bluuebunny/crossref_metadata_2025 huggingface dataset:
The hypothalamic-pituitary-gonadal axis: immune function and autoimmunity
Authors: Tanriverdi F , Silveira LF , MacColl GS , Bouloux PM
Published: December 2004

GnRH and sex steroids play an important role in immune system modulation and development. GnRH and the GnRH receptor are produced locally by immune cells, suggesting an autocrine role for GnRH. Experimental studies show a stimulatory action of exogenous GnRH on the immune response. The immune actions of GnRH in vivo are, however, less well established. Oestrogen and androgen receptors are expressed in primary lymphoid organs and peripheral immune cells. Experimental data have established that oestrogens enhance the humoral immune response and may have an activating role in autoimmune disorders. Testosterone enhances suppressor T cell activity. Although there are some clinical studies consistent with these findings, the impact of sex steroids in autoimmune disease pathogenesis and the risk or benefits of their usage in normal and autoimmune-disordered patients remain to be elucidated. There are neither experimental nor clinical data evaluating functional GnRH-sex steroid interactions within the human immune system, and there is a paucity of data relating to GnRH analogues, hormone replacement therapy and oral contraceptive and androgen action in autoimmune diseases. However, a growing body of experimental evidence suggests that an extra-pituitary GnRH immune mechanism plays a role in the programming of the immune system. The implications of these findings in understanding immune function are discussed.
Cloning of a proopiomelanocortin cDNA from the pituitary gland of the sea bass (Dicentrarchus labrax) and assessment of mRNA expression in different tissues by means of real-time PCR
Authors: Varsamos S , Wendelaar Bonga SE , Flik G , Quere R , Commes T
Published: December 2004

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.
The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion
Authors: Ball DK , Rachfal AW , Kemper SA , Brigstock DR
Published: December 2004

Connective tissue growth factor (CTGF) is a 349-residue mosaic protein that contains four structural modules implicated in protein-protein interactions. To address the functionality of residues 247-349 (containing module 4 alone), this region of CTGF was produced as a maltose binding protein (MBP) fusion protein in E. coli. After removal of MBP, recombinant CTGF commenced at Glu(247), was of M(r) 10 000, was immunoreactive with anti-CTGF[247-260], bound strongly to heparin, and promoted dose-dependent adhesion of fibroblasts, myofibroblasts, endothelial cells, and epithelial cells. An 8 kDa presumptive C-terminally truncated form of CTGF commencing at Glu(247) also promoted cell adhesion. CTGF-mediated cell adhesion was abolished by heparin or EDTA. These data demonstrate the presence of heparin-binding and cell-adhesion motifs within the C-terminal 103 residues of CTGF and show that CTGF-mediated cell adhesion is heparin-and divalent cation-dependent. Thus, CTGF isoforms comprising essentially module 4 are intrinsically functional in the absence of the other constituent modules of CTGF.
Fibroblast growth factor 7, secreted by breast fibroblasts, is an interleukin-1beta-induced paracrine growth factor for human breast cells
Authors: Palmieri C , Roberts-Clark D , Assadi-Sabet A , Coope RC , O'Hare M , Sunters A , Hanby A , Slade MJ , Gomm JJ , Lam EW , Coombes RC
Published: December 2004

Keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7) is known to be a potent growth factor for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related growth factor FGF10. We also determined the factors that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells. Using an FGF7-specific antibody which does not react with the FGF7 heparan sulphate proteoglycan (HSPG)-binding site, we showed epithelial and myoepithelial immunohistochemical staining in normal breast sections, and epithelial staining in breast carcinomas. Stromal staining was also detected in some lobular carcinomas as well as a subset of invasive ductal carcinomas. FGF10 and FGF receptor (FGFR)2 immunostaining showed a similar epithelial expression pattern, whereas no stromal staining was observed. We purified normal breast stromal, epithelial and myoepithelial cells and showed that FGF7 stimulated proliferation of both epithelial cell types, but not stromal fibroblasts. We also examined the effects of FGF7 on Matrigel-embedded organoids, containing both epithelial and myoepithelial cells, and showed FGF7 induced an increase in cellular proliferation. Furthermore, conditioned medium derived from stromal cells was shown to increase the proliferation of normal and neoplastic breast epithelial cells, which could be abolished by a neutralising antibody to FGF7. Finally, we showed that interleukin-1beta, but not oestradiol or other oestrogen receptor ligands, caused a dose-related FGF7 release. Further results also indicate that the epithelial localisation of FGF7 and FGF10 in breast tissue sections is likely to be due to their binding to their cognate receptor. In summary, our findings suggest that FGF7 is a paracrine growth factor in the breast. FGF7 is produced by the breast stromal fibroblasts and has profound proliferative and morphogenic roles on both epithelial and myoepithelial cells.
Exogenous leptin controls the development of the small intestine in neonatal piglets
Authors: Wolinski J , Biernat M , Guilloteau P , Westrom BR , Zabielski R
Published: December 2004

Leptin, a hormone produced and secreted by adipose tIssue, muscles and stomach, is involved in the regulation of adipose tIssue mass, food intake and body weight in neonatal animals. It is also produced in the mammary glands and secreted into the colostrum and milk. Since leptin receptors are widely distributed in the small intestine mucosa, the aim of the present study was to investigate the effect of exogenous leptin on the development of the small intestine in neonatal piglets. Male neonatal piglets were fed with sow's milk or artificial milk formula. Every 8 h the latter received either vehicle or leptin (2 or 10 microg/kg body weight). The animals were either killed after 6 days of treatment and the small intestine sampled for histology and brush border enzyme activities or were tested for marker molecule (Na-fluorescein and BSA) absorption in vivo. Feeding milk formula slowed the maturation of small intestinal mucosa compared with feeding sow's milk. However, after leptin treatment the length of the small intestine was increased, and intestinal villi length, but not crypt size, was reduced compared with controls. The mitotic index was increased and the percentage of vacuolated enterocytes was reduced in the entire small intestine. Enterocyte brush border protease and lactase activities were reduced in the jejunum. Na-fluorescein marker molecule absorption did not change but that of BSA was reduced 3.8-fold. In conclusion, exogenous leptin administered in physiological doses reversed the maturation of the small intestinal mucosa to the range found in sow-reared piglets.
Expression of islet neogenesis-associated protein in islets of normal hamsters
Authors: Flores LE , Garcia ME , Borelli MI , Del Zotto H , Alzugaray ME , Maiztegui B , Gagliardino JJ
Published: December 2004

The aim of the present study was to test the possible presence and expression of islet neogenesis-associated protein (INGAP) in islet cells of normal adult hamsters. Pancreata from normal male Syrian hamsters were removed to perform the following studies. (i) Western blot analysis using the cytosolic fraction from homogenates of isolated islets, exocrine tIssue and whole pancreas, and rabbit INGAP-specific antibody. (ii) Immunohistochemical identification of INGAP-positive cells in fixed sections of intact pancreata, fresh and 72 h cultured islets (isolated by collagenase digestion), and smears of exocrine pancreatic cells, using the same INGAP-specific antibody and streptavidin-biotin complex. (iii) RT-PCR using total RNA extracted from isolated islets and from exocrine tIssue as template, and a specific pair of primers. (iv) Control of the sequence of the PCR products. INGAP protein was identified by Western blot in the cytosolic fraction of homogenates from fresh isolated islets, exocrine cells and whole fresh pancreas. INGAP-immunopositive cells were observed in duct, exocrine and islet cells in either fixed intact or digested pancreatic tIssue. INGAP mRNA was identified in samples of total RNA from fresh and cultured isolated islets and from exocrine cells. Our data demonstrate that INGAP is present and expressed in islets and in exocrine pancreatic cells of normal hamsters. The ubiquitous localization of INGAP suggests its possible role in the physiological process of islet growth and its protective effect upon streptozotocin-induced diabetes.
Ovarian and hormonal response of female goats to active immunization against inhibin
Authors: Medan MS , Watanabe G , Sasaki K , Nagura Y , Sakaime H , Fujita M , Sharawy S , Taya K
Published: December 2004

This study was conducted to evaluate the effect of active immunization against inhibin on hormonal levels and the ovulation rate in goats. Ten adult Shiba goats (Capra hircus) in two groups were used in this study. The first group was injected with inhibin vaccine (immunized, n=5) and the second group was injected with Freund's adjuvant (control, n=5) followed by three booster injections at 4-week intervals. After the third booster injection, three consecutive periods of oestrus were induced using prostaglandin F(2alpha) at intervals of 11 days. Blood samples were collected at 2-6 h intervals and the ovaries were monitored using B-mode ultrasonography. All inhibin-immunized goats generated antibodies that bound (125)I-labelled bovine inhibin and their FSH concentrations were significantly higher than corresponding values in the control group. Also, inhibin-immunized goats had significantly higher preovulatory oestradiol-17beta (P<0.01) and higher concentrations of progesterone in the luteal phase (P<0.05). Immunization of goats against inhibin resulted in a significant (P<0.01) increase in ovulation rate (control: 1.7+/-0.3 vs immunized: 7.6+/-1.1). These results demonstrate that active immunization against inhibin enhances ovarian follicular development and ovulation rate by promoting an increase in pituitary FSH secretion. Therefore, immunization against inhibin may be a useful alternative to the conventional approach of superovulation in goats.
Localization of estrogen receptors-alpha and -beta and androgen receptor in the human growth plate at different pubertal stages
Authors: Nilsson O , Chrysis D , Pajulo O , Boman A , Holst M , Rubinstein J , Martin Ritzen E , Savendahl L
Published: December 2004

Sex steroids are required for a normal pubertal growth spurt and fusion of the human epiphyseal growth plate. However, the localization of sex steroid receptors in the human pubertal growth plate remains controversial. We have investigated the expression of estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR) in biopsies of proximal tibial growth plates obtained during epiphyseal surgery in 16 boys and eight girls. All pubertal stages were represented (Tanner stages 1-5). ERalpha, ERbeta and AR were visualized with immunohistochemistry and the number of receptor-positive cells was counted using an image analysis system. Percent receptor-positive chondrocytes were assessed in the resting, proliferative and hypertrophic zones and evaluated for sex differences and pubertal trends. Both ERalpha- and ERbeta-positive cells were detected at a greater frequency in the resting and proliferative zones than in the hypertrophic zone (64+/-2%, 64+/-2% compared with 38+/-3% for ERalpha, and 63+/-3%, 66+/-3% compared with 53+/-3% for ERbeta), whereas AR was more abundant in the resting (65+/-3%) and hypertrophic zones (58+/-3%) than in the proliferative zone (41+/-3%). No sex difference in the patterns of expression was detected. For ERalpha and AR, the percentage of receptor-positive cells was similar at all Tanner pubertal stages, whereas ERbeta showed a slight decrease in the proliferative zone during pubertal development (P<0.05). In summary, our findings suggest that ERalpha, ERbeta and AR are expressed in the human growth plate throughout pubertal development, with no difference between the sexes.
Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells
Authors: Stirland JA , Seymour ZC , Windeatt S , Norris AJ , Stanley P , Castro MG , Loudon AS , White MR ,   Davis
Published: December 2004

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.
Dual mode of cortisol action on GH/IGF-I/IGF binding proteins in the tilapia, Oreochromis mossambicus
Authors: Kajimura S , Hirano T , Visitacion N , Moriyama S , Aida K , Grau EG
Published: December 2004

Glucocorticoids are known to impede somatic growth in a wide range of vertebrates. In order to clarify the mechanisms through which they may act in an advanced teleost fish, we examined the effects of cortisol administration on the growth hormone (GH)/insulin-like growth factor-I (IGF-I)/IGF-binding protein (IGFBP) system in the tilapia (Oreochromis mossambicus). In a short-term experiment, fish were injected intraperitoneally with cortisol (2 or 10 microg/g), and killed at 2, 4, 8 and 24 h after the injection. In a longer-term experiment, fish were killed 24 and 48 h after cortisol injection (2, 10 and 50 microg/g). Cortisol at doses of 2 and 10 microg/g significantly increased IGFBPs of four different sizes (24, 28, 30, and 32 kDa) in the plasma within 2 h without altering plasma levels of IGF-I or GH. On the other hand, cortisol at doses of 10 and 50 microg/g significantly reduced plasma IGF-I levels after 24 and 48 h. IGF-I mRNA levels in the liver were also significantly reduced by cortisol at doses of 10 and 50 microg/g after 48 h, suggesting that a decrease in plasma IGF-I levels is mediated through the attenuation of IGF-I gene expression in the liver. In contrast, no significant change was observed in plasma or pituitary contents of GH at any time point examined, which would appear to indicate that cortisol reduces IGF sensitivity to GH (GH-resistance). These results clearly indicate that cortisol induces a rapid increase in plasma IGFBPs and a more delayed decrease in IGF-I production. The dual mode of cortisol action may contribute to the inhibitory influence of cortisol on somatic growth in teleosts.
Inhibition of TNF-alpha production by pentoxifylline does not prevent endotoxin-induced decrease in serum IGF-I
Authors: Colson A , Willems B , Thissen JP
Published: December 2004

Sepsis and endotoxin (LPS or lipopolysaccharide) injection induce a state of growth hormone (GH) resistance leading to decreased circulating insulin-like growth factor (IGF)-I. Because the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta inhibit the GH-stimulated IGF-I expression in vitro, it was tempting to speculate that these two cytokines might play an important role in the reduction of circulating IGF-I levels caused by LPS. Pentoxifylline, a methylxanthine usually used in the treatment of peripheral arterial circulatory disorders, has been reported to inhibit TNF-alpha synthesis. The goal of our study was to investigate whether inhibition of TNF-alpha production by pentoxifylline could prevent the decrease in IGF-I and the GH resistance caused by LPS injection. Because previous studies demonstrated that pentoxifylline can reduce muscle catabolism induced by sepsis, we also assessed whether pentoxifylline could exert its anticatabolic effect by preventing the decrease in circulating IGF-I. LPS injection in rats decreased serum IGF-I (-45% at 12 h; P<0.01 vs time 0) and its liver mRNA (-67% at 12 h; P<0.01 vs time 0) while it induced circulating TNF-alpha and IL-1beta and their hepatic expression (P<0.01). Pretreatment of LPS-treated animals by pentoxifylline abolished the LPS-induced rise in serum TNF-alpha (-98% at 90 min; P<0.001 vs LPS alone) and to a lesser extent in serum IL-1beta (-44% at 3 h; not significant vs LPS alone). Despite its dramatic inhibitory effect on TNF-alpha induction, however, pentoxifylline failed to suppress both the decrease in IGF-I and the GH resistance induced by LPS in rats. These results suggest that mediators other than TNF-alpha, in particular IL-1beta or IL-6, could contribute to the GH resistance induced by LPS. They also suggest that the anticatabolic effect of pentoxifylline is not due to prevention of the decline of circulating IGF-I.
Skeletal changes in type-2 diabetic Goto-Kakizaki rats
Authors: Ahmad T , Ohlsson C , Saaf M , Ostenson CG , Kreicbergs A
Published: December 2004

We characterized appendicular and axial bones in rats with type-2 diabetes in five female Goto-Kakizaki (GK) rats, a strain developed from the Wistar rat showing spontaneous type-2 diabetes, and five age- and sex-matched non-diabetic Wistar rats. The humerus, tibia, metatarsals and vertebral bodies were analysed by peripheral quantitative computerized tomography (pQCT). In diabetic rats, the height of the vertebral bodies and length of the humerus were decreased while the length of the metatarsals was increased. A decreased cross-sectional area was found in the vertebral end-plate region and the tibial metaphysis. Notably, the diaphysis in all long bones showed expansion of periosteal and endosteal circumference. In tibia this resulted in increased cortical thickness, whereas in humerus and metatarsal it was unchanged. Areal moment of inertia was increased in all diaphyses suggesting greater bending strength. The most conspicuous finding in diabetic rats pertained to trabecular osteopenia. Thus, trabecular bone mineral density was significantly reduced in all bones examined, by 33-53%. Our pQCT study of axial and appendicular bones suggests that the typical feature of diabetic osteopathy in the GK rat is loss of trabecular bone and expansion of the diaphysis. The loss of metaphyseal trabecular bone if also present in diabetic patients may prove to underlie the susceptibility to periarticular fracture and Charcot arthropathy. The findings suggest that the risk of fracture in diabetes varies according to the specific sub-regions of a bone. The approach described may prove to be useful in the early detection of osteopathy in diabetic patients who may be amenable to preventive treatment.
Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex
Authors: Suwa T , Chen M , Hawks CL , Hornsby PJ
Published: December 2004

The mechanisms underlying the differentiation of the adrenal cortex into zones are unclear. Microarray studies on RNA from microdissected zona reticularis (ZR) and zona fasciculata/zona glomerulosa (ZF/ZG) derived from adult human adrenal glands showed that a gene of the dickkopf family (DKK), DKK3, is differentially expressed in the zones. The Dickkopf proteins are morphogens involved in Wnt signalling. Northern blotting showed higher DKK3 transcript levels in ZF/ZG than ZR samples. In situ hybridization on adult human adrenal gland sections showed that DKK3 expression was much higher in the ZG than in the ZF or ZR. DKK3 expression was also higher in the medulla. We screened for expression of other members of the DKK family and the related Wingless-type mouse mammary tumor virus integration site gene family (WNT), frizzled (FZD), and dishevelled (DVL) gene families. Among dickkopf family members, only DKK3 was expressed at a detectable level in both human and mouse adrenocortical RNA samples. Consistent with previously published data on the effects of Wnt4 gene disruption in the mouse, we found only WNT4 expression within the WNT family in both human and mouse RNA. Northern blotting showed that WNT4 was expressed at a higher level in ZF/ZG cells than in ZR. The higher level of DKK3 and WNT4 expression in ZF/ZG cells was confirmed by real-time PCR. In the frizzled and dishevelled families we found FZD1, FZD2 and DVL3 transcripts in human adrenocortical RNA, and FZD2 and DVL3 in mouse adrenocortical RNA. These data show that a variety of genes of the Wnt signalling pathways are expressed in the adrenal cortex. The zonal distribution of DKK3 expression suggests that it could be involved in zonal differentiation or growth.
Thyroid hormone responsiveness in N-Tera-2 cells
Authors: Chan S , McCabe CJ , Visser TJ , Franklyn JA , Kilby MD
Published: December 2004

N-TERA-2 cl/D1 (NT2) cells, a human embryonal cell line with characteristics of central nervous system precursor cells, were utilised to study thyroid hormone action during early neuronal growth and differentiation. Undifferentiated NT2 cells expressed mRNAs encoding thyroid hormone receptors (TRs) alpha1, alpha2 and beta1, iodothyronine deiodinases types 2 (D2) and 3 (D3) (which act as the pre-receptor regulators), and the thyroid hormone-responsive genes myelin basic protein (MBP) and neuroendocrine specific protein A (NSP-A). When terminally differentiated into post-mitotic neurons (hNT), TRalpha1 and TRbeta1 mRNA expression was decreased by 74% (P=0.05) and 95% (P<0.0001) respectively, while NSP-A mRNA increased 7-fold (P<0.05). However, mRNAs encoding TRalpha2, D2, D3 and MBP did not alter significantly upon neuronal differentiation and neither did activities of D2 and D3. With increasing 3,5,3'-triiodothyronine (T(3)) concentrations, TRbeta1 mRNA expression in cultured NT2 cells increased 2-fold at 10 nM T(3) and 1.3-fold at 100 nM T(3) (P<0.05) compared with that in T(3)-free media but no change was seen with T(3) treatment of hNT cells. D3 mRNA expression in NT2 cells also increased 3-fold at 10 nM T(3) (P=0.01) and 2.4-fold at 100 nM T(3) (P<0.05) compared with control, but there was no change in D3 enzyme activity. In contrast there was a 20% reduction in D3 mRNA expression in hNT cells at 10 nM T(3) (P<0.05) compared with control, with accompanying reductions in D3 activity with increasing T(3) concentrations (P<0.05). There was no significant change in the expression of the TRalpha isoforms, D2, MBP and NSP-A with increasing T(3) concentrations in either NT2 or hNT cells. Undifferentiated NT2 and differentiated hNT cells show differing patterns of T(3)-responsiveness, suggesting that there are different regulatory factors operating within these cell types.
The role of apoptosis-inducing receptors of the tumor necrosis factor family in thyroid cancer
Authors: Mitsiades CS , Poulaki V , Mitsiades N
Published: December 2004

The tumor necrosis factor (TNF) family comprises several ligands, such as the prototype TNF-alpha, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), which trigger apoptosis in susceptible cells by activating respective cell-surface receptors. The study of these cell death pathways has attracted significant attention in several fields, including that of thyroid cancer, because they participate in immune system function, as an arm of cell-mediated cytotoxicity, and because recombinant ligands are available for pharmacological use. TNF-alpha is a pluripotent cytokine that induces both pro-apoptotic and anti-apoptotic effects on thyroid carcinoma cells. FasL triggers apoptosis in other tumor types, but thyroid carcinoma cells are resistant to this effect. On the other hand, TRAIL potently and selectively kills thyroid carcinoma cells. Consequently, TRAIL is the only member of the family with significant anticancer activity and an acceptable toxicity profile to be used as a novel therapy for thyroid cancer. The caspase inhibitor FLIP plays a significant role in negatively regulating receptor-induced apoptosis. Thelper 1-type cytokines, such as interferon-gamma, TNF-alpha and interleukin-1beta increase the sensitivity of both normal and neoplastic thyrocytes to FasL and TRAIL. On the other hand, IGF-I and other growth/survival factors produced in the local tumor microenvironment activate the phosphatidylinositol 3-kinase/Akt kinase pathway and exert an anti-apoptotic effect by upregulating several apoptosis inhibitors, including FLIP. Pharmacological modulation of apoptosis induced by FasL and TRAIL/Apo2L holds promise of therapeutic applications in human malignancies.
Parathyroid hormone (PTH) down-regulates PTH/PTH-related protein receptor gene expression in UMR-106 osteoblast-like cells via a 3',5'-cyclic adenosine monophosphate-dependent, protein kinase A-independent pathway
Authors: Kawane T , Mimura J , Yanagawa T , Fujii-Kuriyama Y , Horiuchi N
Published: December 2004

Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.
Expression and cellular localization of insulin-like growth factor-II protein and mRNA in Sparus aurata during development
Authors: Radaelli G , Patruno M , Maccatrozzo L , Funkenstein B
Published: December 2004

The spatial localization of IGF-II protein and mRNA was investigated during larval and postlarval developmental stages of the gilthead sea bream (Sparus aurata) by immunohistochemistry and in situ hybridization, using specific antisera and riboprobes. Steady-state levels of IGF-II mRNA in larvae were determined by Northern blot analysis and were found to be increased. Immunoreactivity towards IGF-II was found in larval skin, muscle, gills, gut, olfactory epithelium and kidney. After metamorphosis, the strongest immunoreactivity was found in red skeletal muscle. Positive reaction with IGF-II antibodies was also found in the olfactory epithelium and in the epithelia of pharynx, oesophagus, stomach and kidney. In the adult, the most intense signal was observed in the red and pink musculature and in heart musculature. Immunostaining was also found in saccus vasculosus, thymus, spleen and ovary. IGF-II mRNA was detected by in situ hybridization in the brain, olfactory epithelium, eye, pharynx, skeletal musculature and liver. The spatial distribution of IGF-II shown in this study is consistent with previous findings on the cellular localization of IGF type 1 receptor in the sea bream and supports a role for IGF-II during development and growth of sea bream. Furthermore, these results suggest that IGF-II acts in an autocrine/paracrine manner.
Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours
Authors: Vax VV , Bibi R , Diaz-Cano S , Gueorguiev M , Kola B , Borboli N , Bressac-de Paillerets B , Walker GJ ,   Dedov, Grossman AB , Korbonits M
Published: December 2004

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.
Glucocorticoid action and novel mechanisms of steroid resistance: role of glucocorticoid receptor-interacting proteins for glucocorticoid responsiveness
Authors: Wikstrom AC
Published: December 2004

Glucocorticoids are widely used to treat inflammatory and malignant diseases. However, many individuals show a lack of therapeutic response and unwanted side-effects. Various known and unknown parameters determine glucocorticoid responsiveness, among them glucocorticoid receptor (GR)-interacting proteins. Several of the proteins interacting with GR also participate in other signal transduction pathways such as the AP-1 pathway and the nuclear factor-kappaB pathway. We suggest that a closer study of GR-interacting proteins may shed new light on mechanisms determining glucocorticoid sensitivity. In this commentary, the general mechanisms of GR action will be addressed and a proteomic-based method to study GR-interacting proteins will be described in brief.
Glucocorticoid resistance in inflammatory bowel disease
Authors: Farrell RJ , Kelleher D
Published: December 2004

Glucocorticoids are potent inhibitors of T cell activation and proinflammatory cytokines and are highly effective treatment for active inflammatory bowel disease (IBD). However, failure to respond, acutely or chronically, to glucocorticoid therapy is a common indication for surgery in IBD, with as many as 50% of patients with Crohn's disease (CD) and approximately 20% of patients with ulcerative colitis (UC) requiring surgery in their lifetime as a result of poor response to glucocorticoids. Studies report that approximately one-third of patients with CD are steroid dependent and one-fifth are steroid resistant while approximately one-quarter of patients with UC are steroid dependent and one-sixth are steroid resistant. While the molecular basis of glucocorticoid resistance has been widely assessed in other inflammatory conditions, the pathophysiology of the glucocorticoid resistance in IBD is poorly understood. Research in IBD suggests that the phenomenon of glucocorticoid resistance is compartmentalised to T-lymphocytes and possibly other target inflammatory cells. This review focuses on three key molecular mechanisms of glucocorticoid resistance in IBD: (i) decreased cytoplasmic glucocorticoid concentration secondary to increased P-glycoprotein-mediated efflux of glucocorticoid from target cells due to overexpression of the multidrug resistance gene (MDR1); (ii) impaired glucocorticoid signaling because of dysfunction at the level of the glucocorticoid receptor; and (iii) constitutive epithelial activation of proinflammatory mediators, including nuclear factor kappa B, resulting in inhibition of glucocorticoid receptor transcriptional activity. In addition, the impact of disease heterogeneity on glucocorticoid responsiveness and recent advances in IBD pharmacogenetics are discussed.
Propylthiouracil-induced hypothyroidism is associated with increased tolerance of the isolated rat heart to ischaemia-reperfusion
Authors: Pantos C , Malliopoulou V , Mourouzis I , Sfakianoudis K , Tzeis S , Doumba P , Xinaris C , Cokkinos AD , Carageorgiou H , Varonos DD , Cokkinos DV
Published: December 2004

The present study investigated the response of the hypothyroid heart to ischaemia-reperfusion. Hypothyroidism was induced in Wistar rats by oral administration of propylthiouracil (0.05%) for 3 weeks (HYPO rats), while normal animals (NORM) served as controls. Isolated hearts from NORM and HYPO animals were perfused in Langendorff mode and subjected to zero-flow global ischaemia followed by reperfusion (I/R). Post-ischaemic recovery of left ventricular developed pressure was expressed as % of the initial value (LVDP%). Basal expression of protein kinase C epsilon (PKCepsilon) and PKCdelta and phosphorylation of p46 and p54 c-jun NH(2)-terminal kinases (JNKs) in response to I/R were assessed by Western blotting. LVDP% was found to be significantly higher in HYPO hearts than in NORM. At baseline, PKCepsilon expression was 1.4-fold more in HYPO than in NORM hearts, P<0.05, while PKCdelta was not changed. Furthermore, basal phospho-p54 and -p46 JNK levels were 2.2- and 2.6-fold more in HYPO than in NORM hearts, P<0.05. In response to I/R, in NORM hearts, phospho-p54 and -p46 JNK levels were 5.5- and 6.0-fold more as compared with the baseline values, P<0.05, while they were not significantly altered in HYPO hearts. HYPO hearts seem to display a phenotype of cardioprotection against ischaemia-reperfusion and this is associated with basal PKCepsilon overexpression and attenuated JNK activation after I/R.
Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis
Authors: Vanttinen T , Liu J , Kuulasmaa T , Kivinen P , Voutilainen R
Published: December 2004

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.
Thyroid hormone modulates ClC-2 chloride channel gene expression in rat renal proximal tubules
Authors: Santos Ornellas D , Grozovsky R , Goldenberg RC , Carvalho DP , Fong P , Guggino WB , Morales M
Published: December 2004

Thyroid hormones has its main role in controlling metabolism, but it can also modulate extracellular fluid Volume (ECFV) through its action on the expression and activity of Na(+) transporters. Otherwise, chloride is the main anion in the ECFV and the influence of thyroid hormones in the regulation of chloride transporters is not yet understood. In this work, we studied the effect of thyroid hormones in the expression of ClC-2, a cell Volume-, pH- and voltage-sensitive Cl(-) channel, in rat kidney. To analyze the modulation of ClC-2 gene expression by thyroid hormones, we used hypothyroid (Hypo) rats with or without thyroxine (T(4)) replacement and hyperthyroid (Hyper) rats as our experimental models. Total RNA was isolated and the expression of ClC-2 mRNA was evaluated by a ribonuclease protection assay, and/or semi-quantitative RT-PCR. Renal ClC-2 expression decreased in Hypo rats and increased in Hyper rats. In addition, semi-quantitative RT-PCR of different nephron segments showed that these changes were due exclusively to the modulation of ClC-2 mRNA expression by thyroid hormone in convoluted and straight proximal tubules. To investigate whether thyroid hormones action was direct or indirect, renal proximal tubule primary culture cells were prepared and subjected to different T(4) concentrations. ClC-2 mRNA expression was increased by T(4) in a dose-dependent fashion, as analyzed by RT-PCR. Western blotting demonstrated that ClC-2 protein expression followed the same profile of mRNA expression.
Endocrine responses to short-term feed deprivation in weanling pigs
Authors: Salfen BE , Carroll JA , Keisler DH
Published: December 2004

The study objective was to assess endocrine and tIssue responses to feed deprivation in weaned pigs. In experiment 1, eight crossbred castrated male pigs were either fed on a continual basis (CON; n=4) or were feed deprived for 24 h and then re-fed until 30 h (FD; n=4). Relative serum concentrations of ghrelin tended to be lower in FD pigs at 12 h (P=0.08) when compared with CON pigs, but was higher at 24 h and 30 h compared with 12 h (P<0.05). Serum IGF-I was lower in FD pigs from 12 to 30 h as compared with CON pigs (P<0.05) and increased following re-feeding (P<0.06). Experiment 2 consisted of 32 pigs that were either fed for 72 or 96 h (CON72 and CON96; n=16), feed deprived for 72 h (FD72; n=8), or FD72 and then re-fed from 72-96 h (FD72/RF24; n=8). Ghrelin in the FD72 and FD72/RF24 groups was lower at 12 h (P<0.03) compared with CON pigs, but then increased from 12 h to 36 h (P<0.01). Serum IGF-I and leptin decreased following feed deprivation (P<0.001) and remained low until re-feeding. Cortisol was elevated from 12 h to 72 h during feed deprivation (P<0.01) but was not different from CON96 pigs following refeeding (P>0.88). Expression of ghrelin mRNA tended to be lower in the FD72 pigs' stomachs, pituitary glands, and hypothalami (P=0.06, 0.07, and 0.08 respectively) compared with CON pigs. These results provide evidence that feed deprivation is accompanied by multiple changes in the endocrine and neuroendocrine axis which influences feed intake, somatotropic response, and hypothalamic-pituitary-adrenal axis hormone concentrations.
Frequent overexpression of cyclin D1 in sporadic pancreatic endocrine tumours
Authors: Guo SS , Wu X , Shimoide AT , Wong J , Moatamed F , Sawicki MP
Published: December 2004

Pancreatic endocrine tumours (PETs) occur sporadically or are inherited as part of the multiple endocrine neoplasia type-1 syndrome. Little is known about the molecular events leading to these tumours. Cyclin D1, a key regulator of the G1/S transition of the cell cycle, is overexpressed in a variety of human cancers as well as certain endocrine tumours. We hypothesized that similar to other endocrine tumours, cyclin D1 is overexpressed in human sporadic PETs. Cyclin D1 protein overexpression was found in 20 of 31 PETs (65%) when compared with normal pancreatic tIssue. Furthermore, Northern blot analysis suggests that cyclin D1 up-regulation occurs at the post-transcriptional level in some PETs. Because the key cell growth signalling pathways p42/p44/ERK (extracellular signal-regulated kinase), p38/MAPK (mitogen-activated protein kinase), and Akt/PKB (protein kinase B) can regulate cyclin D1 protein expression in other cell types, pancreatic endocrine tumours were analysed with phospho-specific antibodies against the active forms of these proteins to elucidate a tIssue-specific regulatory mechanism of cyclin D1 in PETs. We found frequent activation of the p38/MAPK and Akt pathways, but down-regulation of the ERK pathway, in cyclin D1 overexpressing PETs. This study demonstrates that cyclin D1 overexpression is associated with human sporadic PET tumorigenesis, and suggests that this up-regulation may occur at the post-transcriptional level. These findings will direct future studies of PETs towards cell cycle dysregulation and the identification of key growth factor pathways involved in the formation of these tumours.
Thyroid hormone modulation of brain in vivo tyrosine hydroxylase activity and kinetics in the female catfish Heteropneustes fossilis
Authors: Chaube R , Joy KP
Published: December 2004

In the female catfish Heteropneustes fossilis, administration of thyroxine (T(4))(,) 1 micro g/g body weight, i.p., in both gonadal resting and preparatory phases for 7, 14 and 21 days caused hyperthyroidism, as evidenced from a duration-dependent significant increase in serum triiodothyronine (T(3)), and of tyrosine hydroxylase (TH) activity in telencephalon, hypothalamus-pituitary and medulla oblongata (Newman-Keuls' test; P<0.05). Hypothyroidism induced by adding 0.03% thiourea to aquarium water holding the catfish for 7, 14 and 21 days decreased serum T(3) levels in a duration-dependent manner (Newman-Keuls' test; P<0.05) and inhibited TH activity in the brain regions. T(4) replacement in 21day thiourea-treated fish restored and even elevated significantly serum T(3) levels as well as brain TH activity in a duration-dependent manner. In general, the changes in enzyme activity were higher in the forebrain regions than medulla oblongata and in the resting phase than preparatory phase. Kinetic studies by Lineweaver-Burk plots showed that the stimulatory effect following T(4) administration and T(4) replacement on TH activity was due to increased affinity of the enzyme for its cofactor (6,7-dimethyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine), as evident from a significant decrease in apparent Michaelis-Menten constant (K(m)) and an increase in apparent velocity maximum (V(max)). The TH inhibition due to the thiourea treatment can be related to decreased affinity of the enzyme for its cofactor, as evident from a significant increase in apparent K(m) value and a significant decrease in V(max). These data clearly show that circulating levels of T(4)/T(3) modulate brain TH activity by altering the kinetic properties of the enzyme, which, in turn, influence catecholaminergic activity and dependent functions.