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Charles E. Inturrisi - One of the best experts on this subject based on the ideXlab platform.

  • acute and persistent suppression of preproenkephalin mrna expression in the striatum following developmental hypoxic ischemic injury
    Journal of Neurochemistry, 2008
    Co-Authors: Robert E Burke, Steven O Franklin, Charles E. Inturrisi
    Abstract:

    The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative Solution Hybridization assay for preproenkephalin mRNA in conjunction with in situ Hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ Hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ Hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.

  • region specific changes in nmda receptor mrna induced by chronic morphine treatment are prevented by the co administration of the competitive nmda receptor antagonist ly274614
    Molecular Brain Research, 2003
    Co-Authors: Hongbo Zhu, Marina Brodsky, Laurel A Gorman, Charles E. Inturrisi
    Abstract:

    The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative Solution Hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.

  • Attenuation of acute morphine withdrawal in the neonatal rat by the competitive NMDA receptor antagonist LY235959.
    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 2002
    Co-Authors: Kathy L. Jones, Hongbo Zhu, Shirzad Jenab, Charles E. Inturrisi, Gordon A. Barr
    Abstract:

    The present study examined the ability of LY235959, a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, to attenuate behaviors and c-fos mRNA expression associated with acute morphine withdrawal in the infant rat. Rat pups were given a single dose of morphine (10.0 mg/kg, s.c.) or saline. Two hours later, pups were removed from the dam and injected with either LY235959 (10.0 mg/kg, s.c.) or saline. Fifteen minutes later acute morphine withdrawal was precipitated with naltrexone (10.0 mg/kg, s.c.) and behaviors were recorded every 15 s for the next 60 min. Immediately after behavioral testing, brain and spinal cord were assayed for c-fos mRNA analysis by Solution Hybridization. The intensity of the morphine withdrawal syndrome was reduced in pups pre-treated with LY235959. Withdrawal behaviors such as head moves, moving paws, rolling, and walking were decreased, and vocalizations were completely eliminated in pups pre-treated with LY2359559. Acute morphine withdrawal increased c-fos mRNA expression in the brain and the spinal cord, which was attenuated by pre-treatment of LY235959. Thus, in the 7-day-old rat, as in the adult, NMDA receptors play a role in the behavioral and molecular manifestations of acute morphine withdrawal.

  • the effect of the irreversible μ opioid receptor antagonist clocinnamox on morphine potency receptor binding and receptor mrna
    European Journal of Pharmacology, 1995
    Co-Authors: Kawa Chan, Charles E. Inturrisi, Marina Brodsky, Trong Davis, Steve Franklin, Byron C Yoburn
    Abstract:

    In these experiments, the effect of the irreversible mu-opioid receptor antagonist clocinnamox on the potency of morphine, opioid receptor binding and mu-opioid receptor mRNA was examined. Mice were injected with clocinnamox (0.32-12.8 mg/kg) and the analgesic potency of morphine was examined 24 h later. Clocinnamox produced a dose-dependent decrease in the potency of morphine; and at the higher dose of clocinnamox the maximal analgesic effect was not observed following doses of morphine in excess of 500 mg/kg s.c. In saturation binding studies in brain, clocinnamox (0.32-25.6 mg/kg) dose-dependently decreased mu-opioid ([3H][D-Ala2,MePhe4,Gly-ol5]enkephalin; DAMGO) receptor Bmax with relatively minimal effects on Kd. Binding to delta-opioid receptor ([3H][D-Pen2,D-Pen5]enkephalin; DPDPE) and kappa-opioid receptor ([3H](5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec -8-yl) benzeneacetamide; U69,593) was not affected by clocinnamox. The effect of clocinnamox was time-dependent in that the greatest changes in morphine potency and mu-opioid receptor density were observed within 24 h of administration and decreased with time (336 h). Although mu-opioid receptor density was decreased to less than 30% of control 24 h following clocinnamox (12.8 mg/kg) and had increased to 80% by 5 days, a Solution Hybridization assay for mu-opioid receptor mRNA transcript revealed no changes in the steady-state levels of this mRNA. These studies indicate that clocinnamox is an irreversible antagonist at the mu-opioid receptor since it appears to selectively affect receptor density with minimal effects on affinity. Furthermore, clocinnamox produces time- and dose-dependent changes in Bmax and these changes appear to be unrelated to changes in mu-opioid receptor mRNA. It is possible that the repopulation of brain by mu-opioid receptors following clocinnamox is mediated by an existing pool of receptors that are activated following treatment.

Domingo C Salazargarcia - One of the best experts on this subject based on the ideXlab platform.

  • the efficacy of whole human genome capture on ancient dental calculus and dentin
    American Journal of Physical Anthropology, 2019
    Co-Authors: Kirsten A Ziesemer, Jazmin Ramosmadrigal, Allison E Mann, Bernd W Brandt, Krithivasan Sankaranarayanan, Andrew T Ozga, Menno L P Hoogland, Courtney A Hofman, Domingo C Salazargarcia
    Abstract:

    Objectives: Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. Materials and methods: Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in-Solution Hybridization capture and high-throughput sequencing. Results: Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four-fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on-target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum-level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off-target sequences in human genome-enriched calculus libraries may still be useful for oral microbiome reconstruction. Discussion: While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.

Peter Neubauer - One of the best experts on this subject based on the ideXlab platform.

  • Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates
    Microbial Cell Factories, 2003
    Co-Authors: Jari Rautio, Kim Bundvig Barken, Juhani Lahdenperä, Antje Breitenstein, Søren Molin, Peter Neubauer
    Abstract:

    Background A rapid microtiter plate based sandwich Hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following Solution Hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. Results The sensitivity of the assay was approximately 1.2 × 10^9 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. Conclusions The here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

  • sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates
    Microbial Cell Factories, 2003
    Co-Authors: Jari Rautio, Kim Bundvig Barken, Juhani Lahdenperä, Antje Breitenstein, Søren Molin, Peter Neubauer
    Abstract:

    Background A rapid microtiter plate based sandwich Hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following Solution Hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules.

Iain C A F Robinson - One of the best experts on this subject based on the ideXlab platform.

  • steroid regulation of growth hormone gh receptor and gh binding protein messenger ribonucleic acids in the rat
    Endocrinology, 1995
    Co-Authors: Britt G Gabrielsson, Danielle Carmignac, David Flavell, Iain C A F Robinson
    Abstract:

    In the rat, the GH receptor (GHR) and the GH-binding protein (GHBP), which arise from alternative splicing of the same gene, show a sexually dimorphic and GH-dependent expression pattern. Multiple alternative 5'-untranslated regions (UTRs) are present in GHR and GHBP transcripts in the rat, one of which, GHR1, has recently been shown to be liver specific and found at higher levels in females. We have measured the hepatic GHR1, GHR, and GHBP transcript levels, by RNase protection and Solution Hybridization assay, in animals with differing hormonal status, in which hepatic GHR binding and plasma GHBP have been previously assayed. Estradiol (E2) induced GHR1 in males, whereas ovariectomy or the antiestrogen tamoxifen reduced GHR1 expression in females. The induction of GHR1 by E2 was GH dependent, being lower in GH-deficient dwarf rats and absent in hypophysectomized rats, paralleling previous measurements of plasma GHBP and hepatic GHR binding in these animals. Significant changes in GHR1 could explain the ...

Mary Jeanne Kreek - One of the best experts on this subject based on the ideXlab platform.

  • effects of extended access to high versus low cocaine doses on self administration cocaine induced reinstatement and brain mrna levels in rats
    Psychopharmacology, 2004
    Co-Authors: V. P. Yuferov, John R Mantsch, Annemarie Mathieukia, Mary Jeanne Kreek
    Abstract:

    The investigation of rodent cocaine self-administration (SA) under conditions that promote escalating patterns of intake may provide insight into the loss of control over drug use that is central to human addiction. This study examines the effects of daily long-access (LgA) SA of high or low cocaine doses on drug intake, extinction, reinstatement, and brain mRNA levels. Three groups of male Sprague-Dawley rats were trained to self-administer cocaine during multiple-dose sessions. Short-access (ShA) rats were tested daily for multi-dose SA then remained in the chambers for 7 h with no cocaine available. LgA rats had access to low (0.5 mg/kg per infusion; LgA-LD) or high (2.0 mg/kg per infusion; LgA-HD) cocaine doses for 7 h after multi-dose SA. After 14 days, responding was extinguished, cocaine-induced reinstatement was determined, and preproenkephalin (ppENK), preprodynorphin (ppDYN), corticotropin releasing factor (CRF) and dopamine D2 receptor (D2R) mRNA levels were measured in various brain regions using a quantitative Solution Hybridization RNase protection assay. Whereas SA was not altered in ShA rats and only increased during the “loading phase” in LgA-LD rats, a general escalation of intake was found in LgA-HD rats. LgA, particularly LgA-HD, rats were more susceptible to reinstatement than ShA rats. Caudate-putamen ppENK and nucleus accumbens D2R mRNA levels were elevated in LgA-HD rats. Overall, D2R mRNA levels were positively correlated with reinstatement. The escalation of cocaine SA under LgA conditions is dose-dependent and is associated with heightened susceptibility to drug-induced relapse. The characterization of neurobiological alterations that accompany escalated SA should facilitate the identification of mechanisms underlying the onset of human addiction.

  • effects of the androgenic anabolic steroid nandrolone decanoate on adrenocorticotropin hormone corticosterone and proopiomelanocortin corticotropin releasing factor crf and crf receptor1 mrna levels in the hypothalamus pituitary and amygdala of the rat
    Neuroscience Letters, 2000
    Co-Authors: Stefan D Schlussman, Yan Zhou, Pia Johansson, Anne Kiuru, Fred Nyberg, Mary Jeanne Kreek
    Abstract:

    There is increasing abuse of androgenic anabolic steroids (AAS) by non-athletes. AAS abuse has been associated with psychiatric symptoms such as mania, major depression and aggression and the development of dependence. Little is known about the effects of AAS on hypothalamic-pituitary-adrenal axis function or corticotropin releasing factor, which may be involved in mediating some of the psychiatric symptoms associated with AAS abuse. Male Sprague-Dawley rats received one daily intra-muscular injection of the AAS nandrolone decanoate (ND, 15 mg/kg) or vehicle for 3 days. Animals were sacrificed either 1 h or 24 h after the last injection, brain regions dissected and trunk blood collected. Corticotropin releasing factor (CRF), CRF receptor1 (CRF-R1) and proopiomelanocortin (POMC) mRNAs were measured with Solution Hybridization/RNase protection. Circulating levels of corticosterone and adrenocorticotropin hormone (ACTH) were determined using radioimmunoassays. One hour following the last injection, ND significantly increased circulating levels of both corticosterone and ACTH levels. In the amygdala, CRF mRNA levels were unchanged 1 h after the last injection of ND but were significantly reduced at 24 h. The same was found for hypothalamic POMC. No significant AAS effects were observed on: hypothalamic CRF mRNA; POMC mRNA in the amygdala or CRF R1 mRNA in the anterior pituitary.

  • chronic food restriction and streptozotocin induced diabetes differentially alter prodynorphin mrna levels in rat brain regions
    Molecular Brain Research, 1997
    Co-Authors: Yemiliya Berman, Mary Jeanne Kreek, Lakshmi A Devi, Rudolph Spangler, Kenneth D Carr
    Abstract:

    It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative Solution Hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/μg total RNA), bed nucleus of the stria terminalis (4.18 pg/μg), and in caudate nucleus (3.51 pg/μg). Lower levels were observed in the lateral hypothalamus (1.87 pg/μg) and central nucleus of the amygdala (1.22 pg/μg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need.

  • quantitation of preproenkephalin mrna levels in brain regions from male fischer rats following chronic cocaine treatment using a recently developed Solution Hybridization assay
    Molecular Brain Research, 1992
    Co-Authors: Andrea D Branch, Ellen M Unterwald, Susan E Lee, Mary Jeanne Kreek
    Abstract:

    Abstract Quantitative Solution Hybridization assays were used to determine the picogram amounts of preproenkephalin mRNA (PPenk mRNA) and the microgram quantities of total rat RNA in extracts of eight brain regions from rats which had received three daily intraperitoneal injections of cocaine (10 or 30 mg/kg/day) or saline for 14 days. The young adult male Fischer rats were sacrificed 30 min after the final injection. The highest density of PPenk mRNA (pg PPenk mRNA/μg total cellular RNA) was found in extracts of striatum (34.08 ± 1.79 pg/μg for 11 saline-treated rats), followed by extracts of nucleus accumbens (10.08 ± 0.81 pg/μg), and extracts of hypothalamus (2.99 ± 0.31 pg/μg). Extracts of frontal cortex (1.78 ± 0.24 pg/μg), pituitary (1.39 ± 0.08 pg/μg), central grey (1.31 ± 0.16 pg/μg), and cerebellum (1.24 ± 0.09 pg/μg) had intermediate values. Extracts of hippocampus (0.53 ± 0.03 pg/μg) had the lowest density. No significant differences were found among the treatment groups in any brain area investigated. Therefore, chronic cocaine treatment as administered in this protocol did not alter expression of the gene encoding proenkephalin.