bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of insulin-like growth factor (IGF) and IGF binding protein biosynthesis by hypoxia in cultured vascular endothelial cells
Authors: Tucci M , Nygard K , Tanswell BV , Farber HW , Hill DJ , Han VK
Published: December 2004

Endothelial cells (EC) are hypoxia-tolerant and their capacity to proliferate in low oxygen tension is essential to maintain vascular endothelium integrity. The present study addresses whether hypoxia alters the expression of insulin-like growth factor (IGF) and IGF binding protein (IGFBP) genes in bovine aortic EC (BAEC) and bovine pulmonary artery EC (BPAEC). EC were cultured in normoxic (21%) conditions and exposed to 0% oxygen for 24, 48, or 72 h; some cells were reoxygenated by exposure to 21% oxygen for 24 or 48 h following hypoxia. IGF-I peptide and mRNA levels were very low in both cell types, and decreased further with exposure to hypoxia. Ligand blotting showed that both cell types synthesized 24 kDa (IGFBP-4), 30 kDa (IGFBP-5 and/or IGFBP-6), 43 kDa and 48 kDa IGFBPs (IGFBP-3 glycosylation variants). IGFBP-4 was the predominant IGFBP expressed by both cell types and did not change with exposure to hypoxia. Hypoxia caused a significant increase in IGFBP-3 secretion in BPAEC but not in BAEC. IGFBP-3 stable mRNA levels in BPAEC were increased correspondingly. IGFBP-5 was expressed only in BAEC and decreased with exposure to hypoxia. IGFBP-6 mRNA expression was low and increased in both cell types with exposure to hypoxia. These results demonstrate that EC IGFBP baseline expression as well as its expression in hypoxia vary in different vascular beds and suggest that the IGFBPs may be the dominant paracrine regulators of proliferation of EC as well as maintenance of endothelium integrity during hypoxia.
beta-Naphthoflavone abolishes interrenal sensitivity to ACTH stimulation in rainbow trout
Authors: Wilson JM , Vijayan MM , Kennedy CJ , Iwama GK , Moon TW
Published: December 2004

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.
Rab3B is essential for GnRH-induced gonadotrophin release from anterior pituitary cells
Authors: Tasaka K , Masumoto N , Mizuki J , Ikebuchi Y , Ohmichi M , Kurachi H , Miyake A , Murata Y
Published: December 2004

Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca2+ concentration ([Ca2+]). Rab3B, a member of the small GTP-binding protein Rab family, is known to be involved in Ca(2+)-regulated exocytosis in pituitary cells. However, it is not known whether Rab3B functions in the physiological process regulated by GnRH in gonadotrophs. In this study using antisense oligonucleotide against Rab3B (AS-Rab3B) we determined that Rab3B is involved in GnRH-induced gonadotrophin release. Rab3B immunopositive cells were reduced in 24% of pituitary cells by AS-Rab3B. This treatment did not affect the population of gonadotrophs or the intracellular contents of gonadotrophins. However, AS-Rab3B significantly inhibited the total amount of basal and GnRH-induced gonadotrophin released from pituitary cells. These results show that Rab3B is involved in basal and GnRH-induced gonadotrophins release but not the storage of gonadotrophins. Next, the changes in [Ca2+] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca2+] was not different in the two groups, but exocytosis was significantly inhibited in Rab3B-negative cells. These results suggest that Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs.
Granulins: the structure and function of an emerging family of growth factors
Authors: Bateman A , Bennett HP
Published: December 2004

The granulin/epithelin motif defines a family of structurally unique proteins, of great evolutionary antiquity, which have been implicated as regulators of cell growth. Recurrent in granulin research are the surprising parallels between the granulin and EGF systems. Both are cysteinerich peptides of approximately 6 kDa that can modify cell growth. They show similar, but not identical, biological activities, although granulin/epithelin peptides do not bind EGF receptors; the three-dimensional folds of granulin and EGF are partially superimposible; and the precursors for mammalian granulin/epithelins and EGF are both organized as multiple repeats of conserved cysteine modules. Given the dissimilarity between amino acid sequences of members of the granulin/epithelin family and EGF-related peptides, the parallelism between the two systems probably represents convergent evolution towards related solutions to common biological problems. The granulin/epithelin precursor gene is expressed throughout the body, but its expression is predominantly in epithelial and haematopoietic cells. There is a great deal of versatility in the means by which cells process and handle the granulin/epithelin precursor. In some instances, the precursor is secreted intact (Zhou et al. 1993), and in others it is stored in a vesicular organelle, such as the sperm acrosome (Baba et al. 1993a). It may be processed into small 6-kDa peptides, which, in the neutrophil, can also be stored in vesicles (Bateman et al. 1990, Couto et al. 1992). The 6-kDa peptide forms, the intact precursor, and related proteins such as TGFe, regulate the growth of epithelial and mesenchymal cells. Epithelial cells express putative receptors for granulin/epithelin peptides and TGFe (Culouscou et al. 1993, Parnell et al. 1995). Thus, although much remains to be clarified, granulin/epithelin polypeptides and related proteins are emerging as widely distributed potential autocrine and paracrine growth modulating factors for epithelial and mesenchymal cells.
The role of gonadectomy and testosterone replacement on thymic luteinizing hormone-releasing hormone production
Authors: Azad N , LaPaglia N , Agrawal L , Steiner J , Uddin S , Williams DW , Lawrence AM , Emanuele NV
Published: December 2004

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.
Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages
Authors: Lukyanenko YO , Carpenter AM , Brigham DE , Stocco DM , Hutson JC
Published: December 2004

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.
Thyroid hormone stimulates progesterone release from human luteal cells by generating a proteinaceous factor
Authors: Datta M , Roy P , Banerjee J , Bhattacharya S
Published: December 2004

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.
Role of hyperinsulinemia on hepatic insulin receptor concentration and autophosphorylation in the presence of high growth hormone levels in transgenic mice overexpressing growth hormone gene
Authors: Dominici FP , Balbis A , Bartke A , Turyn D
Published: December 2004

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.
Differential expression of two pro-opiomelanocortin mRNAs during temperature stress in common carp (Cyprinus carpio L.)
Authors: Arends RJ , van der Gaag R , Martens GJ , Wendelaar Bonga SE , Flik G
Published: December 2004

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.
The relatively high frequency of p53 gene mutations in multiple and malignant phaeochromocytomas
Authors: Yoshimoto T , Naruse M , Zeng Z , Nishikawa T , Kasajima T , Toma H , Yamamori S , Matsumoto H , Tanabe A , Naruse K , Demura H
Published: December 2004

To explore the clinical significance of p53 in the pathogenesis of adrenal neoplasms, we investigated the incidence of p53 gene mutations in functioning human adrenal tumours using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen p53 exons 4 to 9. We examined 29 adrenocortical adenomas (primary aldosteronism, n=17; Cushing's syndrome, n=12, all benign), and 33 phaeochromocytomas (benign solitary, n=18; benign multiple, n=5; malignant, n=10) in Japanese and Chinese patients. PCR-SSCP did not show any abnormal band-shifts in any of the adrenocortical adenoma and benign solitary phaeochromocytoma tissues. In contrast, six phaeochromocytoma tissues (two cases benign multiple, four cases malignant) showed PCR-SSCP band-shifts. Subsequent DNA sequencing analysis of the shifted bands revealed six cases with nine mutations or intronic sequence alterations: three cases contained sequence alterations within intronic regions, three cases with silent mutation (sequence alteration in codon without amino acid alteration), and three cases contained missense mutations (one case each in exons 5, 6 and 9). Immunohistochemical staining demonstrated that two of three cases with missense mutations and one case with an intronic sequence alteration over-expressed p53 protein in tumour cell nuclei. We observed no association between p53 gene mutation and p21/WAF1/Cip-1 expression. The relatively high incidence of p53 gene mutations or intronic sequence alteration in multiple and malignant phaeochromocytomas, but not in benign solitary cases, suggests that p53 mutation could play some role in the pathogenesis of multiple and/or malignant phaeochromocytomas.
Interleukin-1alpha regulates G1 cell cycle progression and arrest in thyroid carcinoma cell lines NIM1 and NPA
Authors: Zeki K , Morimoto I , Arao T , Eto S , Yamashita U
Published: December 2004

This study provides the first report that the same cytokine (interleukin-1 (IL-1)) can induce opposite effects on cyclin-dependent kinases (Cdks) and Cdk inhibitors (Cdkis) in the G1 phase even in the same type of cancer cells (papillary thyroid carcinoma cells). Cell cycle analysis revealed an increase in NIM1 cells and a decrease in NPA cells in the S and G2+M phases after treatment with IL-1alpha. The addition of IL-1alpha to NIM1 cells reduced the expression of p16 and p21 protein and induced the expression of Cdk2 and Cdk4 protein, which leads to the phosphorylation of retinoblastoma protein. The addition of IL-1alpha to NPA cells induced the expression of p27 protein and reduced the expression of Cdk2 protein, which leads to induction of p107 protein expression. It is of interest that p21 protein expression was not observed in NPA cells. These results suggest that several Cdks and Cdkis play a regulatory role in the G1 cell cycle progression and arrest induced by IL-1alpha in thyroid carcinoma cell lines.
Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG
Authors: Kovalevskaya G , Birken S , Kakuma T , O'Connor JF
Published: December 2004

Human chorionic gonadotropin (hCG) exhibits molecular heterogeneity in both its protein and carbohydrate moieties. This communication describes changes in hCG isoforms detected directly in clinical samples. These isoforms, quantified in blood or urine specimens, show a progression of change throughout normal pregnancy. Early pregnancy produces a type of hCG that resembles, in terms of immunoreactivity, a major form of hCG excreted in choriocarcinoma. The isoforms predominate for the first 5-6 weeks of gestation and then diminish, being replaced with the hCG isoforms which predominate throughout the remainder of pregnancy. The alteration in hCG isoform content occurs in both blood and urine. The progression of isoforms is best delineated by calculating the change in the ratio of the two forms, as many hCG assays either do not detect or fail to discriminate among these isoforms. An analogous pattern of hCG isoforms was observed in patients with in vitro fertilization pregnancies. hCG isolated from the pituitary displayed binding characteristics similar to those of the hCG derived from normal pregnancy urine. The early pregnancy hCG isoforms appear to have a differential expression in normal pregnancy as opposed to pregnancies which will not carry to term, suggesting that a determination of the relative balance of hCG isoforms may have diagnostic application in predicting pregnancy outcome.
Experimental cryptorchidism induces a change in the pattern of expression of LH receptor mRNA in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate
Authors: Tena-Sempere M , Kero J , Rannikko A , Huhtaniemi I
Published: December 2004

In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression.
Insulin-like growth factors prevent cytokine-mediated cell death in isolated islets of Langerhans from pre-diabetic non-obese diabetic mice
Authors: Hill DJ , Petrik J , Arany E , McDonald TJ , Delovitch TL
Published: December 2004

Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) contribute to the initial stages of the autoimmune destruction of pancreatic beta cells. IL-1beta is released by activated macrophages resident within islets, and its cytotoxic actions include a stimulation of nitric oxide (NO) production and the initiation of apoptosis. Insulin-like growth factors (IGFs)-I and -II prevent apoptosis in non-islet tissues. This study investigated whether IGFs are cytoprotective for isolated islets of Langerhans from non-obese diabetic mice (NOD) mice exposed to cytokines. Pancreatic islets isolated from 5-6-week-old, pre-diabetic female NOD mice were cultured for 48 h before exposure to IL-1beta (1 ng/ml), TNF-alpha (5 ng/ml), IFN-gamma (5 ng/ml) or IGF-I or -II (100 ng/ml) for a further 48 h. The incidence of islet cell apoptosis was increased in the presence of each cytokine, but this was significantly reversed in the presence of IGF-I or -II (IL-1beta control 3.5+/-1.6%, IL-1beta 1 ng/ml 27.1+/-5.8%, IL-1beta+IGF-I 100 ng/ml 4.4+/-2.3%, P<0.05). The majority of apoptotic cells demonstrated immunoreactive glucose transporter 2 (GLUT-2), suggesting that they were beta cells. Islet cell viability was also assessed by trypan blue exclusion. Results suggested that apoptosis was the predominant cause of cell death following exposure to each of the cytokines. Co-incubation with either IGF-I or -II was protective against the cytotoxic effects of IL-1beta and TNF-alpha, but less so against the effect of IFN-gamma. Exposure to cytokines also reduced insulin release, and this was not reversed by incubation with IGFs. Immunohistochemistry showed that IGF-I was present in vivo in islets from pre-diabetic NOD mice which did not demonstrate insulitis, but not in islets with extensive immune infiltration. Similar results were seen for IGF-binding proteins (IGFBPs). These results suggest that IGFs protect pre-diabetic NOD mouse islets from the cytotoxic actions of IL-1beta, TNF-alpha and IFN-gamma by mechanisms which include a reduction in apoptosis.
Cell-specific localization of G protein alpha-subunits in the islets of Langerhans
Authors: Skoglund G , Basmaciogullari A , Rouot B , Marie JC , Rosselin G
Published: December 2004

G protein alpha-subunits are involved in the transduction of receptor-mediated regulation of insulin and glucagon secretions. To get further insight into the status of G proteins in alpha- and beta-cells of the Langerhans islets, we have used immunohistochemistry to study the distribution of alpha-subunits in pancreas sections from the rat. Our results show that only insulin-immunoreactive beta-cells display immunoreactivity for selective antibodies directed against the different members of the Galphas and Galpha12-families (alphas, alphaolf, and alpha12, alpha13 respectively). Immunoreactivities for antibodies directed against members of the Galphaq- and Galphai-families showed a more diverse localization: alpha11 and alphao2 were only detected in glucagon-immunoreactive alpha-cells, whereas alphai1 was detected in all beta-cells but only in a few alpha-cells. Even though beta-cells showed immunoreactivities for alphao-non-isoform-selective antibodies, we could not identify the isoform(s) present using selective alphao1 and alphao2 antibodies. Other members of the Galphai- and Galphaq-families (alphai3, alphat2, alphaz and alphaq) were detected in both alpha- and beta-cells. In conclusion, our findings demonstrate a clear difference in the localization of G protein alpha-subunits between alpha- and beta-cells, suggesting the involvement of specific receptor transduction pathways for the neuronal/hormonal regulation of alpha- and beta-cell functions.
A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript
Authors: Millar R , Conklin D , Lofton-Day C , Hutchinson E , Troskie B , Illing N , Sealfon SC , Hapgood J
Published: December 2004

Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.
Enteral IGF-I enhances fetal growth and gastrointestinal development in oesophageal ligated fetal sheep
Authors: Kimble RM , Breier BH , Gluckman PD , Harding JE
Published: December 2004

Infants with upper gut atresia often have impaired intrauterine growth and gut function. IGF-I is important in fetal growth and is contained in amniotic fluid. We therefore wanted to test the hypothesis that IGF-I infused into fetal gut would reverse the effects of an upper gut obstruction on gut structure and growth in fetal sheep. At 90 days gestation fetuses (n=6 per group) underwent oesophageal ligation, followed by continuous infusion of IGF-I (1-8 microgram/day) or saline into the gut beyond the ligation until 137 days. Controls underwent sham ligation only. Oesophageal ligation tended to reduce fetal body and organ weights. IGF-I treatment prevented this reduction and increased body length and spleen weight above those of controls. The decrease in bowel wall thickness induced by oesophageal ligation was also prevented by IGF-I treatment. Amniotic fluid IGF-I concentrations did not change over gestation and were higher in the IGF-I treated group. No change in fetal plasma IGF-I concentrations were detectable. We conclude that enterally administered IGF-I may enhance fetal growth and gut development in utero and that IGF-I in amniotic fluid may play a physiological role in gut development in the fetus.
Calciotrophic hormones during experimental hypocalcaemia and hypercalcaemia in spontaneously diabetic rats
Authors: Verhaeghe J , van Bree R , van Herck E , Jans I , Zaman Z , Bouillon R
Published: December 2004

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) concentrations have been found to be decreased in diabetic humans and rats. To investigate further the regulation of plasma Ca in diabetes, first we measured Ca(2+), P, Mg, parathyroid hormone(1-34) (PTH), and total and free 1,25(OH)(2)D(3) in male spontaneously diabetic rats 7 and 28 days after the onset of glycosuria. Secondly, we studied changes in the levels of PTH and 1,25(OH)(2)D(3) in response to hypocalcaemia induced by an i.v. infusion of EGTA (2.5%, wt/vol.) for 24 h, and changes in the levels of 1,25(OH)(2)D(3) in response to an i.v. infusion of rat PTH (10 microgram over 24 h) without or with concomitant EGTA infusion (producing hypercalcaemia or normo/hypocalcaemia respectively), in diabetic and control rats. Ca(2+), P, Mg and PTH concentrations remained within the control ranges after 7 and 28 days of glycosuria; 1,25(OH)(2)D(3) concentrations were decreased after 7, but not after 28, days of glycosuria. PTH concentrations showed a similar rise during EGTA-induced hypocalcaemia in control and diabetic rats compared with saline-infused rats, whereas 1,25(OH)(2)D(3) concentrations were unchanged in both groups. Total and free 1,25(OH)(2)D(3) levels were comparably (about 3-fold) increased during PTH, but not during combined PTH and EGTA infusion in control and diabetic rats. Total 1, 25(OH)(2)D(3) concentrations were lower in the diabetic groups infused with saline or PTH than in their respective controls, and there was a similar trend in the PTH+EGTA-infused group; free 1, 25(OH)(2)D(3) levels, however, were normal or increased in the diabetic groups, confirming our previous data. The novel finding of this study is that, despite severe insulin deficiency and altered 1, 25(OH)(2)D(3) levels, the in vivo response of PTH levels to hypocalcaemia and the in vivo response of 1,25(OH)(2)D(3) levels to PTH in diabetic rats are comparable with those found in nondiabetic rats.
Secretion of tissue inhibitors of matrix metalloproteinases by human fetal membranes, decidua and placenta at parturition
Authors: Riley SC , Leask R , Denison FC , Wisely K , Calder AA , Howe DC
Published: December 2004

At parturition, breakdown of extracellular matrix in the fetal membranes may play a part in the rupture of the membranes and in the aetiology of premature rupture, in addition to having a regulatory role in the cell-cell interactions and signalling at the feto-maternal interface to stimulate myometrial contractility. The matrix metalloproteinases (MMPs) are important enzymes for the breakdown of extracellular matrix and their activity is regulated by a family of endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs). At parturition, alteration in the balance between MMPs and TIMPs may mediate this extracellular matrix breakdown during rupture of fetal membranes. The aims of this study were to determine if the intrauterine secretion of TIMPs changes at labour, and to characterise their cellular sources. A broad range of TIMP activities (27-30 kDa, 24 kDa and 21 kDa) were detected by reverse zymography in term amniotic fluid. There was a significant (P<0.05) decrease in the amount of TIMPs in amniotic fluid and their release with the onset of labour. The TIMPs were characterised by immunoblot as TIMPs-1, -2, -3 and -4. High levels of TIMPs were secreted by explants of chorio-decidua, decidua parietalis and placenta, with less being released by amnion. Immunolocalisation studies revealed a specific distribution pattern for each of the TIMP isoforms. Trophoblast cells of chorion laeve, decidua parietalis and placental syncytiotrophoblast demonstrated specific immunoreactivity for all four isoforms. TIMPs were also found bound to selective regions of extracellular matrix. The decrease in TIMPs during labour may permit increased breakdown of extracellular matrix in the fetal membranes and decidua at parturition, thus altering cell signalling at the feto-maternal interface and facilitating membrane rupture.
Post-ovulatory secretion of pituitary gonadotropins GtH I and GtH II in the rainbow trout (Oncorhynchus mykiss): regulation by steroids and possible role of non-steroidal gonadal factors
Authors: Chyb J , Mikolajczyk T , Breton B
Published: December 2004

In order to determine the factors of ovarian origin which can modulate the postovulatory secretion of the FSH-like gonadotropin (GtH I) and the LH-like gonadotropin (GtH II), freshly ovulated female rainbow trout were divided into two groups. In the first group the fish were stripped in order to eliminate the eggs and ovarian fluid from the body cavity, while in the second group the eggs were kept in the body cavity. Subsequently, fish from both groups were implanted with testosterone (10 mg/kg), 17beta-estradiol (10 mg/kg) or 17,20beta-ddihydroxy-4-regnen-3-one (17,20betaP) (1 mg/kg) or injected every 2 days with desteroidized ovarian fluid (1.5 ml/kg). The secretion of GtH I dramatically increased in stripped fish, reaching its maximum levels 2 weeks after ovulation. The preservation of eggs in the body cavity led to the suppression of this increase. The profiles of GtH II secretion were opposite to those encountered for GtH I because the increase of GtH II was observed only in unstripped fish. The administration of steroids showed that testosterone is able to inhibit GtH I release and stimulate that of GtH II in stripped fish, having no effect on the release of these gonadotropins in non-stripped animals. 17beta-Estradiol failed to modify GtH I secretion, however it decreased the release of GtH II in fish containing retained eggs in the body cavity. 17,20betaP had a delayed stimulating influence on GtH I release in unstripped fish. Finally, multiple injections of desteroidized ovarian fluid into stripped fish led to a significant decrease of GtH I release and to an increase of GtH II secretion. This study demonstrates that factors, which are present in ovarian fluid, modulate the post-ovulatory secretion of both gonadotropins--their net action is negative on GtH I and positive on GtH II. Among the steroids, testosterone is of major importance, being able to inhibit GtH I release and to stimulate that of GtH II. We also show that non-steroidal factors present in the ovarian fluid can influence the release of both gonadotropins, which indirectly supports the previous findings about the existence of inhibin/activin-like factors in fish.
Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats
Authors: de Dios I , Garcia-Montero AC , Orfao A , Manso MA
Published: December 2004

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.
GnRH and oxytocin have nonidentical effects on the cellular LH response by gonadotrophs at pro-oestrus
Authors: Evans JJ , Youssef AH , Abbas MM , Schwartz J
Published: December 2004

For full fertility in the female ovulation is necessary, which is dependent on the production of a surge of LH. An understanding of the processes which result in the high levels of LH requires delineation of the activities of individual component cells. In this study the responses of gonadotrophs to two signalling hypothalamic peptides, GnRH and oxytocin, were investigated. A cell immunoblot method was used to identify and distinguish between cells which secrete LH and those which contain LH but do not secrete the glycohormone. Rats were killed on the morning of pro-oestrus, the pituitary collected and the cells dispersed onto a protein-binding membrane for study. Cells were then incubated with GnRH and oxytocin, after which the membranes including the attached cells were stained by immunocytochemistry for LH. GnRH increased the total number of immunopositive cells which were present in a concentration-dependent manner. The most prominent change after 2 h incubation was in the number of secreting cells, whereas after 4 h there was also a marked increase in numbers of nonsecreting cells. Oxytocin also increased the total number of immunopositive cells in a concentration-responsive manner, however the profile of action of oxytocin was different from that observed for GnRH. Oxytocin had a relatively greater effect on numbers of immunopositive nonsecreting cells. Thus, the results reveal the potential for gonadotrophs to be flexibly and appropriately modulated by selected hypothalamic peptides. When cells were preincubated with oxytocin prior to GnRH there was not an additive increase in the numbers of immunopositive cells, suggesting that the two agonists act, in a nonidentical manner, on similar cells. The increase in the total number of immunopositive cells implies that there was a production of LH or post-translational processing, induced by exposure to GnRH or oxytocin. The results confirmed the heterogeneity of gonadotrophs and the existence of functionally distinguishable subpopulations, and revealed a difference between the effects of GnRH and oxytocin on expression and secretion of LH.
Estrogen mediates the sex difference in post-burn immunosuppression
Authors: Gregory MS , Duffner LA , Faunce DE , Kovacs EJ
Published: December 2004

Previous studies in our laboratory have demonstrated that cell-mediated immune function was suppressed in female, but not male, mice at 10 days after burn injury and was mediated, in part, by increased production of interleukin-6 (IL-6). Because 17beta-estradiol (E(2)) influences immune function after trauma and the hormone is known to regulate IL-6 production, the effect of E(2) on immune function after thermal injury was examined. Increased circulating concentrations of E(2) corresponded with suppressed delayed-type hypersensitivity (DTH) and splenocyte-proliferative responses, and increased circulating concentrations of IL-6 in female mice after burn. Ovariectomy restored the suppressed DTH response and decreased IL-6 concentrations, and administration of exogenous E(2) to both ovariectomized females and intact male mice resulted in a suppressed DTH response. In addition, in vitro treatment with E(2) suppressed splenocyte proliferation in a macrophage-dependent manner and enhanced macrophage production of IL-6. These results strongly suggest that the sex difference in cell-mediated immunity 10 days after burn injury is mediated by altered concentrations of E(2), which in turn modulate key macrophage-derived immunoregulatory cytokines.
Stimulation of endogenous GH and interleukin-6 receptors selectively activates different Jaks and Stats, with a Stat5 specific synergistic effect of dexamethasone
Authors: von Laue S , Finidori J , Maamra M , Shen XY , Justice S , Dobson PR , Ross RJ
Published: December 2004

The interaction of GH, interleukin (IL)-6 and glucocorticoids is likely to be important in regulating the GH-insulin-like growth factor (IGF)-I axis. The signalling cascades activated by GH and IL-6 appear to be very similar, as demonstrated by studies using overexpression of the receptor and other components of the Jak-Stat and mitogen-activated protein (MAP) kinase pathways. Here we show that the human embryonic kidney cell line 293 (HEK293) expresses GH and IL-6 receptors endogenously. To determine which specific pathways might be activated by the two cytokines, at physiological levels of all components, we studied GH and IL-6 mediated signal transduction both under basal conditions and in the presence of overexpressed receptors and Stat proteins. Our results suggest a receptor specificity of Jak2 for GH receptors, and Jak1 for IL-6 receptors. Stat activation in response to GH and IL-6 was determined by reporter gene induction. Both GH and IL-6 were able to induce the reporter gene containing the Stat5 responsive element (LHRE) but the IL-6 response appeared to be mediated mainly through Stat3 activation. In contrast, the reporter gene containing the Stat3 responsive element (SIE) was IL-6 specific. The levels of gene induction by GH and IL-6 were not altered by the co-stimulation with GH and IL-6, suggesting that there is little cross-talk at the Jak-Stat activation level between the two cytokines. Neither GH nor IL-6 activated the MAP-kinase responsive serum response element (SRE), unless GH receptors or gp130 were overexpressed. Transfection of Stat3 or Stat5 expression vectors enhanced the response to GH and IL-6. Stimulation with dexamethasone synergistically enhanced GH activation of the LHRE reporter gene but had no effect on the IL-6 activation of the same reporter or on the SIE reporter gene. Thus, our studies suggest that while each cytokine, GH and IL-6, may activate various members of the Jak-Stat pathway in overexpression studies, specific activation of Stat3 by IL-6 and of Jak2 and Stat5 by GH can be observed in HEK293 cells and that in this system the synergistic effect of dexamethasone appears specific for Stat5.
Placental lactogen-I gene activation in differentiating trophoblast cells: extrinsic and intrinsic regulation involving mitogen-activated protein kinase signaling pathways
Authors: Peters TJ , Chapman BM , Wolfe MW , Soares MJ
Published: December 2004

Trophoblast giant cells are one of the primary endocrine cell types of the rodent placenta. Placental lactogen-I (PL-I) is the initial prolactin (PRL) family member expressed as trophoblast giant cells differentiate. In this report, we use the Rcho-1 trophoblast cell line as a model for studying the regulation of PL-I gene expression during trophoblast giant cell differentiation. Evidence is provided for trophoblast cell expression of epidermal growth factor receptor (EGFR), ErbB2, fibroblast growth factor receptor 1 (FGFR1), transforming growth factor-alpha, and heparin-binding EGF. EGF and FGF-2 stimulated PL-I mRNA and protein accumulation and PL-I promoter activity in a concentration-dependent manner. These latter growth factor actions on PL-I promoter activities were specifically inhibited by cotransfection with dominant negative constructs for EGFR and FGFRs respectively. Utilization of the mitogen-activated protein kinase (MAPK) pathway by EGF and FGF-2 in trophoblast cells was demonstrated by growth factor stimulation of a Gal4 DNA binding/Elk1 transactivational domain fusion construct, and more specifically by activation of extracellular signal regulated kinase and p38 MAPK. PL-I gene activation was also sensitive to disruption of MAPK and activation protein-1 (AP-1) signaling pathways. In conclusion, autocrine/paracrine pathways involving EGFR and FGFR1, MAPK and AP-1 are shown to participate in the regulation of the PL-I gene in differentiating trophoblast cells.