocrinology (1997) 152, 371–377
Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes
Authors: Montiel M , Caro M C , Jiménez E
Published: December 2004

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5.

Journal of Endocrinology (1997) 152, 407–412
IGF-II, IGF-binding proteins and IGF receptors in pancreatic β-cell lines
Authors: Katz L E L , Bhala A , Camron E , Nunn S E , Hintz R L , Cohen P
Published: December 2004

The IGFs are mitogenic agents which are closely linked to regulatory processes in carbohydrate metabolism. Because limited information is available on the occurrence of the IGF system in the pancreatic β-cell milieu, we evaluated the presence of IGFs, IGF receptors, and IGF-binding proteins (IGFBPs) in the β-cell lines βTC3 and HIT T-15. Serum-free conditioned media (SFCM) from βTC3 cells contained IGF-II at concentrations greater than 100 ng/ml. High (15 kDa) and low (7·5 kDa) molecular weight IGF-II were detected both by column chromatography followed by RIA and by immunoblotting. GH (10–1000 ng/ml) conditioning of βTC3 cells stimulated IGF-II secretion in a dose-dependent manner. IGF-II mRNA was detected in βTC3 cells using Northern blots, and also showed a GH-dependent relationship. IGF-II peptide was detected in SFCM from HIT cells, albeit at lower concentrations. To evaluate the presence of IGF receptors in β-cell lines, affinity cross-linking studies were performed on βTC3 cells, demonstrating type I IGF receptors which bound iodinated IGF-II with high affinity, iodinated IGF-I with lesser affinity, and had minimal appreciable binding to iodinated insulin. Type II IGF receptors were not detected. SFCM from βTC3 and HIT cells was subjected to Western ligand blotting, which disclosed the presence of two major IGFBPs of 29 kDa and 24 kDa, characteristic of IGFBP-2 and IGFBP-4. The identity of the specific IGFBPs was confirmed by immunoprecipitation and Northern blotting. Varying the glucose concentration had no significant effect on the levels of IGFBPs, nor did preconditioning with GH, IGF-I, IGF-II, insulin, or glucagon. Levels of both IGFBPs in βTC3 cell-conditioned media increased in the presence of dexamethasone at concentrations of 10−6 m or greater. In summary, we present evidence that β-cell lines comprise an environment for GH and IGF action. We speculate that IGFs, their receptors and binding proteins function as a complex interactive system which regulates β-cell growth and function.

Journal of Endocrinology (1997) 152, 455–464
Protein kinase C dependent induction of pp125FAK in monocytes by colony stimulating factor-GM: evidence for a synergistic effect by the cytokine and 1,25 dihydroxyvitamin D3
Authors: Kanan Raed M , Rathod Hersha , Datta Harish K
Published: December 2004

The differentiation of monocytes into osteoclasts has been recently achieved in vitro in a suitable milieu containing morphogens that includes 1,25 dihydroxyvitamin D3, colony stimulating factors, interleukins and the presence of cells of osteoblastic lineage. However, the precise role of these factors in the osteoclastic differentiation process has not yet been examined. Since our previous studies have shown that osteoclasts express a much higher level of focal adhesion kinase (pp125FAK) than cells of macrophage/monocytic lineage, the present study was carried out to ascertain which morphogens are involved in increasing the expression of the kinase during the differentiation of monocytes to osteoclasts. We demonstrate that a marked increase in the expression of pp125FAK occurs only after prolonged exposure to hCSF-GM and combination of hCSF-GM and 1,25 (OH)2 D3. The hCSF-GM was found to be a more potent stimulator of pp125FAK induction than 1,25 (OH)2 D3; interestingly, the presence of both hCSF-GM and 1,25 (OH)2 D3 showed co-operative effect. Furthermore, the presence of a protein kinase C inhibitor, bisindolylmaleimide (GF 109203X), blocked hCSF-GM-mediated induction of focal adhesion kinase, implicating an important role for protein kinase C in the induction of pp125FAK.
IGF-I has a dual effect on insulin release from isolated, perifused adult rat islets of Langerhans
Authors: Hill D J , Sedran R J , Brenner S L , McDonald T J
Published: December 2004

We examined and compared the actions of IGF-I and -II on the release of insulin from isolated, intact rat islets of Langerhans within a perifusion system. Islets were isolated from adult male rats by collagenase digestion and Ficoll gradient separation, and were maintained in tissue culture for 48 h before perifusion. Following an equilibration period, islets were perifused with medium containing 2·7 mm glucose from 0 to 30 min, and 2·7 11·1 or 16·7 mm glucose from 30 to 90 min. All chambers then received medium with 2·7 mm glucose from 90 to 120 min. Various doses (6·7–53 nm) of IGF-I, des(1–3) IGF-I or IGF-II were given either as a pulse between 30 and 35 min, or continuously from 30 to 90 min. Insulin was measured in effluent medium by RIA. When 11·1 mm glucose was administered after 30 min an immediate increase in insulin release occurred, from a baseline of 1–3 pmol/fraction to approximately 7 pmol/fraction. The elevated rate of release was maintained until 90 min, and fell when the glucose concentration was lowered. Glucose at 16·7 mm was a less effective insulin secretogogue than was 11·1 mm. When islets received a pulse infusion of IGF-I (13·3 nm) at 30 min in the presence of 11·1 mm glucose, a statistically significant increase (P<0·005) in insulin release occurred, of approximately 10 pmol/fraction in excess of that seen with glucose alone. The IGF-I-stimulated insulin release was still higher than controls at 115 min. When the concentration of IGF-I was altered between 6·7 nm and 53 nm, maximum insulin release was achieved with 13·3 nm IGF I, both lower and higher concentrations being less effective. A significant inhibition of insulin release occurred with 53 nm IGF-I compared with glucose alone. IGF-II (13·3 nm) did not significantly increase insulin release, while 53 nm IGF-II significantly inhibited release of insulin relative to controls. Des(1–3) IGF-I (13·3 nm), which has a reduced binding affinity for IGF-binding proteins (IGFBPs), administered with 11·1 mm glucose caused an immediate increase in insulin release, which fell to control values within 30 min. Western ligand blot analysis identified four IGFBP species in perifused islets, of 46 kDa, 35 kDa, 28 kDa and 19 kDa respectively, of which the 28 kDa species was identified immunologically as IGFBP-1. When IGF-I was administered continuously from 30 to 90 min it inhibited glucose-stimulated insulin release at all concentrations used. The results suggest that under perifusion conditions, IGF-I can act both as a potent insulin secretogogue, augmenting the actions of glucose, and as an inhibitor of insulin release, depending on concentration and kinetics of administration.

Journal of Endocrinology (1997) 153, 15–25
A micromethod for quantitative determination of iodoamino acids in thyroglobulin
Authors: Baudry N , Mallet B , Lejeune P J , Vinet L , Franc J L
Published: December 2004

We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), diiodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water–acetonitrile–acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell–Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV).

This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0·5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine.

Journal of Endocrinology (1997) 153, 99–104
Analysis of a protein kinase C-α mutation in human pituitary tumours
Authors: Schiemann U , Assert R , Moskopp D , Gellner R , Hengst K , Gullotta F , Domschke W , Pfeiffer A
Published: December 2004

It is generally accepted that protein kinase C-α (PKC-α) is an important enzyme in the cellular regulation of growth and differentiation by phosphorylating proteins. Recent studies have described a point mutation of PKC-α (position 908 of the genetic sequence, codon GAC becoming GGC) in invasive human pituitary tumours which leads to an exchange of amino acids in the protein. We investigated 11 human pituitary tumours to evaluate the data obtained previously. cDNA was subcloned and up to ten individual clones were sequenced from each tumour, resulting in 85 clones analyzed in total. All of the pituitary adenomas showed a normal wild-type sequence of PKC-α DNA. Even if the tumour was 'invasive' (infiltration of the dura mater) no mutation at position 908 of the sequence was found. Moreover, using Western blot analyses we did not observe any differences in PKC-α protein expression in invasive as compared with non-invasive pituitary adenomas. Until now we have been unable to confirm the data of other investigators, suggesting that mutated PKC-α is an inconsistent feature of invasive pituitary tumours.

Journal of Endocrinology (1997) 153, 131–137
Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat
Authors: Aguilera G , Jessop D S , Harbuz M S , Kiss A , Lightman S L
Published: December 2004

The expression of corticotropin releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) and CRH receptor mRNA in the PVN and anterior pituitary was studied during development of adjuvant-induced arthritis in Piebald–Viral–Glaxo rats, using in situ hybridization techniques. As previously shown with i.p. hypertonic saline injection, basal and immobilization stress-stimulated CRH mRNA levels in the PVN were significantly lower than in controls 14 days after adjuvant injection. However, 7 days after injection, preceding the onset of inflammation, the increase of CRH mRNA following immobilization was significantly higher than in control rats. In contrast to other chronic stress paradigms, inflammation stress failed to induce type-1 CRH receptor (CRH-R1) mRNA in the PVN, either at 7 days, or at 14 days after adjuvant injection, when inflammation is present. The ability of acute immobilization to induce CRH-R1 mRNA in the PVN was not affected 14 days after adjuvant injection but parallel to the CRH peptide mRNA response it was markedly potentiated at 7 days. Pro-opiomelanocorpin (POMC) mRNA levels in the anterior pituitary increased significantly 14 days after adjuvant injection, and they were unaffected by 1 h immobilization. While CRH binding in the pituitary decreased significantly 14 days after adjuvant injection, CRH-Rl mRNA was unchanged. This study shows biphasic hypothalamic responses to acute stress during development of adjuvant-induced arthritis, with enhanced CRH peptide and CRH-Rl mRNAs responses at 7 days, preceding the onset of inflammation, and blunted CRH mRNA responses at 14 days at the height of the inflammatory response. The lack of CRH receptor expression in the PVN in this model of chronic inflammation stress associated to low hypothalamic CRH peptide levels supports the view that positive feedback regulation by CRH is necessary to maintain enhanced CRH expression during chronic stress.

Journal of Endocrinology (1997) 153, 185–191
Magnetic resonance imaging as a monitor of kidney enlargement and regression in experimental diabetes in rats
Authors: Christiansen T , Stødkilde-Jørgensen H , Flyvbjerg A
Published: December 2004

The aim of the present study was to use magnetic resonance imaging as a non-invasive measure of renal enlargement and regression in experimental diabetes in rats. Rats were randomized into three groups: (1) non-diabetic control animals, (2) diabetic animals, untreated for 30 days after streptozotozin treatment and (3) diabetic animals treated with insulin from day 21 to day 30 after streptozotocin administration. Magnetic resonance images were obtained from anaesthetized rats and kidney, cortex, medulla and pelvis volumes were calculated throughout the experiment until day 30. In untreated diabetic animals, a marked increase in kidney volume was seen 4 days after the induction of diabetes (P<0·05) and after 30 days this increase amounted to 73%, with similar increases in the cortex (63%) and medulla (69%). No change was seen in the pelvis volume. Insulin administration on day 21 to untreated diabetic rats abruptly reduced kidney volume by 21%, cortex volume by 27% and medulla volume by 18%, and after 4 days all three parameters were similar to those seen in non-diabetic controls. In conclusion, the present study has demonstrated the applicability of magnetic resonance imaging as a non-invasive measure of renal enlargement and regression in experimental diabetes in rats. In untreated hyperglycaemic diabetic rats, with manifest diabetic renal enlargement, acute regression in renal volume was observed concomitant with insulin-induced normoglycaemia.

Journal of Endocrinology (1997) 153, 193–198
Rapidly reversible binding of rabbit prolactin to the rabbit prolactin receptor accounts for the differences between homologous and heterologous binding
Authors: Petridou B , Cahoreau C , Djiane J
Published: December 2004

The binding of radioiodinated rabbit (rb) prolactin (PRL) to rabbit mammary membranes is low and its affinity constant, 0·02 nm−1, calculated from heterologous inhibition assays, is about 300 times lower than that of ovine (o) PRL. Although the differences between homologous and heterologous binding are well documented in different species, the reasons for such differences are still unknown.

Here we show that the low affinity of rbPRL for the native receptor does not affect its in vitro bioactivity compared with that of oPRL. We also show that rbPRL displays high specific binding to the baculovirus-expressed recombinant receptor and further establish that its lower affinity for binding to the homologous receptor is due to its faster and more complete dissociation compared with that of oPRL. Hormone binding affinity for full-length and carboxy-terminal truncated rbPRL receptor mutants expressed in mammalian or in baculovirus-infected cells was not affected by partial truncation of the cytoplasmic domain of the receptor, whereas the affinity for oPRL increased and that for rbPRL decreased upon truncation of both the cytoplasmic and membrane domains. The affinity of rbPRL for the native receptor is two orders of magnitude lower than that for the recombinant receptor. Affinity cross-linking and binding experiments showed that this difference in affinities is not related to selective cleavage of the native microsomal receptor during the binding reaction; however, this difference may be related to cell context-dependent differences in the oligomerization state of the receptor.

Thus, obviously, the cloned receptor is alone sufficient for binding to rbPRL without requiring any receptor-associated protein. The lower affinity for rbPRL binding to its homologous receptor in comparison with higher affinity binding of oPRL to the same receptor is attributable to differences in their dissociation kinetics and in the conformational requirements of the receptor–hormone interaction site for binding to the two hormones.

Journal of Endocrinology (1997) 153, 207–219
Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB
Authors: Evans L W , Muttukrishna S , Knight P G , Groome N P
Published: December 2004

Monoclonal antibodies, specific for the βa and βb subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0·19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentrations of activin-AB were between 1·3% and 2·67%. The between-plate coefficient of variation was 5·5%. Crossreactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting <0·2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-AB levels. As we have reported previously for activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an Mr of >700 000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an Mr of ∼25 000 consistent with the complete dissociation from binding proteins.

Activin-A was detected in relatively high concentrations in human FF (∼5 ng/ml), homogenized placental extracts (4·35–95·5 ng/g), sera from pregnant women (>4 ng/ml) and amniotic fluid (3–13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ml), normal cycle serum (100–200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml) and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (<0·19 ng/ml) in all of these samples with the exception of human FF (∼7 ng/ml).

In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Journal of Endocrinology (1997) 153, 221–230