GPR56, a novel secretin-like human G-protein-coupled receptor gene.

A novel gene product, GPR56, with homology to the seven transmembrane-domain receptor superfamily, has been cloned by PCR amplification using degenerate oligonucleotide primers and subsequent screening of a human heart cDNA library. The isolated 2.8-kb cDNA clone encodes a protein of 693 amino acids that shows highest identity (32%) to HE6, a member of a subclass of the class B secretin-like G-protein-coupled receptors. Northern analysis of various human tissues revealed a wide distribution of the transcript with highest levels found in thyroid gland, brain, and heart. In situ hybridization analysis of human thyroid gland as well as rat heart and brain tissue confirms these results and identifies the hippocampus and hypothalamic nuclei as brain areas with particularly high expression of GPR56 mRNA. The high level of mRNA expression, its wide distribution, and the mucin-like extracellular domain of the receptor protein suggest a possible role for this receptor in cell-cell interaction processes. The human gene for GPR56 has been isolated and its exon-intron structure determined. The total length of the human GPR56 gene is approximately 15 kb, and it consists of 13 exons. Fluorescence in situ hybridization, PCR analysis of somatic cell hybrids, and interspecific mouse backcross mapping have localized the genes to human chromosome 16q13 and mouse chromosome 8.

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.

Assignment of the Y4 receptor gene (PPYR1) to human chromosome 10q11.2 and mouse chromosome 14.

The human and mouse genes for the neuropeptide Y4 receptor have been isolated, sequenced, and shown to contain no introns within the coding region of the gene. Nonisotopic in situ hybridization and interspecific mouse backcross mapping have localized the genes to human chromosome 10q11.2 and mouse chromosome 14. Five nucleotide variants, which do not alter the protein sequence, have been identified within the coding region of the human receptor gene. The human Y4 subtype is most closely related to the Y1-receptor subtype (42%), suggesting that it evolved from an ancestral Y1-like receptor via an RNA-mediated transpositional event.

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.

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.

Release of proinsulin from the human fetal beta cell.

beta cells in the human fetal pancreas are immature in that they release little or no insulin in response to nutrients, such as glucose. The aim of this study was to examine further the immaturity of these cells, specifically regarding the storage and release of the precursor of insulin, proinsulin. Explants of human fetal pancreas were cultured in vitro for 3 weeks. Levels of proinsulin remained relatively constant throughout at 0.04 +/- 0.002 (S.E.M.) pmol/mg per day with a molar ratio of proinsulin to insulin of 2.2 +/- 0.11%. This low ratio was slightly greater than that observed in culture medium conditioned by adult human islets (0.3 +/- 0.1%), but similar to that found in acid-ethanol extracts of cultured explants (1.4 +/- 0.3%). Passaging of human fetal pancreas for 3 months in diabetic nude mice, which should have caused some maturation of the fetal beta cell, did not change the proportion of proinsulin present. Culture of explants in the presence of 12-O-tetra-decanoylphorbol-13-acetate resulted in some inhibition of proinsulin release, but much less than that for insulin, so that the molar ratio increased to 15.4 +/- 1.6% from the control 3.5 +/- 0.3%. Static stimulation of cultured explants with 10 mmol Ca2+/l, 10 mmol theophylline/l, and these two agents together caused 15-, 4- and 10-fold enhancement respectively of proinsulin release; glucose, leucine, arginine and KCl had no effect. In contrast, all these agents caused significant insulin release, the last four to a much smaller extent (less than or equal to three fold) than the first three (10-, 19- and 65-fold respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of international sensitivity index (ISI) of thromboplastins on precision of international normalised ratios (INR)

The contribution of the thromboplastin international sensitivity index (ISI) to the interlaboratory coefficient of variation (CV) of the international normalised ratio (INR) with individual reagents was assessed. In theory the precision of the INR should increase with lower ISI values. An empirical relation has been established between the ISI, the INR, and its CV for two rabbit thromboplastins used in sufficient numbers for analysis in the United Kingdom. This was based on the cumulative data from the United Kingdom National External Quality Assessment Scheme (NEQAS) surveys over two years beginning in 1986. The actual precision achieved in NEQAS for the two reagents depends on the ISI value of the thromboplastin and it agreed closely with the figure predicted by the empirical model. The findings show that the ISI value of a thromboplastin strongly influences the interlaboratory variability of the INR obtained with it. The CV of the INR approximates to the CV of the prothrombin ratio multiplied by the ISI. Manufacturers of thromboplastin should therefore be encouraged to produce reagents showing good precision of results reported as simple prothrombin ratios and a low ISI value to avoid impairment in precision when ratio results are transformed to INR.

Survey of prothrombin time in National External Quality Assessment Scheme exercises (1980-87).

National External Quality Assessment Scheme surveys on the prothrombin time test carried out in hospitals in the United Kingdom have been performed at regular intervals since 1972. Performance has been assessed by comparing observed variability between hospitals with that predicted by a statistical model. The model was based on results from 53 survey plasmas issued between 1980 and 1987. These showed a linear correlation between logarithms of mean and standard deviation of reported ratios. Precision improved until the human brain thromboplastin, Manchester Comparative Reagent, was withdrawn in January 1986. There then followed a pronounced overall deterioration which, by October 1987, had not corrected to the levels achieved by 1985. When the recent results from 1986-87 were analysed according to Quick test reagent only one reagent (ISI 1.1) showed an improvement in precision. Performance of the other Quick test reagents, all with higher ISI values, had not regained the standards of precision previously achieved by the human brain reagent.

Calibration of BCT/441, the ICSH reference preparation for thromboplastin.

An international collaborative exercise has been undertaken to calibrate a secondary international reference preparation (IRP) of thromboplastin on behalf of the International Committee for Standardization in Haematology (ICSH). This preparation of British Comparative Thromboplastin (BCT/441) is required because supplies of the WHO primary IRP (BCT/253) are necessarily limited. The calibration was performed at seven centres with only a small degree of interlaboratory variation. As a result of this study an ISI value of 1.04 has been assigned to the preparation. Opportunity was also taken to assess the reliability of a simplified calibration based on lyophilised plasmas. The results of the latter appeared reliable. BCT/441 will be available to officially designated National Control Laboratories for calibration of local thromboplastins to promote prothrombin time standardization in oral anticoagulant control.

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