Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7846-7854.

5-HT(1A/1B), 5-HT(6), and 5-HT(7) serotonergic receptors recruitment in tonic-clonic seizure-induced antinociception: Role of dorsal raphe nucleus

Pharmacological studies have been focused on the involvement of different neural pathways in the organization of antinociception that follows tonic-clonic seizures, including 5-hydroxytryptamine (5-HT)-, norepinephrine-, acetylcholine- and endogenous opioid peptide-mediated mechanisms, giving rise to more in-depth comprehension of this interesting post-ictal antinociceptive phenomenon. The present work investigated the involvement of 5-HT(1A/1B), 5-HT(6), and 5-HT(7) serotonergic receptors through peripheral pretreatment with methiothepin at doses of 0.5, 1.0, 2.0 and 3.0 mg/kg in the organization of the post-ictal antinociception elicited by pharmacologically (with pentylenetetrazole at 64 mg/kg)-induced tonic-clonic seizures. Methiothepin at 1.0 mg/kg blocked the post-ictal antinociception recorded after the end of seizures, whereas doses of 2.0 and 3.0 mg/kg potentiated the post-ictal antinociception. The nociceptive thresholds were kept higher than those of the control group. However, when the same 5-hydroxytryptamine receptors antagonist was microinjected (at 1.0, 3.0 and 5.0 mu g/0.2 mu L) in the dorsal raphe nucleus, a mesencephalic structure rich in serotonergic neurons and 5-HT receptors, the post-ictal hypo-analgesia was consistently antagonized. The present findings suggest a dual effect of methiothepin, characterized by a disinhibitory effect on the post-ictal antinociception when peripherally administered (possibly due to an antagonism of pre-synaptic 5-HT(1A) serotonergic autoreceptors in the pain endogenous inhibitory system) and an inhibitory effect (possibly due to a DRN post-synaptic 5-HT(1B), 5-HT(6), and 5-HT(7) serotonergic receptors blockade) when centrally administered. The present data also Suggest that serotonin-mediated mechanisms of the dorsal raphe nucleus exert a key-role in the modulation of the post-ictal antinociception. (C) 2009 Elsevier Inc. All rights reserved.

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1 beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1 beta induced iNOS protein expression. Thus, activation of P2X 7 receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1 beta-mediated release of nitric oxide by iNOS.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

Endothelin-1 Induces Contraction of Female Rat Internal Pudendal and Clitoral Arteries through ET(A) Receptor and Rho-Kinase Activation

Introduction. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, acts mainly through the Gprotein-coupled ET(A) receptor (ET(A)R). Increased vascular ET-1 production and constrictor sensitivity have been observed in various cardiovascular diseases, including hypertension, as well as erectile dysfunction. The internal pudendal artery (IPA) supplies blood to the vagina and clitoris. Inadequate blood flow through the IPA may lead to insufficient vaginal engorgement and clitoral tumescence. Aim. Characterize the effects of ET-1 on the IPA and clitoral artery (CA). Methods. IPA and CA from female Sprague Dawley rats (225-250 g) were mounted in myograph chambers. Arterial segments were submitted to increasing concentrations of ET-1 (10-10-10-6 M). Segments were incubated with the ET(A)R antagonist, atrasentan (10-8 M) or the Rho-kinase inhibitor, Y-27632 (10-6 M) 30 minutes prior to agonist exposure. All E(max) values are expressed as % KCl-induced maximal contraction. ET(A)R, RhoA, and Rho-kinase expression from IPA was evaluated by Western blot. mRNA of preproET-1, ET(A)R, ET(B)R, RhoA, and Rho-kinase were measured by real time PCR. Main Outcome Measures. ET-1 constrictor sensitivity in IPA and CA, protein expression and messenger RNA levels of ET-1-mediated constriction components. Results. ET-1 concentration-dependently contracted IPA (% Contraction and pD2, respectively: 156 +/- 18, 8.2 +/- 0.1) and CA (163 +/- 12, 8.8 +/- 0.08), while ET(A)R antagonism reduced ET-1-mediated contraction (IPA: 104 +/- 23, 6.4 +/- 0.2; CA: 112 +/- 17, 6.6 +/- 0.08). Pretreatment with Y-27632 significantly shifted ET-1 pD2 in IPA (108 +/- 24, 7.9 +/- 0.1) and CA (147 +/- 58 and 8.0 +/- 0.25). Protein expression of ET(A)R, ET(B)R, RhoA, and Rho-kinase were detected in IPA. IPA and CA contained preproET-1, ET(A)R, ET(B)R, RhoA, and Rho-kinase message. Conclusion. We observed that the IPA and CA are sensitive to ET-1, signaling through the ET(A)R and Rho-kinase pathway. These data indicate that ET-1 may play a role in vaginal and clitoral blood flow and may be important in pathologies where ET-1 levels are elevated. Allahdadi KJ, Hannan JL, Tostes RC, and Webb RC. Endothelin-1 induces contraction of female rat internal pudendal and clitoral arteries through ETA receptor and Rho-kinase activation. J Sex Med 2010;7:2096-2103.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage

The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes - nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregutated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa. (C) 2009 Elsevier GmbH. All rights reserved.

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Dentine caries inhibition through CO(2) laser (10.6 mu m) irradiation and fluoride application, in vitro

Objective: The purpose of the study was to investigate whether dentine irradiation with a pulsed CO(2) laser (10.6 mu m) emitting pulses of 10 ms is capable of reducing dentine calcium and phosphorus losses in an artificial caries model. Design: The 90 dentine slabs obtained from bovine teeth were randomly divided into six groups (n = 15): negative control group (GC); positive control group, treated with fluoride 1.23% (GF); and laser groups irradiated with 8 J/cm(2) (L8); irradiated as in L8 + fluoride 1.23% (L8F); irradiated with 11j/cm(2) (L11); irradiated as in L11 + fluoride 1.23% (L11F). After laser irradiation the samples were submitted to a pH-cycling model for 9 days. The calcium and phosphorous contents in the de- and remineralization solutions were measured by means of inductively coupled plasma optical emission spectrometer - ICP-OES. Additionally intra-pulpal temperature measurements were performed. The obtained data were analysed by means of ANOVA and Tukey`s test (alpha = 0.05). Results: In the demineralization solutions the groups L11F and GF presented significantly lower means of calcium and phosphorous losses than the control group; and in L11F means were significantly lower than in the fluoride group. Both irradiation parameters tested caused intrapulpal temperature increase below 2 degrees C. Conclusion: It can be concluded that under the conditions of this study, CO(2) laser irradiation (10.6 mu m) with 11J/cm(2) (540 mJ and 10 Hz) of fluoride treated dentine surfaces decreases the loss of calcium and phosphorous in the demineralization process and does not cause excessive temperature increase inside the pulp chamber. (C) 2010 Elsevier Ltd. All rights reserved.

A Role for the Substance P/NK-1 Receptor Complex in Cell Proliferation in Oral Squamous Cell Carcinoma

Objective: To investigate the presence and distribution of substance P (SP) and neurokinin I receptor (NK-IR) in oral squamous cell carcinoma (OSCC) and their relationship with proliferation. Patients and Methods: Ninety OSCCs from 73 patients were immunohistochemically analyzed using monoclonal antibodies against SP, NK-IR and Ki-67 in a case and control study. Results: Seventy-one percent (n=49) of cases expressed SP on tumour cell membrane, 81.3% (n=69) in cytoplasm, 39.4% (n=28) in nucleus, 81.6% (n=71) in infiltrating lymphocytes, and 58.1% (n=43) in peritumoural or intratumoural blood vessels; 14% (n=12) of cases expressed NK-1R on tumour cell membrane, 50% (n=43) in cytoplasm, 48.3% (n=42) in infiltrating lymphocytes and 22.5% (n=18) in tumour blood vessels. All cases expressed Ki-67, which was expressed in >25% of tumour cells in 79.8% of cases (n=63). Direct significant associations were observed in SP expression between different tissue levels (p<0.01), between SP and NK-IR tumour cell membrane expression (p<0.01), and between joint,SP and NK-IR expression in tumour cell cytoplasm and a higher expression of Ki-67 (p<0.05). Conclusion: The ubiquitous presence of SP strongly suggests a role for SP/NK-1R complex in tumour development and progression and possibly for NK-IR antagonists, such as L-773060, in the management of patients with oral cancer.

The Effect of TAK-778 on Gene Expression of Osteoblastic Cells Is Mediated Through Estrogen Receptor

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10-5 M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-P receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders. Exp Biol Med 234:190-199, 2009

Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of CH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.