Cloning and expression of a novel rat GABAA receptor

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Functional vasoactive intestinal polypeptide (VIP) receptors in human neuroblastoma subclones that contain VIP precursor mRNA and release VIP-like substances

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP). Conditioned medium from SH-IN cells competitively displaced 125I-VIP binding and increased cAMP levels in SH-EP cells, indicating that all of the necessary components for a potential autocrine action of VIP exist in SK-N-SH cells. After numerous cell passages, the SH-EP subclone converted to a distinct phenotype in which VIP precursor mRNA and VIP immunoreactivity in the cell and medium were no longer detectable. In correlation, the VIP receptor number increased, and the EC50 for VIP stimulation of cAMP production shifted to a lower concentration. This points to the possibility that the continuous presence of endogenous VIP in earlier passage SH-EP cells causes a modification in VIP receptor number and cell responsiveness to VIP.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin

Summary Anti-mitochondrial autoantibody and fluorescent derivatives of insulin stain phase-dense mitochondria in acetone-fixed monolayers of fibroblasts. Double fluorochrome studies show mitochondria in close topographic association with intermediate filaments. In cells treated with vinblastine or colchicine, mitochondria are relocated in sites closely associated with coils of perinuclear intermediate filaments. In contrast, autoantibody to polyribosomes stains granules aligned in the long axis of well spread embryonic cells, in the direction of actin-containing fibrils, an arrangement that is lost in cells pretreated with the actin filament disrupting drug cytochalasin B. In more mature fibroblasts, antiribosomal antibody reacts with phase-dense rough endoplasmic reticulum and this staining pattern is not affected by cytochalasin B. The observations suggest that mitochondria are associated with intermediate filaments and that free polyribosomes, but not polyribosomes attached to rough endoplasmic reticulum, are associated with cytoplasmic actin.Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance and Dr. H.A. Ward and staff for preparation of fluorescent conjugates     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Amiloride does not alter NaCl avoidance in Fischer-344 rats

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any preference for NaCl at any concentration in two- bottle preference tests. Because 100 microM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068-0.273 M) in F-344 rats with and without 100 microM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.      

Using the longest significance run to estimate region-specific p-values in genetic association mapping studies

Background  

Association testing is a powerful tool for identifying disease susceptibility genes underlying complex diseases. Technological advances have yielded a dramatic increase in the density of available genetic markers, necessitating an increase in the number of association tests required for the analysis of disease susceptibility genes. As such, multiple-tests corrections have become a critical issue. However the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. Alternatively, we propose here the application of the longest significant run (LSR) method to estimate a region-specific p-value to provide an index for the most likely candidate region.