ABSTRACT
RATIONALE AND OBJECTIVE: The aim of this study was to investigate the possible facilitating effect of the partial NMDA receptor agonist D-cycloserine (DCS) on memory consolidation of conditioned sexual responses and to examine the capability of DCS to reduce context-specificity of learning. METHODS: In a randomized placebo-controlled double-blind trial, 50 healthy females were exposed to a differential conditioning procedure. Two pictures of a male abdomen were used as conditional stimuli (CSs), of which one (the CS+) was followed by the unconditional stimulus (US), a genital vibrotactile stimulus. After the conditioning session on day 1, participants received either 125 mg of DCS or a placebo. The effects of DCS on affect, sexual arousal and US expectancy in response to the CS+ and CS- were examined 24 h after the conditioning procedure. RESULTS: A main effect of DCS was found on affect at the first test trials (p = 0.04, ηp2 = 0.09), and a similar non-significant but trend level effect was found for sexual arousal (p = 0.06, ηp2 = 0.07), which appeared to persist over a longer time (p = 0.07, ηp2 = 0.08). Unexpectedly, ratings of positive affect and sexual arousal in response to both the CS+ and the CS- were higher in the DCS condition compared to the control condition, possibly indicating that DCS administration reduced stimulus specificity. Since the results did not show clear evidence for context learning, we were not able to test effects on context-specificity of learning. CONCLUSION: Although largely inconclusive, the results provide tentative support for a facilitating effect of DCS on affect and sexual arousal in response to stimuli that were presented in a sexual conditioning procedure, however, no conclusions can be drawn about effects of DCS on sexual reward learning, since the design and results do not lend themselves to unambiguous interpretation.
Subject(s)
Conditioning, Classical/drug effects , Cycloserine/pharmacology , Memory Consolidation/drug effects , Receptors, N-Methyl-D-Aspartate/agonists , Sexual Behavior/drug effects , Sexual Behavior/psychology , Adult , Clitoris/drug effects , Clitoris/physiology , Conditioning, Classical/physiology , Double-Blind Method , Emotions/drug effects , Emotions/physiology , Extinction, Psychological/drug effects , Extinction, Psychological/physiology , Female , Humans , Male , Memory Consolidation/physiology , Photic Stimulation/methods , Reward , Sexual Behavior/physiology , Vibration , Young AdultABSTRACT
For more than a decade, researchers have been trying to develop non-invasive imaging techniques for the in vivo measurement of viable pancreatic beta cells. However, in spite of intense research efforts, only one tracer for positron emission tomography (PET) imaging is currently under clinical evaluation. To many diabetologists it may remain unclear why the imaging world struggles to develop an effective method for non-invasive beta cell imaging (BCI), which could be useful for both research and clinical purposes. Here, we provide a concise overview of the obstacles and challenges encountered on the way to such BCI, in both native and transplanted islets. We discuss the major difficulties posed by the anatomical and cell biological features of pancreatic islets, as well as the chemical and physical limits of the main imaging modalities, with special focus on PET, SPECT and MRI. We conclude by indicating new avenues for future research in the field, based on several remarkable recent results.
Subject(s)
Insulin-Secreting Cells/diagnostic imaging , Molecular Imaging/methods , Animals , Humans , Insulin-Secreting Cells/transplantation , Islets of Langerhans Transplantation/diagnostic imaging , Magnetic Resonance Imaging/methods , Mice , Positron-Emission Tomography/methods , Rats , Tomography, Emission-Computed, Single-Photon/methodsABSTRACT
INTRODUCTION: A national surveillance programme (ONAP project) was created in France in 1996 by two professional societies to estimate the incidence and identity the characteristics of occupational asthma. MATERIALS AND METHODS: In 2001 and 2002 chest physicians and occupational physicians in Alsace were intensively solicited for better voluntary reporting of cases of occupational asthma. The objective of this study was to evaluate the consequences of such a procedure on the number of cases reported, with a view to collecting comprehensive data. RESULTS: The mean annual incidence of occupational asthma was estimated at 126 cases per million workers with a female predominance (52.4%). Flours and isocyanates represented 40% of the suspected agents. Isocyanate asthma (21% of the total) was reported mainly in workers in the car supply industry, and seems to be a specific feature of the region. Persulfates represented 5.3% of the cases; latex and aldehydes 2.6%. The study also points to emergent aetiologies and work risks, i.e. quaternary ammonium compounds in disinfecting detergent products used by cleaners and healthcare workers. CONCLUSION: This study, which allowed better assessment of the real incidence of OA in Alsace and better detection of substances and occupations at risk, is an incentive to continue our Surveillance programme.
Subject(s)
Asthma/epidemiology , Occupational Diseases/epidemiology , Registries , Adult , Asthma/chemically induced , Asthma/diagnosis , Asthma/etiology , Female , France/epidemiology , Humans , Incidence , Isocyanates/adverse effects , Male , Middle Aged , Occupational Diseases/chemically induced , Occupational Diseases/diagnosis , Occupational Diseases/etiology , Occupations , Population Surveillance , Risk Factors , Sex FactorsABSTRACT
The Dubin-Johnson syndrome is an inherited disorder characterized by conjugated hyperbilirubinemia. The deficient hepatobiliary transport of anionic conjugates is caused by the absence of a functional multidrug-resistance protein 2 (MRP2, symbol ABCC2) from the apical (canalicular) membrane of hepatocytes. Mechanisms underlying this deficiency may include rapid degradation of mutated MRP2 messenger RNA (mRNA) or impaired MRP2 protein maturation and trafficking. We investigated the consequences of the mutation MRP2Delta(R,M), which leads to the loss of 2 amino acids from the second ATP-binding domain of MRP2. The MRP2Delta(R,M) mutation is associated with the absence of the MRP2 glycoprotein from the apical membrane of hepatocytes. Transfection of mutated MRP2 complementary DNA (cDNA) led to an MRP2Delta(R,M) protein that was only core glycosylated, sensitive to endoglycosidase H digestion, and located in the endoplasmic reticulum (ER) of transfected HEK293 and HepG2 cells. This indicated that deletion of Arg1392 and Met1393 leads to impaired maturation and trafficking of the protein from the ER to the Golgi complex. Inhibition of proteasome function resulted in a paranuclear accumulation of the MRP2Delta(R,M) protein, suggesting that proteasomes are involved in the degradation of the mutant protein. This is the first mutation in Dubin-Johnson syndrome shown to cause deficient MRP2 maturation and impaired sorting of this glycoprotein to the apical membrane.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Gene Deletion , Jaundice, Chronic Idiopathic/genetics , Jaundice, Chronic Idiopathic/metabolism , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins , Protein Processing, Post-Translational , ATP Binding Cassette Transporter, Subfamily B/genetics , Amino Acid Sequence/genetics , Cell Line/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cysteine Endopeptidases/physiology , Fluorescent Antibody Technique , Green Fluorescent Proteins , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Humans , Indicators and Reagents , Leupeptins/pharmacology , Luminescent Proteins , Microscopy, Confocal , Microscopy, Immunoelectron , Molecular Sequence Data , Multidrug Resistance-Associated Protein 2 , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/physiology , Mutation/genetics , Proteasome Endopeptidase Complex , Tissue DistributionABSTRACT
The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Aniline Compounds/pharmacokinetics , Fluorescent Dyes/pharmacokinetics , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins , Xanthenes/pharmacokinetics , Aniline Compounds/antagonists & inhibitors , Bile Acids and Salts/antagonists & inhibitors , Bile Acids and Salts/pharmacokinetics , Biological Transport/drug effects , Biological Transport/physiology , Cell Membrane/metabolism , Cyclosporine/pharmacology , Fluorescent Antibody Technique , Humans , Immunoblotting , Multidrug Resistance-Associated Protein 2 , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Tissue Distribution , Tumor Cells, Cultured , Vacuoles/metabolism , Xanthenes/antagonists & inhibitorsABSTRACT
The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Gene Expression Regulation , Mitochondrial Proteins , Multidrug Resistance-Associated Proteins , Ribosomal Proteins/genetics , Saccharomyces cerevisiae Proteins , 2-Acetylaminofluorene/pharmacology , Base Sequence , Cloning, Molecular , DNA, Complementary , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Hydroxamic Acids/pharmacology , Immunoblotting , Molecular Sequence Data , Multidrug Resistance-Associated Protein 2 , Nocodazole/pharmacology , Promoter Regions, Genetic , RNA, Messenger/genetics , Transfection , Tumor Cells, CulturedABSTRACT
The polarized rat hepatoma/human fibroblast hybrid cell line, WIF-B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF-B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190-kd membrane glycoprotein mediating adenosine triphosphate (ATP)-dependent transport of glucuronides, glutathione S-conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF-B cells was shown by reverse-transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789-bp fragment. Immunoblotting, using antibodies reacting with the amino-terminal or with the carboxyl-terminal sequence of rat Mrp2, detected the 190-kd glycoprotein in WIF-B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF-B cells with a membrane-permeable ester of the calcium-dependent fluorescent indicator, Fluo-3, was followed by Mrp2-mediated secretion of the amphiphilic anion, Fluo-3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP-dependent transport into Mrp2-containing plasma membrane vesicles in comparison with Mrp2-deficient vesicles established that Fluo-3 is transported by Mrp2 with a Km value of 3.7 micromol/L. Our results indicate that the polarized WIF-B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo-3.
Subject(s)
Carrier Proteins/genetics , Gene Expression , Liver Neoplasms, Experimental/metabolism , Adenosine Triphosphate/pharmacology , Aniline Compounds/metabolism , Animals , Anion Transport Proteins , Biological Transport, Active/drug effects , Cell Membrane/metabolism , Fibroblasts/metabolism , Fluorescent Antibody Technique , Fluorescent Dyes , Humans , Hybrid Cells , Immunoblotting , Male , Polymerase Chain Reaction , RNA, Messenger/analysis , Rats , Rats, Wistar , Tumor Cells, Cultured , Xanthenes/metabolismABSTRACT
A membrane glycoprotein of 190 kDa has been identified previously by photoaffinity labeling as a candidate for the ATP-dependent export pump for leukotriene C4 in mastocytoma cells [Leier, I., Jedlitschky, G., Buchholz, U. & Keppler, D. (1994) Eur. J. Biochem. 220, 599-606]. The present study indicates that this protein represents the murine homolog of the human multidrug resistance protein (MRP). In immunoblot analyses several polyclonal anti-MRP antibodies and one monoclonal antibody recognized the protein of 190-kDa in plasma membranes of mastocytoma cells. Immunoprecipitation after photoaffinity labeling with [3H]leukotriene C4 precipitated the labeled 190-kDa glycoprotein. Deglycosylation by glycopeptide N-glycosidase F of mastocytoma membrane proteins was performed in comparison with membranes from MRP-overexpressing cells and resulted in a reduction of the molecular mass of 190 kDa by about 20 kDa in all membrane preparations. The expression of the murine mrp gene in the mastocytoma cells was analyzed by amplification and sequencing of two mrp cDNA fragments in the first nucleotide binding domain (182 bp) and in a domain proximal to the 3'-end (291 bp). The deduced amino acid sequences of these fragments were identical with murine Mrp and 86.7% and 89.7% identical with the corresponding sequences of human MRP. These results indicate that the ATP-dependent release of leukotriene C4 by murine mastocytoma cells is mediated by murine Mrp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Leukotriene C4/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , DNA Primers , Glycopeptides/chemistry , Glycopeptides/isolation & purification , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Mast-Cell Sarcoma , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
ATP-dependent transport of glutathione and glucuronate conjugates from hepatocytes into bile is mediated by a distinct member of the ATP-binding cassette superfamily. We have cloned and sequenced the canalicular isoform of the multidrug resistance protein from rat liver, and termed it cMrp. This membrane glycoprotein is composed of 1541 amino acids with an identity of 47.8% with the human multidrug resistance protein (MRP) and of 41.9% with the yeast cadmium factor (YCF1). The carboxyl-terminal 130 amino acids of the human hepatocyte canalicular isoform of MRP (cMRP) were 80.2% identical with rat cMrp. cMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/TR- mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane. Immunoblotting using an antibody raised against the carboxyl terminus of cMrp detected the glycoprotein of about 190 kDa only in the canalicular membrane from normal liver. Double immunofluorescence and confocal laser scanning microscopy localized cMrp exclusively to the canalicular membrane domain of hepatocytes and demonstrated its loss in the hyperbilirubinemic mutant rat. The results identify cMrp as a canalicular transport protein with a novel sequence and with a function similar to the one of the MRP.