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Biomedical subjects

J Shine

Publications and source records attributed to J Shine.

At least 19 recordsLinked to original sources
PubMed1997

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene.

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.

Amino Acid Sequence↗
PubMed1997

Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation.

The human y1 and y5 receptor genes are transcribed in opposite directions from a common promoter region on chromosome 4q31-q32. One of the alternately spliced 5' exons of the y1 receptor gene (1C) is also an integral part of the coding region of a novel neuropeptide Y receptor, Y5. Exon 1C of the y1 receptor gene, if translated from the opposite strand, encodes sequences corresponding to the large third intracellular loop of the Y5 receptor. The close proximity of the two neuropeptide Y receptor genes suggests that they have evolved from a gene duplication event with the small intron interrupting the coding sequence of the y1 gene being converted into a functional sequence within the y5 gene, while the reverse complementary sequence was utilized as an alternatively spliced 5' exon for the y1 gene. The transcription of both genes from opposite strands of the same DNA sequence suggests that transcriptional activation of one will have an effect on the regulation of gene expression of the other. As both Y1 and Y5 receptors are thought to play an important role in the regulation of food intake, coordinate expression of their specific genes may be important in the modulation of NPY activity.

Alternative Splicing↗
PubMed1997

Activity of centrally truncated analogues of neuropeptide Y at Y1 and Y2 receptor subtypes in vivo.

Neuropeptide Y (NPY), a sympathetic cotransmitter, has both prejunctional and postjunctional actions in the cardiovascular system. In anaesthetized rats, the bioassay system used here, NPY attenuates cardiac vagal action (a prejunctional or Y2 action) and increases blood pressure (a postjunctional or Y1 action). Several NPY analogues were tested against NPY. In these, centrally located amino acid sequences of various lengths were removed, and replaced with simpler 'spacers'. As the parent NPY molecule is considered to exist in a U-shape, these central truncations were intended to shorten the depth of the U, while maintaining the integrity of its two ends. The centrally truncated NPY analogues examined here retain activity at both receptor subtypes in vivo. These findings indicate that the U-shape of the parent molecule probably exists to assist stability, but that receptor binding occurs through sequences closer to the termini.

Amino Acid Sequence↗
PubMed1997

Mechanics of the anterior drawer and talar tilt tests. A cadaveric study of lateral ligament injuries of the ankle.

We analyzed the changes in lateral ligament forces during anterior drawer and talar tilt testing and examined ankle joint motion during testing, following an isolated lesion of the anterior talofibular ligament (ATFL) or a combined lesion of the ATFL and calcaneofibular ligament (CFL). 8 cadaver specimens were held in a specially designed testing apparatus in which the ankle position (dorsiflexion-plantarflexion and supination-pronation) could be varied in a controlled manner. Ligament forces were measured with buckle transducers, and joint motion was measured with an instrumented spatial linkage. An anterior drawer test was performed using an 80 N anterior translating force, and a talar tilt test was performed using a 5.7 Nm supination torque with intact ligaments, after sectioning of the ATFL, and again after sectioning of the CFL. The tests were repeated at 10 degrees dorsiflexion, neutral, and 10 degrees and 20 degrees plantarflexion. In the intact ankle, the largest increases in ATFL force were observed during testing in plantarflexion, whereas the largest increases in CFL force were observed in dorsiflexion. Isolated ATFL injury caused only small laxity changes, but a pronounced increase in laxity was observed after a combined CFL and ATFL injury.

Adult↗
PubMed1996

Molecular cloning and characterisation of the mouse preprogalanin gene.

Using a probe obtained by PCR amplification from mouse genomic DNA, a genomic clone was isolated covering the entire mouse preprogalanin gene. The mouse gene has an exon:intron organisation very similar to that of the rat and human genes. The first exon is noncoding while exons 2-5 carry the coding region. Exon 6 also encodes the stop codon and a polyadenylation signal. The deduced amino-acid sequence of mouse preprogalanin is 94% and 68% identical to the rat and human peptide, respectively. The amino-acid sequence of mouse galanin was confirmed by RT-PCR amplification of mouse brain RNA. The cloning of the mouse galanin gene should allow elucidation of the regulatory characteristics of its promoter and facilitate transgenic approaches to the analysis of galanin gene function in this species.

Amino Acid Sequence↗
PubMed1996

Intron 17 of the human retinoblastoma susceptibility gene encodes an actively transcribed G protein-coupled receptor gene.

The human retinoblastoma susceptibility gene, a member of the tumor suppressor gene family, is located on chromosome 13q14.12-13q14.2 and consists of 27 exons that are distributed over 180 kb. This study shows that intron 17, the largest in size, consisting of nearly 72,000 bp, contains an open reading frame encoding a novel G protein-coupled receptor in the reverse orientation relative to the transcription of the retino-blastoma susceptibility gene. Correction of a frameshift mutation revealed that this novel G protein-coupled receptor is the human homolog of a chicken T-cell-specific receptor cDNA. This is an additional description of an actively transcribed protein-encoding gene positioned within an intron of another gene, suggesting that introns can have important structural functions.

Amino Acid Sequence↗
PubMed1996

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.

Adenosine↗
PubMed1995

Expression of the neuropeptide Y Y1 receptor in human embryonic kidney 293 cells: ligand binding characteristics, in situ hybridization and receptor autoradiography.

The human neuropeptide Y Y1 receptor cDNA was transfected into human embryonic kidney 293 (HEK 293) cells and used to determine the selectivity of newly developed Y1 and Y2 radioligands in a model which expresses a single NPY receptor subtype. The Y1 receptor probe, [125I][Leu31,Pro34]PYY, binds with high afinity (KD of 0.4-0.6 nM) to Y1-transfected HEK 293 cells whereas the Y2 radioligand, [125I]PPY3-36 failed to demonstrate any significant labelling. Only non-selective (PYY) or selective Y1 receptor agonists behaved as potent competitors for [125I][Leu31,Pro34]PYY binding in transfected cells. Additionally, the efficacy of the transfection method used was evaluated at both the transcriptional and translational levels. In situ hybridization revealed the heterogeneous distribution of the NPY Y1 receptor mRNA expressed in transfected HEK 293 cells. Similarly, the levels of NPY Y1 binding sites per transfected cell varied as shown using [125I][Leu31,Pro34]PYY receptor autoradiography. Taken together, these results demonstrate that HEK 293 cells transfected with the NPY Y1 receptor cDNA expressed both the related receptor mRNA and protein albeit at different levels depending upon each transfected cell. Additionally, these data further establish the selectivity of the newly developed Y1 and Y2 radioligands.

Autoradiography↗
PubMed1995

Multiple promoters regulate tissue-specific expression of the human NPY-Y1 receptor gene.

Several cDNA clones encoding the human neuropeptide Y-Y1 receptor have been isolated that contain differing sequences at their 5'-ends. The divergence occurs at a splice junction in the 5'-untranslated region, suggesting that at least three forms of the neuropeptide Y-Y1 receptor transcript are generated by alternative splicing at this site. Genomic clones have been isolated that encompass the alternatively spliced 5'-exons. The exons are found 6.4, 18.4, and 23.9 kilobases upstream of exon 2. In the corresponding promoter regions of the various exons, possible response elements for the glucocorticoid receptor, as well as potential binding sites for the AP-1, AP-2, and NF-kappa B transcription factors are found. Analysis of NPY-Y1 transcripts in various cell types demonstrates the tissue-specific activation of the three promoters.

Alternative Splicing↗
PubMed1995

Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production.

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1995

Gene duplication of the human peptide YY gene (PYY) generated the pancreatic polypeptide gene (PPY) on chromosome 17q21.1.

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are structurally related but functionally diverse peptides, encoded by separate genes and expressed in different tissues. Although the human NPY gene has been mapped to chromosome 7, we demonstrate here that the genes for human PYY and PP (PPY) are localized only 10 kb apart from each other on chromosome 17q21.1. The high degree of homology between the members of this gene family, both in primary sequence and exon/intron structure, suggests that the NPY and the PYY genes arose from an initial gene duplication event, with a subsequent tandem duplication of the PYY gene being responsible for duplication of the PYY gene being responsible for the creation of the PPY gene. A second weaker hybridization signal also found on chromosome 17q11 and results obtained by Southern blot analysis suggest that the entire PYY-PPY region has undergone a further duplication event.

Amino Acid Sequence↗
PubMed1995

Seminalplasmin: recent evolution of another member of the neuropeptide Y gene family.

Seminalplasmin, the major basic protein of bull semen, an important regulator of calcium transport in bovine sperm and a positive modulator of the zona pellucida-induced acrosome reaction, is shown to be a recently created member of the neuropeptide Y gene family. Sequence analysis of the bovine peptide YY-pancreatic polypeptide gene cluster reveals an unexpected and extensive homology between seminalplasmin and the neuropeptide Y gene family, at the level of both gene structure and primary amino acid and nucleotide sequences. The extremely high degree of homology to the peptide YY gene, in both coding and especially noncoding regions, suggests that the seminalplasmin gene has arisen by a very recent gene duplication of the bovine peptide YY gene. Despite the more than 95% nucleotide sequence identity, a few specific mutations in the seminalplasmin gene have resulted in both the loss of the amino- and carboxyl-terminal cleavage sites characteristic of all other members of the neuropeptide Y family and the acquisition of a function apparently unrelated to the neurotransmitter/endocrine role of peptide YY.

Amino Acid Sequence↗
PubMed1995

Characterization of the 5'-flanking region of the human preprogalanin gene.

Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and -122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO-K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor.

Animals↗
PubMed1995

Ligand binding analysis of human neuropeptide Y1 receptor mutants expressed in E. coli.

Site-directed mutants of the human neuropeptide Y1 (NPY Y1) receptor expressed as a maltose binding protein fusion protein in E. coli show identical ligand binding parameters compared with the same mutants expressed in mammalian cells using a vaccinia virus expression system. However, it was remarkable that two receptor mutants, which were initially classified as non-binding when expressed in an eukaryotic expression system, could actually be revealed to have wild-type binding activity when expressed in E. coli. Re-expression and retesting of these mutants in mammalian cells confirmed this result. This shows that bacterial expression can be used as a fast, versatile and valuable alternative to mammalian expression systems for the analysis of ligand binding sites in G-protein coupled receptors.

ATP-Binding Cassette Transporters↗
PubMed1994

Isolation and chromosomal localization of a novel human G-protein-coupled receptor (GPR3) expressed predominantly in the central nervous system.

Degenerate oligonucleotide primers designed against known G-protein-coupled receptors were used in polymerase chain reaction amplification to isolate a novel receptor sequence (R4) from a rat insulinoma cell line and its human homolog (GPR3) from a human neuroblastoma cDNA library. The novel human receptor sequence is expressed in low abundance predominantly in the central nervous system and at low levels in the lung and kidney. The gene encoding GPR3 is intronless within the coding region, contains at least one intron in the 5'-untranslated region, and has been localized to chromosome 1p34.3. The activating ligand for the homologous receptors R4 and GPR3 is not known, but sequence similarity with the closely related orphan rat receptor R334 [FEBS Lett. 292:243 (1991)] suggests that R334 and the homologous receptors R4 and GPR3 probably represent two discrete molecular subtypes that interact with the same or closely related ligands.

Amino Acid Sequence↗