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1.
Handb Exp Pharmacol ; (173): 405-22, 2006.
Article in English | MEDLINE | ID: mdl-16594628

ABSTRACT

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.


Subject(s)
Nucleic Acids/chemistry , Nucleic Acids/drug effects , RNA, Complementary/drug effects , Animals , Humans , RNA/drug effects , RNA, Antisense/pharmacology , RNA, Small Interfering/pharmacology
2.
Pharmacopsychiatry ; 37(4): 171-4, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15467974

ABSTRACT

BACKGROUND: Post-mortem studies with brain samples of schizophrenic patients led revealed altered GABA-ergic markers like reduced expression of the GABA transporter GAT-1. Whether this effect is due to the pathophysiology of schizophrenia or to antipsychotic treatment has not been investigated. We therefore established an animal trial of long-term antipsychotic treatment to address this question. METHODS: A total of 33 adult male rats were investigated in three cohorts of 11 animals. One group received clozapine (45 mg/kg/ day), another group haloperidol (1.5 mg/kg/day), and the third one pH-adapted water over a period of 6 months. In situ hybridization with cRNA probes specific for GABA transporters VGAT, GAT-1 and GAT-3 were performed in comparison to control animals. RESULTS: While GAT-1 was upregulated, VGAT expression declined in cortical and limbic brain regions, whereby haloperidol showed a greater effect than clozapine. GAT-3 expression was suppressed in parietal and temporal cortex. CONCLUSIONS: We thus conclude that long-term antipsychotic treatment alters GABA transporter expression in rat. The upregulation of GAT-1 contrasts with the post-mortem finding of reduced GAT-1 expression in schizophrenic patients. Our results facilitate the distinction between disease dependent changes of GABAergic markers and medication effects.


Subject(s)
Antipsychotic Agents/pharmacology , Clozapine/pharmacology , Haloperidol/pharmacology , Membrane Transport Proteins/drug effects , Membrane Transport Proteins/metabolism , Amino Acid Transport Systems/drug effects , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems/metabolism , Animals , Antipsychotic Agents/administration & dosage , Brain/anatomy & histology , Brain/drug effects , Brain/metabolism , Clozapine/administration & dosage , Drug Administration Schedule , GABA Plasma Membrane Transport Proteins , Haloperidol/administration & dosage , In Situ Hybridization , Male , Membrane Transport Proteins/genetics , RNA, Complementary/drug effects , RNA, Complementary/genetics , Rats , Rats, Sprague-Dawley , Time Factors , Vesicular Inhibitory Amino Acid Transport Proteins , Vesicular Transport Proteins/drug effects , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , Water/administration & dosage
3.
Eur J Pharmacol ; 471(1): 9-20, 2003 Jun 13.
Article in English | MEDLINE | ID: mdl-12809947

ABSTRACT

L-[-]-2,3,5,6-Tetrahydro-6-phenylimidazo[2,1b]-thiazole hydrochloride (levamisole) is an anthelmintic that targets the nicotinic acetylcholine receptors of parasitic nematodes. We report here the effects of levamisole on human neuronal alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptors, heterologously expressed in Xenopus oocytes and studied with the voltage clamp method. Applied alone, levamisole was a very weak partial agonist for the two subunit combinations. When co-applied with acetylcholine, micromolar concentrations of levamisole potentiated responses, while millimolar concentrations inhibited them; these effects were complex functions of both acetylcholine and levamisole concentrations. The differences in the levamisole effects on the two receptor combinations suggest that the effects are mediated by the beta subunit. Several combinations of agonist and anthelmintic gave the dual potentiation/inhibition behavior, suggesting that the modulatory effects are general. Levamisole inhibition showed macroscopic characteristics of open channel block. Several results led us to conclude that levamisole potentiation occurs through noncompetitive binding to the receptor. We propose pseudo-site binding for noncompetitive potentiation by levamisole.


Subject(s)
Allosteric Regulation/drug effects , Levamisole/pharmacokinetics , Receptors, Nicotinic/drug effects , Acetylcholine/administration & dosage , Acetylcholine/pharmacokinetics , Action Potentials/drug effects , Animals , Drug Combinations , Drug Synergism , Female , Gene Expression , Humans , Levamisole/administration & dosage , Neurons/chemistry , Oocytes/drug effects , Patch-Clamp Techniques , RNA, Complementary/drug effects , Receptors, Nicotinic/genetics , Xenopus laevis
4.
Can J Physiol Pharmacol ; 81(2): 177-85, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12710532

ABSTRACT

Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smooth muscle cell (VSMC) growth, but which of its Y1-Y5 receptors are involved remains unclear. In quiescent rat VSMCs, NPY receptor mRNAs were undetectable (reverse transcription-polymerase chain reaction), but Y1, Y2, and Y5 expression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold [3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nM concentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low affinity peak by 50% and fully blocked NPY's response when combined. In VSMCs lacking NPY receptors and responsiveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCs with low or no NPY responsiveness, pre-exposure to beta-adrenergic receptor agonist (isoproterenol), forskolin, or dibutyryl cAMP augmented NPY's mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenol only). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is low in nonproliferating cells, amplification of NPY's mitogenic responses requires upregulation of at least the Y1 receptor by NPY itself or beta-adrenergic, cAMP-dependent activation.


Subject(s)
Growth Substances/metabolism , Muscle, Smooth, Vascular/cytology , Neuropeptide Y/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Catecholamines/metabolism , Cell Division/physiology , Cells, Cultured , Cyclic AMP/metabolism , DNA/biosynthesis , DNA, Complementary/genetics , Drug Synergism , In Vitro Techniques , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Neuropeptide Y/antagonists & inhibitors , RNA, Complementary/drug effects , RNA, Complementary/isolation & purification , RNA, Messenger/genetics , Rats , Receptors, Adrenergic, beta/physiology , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide Y/antagonists & inhibitors , Receptors, Neuropeptide Y/drug effects , Receptors, Neuropeptide Y/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Swine , Thymidine/metabolism , Transfection
5.
Bioorg Med Chem Lett ; 12(3): 365-70, 2002 Feb 11.
Article in English | MEDLINE | ID: mdl-11814798

ABSTRACT

The design and synthesis of synthetically modified cyclic A-site 16S rRNA construct is reported. The binding characteristics of several members of the aminoglycoside antibiotics with this novel class of synthetically modified A-site 16S rRNA constructs were subsequently investigated.


Subject(s)
Anti-Bacterial Agents/chemistry , RNA, Ribosomal, 16S/metabolism , Aminoglycosides , Anisotropy , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/metabolism , Base Pairing , Binding Sites , Cyclization , Drug Design , Indicators and Reagents , Kinetics , Nucleic Acid Conformation , RNA, Complementary/drug effects , Structure-Activity Relationship
6.
Brain Res ; 803(1-2): 153-60, 1998 Aug 24.
Article in English | MEDLINE | ID: mdl-9729352

ABSTRACT

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.


Subject(s)
Neurotransmitter Agents/pharmacology , Receptors, AMPA/metabolism , Receptors, Kainic Acid/metabolism , Recombinant Proteins/metabolism , Adrenal Cortex Hormones , Animals , Dose-Response Relationship, Drug , Kainic Acid/antagonists & inhibitors , Kainic Acid/pharmacology , Microinjections , Neurotransmitter Agents/physiology , Oocytes/cytology , Oocytes/drug effects , Pregnenolone/pharmacology , RNA, Complementary/administration & dosage , RNA, Complementary/drug effects , RNA, Complementary/pharmacology , Rats , Receptors, AMPA/drug effects , Receptors, AMPA/genetics , Receptors, Kainic Acid/drug effects , Receptors, Kainic Acid/genetics , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Proteins/drug effects , Xenopus , GluK2 Kainate Receptor
7.
Antimicrob Agents Chemother ; 37(9): 1945-54, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8239610

ABSTRACT

Phosphorothioate oligonucleotides complementary to mRNA of the human cytomegalovirus (HCMV) DNA polymerase gene or to RNA transcripts of the major immediate-early regions 1 and 2 (IE1 and IE2) of HCMV were evaluated for antiviral activity in a 96-well immunoassay with primary human dermal fibroblasts as host cells. Oligonucleotides complementary to RNA of the IE2 region exhibited the most potent antiviral activity. One of these oligonucleotides, ISIS 2922, was at least 30-fold more potent than the nucleoside analog, ganciclovir, with a 50% effective concentration of 0.37 microM in the 96-well immunoassay. In an infectious virus yield reduction assay, ISIS 2922 and ganciclovir reduced production of infectious virus by 2 log units at concentrations of 2.2 and 36 microM, respectively. A control oligonucleotide showed no inhibition of virus production at concentrations as high as 3 microM. ISIS 2922 reduced IE protein synthesis in HCMV-infected cells in a dose-dependent manner which correlated with antiviral activity. The antiviral activity of ISIS 2922 was not due to oligonucleotide-induced cytotoxicity since effects on cell viability or proliferation were observed only at concentrations well in excess of effective antiviral concentrations. The specificity and potency of ISIS 2922 suggest that it may be useful for the treatment of cytomegalovirus disease in humans.


Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Genes, Immediate-Early , RNA, Complementary/drug effects , RNA, Viral/drug effects , Thionucleotides/pharmacology , Base Sequence , Cytomegalovirus/genetics , Cytomegalovirus/metabolism , Cytopathogenic Effect, Viral/drug effects , DNA-Directed DNA Polymerase/genetics , Fibroblasts/drug effects , Humans , Immunoassay , Microbial Sensitivity Tests , Molecular Sequence Data , Transcription, Genetic , Viral Plaque Assay
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