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Biomedical subjects

P Schloss

Publications and source records attributed to P Schloss.

At least 19 recordsLinked to original sources
PubMed1995

The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C.

Treatment of human embryonic kidney 293 cells expressing the rat gamma-aminobutyric acid (GABA) transporter 1 (GAT1) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to decrease the velocity of specific [3H]GABA uptake. This downregulation varied with extracellular GABA concentration and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and staurosporine. An about 50% reduction of uptake velocity by PMA treatment was observed at GABA concentrations > 1 microM, whereas only a minor effect was seen at low substrate concentrations. These data indicate that GAT1 activity is downregulated by PKC activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Heterogeneity of antidepressant binding sites on the recombinant rat serotonin transporter SERT1.

Antidepressant drugs block the uptake of serotonin into serotonergic nerve terminals and blood platelets. Here, binding of the tricyclic antidepressant [3H]imipramine to the recombinant rat serotonin transporter SERT1 expressed in human embryonic kidney cells was found to be nonhomogeneous. Scatchard analysis and competition experiments revealed the existence of two distinct antidepressant binding sites. At site 1, [3H]imipramine binding was strictly sodium-dependent with an apparent KD of approximately 10 nM. In contrast, [3H]imipramine binding to site 2 occurred also in the absence of sodium and exhibited a lower affinity. Binding of the nontricyclic antidepressant [3H]citalopram was observed only at site 2. The natural substrate of this carrier, serotonin, competitively inhibited antidepressant binding at both sites; however, its affinity to site 2 was approximately 5-fold lower. These data provide a molecular explanation for the distinct pharmacological actions of different antidepressants.

Animals↗
PubMed1995

Modulation of a recombinant glycine transporter (GLYT1b) by activation of protein kinase C.

Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [3H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.

Amino Acid Transport Systems, Neutral↗
PubMed1994

Na(+)-dependent high-affinity uptake of choline into cultured fibroblasts.

The acetylcholine precursor choline is transported into cholinergic neurons by a high-affinity, sodium-dependent mechanism that is selectively localized to the cholinergic nerve terminal. In addition, a low-affinity, sodium-independent choline uptake system is present in cholinergic and non-cholinergic cells which deliver choline for cell membrane anabolism. Here, we show that uptake of [3H]choline into cultured fibroblast cell lines exhibits high affinity (Km < or = 10 microM), is sodium-dependent, and is blocked by hemicholinium, a classical inhibitor of neuronal high-affinity choline uptake. Our data indicate that sodium-dependent high-affinity choline transport systems are also present in non-cholinergic cells.

Biological Transport, Active↗
PubMed1994

The putative rat choline transporter CHOT1 transports creatine and is highly expressed in neural and muscle-rich tissues.

A rabbit homolog of the putative rat choline transporter CHOT1 has recently been shown to mediate creatine transport (Guimbal and Kilimann, J. Biol. Chem., 1993). Here, the functional properties of the CHOT1 cDNA were studied by heterologous expression in HEK-293 cells. After transfection, the cells displayed a 7-8fold stimulation of [14C]creatine uptake with a Km of 46.2 microM. This transport was inhibited by the creatine uptake inhibitor beta-guanidinopropionate (Ki of 23 microM). Northern blot analysis revealed a major transcript of 4.8 kb in brain, heart, skeletal muscle and kidney. In situ hybridization showed high transcript levels in the cerebellum, hippocampus and other brain regions. High expression was also seen in the rat embryo along the entire neuraxis and in some non-neuronal tissues. These data establish CHOT1 as a widely expressed rat creatine transporter.

Animals↗
PubMed1994

Neurotransmitter transporters: new members of known families.

The identification of two gene families encoding neurotransmitter transporters was a major step towards a better understanding of these proteins and their function in neurotransmission. The recent isolation of additional members of these families underscores their high molecular diversity and implies a delicate regulation of transmitter uptake.

Animals↗
PubMed1993

Extended school year programs: a community-driven curriculum model.

Appropriate education guaranteed to children with disabilities under P.L. 94-142 has been translated by the courts to mean more than 180 days of education. There is now a strong legal precedent establishing the right to an extended school year (summer school) for students with severe disabilities. The courts have left open to interpretation, however, issues related to the program implementation. Descriptions of extended school year programs vary widely and include respite for parents, continuation of the regular school year curriculum, and remediation in skill areas. The only consensus seems to be that these programs should be tailored to individual needs. In the present article a systematic approach to curriculum development for students with severe disabilities enrolled in extended school year programs was described. This approach is (a) community driven, (b) consistent with each student's IEP objectives for the regular school year, (c) tailored to the individual student, and (d) consistent with current best practices of teaching functional skills performed by individuals without disabilities in a variety of integrated nonschool settings. Student progress data were presented.

Activities of Daily Living↗
PubMed1992

Glutamate receptors of Drosophila melanogaster: cloning of a kainate-selective subunit expressed in the central nervous system.

We report the isolation and functional characterization of cDNAs encoding a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits significant homology to mammalian glutamate receptor subunits. Injection of DGluR-I cRNA into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and philanthotoxin. DGluR-I transcripts are differentially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1992

Cross-linking of 125I-alpha-bungarotoxin to Drosophila head membranes identifies a 42 kDa toxin binding polypeptide.

The nicotinic acetylcholine receptor (nAChR) antagonist alpha-bungarotoxin (alpha-Btx) binds to two different classes of high affinity binding sites from the Drosophila central nervous system. We have used the bivalent reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to cross-link 125I-alpha-Btx (M(r) = 8 kDa) to Drosophila head membranes. Upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), one major adduct of M(r) approximately 50 kDa was identified, suggesting that a 42 kDa polypeptide binds the toxin. Adduct formation was inhibited by other cholinergic ligands. Detergent-solubilized receptor complexes containing the cross-linked products were immunoprecipitated by antisera against two nAChR subunits previously identified by molecular cloning, the ALS and ARD proteins, suggesting that the 42 kDa toxin binding polypeptide constitutes a component of the previously described class 1 alpha-Btx binding site.

Animals↗
PubMed1992

Neurotransmitter transporters. A novel family of integral plasma membrane proteins.

The re-uptake of neurotransmitters into the nerve terminal terminates synaptic transmission at most central synapses and constitutes a key step in the modulation of synaptic efficacy. Recently, the cloning of several Na(+)-driven neurotransmitter transporters has resulted in the description of a novel family of homologous membrane proteins, each with 12 transmembrane segments. These transporters constitute major targets of widely used drugs, and modulation of transporter gene expression and/or activity may represent an important substrate for plasticity in the nervous system.

Amino Acid Sequence↗
PubMed1992

Primary structure and functional expression of a choline transporter expressed in the rat nervous system.

Synthesis of the neurotransmitter acetylcholine in cholinergic nerve terminals is regulated by a sodium-driven high-affinity choline uptake system in the plasma membrane. We have isolated cDNAs from rat spinal cord and brainstem which encode a choline transporter (CHOT1). The predicted protein shares considerable amino acid identity and several structural features including twelve putative transmembrane regions with other neurotransmitter transporters. Expression of in vitro transcribed CHOT1 RNA in Xenopus oocytes generated Na(+)-dependent choline uptake, which was not seen in control oocytes. Amplification by polymerase chain reaction (PCR) revealed significant amounts of CHOT1 mRNA in brain, cerebellum, spinal cord and, to a lesser extent, heart, but only very low expression in lung, kidney and muscle.

Amino Acid Sequence↗
PubMed1991

Isolation of cDNAs encoding a novel member of the neurotransmitter transporter gene family.

Degenerate oligonucleotides deduced from two regions of the rat gamma-aminobutyric acid (GABA) transporter were used to amplify related sequences from rat spinal cord by polymerase chain reaction (PCR). The resulting product then allowed isolation of two overlapping cDNA clones with an open reading frame encoding a hydrophobic polypeptide of 630 amino acids. This protein, termed NTT, exhibits 38% sequence homology to the GABA and 48% to the noradrenaline transporters and contains 12 putative transmembrane regions. PCR amplification revealed low-level expression of NTT transcripts in spinal cord, but not in brain and cerebellum.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle.

Insects and other invertebrates use glutamate as a neurotransmitter in the central nervous system and at the neuromuscular junction. A complementary DNA from Drosophila melanogaster, designated DGluR-II, has been isolated that encodes a distant homolog of the cloned mammalian ionotropic glutamate receptor family and is expressed in somatic muscle tissue of Drosophila embryos. Electrophysiological recordings made in Xenopus oocytes that express DGluR-II revealed depolarizing responses to L-glutamate and L-aspartate but low sensitivity to quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. The DGluR-II protein may represent a distinct glutamate receptor subtype, which shares its structural design with other members of the ionotropic glutamate receptor family.

Amino Acid Sequence↗
PubMed1991

Neuronal nicotinic acetylcholine receptors in Drosophila: antibodies against an alpha-like and a non-alpha-subunit recognize the same high-affinity alpha-bungarotoxin binding complex.

ALS and ARD proteins are thought to represent a ligand binding and a structural subunit, respectively, of Drosophila nicotinic acetylcholine receptors (nAChRs). Here, antibodies raised against fusion constructs encompassing specific regions of the ALS and ARD proteins were used to investigate a potential association of these two polypeptides. Both ALS and ARD antisera removed 20-30% of the high-affinity binding sites for the nicotinic antagonist 125I-alpha-bungarotoxin (125I-alpha-Btx) from detergent extracts of fly head membranes. Combinations of both types of antisera also precipitated the same fraction of alpha-Btx binding sites, a result suggesting that both polypeptides are components of the previously defined class I 125I-alpha-Btx binding sites in the Drosophila CNS. 125I-alpha-Btx binding to a MS2 polymerase-ALS fusion protein containing the predicted antagonist binding region showed that the ALS protein indeed constitutes the ligand binding subunit of a nicotinic receptor complex. These data are consistent with neuronal nAChRs in Drosophila containing at least two types of subunits, ligand binding and structural ones.

Animals↗
PubMed1990

Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated alpha-subunit.

Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or alpha 2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR alpha-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila.

Amino Acid Sequence↗
PubMed1989

Central nicotinic acetylcholine receptors in the chicken and Drosophila CNS: biochemical and molecular biology approaches.

Putative neuronal nicotinic acetylcholine receptors (nAChRs) were investigated using biochemical and molecular biology approaches. In the chick visual system, a nicotinic cholinergic binding site was localized on a polypeptide of Mr 57,000 using the potent antagonist, alpha-bungarotoxin (alpha-Btx). Ion flux experiments indicate that this membrane protein is different from the toxin-insensitive nAChR present in vertebrate neurons. Crosshybridization with a Torpedo nAChR cDNA probe allowed isolation of a cholinergic receptor cDNA (ARD) from the Drosophila CNS. Analysis of the corresponding gene, its transcripts and the ARD protein are presented here.

Animals↗
PubMed1988

Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system.

Animals↗