Biomedical subjects

B Olde

Publications and source records attributed to B Olde.

At least 19 recordsLinked to original sources

PubMed2005

Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion.

Free fatty acids (FFA) have generally been proposed to regulate pancreatic insulin release by an intracellular mechanism involving inhibition of CPT-1. The recently de-orphanized G-protein coupled receptor, FFA(1)R/GPR40, has been shown to be essential for fatty-acid-stimulated insulin release in MIN6 mouse insulinoma cells. The CPT-1 inhibitor, 2-bromo palmitate (2BrP), was investigated for its ability to interact with mouse FFA(1)R/GPR40. It was found to inhibit phosphatidyl inositol hydrolysis induced by linoleic acid (LA) (100 muM in all experiments) in HEK293 cells transfected with FFA(1)R/GPR40 and in the MIN6 subclone, MIN6c4. 2BrP also inhibited LA-stimulated insulin release from mouse pancreatic islets. Mouse islets were subjected to antisense intervention by treatment with a FFA(1)R/GPR40-specific morpholino oligonucleotide for 48 h. Antisense treatment of islets suppressed LA-stimulated insulin release by 50% and by almost 100% when islets were pretreated with LA for 30 min before applying the antisense. Antisense treatment had no effect on tolbutamide-stimulated insulin release. Confocal microscopy using an FFA(1)R/GPR40-specific antibody revealed receptor expression largely localized to the plasma membrane of insulin-producing cells. Pretreating the islets with LA for 30 min followed by antisense oligonucleotide treatment for 48 h reduced the FFA(1)R/GPR40 immunoreactivity to background levels. The results demonstrate that FFA(1)R/GPR40 is inhibited by the CPT-1 inhibitor, 2BrP, and confirm that FFA(1)R/GPR40 is indeed necessary, at least in part, for fatty-acid-stimulated insulin release.

Animals↗

PubMed2001

A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors.

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.

Animals↗

PubMed2000

First-generation monoclonal antibodies identifying the human leukotriene B(4) receptor-1.

The leukotriene B(4) receptor (BLTR) is a seven-transmembrane chemoattractant receptor that is important in pro-inflammatory responses. We have produced the first widely applicable monoclonal antibodies against the human BLTR and confirmed the antibody specificity using flow cytometric analysis of three different cell lines stably expressing the recombinant receptor. The antibodies did not cross-react with the recently cloned second LTB(4) receptor, BLTR2, or the Cys LT1 and Cys LT2 receptors. Functional analysis in combination with two-color flow cytometry showed that the BLTR antibodies bind to cells that are activated by LTB(4). The antibodies were shown to recognize BLTR in cell ELISA and immunocytochemistry. Endogenous expression of BLTR in CD15-positive blood leukocytes and in differentiated HL-60 cells was also demonstrated with the antibodies.

3T3 Cells↗

PubMed2000

Cloning and characterization of cDNA encoding a novel human leukotriene B(4) receptor.

By homology screening using BLAST searches of expressed sequence tags (ESTs), we have found a previously unidentified cDNA encoding a putative seven-transmembrane receptor with highest similarity to the leukotriene B(4) receptor, BLTR. Analysis of calcium flow in transfected cells, along with sequence analysis, revealed that the EST encoded a functionally inactive protein, lacking the segment corresponding to the C-terminal part of the putative receptor protein. The missing segment was obtained by PCR amplification of a human leukocyte cDNA library and ligated to the truncated EST cDNA. The novel cDNA encodes a full-length receptor with 39% identity to the previously cloned BLTR. Studies of intracellular calcium flow of transfected HeLa cells exposed to various leukotrienes showed that also the novel BLTR-like receptor can be activated by leukotriene B(4), and it is therefore tentatively named BLTR2.

Aequorin↗

PubMed1999

Regulation of human D1 dopamine receptor function and gene expression in SK-N-MC neuroblastoma cells.

SK-N-MC human neuroblastoma cells express functional D1, but not D5, dopaminergic receptors. Stimulating cells with dopamine or the D1-selective agonist, SKF R-38393, rapidly (t(1/2) = 1 h) resulted in > 95% attenuation of dopamine-mediated accumulation of cyclic AMP, without any change in D1 dopamine receptor levels. Prolonged (> 4 h) exposure of cells to dopamine attenuated D1 receptor levels to 45-50% of control (t(1/2) = 8 h) and was accompanied by a loss of high-affinity binding sites. At the molecular level, the expression of D1 receptor messenger RNA was bimodal: an initial increase (by approximately 60%) of receptor messenger RNA within 2 h of treatment of cells with dopamine was followed by a decline to 50% below control messenger RNA levels. Low concentrations (1-10 nM) of dopamine also potentiated D1 messenger RNA levels (up to 48%), resulting in a twofold increase in receptor levels. Transfection studies with the cloned human D1 promoter construct, pGL-D1P, indicated that the up-regulation of D1 messenger RNA was due to activation of promoter by dopamine. The dopamine-mediated up-regulation of both D1 receptor messenger RNA and promoter was prevented by the D1-selective antagonist, SCH 23390. The results suggest that dopamine regulates D1 receptor gene and protein expression in a bimodal manner, partly through activation of the receptor promoter. Moreover, the effects of dopamine are independent of the second messenger, cyclic AMP.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Molecular mapping of epitopes involved in ligand activation of the human receptor for the neuropeptide, VIP, based on hybrids with the human secretin receptor.

Receptors for the neurotransmitter and neuroendocrine peptides, vasoactive intesinal peptide (VIP) and secretin, both belong to the Type B subfamily of G-protein-coupled receptors. This group is evolutionally as well as structurally distinct from the much larger Type A, or rhodopsin-type, subfamily. We have mapped the ligand-activating epitopes of the human VIP1 receptor by the use of hybrid receptor constructs with the human secretin receptor. Twelve chimeras were synthesized the successively replacing portions of the former receptor with corresponding portions of the latter receptor, or by interchanging the first extracellular loops. Each of the different chimeric receptor DNAs were then expressed in murine reporter cells, and their ability to activate cAMP production was investigated on stimulation with the respective natural peptide ligands. We stimulated the reporter cells with secretion or VIP following transient expression of the receptor chimeras. The experiments indicated that there are two molecular domains of importance for the recognition and activation of these peptides, namely, the inner portion of the extracellular tail and the first extracellular loop of the two receptors.

Adenylyl Cyclases↗

PubMed1997

Molecular cloning and tissue distribution of cDNA encoding a novel chemoattractant-like receptor.

With the application of a homology screening strategy, including PCR amplification and southern blot hybridization, a novel cDNA was cloned from rat liver and anterior pituitary libraries. It was found to encode a 371-amino acid protein which has the characteristics of a heptahelix receptor and shows structural identity to members of the chemoattractant receptor family. A primary receptor message of 3.5 kb size was identified by northern blot hybridization. This RNA species showed high expression in heart and lung, while expression was lower in small intestines, colon, kidney, liver, uterus, and in brain. Another larger RNA species of 6.3 kb appeared in heart and lung. In situ hybridization histochemistry performed on tissue from liver and kidney revealed a mainly vascular distribution of the receptor message.

Amino Acid Sequence↗

PubMed1997

Leukotriene B4 is the functional ligand binding to and activating the cloned chemoattractant receptor, CMKRL1.

We recently described a novel chemoattractant receptor, provisionally named CMKRL1, which has turned out to be the first cloned leukotriene (LT) receptor. Present binding assays using tritiated LTB4 and isolated membranes from COS-7 cells, transiently transfected with cDNA encoding this receptor, yielded a linear Scatchard plot showing expression of only a single, high-affinity receptor population with a mean Kd of 2.1 nM and Bmax of 17.0 pmoles/mg protein. Sham-transfected cells exhibited no specific binding. LTB4 elicited concentration-dependent increases in intracellular calcium measured with Fura-2 in individual CHO cells stably expressing CMKRL1. No response was seen with sham-transfected control cells, or in calcium-free medium which suggests that calcium mainly originates from extracellular sources. The LTB4-induced cellular calcium increment was blocked in the presence of a monoclonal antibody, raised against a synthetic peptide corresponding to the extracellular tail of CMKRL1 and capable of visualizing the receptor by fluorescence immunocytochemistry. Taken together the analyses show that LTB4 is the endogenous ligand for CMKRL1 which is, thus, identical to the LTB4 receptor, designated BLTR according to the NC-IUPHAR nomenclature.

Animals↗

PubMed1997

Molecular cloning and functional expression of a serotonin receptor from Caenorhabditis elegans.

A cDNA encoding a serotonin receptor has been isolated from a Caenorhabditis elegans mixed stage cDNA library. The nematode serotonin receptor, designated 5HT-Ce, was permanently expressed in murine Ltk-cells, where it mediates adenylate cyclase attenuation. Sequence analysis and the pharmacological profiles demonstrate its relatedness not only to Drosophila and Lymnae 5HT receptors but also to mammalian 5HT1a receptors. The 5HT-Ce-gene does not map close to the position of any known serotonergic mutations.

Adenylyl Cyclase Inhibitors↗

PubMed1996

Pollux, a novel Drosophila adhesion molecule, belongs to a family of proteins expressed in plants, yeast, nematodes, and man.

Adhesion molecules have pivotal roles in cellular processes critical to the development and maintenance of multicellular organisms. Here we describe a new member of the adhesive repertoire encoded by the Drosophila pollux (plx) gene. Marked by a novel 74-amino-acid domain, Plx belongs to a highly conserved family with members in plants, yeast, nematodes, and man, including the human oncoprotein TRE17. Essential for viability, plx mutant analysis indicates that larval death is attributable to asphyxiation brought on by fluid-congested tracheal tubes. Ultrastructural examination of mutant tracheae reveals defects in cell-extracellular matrix contacts. During embryogenesis, Plx uniformly covers the apical surface of cellular blastoderm cells. It is later found regionally concentrated along subsets of central nervous system axon pathways and on the apical surface of the trachea's tubular epithelium. Cell attachment assays demonstrate that Plx can serve as a ligand for cell surface integrins. Plx also contains a motor neuron-selective adhesive site, multiple proteoglycan-binding motifs, and a leucine zipper: all suggest possible associations with additional components of the adhesion complex.

Amino Acid Sequence↗

PubMed1994

Effect of some poly(ethylene glycol)-bound and dextran-bound affinity ligands on the partition of synaptic membranes in aqueous two-phase systems.

Ligands with an apparent affinity for various structural elements on the surface of synaptic membrane fragments have been bound to the polymers poly(ethylene glycol) and dextran. The ligand-polymer derivatives have been included in aqueous two-phase systems composed of water, poly(ethylene glycol) and dextran. The uneven distribution of the polymers resulted in the concentration of the polymer-bound ligand in one of the two phases. The effect of the ligand-polymer on the partition of membranes was studied by using synaptic membranes from calf brain, obtained by standard centrifugation methods. By using ligand-containing two-phase systems for nine-step counter-current distribution of membranes, it was shown that the distribution behaviour of various parts of the membrane preparation could be affected. The distribution was followed by determination of opiate binding, acetylcholinesterase, and total membrane (using protein and light-scattering measurements).

Acetylcholinesterase↗

PubMed1991

Subfractions of membranes from calf brain synaptosomes obtained and studied by liquid-liquid partitioning.

Synaptosomes isolated from calf brain cortex were lysed and fragmented by Yeda press treatment. The obtained membranes have previously been fractionated in a counter-current distribution process using a liquid-liquid two-phase system consisting of water, dextran, Ficoll and poly(ethylene glycol) [J. Chromatogr., 358 (1986) 147]. Using the fact that there are discrete membrane populations, a rapid preparative method for isolation of the two main fractions is presented in the present work, as well as a subfractionation of one of them using liquid-liquid extraction with dextran-bound Procion yellow HE-3G. The content of several membrane constituents, i.e. protein, acetylcholinesterase, succinate dehydrogenase and ATPase, as well as opiate binding, were determined for the three fractions. Counter-current distribution of the fractions elucidates their heterogeneity and the effectiveness of the purification.

Acetylcholinesterase↗

PubMed1991

Complementary DNA sequencing: expressed sequence tags and human genome project.

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

Amino Acid Sequence↗

PubMed1990

Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent.

The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.

Animals↗

PubMed1989

Heterogeneity of a crude synaptosomal preparation, studied by affinity partitioning using hexaethonium-poly(ethylene glycol).

The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.

Animals↗

PubMed1988

Partition and counter-current distribution of membrane particles in aqueous dextran-poly(ethylene glycol) two-phase systems with special reference to synaptosomes.

Aqueous two-phase systems composed of water, dextran and poly(ethylene glycol) can be used for the separation of biological particles. The adjustment of the partition of such particles between the two phases and the interface between them has been studied by using a preparation of synaptosomes (from calf brain cortex) also containing free mitochondria. The partition has been affected by variation of polymer concentrations and addition of salts, e.g. phosphates and chloride. The time for separation of the phases showed a bimodal behaviour with an initially rapid formation of bulk phases followed by a slow phase separation. The relative amount of mixed phases at the time of the transition was proportional to the amount of particles included. Counter-current distribution with moderate time for the phase separation was carried out in such way that the interface material travelled with approximately half the speed of the moving upper phase. In this way the distribution of the particles between the upper phase and the interface as well as between the interface and the lower phase could be studied in the same experiment. The heterogeneity of the synaptosome preparation was clearly demonstrated by counter-current distribution at low polymer concentrations while no separation was obtained when the system contained larger amounts of polymers. Possible reasons for this behaviour are discussed.

Animals↗

PubMed1986

Effect of dextran- and poly(ethylene glycol)-bound procion yellow HE-3G on the partition of membranes from calf brain synaptosomes within an aqueous two-phase system.

Membranes obtained by lysis and Yeda-press treatment of synaptosomes (nerve endings) from calf brain cortex have been partitioned within the aqueous phases (and the interface between them) of a Ficoll-dextran-poly(ethylene glycol)-water two-phase system. By introducing the dye Procion yellow HE-3G in the upper phase, bound to poly(ethylene glycol), or in the lower phase, bound to dextran, the partition of the membranes could be strongly affected. The influence on the partition was more pronounced when the dye was bound to dextran. By using a number of two-phase systems in a counter-current distribution process, it was shown that the membrane preparation was inhomogeneous and that the fractions obtained differed in their contents of acetylcholinesterase, succinate dehydrogenase and ATPase. The affinity partitioning effect depended strongly on the concentration of polymer-bound dye. An optimum dye concentration was found when Procion yellow HE-3G was bound to poly(ethylene glycol). When the same dye was bound to dextran, the number of dye molecules per dextran molecule influenced the effectiveness of the extraction.

Acetylcholinesterase↗

PubMed1985

Affinity partitioning and centrifugal counter-current distribution of membrane-bound opiate receptors using naloxone-poly(ethylene glycol).

Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl-cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3'-phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane-bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes.

Animals↗