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R Sprengel

Publications and source records attributed to R Sprengel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.

The arginine residue at position 586 of the GluR-B subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.

Alleles↗
PubMed1995

Gene structure of the murine N-methyl D-aspartate receptor subunit NR2C.

The murine N-methyl D-aspartate receptor subunit NR2C (epsilon-3) is encoded by a unique gene composed of 12 translated and three 5'-untranslated exons that spread over approximately 20 kilobases of genomic sequence. The GC-rich promoter that lacks TATA- and CAAT-positioning elements has two transcriptional start sites separated by 18 base pairs. One of these sites is located in a conserved initiator motif and, together with the first four exons, specifies the 5'-untranslated sequence of 772 nucleotides. In this sequence, two alternative splice variants were detected that show identical expression patterns in adult mouse brain. Comparison of intron positions in genes encoding different members of the glutamate receptor family confirms a close evolutionary relationship of the NR2C and NMDAR1 subunit genes.

Alternative Splicing↗
PubMed1994

A nonlinear regularization approach to early vision.

We propose a new class of approaches to smooth visual data while preserving significant transitions of these data as clues for segmentation. Formally, the given visual data are represented as a noisy (image) function g, and we present a class of continuously formulated global minimization problems to smooth g. The resulting function u can be characterized as the minimizer of a specific nonquadratic functional or, equivalently, as the result of an associated nonlinear diffusion process. Our approach generalizes the well-known quadratic regularization principle while retaining its attractive properties: For any given g, the solution u to the proposed minimization problem is unique and depends continuously on the data g. Furthermore, convergence of approximate solutions obtained by finite element discretization holds true. We show that the nodal variables of any chosen finite element subspace can be interpreted as computational units whose activation dynamics due to the nonlinear smoothing process evolve like a globally asymptotically stable network. A corresponding analogue implementation is thus feasible and would provide a real time processing stage for the transition preserving smoothing of visual data. Using artificial as well as real data we illustrate our approach by numerical examples. We demonstrate that solutions to our approach improve those obtained by quadratic minimization and show the influence of global parameters which allow for a continuous, scale-dependent, and selective control of the smoothing process.

Computer Simulation↗
PubMed1994

Molecular biology of glutamate receptors.

The ligand-gated receptors for L-glutamate play a central role in acute neuronal degeneration. Recently cDNAs have been isolated for subunits of several glutamate receptor subtypes. By sequence homology all these subunits clearly belong to one large gene family. Several subfamilies exist and match roughly previously pharmacologically and electrophysiologically defined subtypes of glutamate receptors. Currently four genes (GluR A, B, C and D) are known that code for the AMPA subtypes of glutamate receptors. Recombinant expression of wild type and mutated sequences identified a critical residue in the putative TM2 channel-lining segment that controls Ca2+ ion permeability. The arginine (R) found in GluR B subunits at that position renders AMPA channels impermeable for Ca2+ ions, whereas glutamine (Q) containing GluR A, C and D subunits give rise to Ca2+ permeable channels. RNA editing converts the genomically encoded glutamine codon into the arginine codon found in GluR B cDNAs for the Q/R site. NMDA subtypes of glutamate receptors are formed after coexpression of the NR1 cDNA with a cDNA of the NR2 family. Depending on the member of the NR2 family used, NMDA receptors with different kinetical and pharmacological properties are generated. Common to all channels of these NMDA receptors is a high permeability for Ca2+ ions and a voltage dependent block by Mg2+ ions. All currently known NMDA receptor subunits have an asparagine at the Q/R homologous position. We found that this residue is critical for Mg2+ block and Ca2+ permeability of NMDA receptor channels.

Amino Acid Sequence↗
PubMed1993

RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency.

A functionally critical position (Q/R site) of the AMPA receptor subunit GluR-B is controlled by RNA editing that operates in the nucleus, since in brain and clonal cell lines of neural origin, unspliced GluR-B transcripts occur edited in the Q/R site CAG codon and, additionally, in intronic adenosines. Transfection of GluR-B gene constructs into PC12 cells revealed that the proximal part of the intron downstream of the unedited exonic site is required for Q/R site editing. This intron portion contains an imperfect inverted repeat preceding a 10 nt sequence with exact complementarity to the exon centered on the unedited codon. Single nucleotide substitutions in this short intronic sequence or its exonic complement curtailed Q/R site editing, which was recovered by restoring complementarity in the respective partner strand. Base conversion in the channel-coding region of GluR-B directed by base paired sequences may be executed by a ubiquitous nuclear adenosine deaminase specific for double-stranded RNA.

Adenosine Deaminase↗
PubMed1993

The unique properties of glutamate receptor channels.

Rapid excitatory neurotransmission in the central nervous system (CNS) is mediated predominantly by synaptically released L-glutamate which activates cation selective channels with different kinetic and ion conductance properties. Studies with cloned glutamate receptor channels helped delineate the functional properties of channels defined in subunit composition. Moreover, molecular studies have revealed novel genetic mechanisms controlling the expression of important structural channel determinants.

Electric Conductivity↗
PubMed1993

The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family.

We have characterized a second member (delta-2) of a new class of subunits for the ligand-gated excitatory amino acid receptor superfamily. The sequence of delta-2 exhibits an average identity of 25% and 18.5% to the non-NMDA and NMDA receptor subunits, respectively. The rat delta-2 gene is expressed predominantly in Purkinje cells of the cerebellum whereas only low levels of delta-1 transcripts are found in the adult brain. However, delta-1 gene expression undergoes a pronounced developmental peak, with particularly high mRNA levels in the caudate putamen of late embryonic/early postnatal stages.

Amino Acid Sequence↗
PubMed1993

Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors.

Follicle stimulating hormone (FSH) plays an important role in the regulation of oogenesis, spermatogenesis and production of steroid hormones. Receptors to FSH, which are uniquely expressed in ovarian granulosa and testicular Sertoli cells, are rapidly lost in tissue culture conditions and upon cell transformation. We have succeeded, by triple transfection of primary rat granulosa cells with SV40 DNA, Ha-ras oncogene and an FSH receptor expression plasmid, to establish stable steroidogenic cell lines expressing FSH receptors. The cell lines respond to rat, ovine and bovine FSH, which stimulate progesterone production at levels comparable to primary granulosa cells obtained from preovulatory follicles. No steroidogenic response is detected upon stimulation with ovine luteinizing hormone or human chorionic gonadotropin. The steroidogenic response is accompanied by de novo appearance of adrenodoxin which serves as a marker for the mitochondrial steroidogenic enzyme system. These cells express approximately 27,000 receptors per cell with a Kd of 100-115 pM. This Kd is close to the value calculated for the native receptor. The ED50 for the steroidogenic response to ovine FSH is 200 pM, suggesting a tight coupling between receptor activation and the steroidogenic response. FSH induces pronounced morphological changes in the established cell lines, which are also characteristic of primary granulosa cells. These FSH responsive cell lines can serve as a useful model for the study of the structure and function of the FSH receptor and the effect of oncogenes on its expression.

Animals↗
PubMed1993

A novel putative G protein-coupled receptor highly expressed in lung and testis.

A putative novel G protein-coupled receptor was isolated using the polymerase chain reaction (PCR) and conventional library screening. Degenerate primers corresponding to the thyroid and the sixth transmembrane regions that are conserved among the G protein-coupled receptors were used to amplify selectively gene fragments of new members of this receptor family from a neuroblastoma-glioma cell line cDNA. One of the PCR amplification products that by sequence analysis appeared to contain a partial fragment of a novel G protein-coupled receptor gene was used as a probe to screen a rat forebrain cDNA library to isolate a full-length clone. Sequence comparison indicates that the new gene belongs to the G protein-coupled receptor family and displays the highest structural similarity with the dog RDC1 orphan receptor and several hormone peptide receptors. The mRNA for this putative receptor is not expressed in any specific region of the brain but seems to be localized in a restricted number of peripheral tissues, including lung, testes, and kidney. We suggest that the 4-24 cDNA clone encodes for a novel G protein-coupled receptor that may belong to hormone peptide receptor family.

Amino Acid Sequence↗
PubMed1993

Structure functional expression and spatial distribution of a cloned cDNA encoding a rat 5-HT1D-like receptor.

Using polymerase chain reaction (PCR) a complementary DNA (cDNA) encoding a 5-hydroxytryptamine (5-HT) receptor was isolated from rat forebrain. The amplified cDNA specifies an open reading frame of 374 amino acids comprising seven putative transmembrane regions. Expression of the cloned cDNA in human embryonic kidney cells (HEK 293) was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes containing the cloned receptor showed high affinity binding of [3H]-5-HT. Competition binding experiments with a variety of serotonin receptor ligands displayed a rank order of affinities corresponding to a 5-HT1D subtype: 5-CT > 5-HT = metergoline > CGS 12066 > methysergide > sumatriptan > mianserin = (-)alpha-Me-5-HT = yohimbine > 8-OH-DPAT > or = rauwolscine > spiperone > DOI > propranolol > or = 2-Me-5-HT > or = ICS 205930. Ketanserin and ritanserin displaced [3H]-5-HT-binding in a biphasic manner. In situ hybridization revealed highest expression of the corresponding mRNA in the pyramidal layer of the olfactory tubercle and the nucleus caudatus and accumbens.

Amino Acid Sequence↗
PubMed1992

The murine NPY-1 receptor gene. Structure and delineation of tissue-specific expression.

The murine gene for the NPY-1 receptor subtype for neuropeptide Y was characterized by DNA sequencing and expression studies. It comprises three exons with a 6,400 bp 5'-untranslated and a 80 bp internal intronic sequence. The 5'-flanking region of this gene lacks TATA or CCAAT consensus sequences in the proximity to the multiple transcription initiation sites. A 1,300 bp genomic fragment of the 5'-flanking region drives the expression of the lacZ reporter gene in NG108-15 cells and primary cultured neurons but not in glial and human embryonic kidney cells. In addition, it contains consensus sequences for various transcription factors including cAMP- and glucocorticoid-responsive elements.

Amino Acid Sequence↗
PubMed1992

Heteromeric NMDA receptors: molecular and functional distinction of subtypes.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Amino Acid Sequence↗
PubMed1992

The murine luteinizing hormone and follicle-stimulating hormone receptor genes: transcription initiation sites, putative promoter sequences and promoter activity.

The putative promoter regions of the murine follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptor genes were isolated and used to map transcription initiation sites for both genes. For the FSH receptor gene, a major transcription initiation site was found 534 nucleotides upstream, and for the LH receptor gene 310 nucleotides upstream of the corresponding translation initiation codons. In addition, several alternative minor transcription initiation sites were observed for both genes. The nucleotide sequences of the promoter regions revealed no canonical promoter elements, such as TATA and CCAAT consensus sites 5' of the main transcriptional start sites. The isolated promoter segments for both receptor genes showed low functional activity as verified in transient expression studies in immature rat granulosa cells using the luciferase coding region as the reporter for promoter activity. Both promoter elements seem to be still under tissue specific control, since neither LH receptor nor FSH receptor promoter activity was detectable in another cell line (CHO) investigated.

Amino Acid Sequence↗
PubMed1992

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

We have recently succeeded in immortalizing rat granulosa cells by co-transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha-hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG-R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Köhler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.

20-alpha-Dihydroprogesterone↗
PubMed1992

Neuropeptide Y1 subtype pharmacology of a recombinantly expressed neuropeptide receptor.

Neuropeptide Y (NPY) is an important central and peripheral modulator of neural and endocrine functions. This neuropeptide interacts with at least two pharmacologically distinct receptors, termed Y1 and Y2. At Y1 receptors, the NPY analog [Leu31,Pro34] NPY, but not the carboxyl-terminal fragment NPY-(18-36), displaces radiolabeled NPY and the sequence-related peptide YY, whereas Y2 receptors exhibit the opposite selectivity. We have used cultured mammalian 293 cells for the high level transient expression of a previously cloned putative neuropeptide receptor of rat brain. We report that this receptor displays the ligand binding properties and selectivity of a Y1 receptor, with a single high affinity site for 125I-NPY (Kd, 0.7 +/- 0.2 nM). The functionality of the recombinantly expressed receptor was demonstrated by an inhibition of adenylyl cyclase and a concomitant mobilization of intracellular Ca2+.

Amino Acid Sequence↗
PubMed1992

Glutamate-gated ion channels in the brain. Genetic mechanism for generating molecular and functional diversity.

L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Most cells are responsive to glutamate which activates cation channels with different pharmacological, kinetic, and ion permeability properties. These channels play important roles in neurotransmission, memory acquisition as well as acute and chronic disorders of the brain. The present report summarizes recent knowledge on AMPA (a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) receptors which mediate fast synaptic neurotransmission.

Animals↗
PubMed1991

RNA editing in brain controls a determinant of ion flow in glutamate-gated channels.

L-glutamate, the principal excitatory transmitter in the brain, gates ion channels mediating fast neurotransmission. Subunit components of two related classes of glutamate receptor channels have been characterized by cDNA cloning and shown to carry either an arginine or a glutamine residue in a defined position of their putative channel-forming segment. The arginine residue in this segment profoundly alters, and dominates, the properties of ion flow, as demonstrated for one channel class. We now show that the genomic DNA sequences encoding the particular channel segment of all subunits harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in mRNAs of three subunits. Multiple genes and alternative exons were excluded as sources for the arginine codon; hence, we propose that transcripts for three subunits are altered by RNA editing. This process apparently edits subunit transcripts of the two glutamate receptor classes with different efficiency and selectivity.

Amino Acid Sequence↗