bluuebunny/crossref_metadata_2025 huggingface dataset:
Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle
Authors: Jankowski M , Wang D , Mukaddam-Daher S , Gutkowska J
Published: January 2005

Cyclic guanosine monophosphate (cGMP), which is implicated in cardiac cell growth and function, is synthesized by cytoplasmic soluble guanylyl cyclase (GC) stimulated via nitric oxide (NO) and by particulate membrane-bound GC activated via natriuretic peptides. We investigated possible cGMP elevation in the left ventricle (LV) of rats developing physiologic LV hypertrophy during gestation. Furthermore, expression of estrogen receptors (ER) and oxytocin receptors (OTR) was evaluated because their activation stimulates NO and atrial natriuretic peptide (ANP) release from the heart. Compared with nonpregnant controls, Sprague-Dawley rats on day 7 of gestation had similar heart weights, but, on days 14 and 21, ventricular mass increased by 12% and 28% respectively (P< 0.05). LV cGMP concentration was elevated at day 14 of gestation (3.25 ± 0.12 vs 4.65 ± 0.17 pmol/g wet weight, P< 0.01) but decreased at day 21 (2.45 ± 0.09 pmol/g, P< 0.05) to increase again on postpartum day 1 (6.01 ± 0.15 pmol/g) and day 4 (9.21 ± 1.79 pmol/g). Changes in endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), OTR and ERα, but not ERβ, proteins paralleled the pregnancy-related cGMP changes in the LV. In contrast, ANP mRNA of the LV remained at control level throughout gestation but increased postpartum, whereas brain natriuretic peptide (BNP) expression declined at term and increased postpartum. The particulate GC natriuretic peptide receptors (GC-A and GC-B) transcripts were already lower at day 14 of gestation. Natriuretic peptide clearance receptor (NPR-C) transcript was not altered on days 7 and 14, but increased at term. We conclude that cGMP concentration in the rat LV is influenced by both NOS and natriuretic peptide systems and may be involved in the changes of LV contractility and hypertrophy that occur during rat gestation.
Neurointermediate pituitary lobectomy decreases the incidence and severity of experimental autoimmune encephalomyelitis in Lewis rats
Authors: Quintanar-Stephano Andrés , Chavira-Ramírez Roberto , Kovacs Kalman , Berczi Istvan
Published: January 2005

Acute experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system, mediated by T lymphocytes. Immunization of Lewis rats with myelin antigens suspended in complete Freund’s adjuvant induces EAE. In a previous study on rats we have found that neurointermediate pituitary lobectomy (NIL) decreased both the humoral and cell-mediated immune responses. Here we investigated the effect of NIL on the incidence and severity of EAE and on the function of the hypothalamic-pituitary-adrenal axis in Lewis rats. NIL, hypophysectomized (Hypox) and sham-operated (Sham) rats were immunized s.c. with guinea-pig brain extract suspended in complete Freund’s adjuvant. Untreated rats were used as controls. Water intake, body weight gain, clinical and histopathologic incidence and severity of EAE were evaluated in the operated groups. On killing, plasma adrenocorticotropin and corticosterone levels were measured and adrenals, thymuses and spleens were weighed. Histopathologic lesions were counted in the brain and spinal cord. Water intake and body weight gain were significantly decreased in Sham and Hypox animals with EAE whereas higher intakes persisted in the NIL group. Plasma levels of adrenocorticotropin were within the normal range whereas corticosterone levels increased in Sham and occasionally in NIL animals. Thymus weights were decreased in NIL and Hypox groups. The clinical and histopathologic incidence and severity of EAE were significantly decreased in NIL animals as compared with Sham and Hypox rats. We concluded that NIL affects the cell-mediated immune response and plays a role in the development and progression of EAE in the Lewis rat.
Gonadotrope oestrogen receptor-α and -β and progesterone receptor immunoreactivity after ovariectomy and exposure to oestradiol benzoate, tamoxifen or raloxifene in the rat: correlation with LH secretion
Authors: Sánchez-Criado J E , de las Mulas J Martín , Bellido C , Aguilar R , Garrido-Gracia J C
Published: January 2005

The selective oestrogen receptor modulator (SERM) tamoxifen (TX) has agonist/antagonist actions on LH secretion in the rat. Whereas in the absence of oestrogens TX elicits progesterone receptor (PR)-dependent GnRH self-priming, it antagonizes oestrogen-stimulatory action on LH secretion. The aim of these experiments was to explore whether TX treatment-induced differential expression of oestrogen receptor (ER)α and ERβ in the gonadotrope may determine its agonist effect on LH secretion. In the first experiment, basal LH secretion, GnRH-stimulated LH secretion and PR-dependent GnRH self-priming were determined in incubated pituitaries from ovariectomized (OVX) rats treated with oestradiol benzoate (EB), TX or raloxifene (RX). Cycling rats in metoestrus or pro-oestrus were used as basic controls. As in pro-oestrus, pituitaries from OVX rats treated with EB exhibited GnRH-stimulated LH secretion, immunohistochemical PR expression and GnRH self-priming. While RX had no effect on these parameters, TX induced PR expression and GnRH self-priming. GnRH self-priming was absent in pituitaries incubated with the antiprogestin ZK299. In the second experiment, we evaluated the immunohistochemical expression of ERα and ERβ in gonadotropes of cycling rats and OVX rats treated with EB, TX or RX. We found that while ERα expression was similar in all six groups, ERα expression was oestrous cycle dependent. Moreover, ERα expression in gonadotropes of TX-treated rats was as high as that found in pro-oestrus, while ERα expression in the gonadotropes of RX-treated rats was lower than in metoestrous or pro-oestrous pituitaries. These results suggest that, in the absence of the cognate ligand, TX, unlike RX, may regulate LH secretion through the ERα subtype in gonadotropes.
Altered expression of IGFs and IGF-binding proteins during intrauterine growth restriction in guinea pigs
Authors: Carter A M , Kingston M J , Han K K , Mazzuca D M , Nygard K , Han V K M
Published: January 2005

The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth. We hypothesized that intrauterine growth restriction (IUGR) in guinea pigs is mediated by the altered expression of IGFs and/or IGF binding protein (BP) mRNAs in tissues and is related to growth of specific tissues. IUGR was induced by unilateral uterine artery ligation on day 30 of gestation, and fetal plasma, amniotic fluid and tissue samples were collected at 55–57 days (term about 68 days) from paired IUGR and control fetuses (n=6). Western ligand blotting and immunoblotting were used to compare IGFBP levels in plasma and amniotic fluid. Total RNA was extracted from placenta and fetal tissues, and the relative abundance of IGF-II and IGFBP-1–6 mRNA was determined by Northern blotting, using species-specific probes where available. IUGR fetuses had decreased (P<0.01, by Student’s t-test) placental weight and body weight with an increase in the brain:liver weight ratio. The principal IGFBPs in fetal plasma migrated at 40–35, 30 and 25 kDa and were identified as IGFBP-3, -2 and -4 respectively. IUGR was associated with elevated plasma IGFBP-2 and IGFBP-4 and reduced IGFBP-3 levels. IGFBPs were detected at low levels in amniotic fluid of control fetuses but at higher levels in IUGR fetuses. In IUGR placentae, there was a small increase in IGFBP-4 mRNA (P<0.05). IGFBP-2 mRNA increased (P<0.001) in liver of IUGR fetuses. IGF-II and IGFBP mRNA expression did not change in fetal muscle. The results are consistent with reduced IGF action, directly or through inhibition by IGFBPs, particularly by circulating and tissue IGFBP-2, as a potential causal factor in decreased growth of the placenta and certain fetal tissues.
The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells
Authors: Velarde M C , Parisek S I , Eason R R , Simmen F A , Simmen R C M
Published: January 2005

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-α, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a ‘feed-forward’ mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.
Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver
Authors: Eleswarapu S , Jiang H
Published: January 2005

Growth hormone (GH) regulates the expression of many genes in the liver, and for some genes this regulation may be mediated through liver-enriched transcription factors (LETFs). As part of the long-term goal to investigate the role of LETFs in GH regulation of gene expression in the liver, in this study we determined the effect of GH administration on the expression of 10 LETFs, including hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3α, HNF-3β, HNF-3γ, HNF-4α, HNF-6, CCAAT/enhancer-binding protein (C/EBP) α, C/EBPβ, and albumin D-element binding protein (DBP) in the bovine liver. Eighteen non-lactating and non-pregnant Angus cows were assigned randomly to three groups (n=6 per group) and each cow received a single intramuscular injection of 500 mg slow-release recombinant bovine GH. Liver biopsy samples were taken from group 1 cows 6 h after GH administration, from group 2 cows 24 h after GH administration, and from group 3 cows 1 week after GH administration. Liver biopsies were also collected from group 3 cows 1 day before GH administration, serving as pre-GH controls. The LETF mRNAs in these liver samples were quantified using ribonuclease protection assays with probes generated from bovine LETF cDNAs cloned by standard reverse transcription–polymerase chain reaction. The levels of HNF-3γ and HNF-6 mRNAs were higher (P< 0.05) in the cows 24 h and 1 week after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-4α mRNA were higher (P< 0.05) in the cows 1 week after GH administration than in the other three groups of cows. The levels of C/EBPα mRNA were higher (P< 0.05) in the cows 24 h after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-3α mRNA were higher (P< 0.05) in the cows 6 h after GH administration but were lower (P< 0.05) in the cows 24 h or 1 week after GH administration compared with those in the untreated cows. The levels of DBP mRNA were higher (P< 0.05) in the cows 6 h after GH administration but were lower (P< 0.05) in the cows 24 h after GH administration compared with those in the untreated cows. The levels of HNF-1α, HNF-3α, and C/EBPβ mRNAs were not different (P>0.05) between groups. The expression of HNF-1β mRNA was not detectable. Thus, the expression of six LETFs including HNF-3γ , HNF-3β, HNF-4α, HNF-6, C/EBPα, and DBP mRNAs in the bovine liver is regulated by GH, and these six LETFs may play a role in mediating GH regulation of gene expression in the liver. Among the 10 LETFs, the response of HNF-3γ to GH is most significant. Cloning and sequencing the promoter region of this gene revealed multiple putative binding elements for signal transducers and activators of transcription 5 (STAT5), suggesting that GH regulation of HNF-3γ expression in the liver may be mediated through direct binding of STAT5 to the HNF-3γ promoter.
Cloning, expression and pharmacological characterization of a vasopressin-related receptor in an annelid, the leech Theromyzon tessulatum
Authors: Levoye A , Mouillac B , Rivière G , Vieau D , Salzet M , Breton C
Published: January 2005

In annelids, it has been established that arginine-vasopressin (AVP)/oxytocin (OT) superfamily peptides are involved in the maintenance of water and electrolyte homeostasis as well as reproduction. At present, there is little information on their receptors. In this study, we report the characterization of a 1.7 kb cDNA for an AVP-related receptor from the leech Theromyzon tessulatum. The open reading frame encodes a 435-aminoacid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% amino-acid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional. This study describes the characterization of a novel AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system.
Insulin sensitizing drugs increase the endogenous dopaminergic tone in obese insulin-resistant women with polycystic ovary syndrome
Authors: Ortega-González C , Cardoza L , Coutiño B , Hidalgo R , Arteaga-Troncoso G , Parra A
Published: January 2005

To investigate whether the long-term administration of metformin or pioglitazone to women with polycystic ovary syndrome (PCOS) could induce changes in their hypothalamic dopaminergic (DA) tone and to analyze whether these changes correlated with modifications in insulin resistance, we originally studied 57 obese hyperinsulinemic, non-diabetic, insulin resistant women with PCOS, but only 34 completed the study. They were randomly divided into two groups: group one (n=17) received pioglitazone (30 mg/day) and group 2 (n=17) received metformin (850 mg, three times a day) over 24 weeks. All women were identically studied before (basal) and 6 months after (T6) drug administration, including clinical evaluations, a 2 h oral glucose tolerance test (75 g) (OGTT) for glucose and insulin measurements, followed a week later by a 2 h intravenous metoclopramide test (10 mg bolus) for prolactin (PRL) determinations. The areas under the insulin (AUC-insulin) and PRL (AUC-PRL) curves were calculated, along with the index of insulin resistance (HOMA-IR) and the indexes of insulin sensitivity (QUICKI and fasting glucose–insulin ratio). At baseline, women in both groups were of similar age, body weight, body mass index (BMI) and Ferriman-Gallwey hirsutism score (F-G score). At completion of the study, body weight and BMI remained unchanged but the F-G score significantly decreased. Fasting serum insulin concentrations and the AUC-insulin significantly decreased by the end of the trial in a similar fashion in both groups, while the AUC-PRL significantly increased at the end of the trial in both groups. At no time were significant correlations between AUC-PRL and AUC-insulin or the indexes HOMA-IR, QUICKI or fasting glucose–insulin ratio observed. The present results suggests that either pioglitazone or metformin administration was associated with a clear improvement in the endogenous hypothalamic DA tone, simultaneously with an amelioration of the insulin resistance status in these obese women with PCOS.
Multistep differentiation of GH-producing cells from their immature cells
Authors: Mogi C , Goda H , Mogi K , Takaki A , Yokoyama K , Tomida M , Inoue K
Published: January 2005

In order to study GH cell differentiation, we used the clonal cell lines called MtT/E and MtT/S cells, which were derived from a rat mammotrophic pituitary tumor. Although MtT/E cells are non-hormone-producing ones, Pit-1 protein is present in their nuclei, which suggests that MtT/E cells are progenitor cells of the Pit-1 cell lineage and have the potential to differentiate into hormone-producing cells. On the other hand, MtT/S cells produce GH; however, the responsiveness to GH-releasing hormone (GHRH) is weak and only a small number of secretory granules are present in their cytoplasm, which suggests that MtT/S cells are premature GH cells. In order to differentiate into GH cells from MtT/E cells as a progenitor cell, we examined several differentiation factors and found that retinoic acid (RA) induced the differentiation of MtT/E cells into GH-producing cells. RA-induced GH cells partially matured with the glucocorticoid treatment; however, the responsiveness to GHRH on GH secretion was incomplete. In order to elucidate the mechanism underlying full differentiation of GH cells, we used MtT/S cells. We treated MtT/S cells with glucocorticoid and found that they differentiated into mature GH cells with many secretory granules in their cytoplasm and they responded well to GHRH. These results suggested that MtT/E and MtT/S cells are progenitor or premature GH cells, and show different responses to differentiation factors. Our data also suggested that GH cells differentiate from their progenitor cells through multistep processes.
Human growth hormone and insulin-like growth factor-I inhibit erythropoietin secretion from the kidneys of adult rats
Authors: Sohmiya Motoi , Kato Yuzuru
Published: January 2005

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) play important roles in erythropoiesis and erythro-poietin (EPO) secretion. We examined the effects of GH and IGF-I on EPO production in adult rat kidney and liver in vivo and in vitro. Male Wistar rats aged 8–10 weeks were used. Recombinant human GH (hGH) was continuously infused (20 μg/kg per h) subcutaneously for 48 h using a micro-osmotic infusion pump. Octreotide (10 μg/kg) was subcutaneously injected every 12 h beginning 12 h before the hGH treatment. GH increased plasma EPO levels earlier than it increased plasma IGF-I levels. At 24 h, the IGF-I content in the liver and kidney was increased from 172.8±14.6 to 232.6±17.8 ng/g tissue (means±S.E.) and from 53.8±3.1 to 112.8±7.2 ng/g tissue, respectively. The EPO content in the liver was increased from 7.5±1.2 to 15.1±1.4 mIU/g tissue at 48 h, whereas the EPO content in the kidney was decreased at 12, 24, and 48 h after the start of hGH treatment. When the kidneys were organ-cultured, hGH considerably decreased EPO levels in the culture medium in a dose-related manner. The addition of anti-hGH IgG blunted the GH-induced inhibition of EPO secretion from the kidneys. IGF-I also decreased EPO levels in the medium in a dose-related manner. The addition of anti-IGF-I IgG blunted the IGF-I-induced inhibition of EPO secretion from the kidneys, whereas the GH-induced inhibition of EPO secretion was not affected. These findings suggest that both hGH and IGF-I have direct inhibitory effects on EPO secretion from adult rat kidneys.
Identification of α-enolase as a nuclear DNA-binding protein in the zona fasciculata but not the zona reticularis of the human adrenal cortex
Authors: Wang Weiye , Wang Lishan , Endoh Akira , Hummelke Geoffrey , Hawks Christina L , Hornsby Peter J
Published: January 2005

In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA–protein complexes was studied using an element in the first intron of the type I and type II 3β-hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethyl-Sepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as α-enolase by mass spectrometry. Although α-enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found α-enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an α-enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.
The effect of a diet supplemented with the n-6 polyunsaturated fatty acid linoleic acid on prostaglandin production in early- and late-pregnant ewes
Authors: Cheng Z , Elmes M , Kirkup S E , Chin E C , Abayasekara D R E , Wathes D C
Published: January 2005

Polyunsaturated fatty acids derived from the diet are incorporated into cell membranes where they act as precursors for prostaglandin (PG) synthesis. Linoleic acid (LA; 18:2 n-6) is a major constituent of plant oils and its consumption in Westernized populations is increasing. This study investigated the influence of LA on PG production by the uterus and placenta. Pregnant ewes were fed a control or an LA-enriched diet. Oxytocin (OT) was injected on day 45 (early) or day 133 (late) of gestation to measure the release of 13,14-dihydro-15-keto PGF2α (PGFM). Ewes were killed on day 46 or day 138 for collection of uterine intercaruncular endometrium and fetal allantochorion. Basal and stimulated PG release from explant cultures was assessed before and after in vitro treatment with OT, lipopolysaccharide (LPS), dexamethasone (DEX) or calcium ionophore (CaI). Expression of cyclooxygenase (COX)-1 and COX-2 was determined by Western blot in endometrium of late-gestation ewes. Circulating PGFM levels in vivo did not differ according to diet but there were highly significant differences in the release of PGs in vitro. Basal production of PGF2αand PGE2 by the endometrium and of PGE2 by the allantochorion were all higher in tissues from LA-supplemented ewes. Endometrial tissues produced more PG following OT and CaI treatment, whereas DEX inhibited production of both PGs at both stages of gestation. In allantochorion collected at day 46 LPS did not significantly alter PGE2 release and DEX increased output, whereas at day 138 LPS was stimulatory but DEX was inhibitory. These data show that a high-LA diet can significantly increase the ability of both endometrium and placental tissues to produce PGs in vitro. This effect of diet may only become apparent after a sustained period of PG release, so was not seen following the brief pulse caused by OT treatment in vivo. As COX protein levels were unaltered, the main influence was likely to be via conversion of LA to arachidonic acid, providing an increased supply of precursor. These results support previous studies which suggest that alterations in dietary polyunsaturated fatty acids may influence the time of labour.
Effect of 17β-estradiol on tumor necrosis factor-α-induced cytotoxicity in the human peripheral T lymphocytes
Authors: Takao Toshihiro , Kumagai Chizuru , Hisakawa Naoko , Matsumoto Reiko , Hashimoto Kozo
Published: January 2005

We determined the effect of 17β-estradiol on tumor necrosis factor α (TNF-α)-induced cytotoxicity in human peripheral T lymphocytes (T cells) using lactate dehydrogenase assay. Treatment with 17β-estradiol (1–100 nM) for 24 h showed dose-dependent reduction of TNF-α-induced cytotoxicity in T cells. To further evaluate the mechanism of 17β-estradiol on TNF-α-induced cytotoxicity in T cells, we identified estrogen receptor (ER) protein in T cells using immunocytochemistry and used the pure ER antagonist ICI 172,780. ERα immunoreactivity was clearly observed in T cells. ERβ immunoreactivity was also detected in some T cells. ICI 172,780 (10−7 M) alone did not affect cytotoxicity in T cells, however, ICI 172,780 (10−7 M) completely abolished 17β-estradiol cytoprotective effects in T cells. TNF-α tended to increase nuclear factor κB (NF-κB) protein levels in nuclear extracts but it did not reach statistical significance by Western blotting. In contrast, NF-κB protein levels in nuclear extracts followed by TNF-α with 17β-estradiol treatment were significantly increased compared with NF-κB protein levels in untreated group. NF-κB blocker pyrrolidinedithiocarbamate (PDTC) (10−4 M) alone did not affect cytotoxicity in T cells. In contrast, PDTC (10−4 M) completely abolished 17β-estradiol cytoprotective effects in T cells. Caspase -3/-7 activity was significantly increased followed by TNF-α, and 17β-estradiol treatment significantly reduced the increment. The present studies suggest the protective effect of 17β-estradiol on TNF-α-induced cytotoxicity through ERs in T cells and that NF-κB activation and caspase suppression may be involved in the mechanism.
A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity
Authors: Yin H , Ukena K , Ubuka T , Tsutsui K
Published: January 2005

We recently identified a novel hypothalamic dodecapeptide inhibiting gonadotropin release in the Japanese quail (Coturnix japonica). This novel peptide was therefore named gonadotropin-inhibitory hormone (GnIH). The GnIH precursor encoded one GnIH and two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln; LPXRF-amide peptides). Identification of the receptor for GnIH is crucial to elucidate the mode of action of GnIH. We therefore identified the receptor for GnIH in the quail diencephalon and characterized its expression and binding activity. We first cloned a cDNA encoding a putative GnIH receptor by a combination of 3′ and 5′ rapid amplification of cDNA ends (RACE) using PCR primers designed from the sequence for the receptor for rat RF-amide-related peptide (RFRP), an orthologous peptide of GnIH. Hydrophobic analysis revealed that the putative GnIH receptor possessed seven transmembrane domains, indicating a new member of the G protein-coupled receptor superfamily. The crude membrane fraction of COS-7 cells transfected with the putative GnIH receptor cDNA specifically bound to GnIH and GnIH-RPs in a concentration-dependent manner. Scatchard plot analysis of the binding showed that the identified GnIH receptor possessed a single class of high-affinity binding sites (Kd=0.752 nM, Bmax=24.8 fmol/mg protein). Southern blotting analysis of reverse transcriptase-mediated PCR products revealed the expression of GnIH receptor mRNA in the pituitary gland and several brain regions including diencephalon in the quail. These results suggest that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit gonadotropin-releasing hormone release.
Frequent serial fecal corticoid measures from rats reflect circadian and ovarian corticosterone rhythms
Authors: Cavigelli S A , Monfort S L , Whitney T K , Mechref Y S , Novotny M , McClintock M K
Published: January 2005

The circadian glucocorticoid rhythm provides important information on the functioning of the hypothalamic-pituitary-adrenal axis in individuals. Frequent repeated blood sampling can limit the kinds of studies conducted on this rhythm, particularly in small laboratory rodents that have limited blood volumes and are easily stressed by handling. We developed an extraction and assay protocol to measure fecal corticosterone metabolites in repeated samples collected from undisturbed male and female adult Sprague–Dawley rats. This fecal measure provides a non-invasive method to assess changes in corticosterone within a single animal over time, with sufficient temporal acuity to quantify several characteristics of the circadian rhythm: e.g. the nadir, acrophase, and asymmetry (saw-tooth) of the rhythm. Males excreted more immunoreactive fecal corticoids than did females. Across the estrous cycle, females produced more fecal corticoids on proestrus (the day of the preovulatory luteinizing hormone surge) than during estrus or metestrus. These results establish a baseline from which to study environmental, psychological, and physiological disturbances of the circadian corticosterone rhythm within individual rats.
Stimulation of a rat uterine stromal cell line in culture reveals a molecular switch for endocrine-dependent differentiation
Authors: Rider V , Potapova T , Dai G , Soares M J
Published: January 2005

Differentiation of uterine stromal cells is critical for the establishment of pregnancy. This study had two purposes: (i) to validate the use of the UIII rat uterine stromal cell model for investigating mechanisms underlying decidual cell differentiation, and (ii) to use this cell model to identify a molecular switch for cellular entry into the decidual cell differentiation pathway. Quiescent rat uterine stromal cells were transfected with a 500 bp segment of the decidual prolactin-related protein (dPRP) promoter ligated to a luciferase reporter gene. Cells were incubated in low-serum medium, or in low-serum medium containing progesterone (1 μM), estradiol 17-β (10 nM), cholera toxin (10 ng/ml) and interleukin-11 (10 ng/ml). Protein extracts were collected 48 h later and luciferase was measured in the cellular lysates. Cholera toxin and interleukin-11 stimulated luciferase expression (P< 0.05) and addition of sex steroids further increased (P< 0.05) dPRP promoter activity. Stromal cells did not proliferate (P< 0.05) under differentiation conditions. Deletion analysis of the dPRP promoter revealed maximal luciferase expression between −250 and −500 bp relative to the transcription start site. Comparison of cyclin E/Cdk2 activity between proliferating and differentiating cells showed a 3-fold increase (P< 0.05) at 12 h in differentiating cells. The results suggest that cyclin E/Cdk2 serves as a molecular switch for uterine stromal cell entry into the decidual cell differentiation pathway.
The molecular basis of tamoxifen induction of mouse uterine epithelial cell proliferation
Authors: Zhang Haifan , McElrath Tim , Tong Wei , Pollard Jeffrey W
Published: January 2005

Tamoxifen, a selective estrogen modulator (SERM) that has found clinical utility in the treatment of breast cancer, is an antagonist in the breast and an agonist in the uterus. These agonist actions in the uterus lead to an increased risk of endometrial cancer. In this study in mice we have analyzed the mechanism of action of tamoxifen in inducing cell proliferation in the uterine luminal epithelia. Tamoxifen induces a wave of DNA synthesis in these epithelial cells with kinetics similar to those seen after 17β-estradiol (E2) treatment. However, by these criteria of mitogenicity, it is much less potent and never achieves full estrogenicity. This uterine epithelial cell proliferation is preceded by the mobilization of cyclin D1 from the cytoplasm to the nucleus which, together with CDK4, phosphorylates members of the Rb-retinoblastoma family of proteins, pRb and p107. Subsequent to this initial nuclear accumulation of cyclin D1, cyclin E and then cyclin A are induced that, together with the activation of CDK2, results in enhanced cyclin E- and cyclin A-dependent CDK2 kinase activity and further phosphorylation of pRb and p107. These actions of tamoxifen parallel those of E2. Tamoxifen also induced the classical estrogen water imbibition response. However, in this it was more potent, producing a maximal response at doses that do not affect DNA synthesis. This suggests that the uterotropic response is not an accurate predictor of the compound’s hyperplasia responses. We can conclude that, in its effects on proliferation, tamoxifen acts as a classical impeded estrogen and this suggests that the AF-1 transcription activation domain of the estrogen receptor that is activated upon both E2 and tamoxifen binding to this receptor regulates these responses in the uterus.
Ernest Starling and ‘Hormones’: an historical commentary
Authors: Henderson John
Published: January 2005

One hundred years ago Ernest Starling (1866–1927), almost surreptitiously, slipped the word ‘hormone’ into the English language. This review, beginning in the middle of the nineteenth century, attempts to trace the growth of ideas in endocrinology up to this important moment.
Homeobox gene repertoires: implications for the evolution of diversity
Authors: Kappen Claudia
Published: January 2005

Homeobox genes are key regulators of morphogenetic processes in developing organisms, and are involved in the formation and differentiation of multiple tissues and cell types. The strong conservation of homeobox gene sequences indicates that many species share a common repertoire of homeobox genes. Experimental evidence provides support for conserved as well as divergent functions, raising the question how homeobox genes evolved in distinct species and in which way they contributed to the evolution of different morphologies. This article examines the composition of homeobox gene repertoires in diverse animals at the whole‐genome level, with the aim to understand the underlying mechanisms of evolution in this multigene family.
HumanXchromosome inactivation
Authors: Chang Samuel C. , Brown Carolyn J.
Published: January 2005

Early in a mammalian female's development, one of her two X chromosomes is silenced, thereby achieving dosage equivalence with the single X chromosome found in males. The process of inactivation is initiated by the untranslated XIST RNA that is expressed solely from the inactive X. Subsequent silencing involves the acquisition of many features of heterochromatin, including late DNA replication, DNA hypermethylation, and specific histone modifications. How the XIST RNA associates in cis with the chromosome from which it is expressed and mediates silencing of one X chromosome remains a focus for ongoing research. The inactive X is not completely refractive to transcription, as approximately 15% of human genes escape the silencing and continue to be expressed from both the active and inactive X chromosomes. The hemizygosity of the X chromosome in males means that X‐linked diseases are distinctively more common in males, and dosage imbalance of other genes may yield additional differences between the sexes.
Approach to common chronic disorders of adulthood
Authors: Scheuner Maren T.
Published: January 2005

Common chronic diseases such as coronary heart disease, stroke, diabetes, and cancer are due to the interaction of genetic and nongenetic risk factors. Recognizing genetic factors that contribute to common chronic diseases can help identify individuals with increased risk, especially at early ages of onset. Understanding genetic susceptibilities for common chronic diseases can also help determine which diseases might develop in an individual, given a particular exposure or behavior. Family history collection and interpretation is the most practical population‐based strategy for identifying individuals with a genetic susceptibility to many common chronic diseases. It represents complex interactions of genetic, environmental, cultural, and behavioral factors shared by family members. Family history characteristics that increase disease risk include early age at diagnosis, two or more close relatives affected with a disease or a related condition, a single family member with two or more related diagnoses, multifocal or bilateral disease, and occurrence of disease in the less‐often affected sex. By recognizing the magnitude of risk associated with these familial characteristics, stratification into different familial risk groups (e.g., low, medium, and high) is possible, which can guide risk‐specific recommendations for disease management and prevention. Clinical genetic evaluation should be considered for individuals with a high familial risk for common chronic diseases or when a Mendelian disorder is suspected. The process includes (1) genetic counseling and education, (2) risk assessment and diagnosis using personal and family medical history, physical examination, and genetic testing, and (3) risk‐appropriate recommendations for management and prevention.
Current approaches to molecular diagnosis
Authors: Mueller O. Thomas
Published: January 2005

Inherited human disorders are caused by different types of mutations, including large genomic rearrangements such as chromosomal translocations or duplications and deletions of genome regions that are visible, using standard or high‐resolution karyotyping procedures. Other rearrangements are smaller, but still encompass relatively large genome areas, such as the microdeletions that are readily detected using FISH technology. However, most disorders are caused by much smaller deletions, substitutions, or rearrangements of DNA that are identified by molecular genetic approaches. This review will summarize the current approaches to diagnostic techniques in a molecular genetic laboratory. A tabular listing of selected inherited syndromes identifies the genes and proteins associated with many syndromes, the types of mutations and common mutations, if any, detection methods, and their inheritance pattern. Special attention is given to disorders of complex etiology, such as those with a trinucleotide repeat expansion, which feature unusual inheritance patterns such as premutation alleles and genetic anticipation, and Prader–Willi and Angelman syndromes, which invoke genetic imprinting mechanisms.
Treatment of monogenic disorders
Authors: Descartes Maria , Biggio Joseph R.
Published: January 2005

The ability to cure or treat genetic disease has not paralleled the advances made in molecular genetics to identify and characterize disease genes. Current treatments are mainly focused on symptom improvement or avoidance and include dietary modification, metabolite replacement, enzymatic blockage, enzyme potentiation, enzymatic bypass, and protein replacement.
Cardiovascular gene therapy
Authors: Bhardwaj Shalini , Roy Himadri , Ylä‐Herttuala Seppo
Published: January 2005

A variety of treatment modalities are presently available for the treatment of cardiovascular diseases either in the form of drug therapy or surgical intervention. A relatively new field of gene therapy for cardiovascular diseases is fast evolving. The diseases in focus are atherosclerosis, coronary and peripheral arterial diseases, coronary restenosis, and vein graft diseases. Promising results have been obtained in animal models of restenosis and vein graft thickening with transfer of genes encoding VEGF, nitric oxide synthatase, thymidine kinase, tissue inhibitors of metalloproteinases, and antisense oligonucleotides against transcription factors or cell cycle regulatory proteins. VEGF and FGF gene transfer have been successfully used in improving blood flow and development of collateral arteries in peripheral vascular disease and myocardial ischemic conditions. Here, we present a synopsis of the potential treatment genes, their therapeutic targets, mode of delivery, human clinical trials, and limitations encountered in the field of cardiovascular gene therapy.
Control of transgene expression in mammalian cells
Authors: Kramer Beat P. , Fussenegger Martin
Published: January 2005

Spacio‐temporal adjustment of therapeutic transgene expression represents a fundamental technology for modern gene therapy since it enables precise in vivo dosing of protein therapeutics to manage an optimal therapeutic range, adaptation to specific disease dynamics, or resetting of molecular interventions as appropriate. Heterologous transcription control systems capitalizing on (1) prokaryotic antibiotic response regulons evolved to manage bacterial adaptation to macrolide, streptogramin, and tetracycline antibiotics, (2) (modified) hormone receptors specific for estradiol, progesterone, or ecdysone derivatives, or (3) chemically induced dimerization of protein domains prevailed in animal studies, and are currently considered for clinical trials. Since sustained administration of clinically licensed, small‐molecule drugs for transgene expression fine‐tuning may result in side effects, systems responsive to nucleic acids or physical parameters including light, temperature, and electromagnetic fields are in preclinical development.
Robotics and automation
Authors: Mardis Elaine R.
Published: January 2005

High‐throughput DNA sequence generation, as we currently know it, was enabled over the past 15 years by a variety of technological developments that rendered it a highly automated process. This article chronicles key milestones in technology and the efforts that utilized these technologies in the pursuit of faster, cheaper DNA sequence generation.
Real‐time
DNA
sequencing
Authors: Hardin Susan H.
Published: January 2005

The design of a novel, real‐time DNA sequencing technology is discussed. An ultimate goal of the technology is to complete human genome sequencing in less than a day for less than $1000.
Synteny mapping
Authors: Gregory Simon G.
Published: January 2005

The level of sequence homology between distantly related species facilitates the construction of a sequence‐ready physical map. The map is rapidly constructed using a highly redundant restriction digest database and alignment of BAC end sequence to a reference genome, and can underpin the generation of genomic sequence or other experimental analyses.
Restriction fragment fingerprinting software
Authors: Soderlund Carol A.
Published: January 2005

A physical map is an ordering of clones and markers. Whole‐genome physical maps may be constructed using restriction fragment fingerprinting. With this approach, two clones are considered overlapping if they contain a similar set of fragments. The FPC (FingerPrinted Contig) software is described, which assembles fingerprinted clones into contigs. Markers can also be used to aid the assembly and results in a partial ordering of the markers. Genetic or radiation hybrid markers are used to anchor the contigs to the chromosomes. A minimal tiling path of clones may be selected for sequencing. FPC may be used with the two salient types of fingerprinting, which are agarose based and HICF (High Information Content Fingerprinting).
The mouse genome sequence
Authors: Jackson Ian J.
Published: January 2005

The mouse is the leading mammalian model organism for the study of genetics. Its genome consists of about 2500 Mb of DNA and contains about 30 000 genes, virtually all of which have close homologs in the human genome. Comparison of the mouse with other mammalian genomes indicates that it is not only the coding sequences of genes that have been maintained in evolution; putative conserved control elements can be identified by aligning multiple genomes. The DNA of the mouse (and rat) is accumulating changes at a much more rapid rate than the human genome. Base substitutions in neutral sequences have accumulated about twice as fast in the mouse when compared to humans, since their last common ancestor 75 million years ago. Furthermore, transposable elements have been more active in the rodent lineage, and there have been more chromosomal rearrangements that alter long‐range relationships between genes.
Eukaryotic parasite genome projects
Authors: Hall Neil
Published: January 2005

Genome sequence data is transforming the field of parasitology, and is proving to be a leading technology in the hunt for new drugs and vaccines for some of the most serious diseases affecting mankind. In this chapter, I will describe some of the key scientific contributions made by the genome projects and draw on some specific examples in protist parasites such as genetics in

Trypanosome brucei

, the discovery of polycistronic transcription

Leishmania major

, and the evolution of the apicoplast in

Plasmodium falciparum

.
Chlamydiae
Authors: Read Timothy D.
Published: January 2005

The chlamydiae are a group of obligate intracellular pathogens with a unique biphasic life cycle. The six genomes sequenced are approximately 1.05–1.2 Mb and show no signs of pathogenicity islands or other recent horizontal transfer events. There are about 750 genes conserved across all genomes, encoding core functions. Potential virulence determinants include type III secretion systems, toxins, variable surface proteins, and tryptophan biosynthesis proteins.
Comparative genomics of the ɛ‐proteobacterial human pathogens

H
elicobacter pylori

and

C
ampylobacter jejuni
Authors: Dorrell Nick , Brendan Wren
Published: January 2005

Helicobacter pylori

and

Campylobacter jejuni

are among the most common bacterial pathogens encountered by humans, but despite the prevalence and importance of the diseases caused by these organisms, they have only been recognized as human pathogens within the last 25 years. However, they are now among the most comprehensively studied organisms, due in no small part to the availability of complete genome sequences for both bacteria. Subsequent genomic studies have started to explain the genetic, ecological, and pathogenic similarities and differences between these two major human pathogens. Understanding the dynamic and complex interplay between these pathogens and the host will place the emphasis on converting this knowledge into appropriate intervention strategies to reduce the burden on human health.
FT‐ICR
Authors: Barrow Mark P. , Burkitt William I. , Derrick Peter J.
Published: January 2005

Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry has become an increasingly significant technique within recent years. The inherently ultrahigh resolution and mass accuracy allow unequivocal assignments of elemental composition to be made for biomolecules. Structural elucidation can be conducted through tandem mass spectrometry experiments. With the advent of electrospray ionization (ESI), FT‐ICR mass spectrometry has become a powerful tool for the investigation of biological macromolecules, including aspects of noncovalent interactions.
Laser‐based microdissection approaches and applications
Authors: Banks Rosamonde E.
Published: January 2005

The analysis and interpretation of either physiological or pathological protein changes in tissue is difficult due to the cellular heterogeneity, with multiple cell types being present. In disease, the situation may be even more complex due to diseased cells being adjacent to normal cells, the presence of distinct microenvironments such as hypoxic areas, and additional infiltrating cells such as lymphocytes. Attempting to understand the biology or pathology of particular cells against such a heterogeneous background is therefore a challenge. Laser‐assisted microdissection systems have now been developed, which allow the enrichment of specific cell types for subsequent analysis. These systems have been reviewed, together with a consideration of some of the technical issues and examples of downstream proteomic applications.