Autoradiography

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Autoradiography

  • receptor autoradiography


  • Selected Abstracts


    Retention, Distribution, and Effects of Intraosseously Administered Ibandronate in the Infarcted Femoral Head,

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007
    James Aya-ay
    Abstract The local distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted femoral heads were studied. Intraosseous administration effectively delivered and distributed ibandronate in the infarcted femoral heads and decreased the femoral head deformity in a large animal model of Legg-Calve-Perthes disease. Introduction: Bisphosphonate therapy has gained significant attention for the treatment of ischemic osteonecrosis of the femoral head (IOFH) because of its ability to inhibit osteoclastic bone resorption, which has been shown to contribute to the pathogenesis of femoral head deformity. Because IOFH is a localized condition, there is a need to explore the therapeutic potential of local, intraosseous administration of bisphosphonate to prevent the femoral head deformity. The purpose of this study was to investigate the distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted head. Materials and Methods: IOFH was surgically induced in the right femoral head of 27 piglets. One week later, a second operation was performed to inject 14C-labeled or unlabeled ibandronate directly into the infarcted head. 14C-ibandronate injected heads were assessed after 48 h, 3 weeks, or 7 weeks later to determine the distribution and retention of the drug using autoradiography and liquid scintillation analysis. Femoral heads injected with unlabeled ibandronate were assessed at 7 weeks to determine the degree of deformity using radiography and histomorphometry. Results: Autoradiography showed that 14C-Ibandronate was widely distributed in three of the four heads examined at 48 h after the injection. Liquid scintillation analysis showed that most of the drug was retained in the injected head, and almost negligible amount of radioactivity was present in the bone and organs elsewhere at 48 h. At 3 and 7 weeks, 50% and 30% of the 14C-drug were found to be retained in the infarcted heads, respectively. Radiographic and histomorphometric assessments showed significantly better preservation of the infarcted heads treated with intraosseous administration of ibandronate compared with saline (p < 0.001). Conclusions: This study provides for the first time the evidence that local intraosseous administration is an effective route to deliver and distribute ibandronate in the infarcted femoral head to preserve the femoral head structure after ischemic osteonecrosis. In a localized ischemic condition such as IOFH, local administration of bisphosphonate may be preferable to oral or systemic administration because it minimizes the distribution of the drug to the rest of the skeleton and bypasses the need for having a restored blood flow to the infarcted head for the delivery of the drug. [source]


    The Bone Lining Cell: Its Role in Cleaning Howship's Lacunae and Initiating Bone Formation

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2002
    V. Everts
    Abstract In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell "rescues" bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process. [source]


    Radioiodinated clioquinol as a biomarker for ,-amyloid: Zn2+ complexes in Alzheimer's disease

    AGING CELL, Issue 1 2006
    Carlos Opazo
    Summary Neocortical ,-amyloid (A,) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with A, and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([125I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [125I]CQ to synthetic A, precipitated by Zn2+ (Kd = 0.45 and 1.40 nm for A,1-42 and A,1-40, respectively), which was fully displaced by free Zn2+, Cu2+, the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [125I]CQ concentrated in a fraction enriched for both A, and Zn, which was modulated by exogenous addition of Zn2+ or DTPA. APP transgenic (Tg2576) mice injected with [125I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [125I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [125I]CQ. A pilot SPECT study of [123I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated A, species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology. [source]


    Tissue Distribution, Autoradiography, and Metabolism of 4-(2,-Methoxyphenyl)-1-[2, -[N -2,-Pyridinyl)- p -[18F]Fluorobenzamido]ethyl]piperazine (p -[18F]MPPF), a New Serotonin 5-HT1A Antagonist for Positron Emission Tomography

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2000
    An In Vivo Study in Rats
    The in vivo behavior of 4-(2,-methoxyphenyl)-1-[2,-[N -(2,-pyridinyl)- p -[18F]fluorobenzamido]ethyl]-piperazine (p -[18F]MPPF), a new serotonin 5-HT1A antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p -[18F]MPPF and in vitro 8-hydroxy-2-(di- n -[3H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p -[18F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT1A receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p -[18F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT1A specificity of p -[18F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT1A binding sites in the brain. [source]


    Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathway

    NEUROGASTROENTEROLOGY & MOTILITY, Issue 9 2010
    S.-c. Huang
    Abstract Background, Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. Methods, We measured relaxation of guinea pig internal anal sphincter strips caused by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. Key Results , In the guinea pig internal anal sphincter, ET-1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or ,-conotoxin GVIA. ET-2 was as potent as ET-1. ET-3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET-1 = ET-2 > ET-3. The ET-1-induced relaxation was inhibited by BQ-123, an ETA antagonist, but not by BQ-788, an ETB antagonist. These indicate that ETA receptors mediate the relaxation. The relaxant response of ET-1 was attenuated by LY 83583, KT 5823, Rp-8CPT-cGMPS, tetraethyl ammonium, 4-aminopyridine and N(omega)-nitro-l-arginine, but not significantly affected by NG -nitro-l-arginine methyl ester, NG -methyl-l-arginine, charybdotoxin, apamin, KT 5720, and Rp-cAMPS. These suggest the involvement of cyclic guanosine 3,,5,-cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized 125I-ET-1 binding to the internal anal sphincter. Binding of 125I-ET-1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ETA and ETB receptors. Conclusions & Inferences, Taken together, these results demonstrate that ETA receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway. [source]


    Neuropathology of Rett syndrome

    DEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 2 2002
    Dawna Duncan Armstrong
    Abstract Rett Syndrome is unlike any other pediatric neurologic disease, and its clinical-pathologic correlation can not be defined with standard histology techniques. Based on hypotheses suggested by careful clinical observations, the nervous system of the Rett child has been explored utilizing morphometry, golgi preparations, computerized tomography, magnetic resonance imaging, chemistry, immunocytochemistry, autoradiography, and molecular biologic techniques. From these many perspectives we conclude that Rett syndrome is not a typical degenerative disorder, storage disorder, nor the result of gross malformation, infectious or neoplastic processes. There remain regions of the brain that have not been studied in detail but the available data suggest that the neuropathology of Rett syndrome can be summarized as follows: the Rett brain is small for the age and the height of the patient; it does not become progressively smaller over three to four decades; it has small dendritic trees in pyramidal neurons of layers III and V in selected lobes (frontal, motor, and temporal); it has small neurons with an increased neuronal packing density; it has an immature expression of microtubular protein-2 and cyclooxygenase; it exhibits a changing pattern of neurotransmitter receptors with an apparent reduction in many neurotransmitters, possibly contributing to some symptomatology. A mutation in Mecp2 causes this unique disorder of brain development. Neuronal mosaicism for normal and mutated Mecp2 produces a consistent phenotype in the classic female patient and a small brain with some preserved islands of function, but with an inability to support hand use and speech. This paper summarizes our current observations about neuropathology of Rett syndrome. MRDD Research Reviews 2002;8:72,76. © 2002 Wiley-Liss, Inc. [source]


    Differentiation of the epidermis of scutes in embryos and juveniles of the tortoise Testudo hermanni with emphasis on beta-keratinization

    ACTA ZOOLOGICA, Issue 3 2005
    L. Alibardi
    Abstract The sequence of differentiation of the epidermis of scutes during embryogenesis in the tortoise Testudo hermanni was studied using autoradiography, electron microscopy and immunocytochemistry. The study was mainly conducted on the epidermis of the carapace, plastron and nail. Epidermal differentiation resembles that described for other reptiles, and the embryonic epidermis is composed of numerous cell layers. In the early stages of differentiation of the carapacial ridge, cytoplasmic blebs of epidermal cells are in direct contact with the extracellular matrix and mesenchymal cells. The influence of the dermis on the formation of the beta-layer is discussed. The dermis becomes rich in collagen bundles at later stages of development. The embryonic epidermis is formed by a flat periderm and four to six layers of subperidermal cells, storing 40,70-nm-thick coarse filaments that may represent interkeratin or matrix material. Beta-keratin is associated with the coarse filaments, suggesting that the protein may be polymerized on their surface. The presence of beta-keratin in embryonic epidermis suggests that this keratin might have been produced at the beginning of chelonian evolution. The embryonic epidermis of the scutes is lost around hatching and leaves underneath the definitive corneous beta-layer. Beneath the embryonic epidermis, cells that accumulate typical large bundles of beta-keratin appear at stage 23 and at hatching a compact beta-layer is present. The differentiation of these cells shows the progressive replacement of alpha-keratin bundles with bundles immunolabelled for beta-keratin. The nucleus is degraded and electron-dense nuclear material mixes with beta-keratin. In general, changes in tortoise skin when approaching terrestrial life resemble those of other reptiles. Lepidosaurian reptiles form an embryonic shedding layer and crocodilians have a thin embryonic epidermis that is rapidly lost near hacthing. Chelonians have a thicker embryonic epidermis that accumulates beta-keratin, a protein later used to make a thick corneous layer. [source]


    Neonatal maternal separation and enhancement of the inspiratory (phrenic) response to hypoxia in adult rats: disruption of GABAergic neurotransmission in the nucleus tractus solitarius

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2008
    Richard Kinkead
    Abstract Neonatal maternal separation (NMS) alters respiratory control development. Adult male rats previously subjected to NMS show a hypoxic ventilatory response 25% greater than controls. During hypoxia, ,-aminobutyric acid (GABA) release within the nucleus tractus solitarius (NTS) modulates the magnitude of the ventilatory response. Because development of GABAergic receptors is sensitive to NMS, we tested the hypothesis that in adults, a change in responsiveness to GABA within the NTS contributes to NMS-related enhancement of the inspiratory (phrenic) response to hypoxia. Pups subjected to NMS were placed in an incubator for 3 h/day for 10 consecutive days [postnatal days 3 to 12]. Controls were undisturbed. Adult (8,10 weeks old) rats were anaesthetized (urethane; 1.6 g/kg), paralysed and artificially ventilated to record phrenic activity. Rats either received a 50-nL microinjection of GABA (5 µm) or phosphate-buffered saline (sham) within the caudal NTS, or no injection prior to being exposed to hypoxia (FiO2 = 0.12; 5 min). NMS enhanced both the frequency and amplitude components of the phrenic response to hypoxia vs controls. GABA microinjection attenuated the phrenic responses in NMS rats only. This result is supported by ligand binding autoradiography results showing that the number of GABAA receptors within the NTS was 69% greater in NMS vs controls. Despite this increase, the phrenic response to hypoxia of NMS rats is larger than controls, suggesting that the higher responsiveness to GABA microinjection within the NTS is part of a mechanism that aims to compensate for: (i) a deficient GABAergic modulation; (ii) enhancement of excitatory inputs converging onto this structure; or (iii) both. [source]


    MDMA self-administration in rats: acquisition, progressive ratio responding and serotonin transporter binding

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007
    Susan Schenk
    Abstract 3,4-Methylenedioxymethamphetamine (MDMA) self-administration has been shown in animals with extensive drug histories, but only a small number of studies have examined high rates of responding maintained by MDMA in previously drug-naļve animals. In the present study, influence of dose (0.25 or 1.0 mg/kg/infusion) on the acquisition of MDMA self-administration was measured during daily 6-h sessions. Dose,effect data were obtained for MDMA (0.25,1.0 mg/kg/infusion) self-administration under a progressive ratio (PR) schedule of reinforcement. The effect of experimenter- or self-administered MDMA on [3H] paroxetine binding in several brain regions was measured. Acquisition of MDMA self-administration was highly variable and not different for 0.25 or 1.0 mg/kg/infusion progressed with approximately 60% of the rats acquiring reliable self-administration during the 15-day test period. The percentage of rats that acquired MDMA self-administration was lower than the percentage of rats that acquired cocaine (0.5 mg/kg/infusion) self-administration, and cocaine self-administration was acquired with a shorter latency. Responding maintained by MDMA was dose dependent, and breakpoints under a PR schedule increased with dose. Radioligand binding and autoradiography demonstrated lower densities of serotonin transporter sites (SERT) in MDMA self-administering rats as compared with controls across brain regions. The reduction in SERT densities was comparable in magnitude to rats treated with experimenter-administered doses of MDMA. These data support the idea that MDMA is a drug with high abuse liability, and long-term self-administration may lead to long-lasting deficits in serotonin neurotransmission. [source]


    Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanisms

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2007
    G. Allaman-Exertier
    Abstract The lateral septal area is rich in vasopressin V1A receptors and is densely innervated by vasopressinergic axons, originating mainly from the bed nucleus of the stria terminalis and the amygdala. Genetic and behavioral studies provide evidence that activation of vasopressin receptors in this area plays a determinant role in promoting social recognition. What could be the neuronal mechanism underlying this effect? Using rat brain slices and whole-cell recordings, we found that lateral septal neurons are under the influence of a basal GABAergic inhibitory input. Vasopressin, acting via V1A but not V1B receptors, greatly enhanced this input in nearly all neurons. The peptide had no effect on miniature inhibitory postsynaptic currents, indicating that it acted on receptors located in the somatodendritic membrane, rather than on axon terminals, of GABAergic interneurons. Cell-attached recordings showed that vasopressin can cause a direct excitation of a subpopulation of lateral septal neurons by acting via V1A but not V1B receptors. The presence in the lateral septum of V1A but not of V1B receptors was confirmed by competition binding studies using light microscopic autoradiography. In conclusion, vasopressin appears to act in the lateral septum in a dual mode: (i) by causing a direct excitation of a subpopulation of neurons, and (ii) by causing an indirect inhibition of virtually all lateral septal neurons. This modulation by vasopressin of the lateral septal circuitry may be part of the neuronal mechanism by which the peptide, acting via V1A receptors, promotes social recognition. [source]


    Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic mice

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2006
    Daniela Noaķn
    Abstract The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4 -EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4 -EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD. [source]


    Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor ,2 F77I point mutation

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2005
    D. W. Cope
    Abstract Zolpidem is a hypnotic benzodiazepine site agonist with some ,-aminobutyric acid (GABA)A receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of ,2 subunit (,2F77I) point mutant mice. Analysis of forebrain GABAA receptor expression with immunocytochemistry, quantitative [3H]muscimol and [35S] t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with [3H]flunitrazepam and [3H]muscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous ,2I77/I77 and ,2F77/F77 mice. However, quantitative immunoblot analysis of ,2I77/I77 hippocampi showed some increased levels of ,2, ,1, ,4 and , subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 µm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, ,2F77/F77) mice by ,,60%, and peak amplitude by ,,20% at 33,34 °C in vitro. The actions of zolpidem (100 nm or 1 µm) were substantially reduced in ,2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 µm) on carbachol-induced oscillations in the CA3 area of ,2I77/I77 mice was significantly different compared with controls. Thus, the ,2F77I point mutation virtually abolished the actions of zolpidem on GABAA receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the ,2 subunit. [source]


    Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005
    Kevin A. Burns
    Abstract Cell proliferation can be detected by the incorporation of tritiated thymidine (3H-dT) or halopyrimidines during DNA synthesis in progenitor cells. Administration of two thymidine analogues at different times can further determine the cell-cycle kinetics of proliferating cells. Traditionally, this was done by combining bromodeoxyuridine (BrdU) immunocytochemistry and 3H-dT autoradiography, or by BrdU and iododeoxyuridine (IdU) double-labeling using two mouse antibodies. However, these methods either require lengthy exposure time or involve complicated histological procedures for differentiating between two antibodies of the same species. Here we report a simple and reliable method of distinguishing BrdU- and IdU-labeled cells by immunofluorescence. This method uses a mouse monoclonal antibody that recognizes both BrdU and IdU and a rat anti-BrdU antibody that has no cross-reactivity with IdU. When combined with species-specific secondary antibodies that are conjugated to different fluorophores, this method identifies BrdU- and IdU-incorporation as doubly and singly labeled cells, respectively. This method has broad applications. First, we demonstrate that this method can distinguish mouse cortical neurons generated on different embryonic days. Second, by administering IdU and BrdU at varying intervals, we used this method to calculate that the length of S-phase of neural progenitor cells in the adult mouse dentate gyrus is approximately 6 h. Finally, we show that a six-fold higher concentration of IdU detects only 10% more cells than the standard dose of BrdU (50 mg/kg) using the double-labeling method. These results suggest that the standard dose of BrdU is sufficient to label the majority of proliferative populations in the S-phase in pulse labeling experiments. [source]


    Reduced plasticity of cortical whisker representation in adult tenascin-C-deficient mice after vibrissectomy

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2004
    Anita Cybulska-Klosowicz
    Abstract The effect of the extracellular matrix recognition molecule tenascin-C on cerebral plasticity induced by vibrissectomy was investigated with 2-deoxyglucose (2DG) brain mapping in tenascin-C-deficient mice. Unilateral vibrissectomy sparing row C of vibrissae was performed in young adult mice. Two months later, cortical representations of spared row C vibrissae and control row C on the other side of the snout were visualized by [14C]2DG autoradiography. In both wild-type and tenascin-C-deficient mice, cortical representation of the spared row was expanded in all layers of the barrel cortex. However, the effect was significantly more extensive in wild-type animals than in the mutant. Elimination of tenascin-C by genetic manipulation thus reduces the effect of vibrissectomy observed in the somatosensory cortex. No increase in number of fibres in the vibrissal nerve of spared vibrissae was seen, and occurrence of additional nerve to the spared follicle was very rare. Thus, in tenascin-C-deficient mice functional plasticity seems to be impaired within the CNS. [source]


    ,2A and ,2C -adrenoceptor regulation in the brain: ,2A changes persist after chronic stress

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2003
    G. Flügge
    Abstract Stress-induced activation of the central nervous noradrenergic system has been suspected to induce depressive disorders. As episodes of depression often occur some time after a stress experience we investigated whether stress-induced changes in the ,2 -adrenoceptor (,2 -AR) system persist throughout a post-stress recovery period. Brains of male tree shrews were analysed after 44 days of chronic psychosocial stress and after a subsequent 10-day recovery period. Expression of RNA for ,2A and ,2C -adrenoceptors was quantified by in situ hybridization, and receptor binding was determined by in vitro receptor autoradiography. Activities of the sympathetic nervous system and of the hypothalamo,pituitary,adrenal axis were increased during chronic stress but normalized during recovery. ,2A -AR RNA in the glutamatergic neurons of the lateral reticular nucleus was elevated significantly after stress and after recovery (by 29% and 17%). In the dorsal motor nucleus of the vagus, subtype A expression was enhanced after recovery (by 33%). In the locus coeruleus, subtype A autoreceptor expression was not changed significantly. Subtype C expression in the caudate nucleus and putamen was elevated by stress (by 5 and 4%, respectively) but normalized during recovery. Quantification of 3H-RX821002 binding revealed receptor upregulation during stress and/or recovery. Our data therefore show: (i) that chronic psychosocial stress differentially regulates expression of ,2 -adrenoceptor subtypes A and C; (ii) that subtype A heteroreceptor expression is persistently upregulated whereas (iii), subtype C upregulation is only transient. The present findings coincide with post mortem studies in depressed patients revealing upregulation of ,2A -ARs. [source]


    Upregulation of [3H]methyllycaconitine binding sites following continuous infusion of nicotine, without changes of ,7 or ,6 subunit mRNA: an autoradiography and in situ hybridization study in rat brain

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002
    Manolo Mugnaini
    Abstract It is well established that exposure of experimental animals to nicotine results in upregulation of the ,4,2-subtype of neuronal nicotinic acetylcholine receptors (nAChRs). The aim of this study was to determine the effect of nicotine on the levels of ,7-nAChRs in rat brain, for which only partial information is available. Rats were infused with nicotine (3 mg/kg/day) or saline for 2 weeks and their brains processed for receptor autoradiography with [3H]methyllycaconitine (MLA), a radioligand with nanomolar affinity for ,7-nAChRs. In control rats binding was high in hippocampus, intermediate in cerebral cortex and hypothalamus, and low in striatum, thalamus and cerebellum. There was high correlation between the distribution of [3H]MLA binding sites and ,7 subunit mRNA (r = 0.816). With respect to saline-treated controls, nicotine-treated rats presented higher [3H]nicotine binding in 11 out of 15 brain regions analysed (average increase 46 ± 6%). In contrast, only four regions showed greater [3H]MLA binding, among which the ventral tegmental area (VTA) and cingulate cortex (mean increase 32 ± 3%). No changes in ,7 mRNA levels were observed after nicotine treatment. Similarly, there was no variation of ,6 subunit transcript in the VTA, a region which may contain MLA-sensitive (non-,7)-,6*-nAChRs (Klink et al., 2001). In conclusion, nicotine increased [3H]MLA binding, although to a smaller extent and in a more restricted regional pattern than [3H]nicotine. The enhancement of binding was not paralleled by a significant change of ,7 and ,6 subunit transcription. Finally, the present results provide the first anatomical description of the distribution of [3H]MLA binding sites in rat brain. [source]


    Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatum

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000
    P. E. Batchelor
    Abstract After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri-wound dopaminergic sprouting, the peri-wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2 weeks in mice. Knock-down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Dopamine transporter immunohisto-chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri-wound sprouting. Quantification of this effect using [H3]-mazindol autoradiography confirmed that peri-wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri-wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury. [source]


    Activation of spinal cannabinoid 1 receptors inhibits C-fibre driven hyperexcitable neuronal responses and increases [35S]GTP,S binding in the dorsal horn of the spinal cord of noninflamed and inflamed rats

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2000
    L. J. Drew
    Abstract The analgesic potential of cannabinoid (CB) receptor agonists is of clinical interest. Improved understanding of the mechanisms of action of cannabinoids at sites involved in the modulation of acute and sustained inflammatory nociceptive transmission, such as the spinal cord, is essential. In vivo electrophysiology was used to compare the effect of the synthetic CB agonist, HU210, on acute transcutaneous electrical-evoked responses of dorsal horn neurons of noninflamed anaesthetized rats and anaesthetized rats with a peripheral carrageenin inflammation. CB receptor G-protein coupling in lumbar spinal cord sections of noninflamed and carrageenin-inflamed rats was studied with in vitro autoradiography of guanylyl 5,-[,-[35S]thio]triphosphate ([35S]GTP,S) binding. Spinal HU210 significantly inhibited the C-fibre-mediated late (300,800 ms) postdischarge response of dorsal horn neurons of noninflamed and carrageenin-inflamed rats; the CB1 receptor antagonist SR141716A blocked the effect of HU210. HU210 had limited effects on A-fibre-evoked dorsal horn neuronal responses of both groups of rats. HU210 significantly increased [35S]GTP,S binding in the dorsal horn of the spinal cord of both groups of rats compared with basal [35S]GTP,S binding; SR141716A blocked these effects. The predominant effect of spinal HU210, via CB1 receptor activation, was on the C-fibre driven postdischarge responses, a measure of neuronal hyperexcitability following repetitive C-fibre stimulation. Sustained, but not enhanced, antinociceptive effects of HU210 following carrageenin inflammation are reported; CB receptor G-protein coupling was not altered by inflammation. These results strengthen the body of evidence suggesting CB agonists may be an important novel analgesic approach for the treatment of sustained pain states. [source]


    Sweat gland epithelial and myoepithelial cells are vitamin D targets

    EXPERIMENTAL DERMATOLOGY, Issue 2 2007
    Nobuo Koike
    Abstract:, Nuclear receptor binding of 1,25(OH)2 -vitamin D3 (vitamin D) in skin keratinocytes of epidermis, hair sheaths and sebaceous glands was discovered through receptor microscopic autoradiography. Extended experiments with 3H-1,25(OH)2 -vitamin D3 and its analog 3H-oxacalcitriol (OCT) now demonstrate nuclear receptor binding in sweat gland epithelium of secretory coils and ducts as well as in myoepithelial cells, as studied in paws of nude mice after i.v. injection. The results suggest genomic regulation of cell proliferation and differentiation, as well as of secretory and excretory functions, indicating potential therapies for impaired secretion as in hypohidrosis of aged and diseased skin. [source]


    Membrane orientation of laminin binding protein

    FEBS JOURNAL, Issue 18 2003
    An extracellular matrix bridging molecule of Leishmania donovani
    Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source]


    Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

    FEBS JOURNAL, Issue 19 2002
    Yoshiko Iida
    We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121,127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation. [source]


    Association of vasopressin 1a receptor levels with a regulatory microsatellite and behavior

    GENES, BRAIN AND BEHAVIOR, Issue 5 2005
    E. A. D. Hammock
    Vasopressin regulates complex behaviors such as anxiety, parenting, social engagement and attachment and aggression in a species-specific manner. The capacity of vasopressin to modulate these behaviors is thought to depend on the species-specific distribution patterns of vasopressin 1a receptors (V1aRs) in the brain. There is considerable individual variation in the pattern of V1aR binding in the brains of the prairie vole species, Microtus ochrogaster. We hypothesize that this individual variability in V1aR expression levels is associated with individual variation in a polymorphic microsatellite in the 5, regulatory region of the prairie vole v1ar gene. Additionally, we hypothesize that individual variation in V1aR expression contributes to individual variation in vasopressin-dependent behaviors. To test these hypotheses, we first screened 20 adult male prairie voles for behavioral variation using tests that measure anxiety-related and social behaviors. We then assessed the brains of those animals for V1aR variability with receptor autoradiography and used polymerase chain reaction to genotype the same animals for the length of their 5, microsatellite polymorphism in the v1ar gene. In this report, we describe the results of this discovery-based experimental approach to identify potential gene, brain and behavior interrelationships. The analysis reveals that V1aR levels, in some but not all brain regions, are associated with microsatellite length and that V1aR levels in those and other brain regions correlate with anxiety-related and social behaviors. These results generate novel hypotheses regarding neural control of anxiety-related and social behaviors and yield insight into potential mechanisms by which non-coding gene polymorphisms may influence behavioral traits. [source]


    Hypoxia modulates cholinergic but not opioid activation of G proteins in rat hippocampus

    HIPPOCAMPUS, Issue 10 2007
    V.S. Hambrecht
    Abstract Intermittent hypoxia, such as that associated with obstructive sleep apnea, can cause neuronal death and neurobehavioral dysfunction. The cellular and molecular mechanisms through which hypoxia alter hippocampal function are incompletely understood. This study used in vitro [35S]guanylyl-5,- O -(,-thio)-triphosphate ([35S]GTP,S) autoradiography to test the hypothesis that carbachol and DAMGO activate hippocampal G proteins. In addition, this study tested the hypothesis that in vivo exposure to different oxygen (O2) concentrations causes a differential activation of G proteins in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. G protein activation was quantified as nCi/g tissue in CA1, CA3, and DG from rats housed for 14 days under one of three different oxygen conditions: normoxic (21% O2) room air, or hypoxia (10% O2) that was intermittent or sustained. Across all regions of the hippocampus, activation of G proteins by the cholinergic agonist carbachol and the mu opioid agonist [D-Ala2, N-Met-Phe4, Gly5] enkephalin (DAMGO) was ordered by the degree of hypoxia such that sustained hypoxia > intermittent hypoxia > room air. Carbachol increased G protein activation during sustained hypoxia (38%), intermittent hypoxia (29%), and room air (27%). DAMGO also activated G proteins during sustained hypoxia (52%), intermittent hypoxia (48%), and room air (43%). Region-specific comparisons of G protein activation revealed that the DG showed significantly less activation by carbachol following intermittent hypoxia and sustained hypoxia than the CA1. Considered together, the results suggest the potential for hypoxia to alter hippocampal function by blunting the cholinergic activation of G proteins within the DG. © 2007 Wiley-Liss, Inc. [source]


    Receptor architecture of human cingulate cortex: Evaluation of the four-region neurobiological model

    HUMAN BRAIN MAPPING, Issue 8 2009
    Nicola Palomero-Gallagher
    Abstract The structural and functional organization of the human cingulate cortex is an ongoing focus; however, human imaging studies continue to use the century-old Brodmann concept of a two region cingulate cortex. Recently, a four-region neurobiological model was proposed based on structural, circuitry, and functional imaging observations. It encompasses the anterior cingulate, midcingulate, posterior cingulate, and retrosplenial cortices (ACC, MCC, PCC, and RSC, respectively). For the first time, this study performs multireceptor autoradiography of 15 neurotransmitter receptor ligands and multivariate statistics on human whole brain postmortem samples covering the entire cingulate cortex. We evaluated the validity of Brodmann's duality concept and of the four-region model using a hierarchical clustering analysis of receptor binding according to the degree of similarity of each area's receptor architecture. We could not find support for Brodmann's dual cingulate concept, because the anterior part of his area 24 has significantly higher AMPA, kainate, GABAB, benzodiazepine, and M3 but lower NMDA and GABAA binding site densities than the posterior part. The hierarchical clustering analysis distinguished ACC, MCC, PCC, and RSC as independent regions. The ACC has highest AMPA, kainate, ,2, 5-HT1A, and D1 but lowest GABAA densities. The MCC has lowest AMPA, kainate, ,2, and D1 densities. Area 25 in ACC is similar in receptor-architecture to MCC, particularly the NMDA, GABAA, GABAB, and M2 receptors. The PCC and RSC differ in the higher M1 and ,1 but lower M3 densities of PCC. Thus, multireceptor autoradiography supports the four-region neurobiological model of the cingulate cortex. Hum Brain Mapp, 2009. © 2008 Wiley-Liss, Inc. [source]


    Effect of Zn deficiency and subsequent Zn repletion on bone mineral composition and markers of bone tissue metabolism in 65Zn-labelled, young-adult rats

    JOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 7-8 2002
    W. Windisch
    Summary The objective of the present study was to investigate the effect of changing skeletal Zn load (mobilization/restoring) on bone mineral composition and bone tissue metabolism. For this purpose, 36 65Zn-labelled, young-adult female rats were fed with either a purified diet with sufficient Zn (21 ,g/g, control) for 26 days, or deficient Zn (1.4 ,g/g) for 12 days followed by 14 days repletion with the control diet. The animals were killed at the onset of the study (reference: n=4), at the end of the Zn deficiency episode (control: n=4; Zn deficiency: n=4), subgroups (n=4) of Zn repleted animals at repletion days 2, 4, 7, 10 and 14, and at day 14 the remaining controls also (n=4). Zn deficiency reduced skeletal Zn concentration from 198 to 155 ,g/g of bone dry matter. About half of mobilized skeletal Zn was refilled within 2 days of repletion and was completely restored until the end of the study. Concentrations of bone ash, Ca, P and Mg remained constant (means in bone dry matter: 51% bone ash, 191 mg Ca/g, 95 mg P/g, 4.4 mg Mg/g). Blood plasma concentrations of osteocalcin and daily urinary excretions of pyridinoline PYD and dexoxypyridinoline DPD were unaffected by treatment (mean: 57 ng/ml, 222 nmol/day, 137 nmol/day). Also daily urinary excretions of Ca, P and Mg remained fairly constant (means: 0.26 mg/day, 16 mg/day, 1.5 mg/day). 65Zn autoradiography of femur sections revealed a pronounced Zn exchange in the area of the metaphysis and epiphysis. We conclude that transient mobilization and restoration of skeletal Zn occurs mainly in trabecular bone, and does not involve major changes in bone mass, macro mineral content, or bone tissue turnover in young-adult rats. [source]


    Retention, Distribution, and Effects of Intraosseously Administered Ibandronate in the Infarcted Femoral Head,

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007
    James Aya-ay
    Abstract The local distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted femoral heads were studied. Intraosseous administration effectively delivered and distributed ibandronate in the infarcted femoral heads and decreased the femoral head deformity in a large animal model of Legg-Calve-Perthes disease. Introduction: Bisphosphonate therapy has gained significant attention for the treatment of ischemic osteonecrosis of the femoral head (IOFH) because of its ability to inhibit osteoclastic bone resorption, which has been shown to contribute to the pathogenesis of femoral head deformity. Because IOFH is a localized condition, there is a need to explore the therapeutic potential of local, intraosseous administration of bisphosphonate to prevent the femoral head deformity. The purpose of this study was to investigate the distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted head. Materials and Methods: IOFH was surgically induced in the right femoral head of 27 piglets. One week later, a second operation was performed to inject 14C-labeled or unlabeled ibandronate directly into the infarcted head. 14C-ibandronate injected heads were assessed after 48 h, 3 weeks, or 7 weeks later to determine the distribution and retention of the drug using autoradiography and liquid scintillation analysis. Femoral heads injected with unlabeled ibandronate were assessed at 7 weeks to determine the degree of deformity using radiography and histomorphometry. Results: Autoradiography showed that 14C-Ibandronate was widely distributed in three of the four heads examined at 48 h after the injection. Liquid scintillation analysis showed that most of the drug was retained in the injected head, and almost negligible amount of radioactivity was present in the bone and organs elsewhere at 48 h. At 3 and 7 weeks, 50% and 30% of the 14C-drug were found to be retained in the infarcted heads, respectively. Radiographic and histomorphometric assessments showed significantly better preservation of the infarcted heads treated with intraosseous administration of ibandronate compared with saline (p < 0.001). Conclusions: This study provides for the first time the evidence that local intraosseous administration is an effective route to deliver and distribute ibandronate in the infarcted femoral head to preserve the femoral head structure after ischemic osteonecrosis. In a localized ischemic condition such as IOFH, local administration of bisphosphonate may be preferable to oral or systemic administration because it minimizes the distribution of the drug to the rest of the skeleton and bypasses the need for having a restored blood flow to the infarcted head for the delivery of the drug. [source]


    Contribution of Nitric Oxide Synthase to Improved Early Graft Patency in Human Saphenous Vein Graft Harvested by a Novel ,No-Touch' Technique

    JOURNAL OF CARDIAC SURGERY, Issue 6 2002
    JCS Tsui
    Aim: Saphenous vein (SV) is the most commonly used conduit in bypass procedures but has a one-year occlusion rate of 15-30%. A new ,no-touch' technique where the SV is harvested with a cushion of surrounding tissue with no distension has led to improved early patency rates of 5% at 18-months. Nitric oxide (NO), synthesised by nitric oxide synthase (NOS) has properties beneficial to graft patency. Our aim was to study the distribution of NOS in SV harvested by this technique and the effect of distension and removal of perivascular tissue on NOS content of SV. Methods: Following ethical committee approval and patients' informed consent, SVs were harvested from ten patients undergoing coronary artery bypass grafting. A segment of vein was harvested by the conventional technique (surrounding tissue stripped and vein distended with saline); another part was stripped but not distended (,control') and the remaining parts harvested by the ,no-touch' technique. Samples of each segment were taken and transverse sections prepared for NOS identification using 3[H]L-NG nitroarginine (NO Arg) autoradiography and NADPH-diaphorase histochemistry. NOS isoforms were studied using standard immunohistochemistry. Endothelial cells and nerves were also identified using immunohistochemistry with CD31 and NF200 respecitvely, to confirm sources of NOS. Morphometric analysis of NADPH-diaphorase staining was carried out to study tissue NOS content. Results: NO Arg binding representing NOS was preserved on the lumen of ,no-touch' vessels whilst that on conventional and control vessels was reduced. NOS was also localised to the medial smooth muscle cells of all vein segments and to the intact adventitia of ,no-touch' segments. This was confirmed by NADPH-diaphorase staining, which revealed a mean reduction of NOS by 19.5% (p < 0.05, ANOVA) in control segments due to stripping of surrounding tissue alone and a reduction of 35.5% (p < 0.01, AVNOVA) in conventional segments due to stripping and distension, compared to ,no-touch' segments. Adventitial NOS sources in ,no-touch' vessels corresponded to vasa vasorum and paravascular nerves. All three NOS isoforms contributed to the preserved NOS in ,no-touch' vessels. Conclusions: Apart from preserved lumenal NOS, NOS sources are also located in the media and adventitia of SV grafts. These are reduced by both adventitial damage and vein distension during conventional vein harvesting. The ,no-touch' technique avoids these procedures, preserving NOS sources. This may result in improved NO availability in SV harvested by this technique, contributing to the improved patency rates reported. [source]


    Effects of estrogen and progesterone treatment on rat hippocampal NMDA receptors: Relationship to Morris water maze performance

    JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2004
    Nahid K. El-Bakri
    Abstract Estrogen modulates NMDA receptors function in the brain. It increases both dendritic spine density and synapse number in the hippocampus, an effect that can be blocked by NMDA antagonist. In this study, we investigated the effect of 17,-estradiol and progesterone treatment on NMDA receptors in ovariectomized rats. Two different doses were used for 10 weeks. Receptor autoradiography was done on brain sections using [3H] MK-801 as a ligand. Our results showed a significant increase in [3H] MK-801 binding in the dentate gyrus, CA3 and CA4 areas of the hippocampus of ovariectomized compared to sham operated rats. In addition, we observed similar changes in CA1. 17,-estradiol treatment in both doses reduced the binding back to the normal level while progesterone treatment did not show any effect. Spatial reference memory was tested on Morris water maze task. Ovariectomy severely impaired spatial reference memory. Estradiol but not progesterone treatment significantly improved the memory performance of the ovariectomized rats. Low dose treatment showed better learning than high dose estrogen treatment. The decrease in the antagonist sites by estradiol treatment could result in an increase in the sensitivity of the hippocampus to the excitatory stimulation by glutamate system and hence the effect of estradiol on learning and memory. The changes of NMDA receptors in the hippocampus support the concept that estrogen-enhancing effect on spatial reference memory could be through the enhancing of NMDA function. [source]


    Enhancement of poly-adenosine diphosphate-ribosylation in human hepatocellular carcinoma

    JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 5 2000
    Fumio Nomura
    Abstract Background: Poly-adenosine diphosphate (ADP)-ribosylation, catalysed by poly(ADP-ribose) polymerase (PARP), is a post-translational modification of nuclear proteins and is involved in a wide range of biological processes including DNA repair, cell proliferation and malignant transformation. Alteration of this reaction in human hepatocellular carcinoma (HCC) is of interest, but has not yet been explored. The aim of this study was to evaluate poly-ADP-ribosylation and to compare the expression of PARP in HCC and adjacent non-tumour tissues. Methods: Tumorous and adjacent non-tumorous tissues were obtained from five consecutive patients with HCC during surgery for tumour resection. Tissue homogenates were subjected to ADP-ribosylation with [32P]-nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by sodium dodecylsulfate,polyacrylamide gel electrophoresis, followed by autoradiography. Expression of PARP was also evaluated by western blotting. Results: Several proteins were ADP-ribosylated in human HCC tissues. Notably, the radiolabelling of a 116-kDa protein was remarkably greater than that in adjacent non-tumorous tissues (86.5 ± 35.2 arbitrary units by densitometry vs 12.2 ± 9.9, mean± SD, n = 5, P < 0.02). The radiolabelling of the 116-kDa protein was decreased in the presence of PARP inhibitors in a concentration-dependent manner. Immunoblot analyses revealed that the radiolabelled protein was PARP and that its expression was significantly greater in HCC than in adjacent non-tumorous tissues (333 ± 204% of non-tumorous tissue, P < 0.05). Conclusions: We found that poly-ADP-ribosylation and PARP expression were significantly increased in human HCC compared with those in adjacent non-tumorous tissues in surgically obtained specimens. [source]


    18F-Labelled vorozole analogues as PET tracer for aromatase

    JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 5 2008
    Maria Erlandsson
    Abstract One- and two-step syntheses for the 18F-labelling of 6-[(S)-(4-chlorophenyl)(1H -1,2,4-triazol-1-yl)methyl]-1-(2-[18F]fluoroethyl)-1H -benzotriazole, [18F]FVOZ, 1 and 6-[(S)-(4-chlorophenyl)(1H -1,2,4-triazol-1-yl)methyl]-1-[2-(2-[18F]fluoroethoxy)ethyl]-1H -benzotriazole, [18F]FVOO, 2 were developed. In the two-step synthesis, the nucleophilic fluorination step was performed by reacting (S)-6-[(4-chlorophenyl)-(1H -1,2,4-triazol-1-yl)methyl]-1H -benzotriazole (VOZ) with either the 18F-labelled ethane-1,2-diyl bis(4-methylbenzenesulfonate) or the oxydiethane-2,1-diyl bis(4-methylbenzenesulfonate). The radiochemical yields were in the range of 9,13% after the 110,120,min total syntheses and the specific radioactivities were 175±7,GBq/µmol and 56,GBq/µmol for compounds 1 and 2, respectively. In the one-step synthesis, the precursor 2-{6-[(4-chlorophenyl)(1H -1,2,4-triazol-1-yl)methyl]-1H -1,2,3-benzotriazol-1-yl}ethyl 4-methylbenzenesulfonate (7) or 1-[2-(2-bromoethoxy)ethyl]-6-[(4-chlorophenyl)(1H -1,2,4-triazol-1-yl)methyl]-1H -benzotriazole (8) was directly labelled via an 18F nucleophilic substitution to give the corresponding tracer. The labelled compounds were obtained in 36,99% radiochemical yield after 75-min syntheses. The specific radioactivities are 100,GBq/µmol for compound 1 and 80,GBq/µmol for compound 2. In vitro autoradiography using frozen rat brains illustrated specific binding in the medial amygdala, the bed nucleus of stria terminalis and the preoptic area, all of which corresponded well to the result of 11C-labelled vorozole. Copyright © 2008 John Wiley & Sons, Ltd. [source]