Biomedical subjects

V E Scott

Publications and source records attributed to V E Scott.

22 records · Page 2Linked to original sources

PubMed1990

Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit.

High affinity acceptors for alpha-dendrotoxin, a selective probe for certain fast activating voltage-dependent K+ channels, were purified approximately 4,000-fold from synaptic plasma membranes of bovine cerebral cortex. Although the preparation possessed a low content of high affinity sites for beta-bungarotoxin, antagonism of alpha-dendrotoxin binding by the latter required high concentrations; this indicates that more than one acceptor subtype has been purified. After deglycosylation of the acceptor, the sizes of its subunits were determined electrophoretically to be Mr 65,000 and Mr 39,000. Solid phase microsequencing of these isolated subunits showed that the smaller one had a blocked N terminus, but the Mr 65,000 protein gave a sequence of 27 residues. This is virtually identical to the N-terminal sequence deduced from cDNA of RCK 5, a K+ channel protein from rat brain known to be susceptible to alpha-dendrotoxin. This first report on the partial sequence of any K+ channel protein confirms that the extensive information acquired to date on the alpha-dendrotoxin acceptors is pertinent to functional neuronal K+ channels.

Amino Acid Sequence↗

PubMed1990

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells.

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.

Animals↗

PubMed1988

Specificities and V genes encoding monoclonal autoantibodies from viable motheaten mice.

Several hundred hybridomas were obtained from 1-2-mo-old viable motheaten (mev) mice. Among the Ig-secreting hybridomas tested, greater than 50% (17/33) exhibited reactivity for autoantigens, supporting the idea that the Ly-1 B cells that predominate in mev mice contain frequent precursors of autoantibody-forming cells. Certain of the specificities of these autoantibodies correlated with the documented pathophysiology of mev mice (antithymocyte, -erythrocyte, -skin, -kidney, and -IgG); others were specific for autoantigens not previously observed in motheaten mice but though to be involved in other autoimmune diseases (e.g., intrinsic factor, transferrin, myelin basic protein, and thyroglobulin). About 2 of 3 (11/17) of the self-reactive antibodies exhibited multispecific binding activity for various autoantigens. Analysis by Northern blotting of the V gene families used in mev autoantibodies showed a random usage of VH families and a biased usage of four Vk gene families. Of 16 autoantibodies tested, 12 used a Vk gene from the Vk1, 4, 10, or 19 families. These patterns of Vk gene usage differ from nonautoimmune control animals. Overall, an immunoregulatory defect operating at a more generalized level than the VH or Vk loci, and due to a single gene mutation, appears to be responsible for the multiple immune abnormalities of mev mice.

Animals↗

PubMedDate not supplied

Fluorescence-based functional assay for sarcolemmal ATP-sensitive potassium channel activation in cultured neonatal rat ventricular myocytes.

INTRODUCTION: Activation of ATP-sensitive K+ channels (K(ATP)) has been shown to induce ischemic preconditioning that serves as a protective mechanism in the heart. A high throughput assay for identifying K(ATP) channel openers would therefore be desirable. METHODS: We describe a cell-based 96-well format fluorescence assay using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) to evaluate membrane potential changes evoked by K(ATP) channel openers and blockers in cultured neonatal rat ventricular myocytes. RESULTS: Pinacidil and its analog P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine), ZD6169 (N-(4-benzoylphenyl)-3,3,3,-trifluoro-2-hydroxy-2-methyl propionamide), and the enantiomers of cromakalim evoked concentration-dependent decreases in DiBAC4(3) fluorescence responses. Pretreatment with the K(ATP) channel blocker, glyburide attenuated opener-evoked decreases in fluorescence responses in a concentration-dependent manner. The rank order potency of openers in cardiac myocytes correlated well, but showed 6-10-fold higher potency in activating vascular smooth muscle K(ATP) channels in A10 cells. DISCUSSION: Our studies demonstrate that the pharmacological modulation of sarcolemmal K(ATP) channels can be readily assessed in a high throughput manner by measuring glyburide-sensitive fluorescence changes in cardiac ventricular myocytes.

Adenosine Triphosphate↗