Search PubMed⌕ Search

Biomedical subjects

D O Morgan

Publications and source records attributed to D O Morgan.

At least 109 records · Page 6Linked to original sources
PubMed1986

Mechanisms of receptor-mediated transmembrane communication.

Our experiments with the hIR protein have been designed to address a very general question of transmembrane receptor structure and function: What are the roles and interactions of the various deduced structural domains of such molecules in the initiation of the response of cells to extracellular signals? All of the evidence to date supports the previous hypothesis based on biochemical data that the IR requires ligand-activated TPK functions to initiate the insulin response by cells (for review, see Kahn 1985). Thus, mutations that compromise hIR TPK activity (site-directed point mutations or deletions) result in a concomitant decrease in at least one aspect of insulin action (glucose uptake; Ellis et al. 1986a). Other studies utilizing microinjection of antibodies to inhibit the receptor kinase have extended this conclusion to include a critical role for the receptor kinase in insulin's ability to stimulate ribosomal protein S6 phosphorylation in CHO cells, glycogen synthetase in hepatoma cells, glucose uptake in adipocytes (Morgan and Roth 1987), and frog oocyte maturation (Morgan et al. 1986). Second, analyses of cell lines that express experimentally truncated hIR TPKs demonstrate that, when membrane-anchored, this TPK domain is in fact capable of autonomous hormone-independent IR function: Such cells exhibit a constitutively elevated, insulin-independent uptake of 2-deoxyglucose (Ellis et al. 1987). Finally, by substitution of a homologous TPK for that of hIR, we find that although such a hybrid is capable of insulin-dependent transmembrane signaling (phosphorylation of the hybrid beta-subunit on tyrosine residues), the hybrid IR.ros molecule does not function as an IR in such cells: It mediates neither short-term (uptake of 2-deoxyglucose) nor long-term (incorporation of [3H]thymidine) effects of insulin (L. Ellis et al., in prep.). Together, these results suggest that (1) the hIR TPK domain conveys a substrate specificity for the insulin response and (2) that a functional hIR extracellular domain alone is not sufficient for generation of the insulin response (e.g., ligand-induced aggregation, or simple delivery of insulin into the cell). With the linking of the extracellular and cytoplasmic domains of the hIR molecule has evolved a cellular mechanism for the control of hIR TPK activity; the result is that cells which express the IR are now insulin responsive, and the physiological responses associated with the hormone are ligand-activated. Thus, the uncontrolled state of autonomous TPK activity, with the associated constitutive physiological response (e.g., as exhibited by the spBam hIR mutant), is circumvented.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Insulin and insulinlike growth factor receptors and responses in cultured human muscle cells.

Specific receptors for insulinlike growth factors I and II (IGF-I and IGF-II) were found on cultured human myoblasts and myotubes. In contrast, myotubes but not myoblasts specifically bound insulin and were stimulated by nanomolar concentrations of insulin to take up deoxyglucose. In addition, in myoblasts, physiological concentrations of IGF-I and -II and, to a lesser extent, insulin stimulated two- to threefold the uptake of the nonmetabolizable amino acid analogue methylaminoisobutyric acid (MAIB). In myotubes, uptake of MAIB was stimulated preferentially by IGF-I. Monoclonal antibodies that preferentially recognize either the insulin receptor or the IGF-I receptor were utilized to examine which receptors mediated the biological effects of these hormones. The effects of insulin on both myoblasts and myotubes appeared to be mediated in part by the insulin receptor and in part by the IGF-I receptor. In myotubes, the effects of IGF-I and -II both appeared to be mediated through the IGF-I receptor. In myoblasts, the effects of the two IGFs appeared to be in part mediated by the IGF-I receptor and in part mediated by either the IGF-II receptor or another type of IGF-I receptor. The present results suggest that cultured human muscle cells provide a useful model system in which to study the biological actions of insulin and the IGFs.

Aminoisobutyric Acids↗
PubMed1985

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.

Animals↗
PubMed1985

Plate binding assay for monoclonal anti-receptor antibodies.

A new procedure has been developed for rapid identification of monoclonal antibodies to the insulin receptor. The technique uses the ability of polyvinyl chloride plates coated with anti-mouse antibodies to specifically adsorb antibodies in hybridoma supernatants. Detection of anti-receptor antibodies is accomplished by allowing the adsorbed anti-receptor antibodies to bind radiolabeled hormone-receptor complexes. The method does not require pure receptor and can be adapted for studies of any receptor or binding protein where a labeled ligand is available. With small amounts of the insulin receptor (20-50 ng), the method could detect antibody concentrations as low as 300 pM. In summary, the assay is sensitive, rapid, and requires small amounts of impure receptor.

Animals↗
PubMed1985

Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle.

Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant. The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection. The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected. The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines.

Animals↗
PubMed1984

Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

Dose-Response Relationship, Drug↗
PubMed1984

Location of neutralizing epitopes defined by monoclonal antibodies generated against the outer capsid polypeptide, VP1, of foot-and-mouth disease virus A12.

The epitopes of six monoclonal antibodies generated against type A12 foot-and-mouth disease virus (FMDV) VP1 or its largest cyanogen bromide fragment (13 kd) were characterized. Five of these monoclonal antibodies neutralized viral infectivity. Solid-phase and competitive antigen binding assays using virion-derived antigens or a biosynthetic VP1 polypeptide identified two distinct neutralizing epitopes. One epitope was located between amino acid residues 145-168 of VP1 and the other between amino acids 169-179. The results indicate that antibodies reacting with two distinct areas of the VP1 polypeptide are capable of neutralizing FMD virus.

Animals↗
PubMed1984

Biochemical map of polypeptides specified by foot-and-mouth disease virus.

Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical polypeptides, were immunoprecipitated with the various antisera. To further establish identity, some proteins were compared by partial protease digestion. Evidence for a membrane association of the polypeptides coded for by the middle genome region is also presented. A biochemical map of the foot-and-mouth disease virus genome was established from the above information.

Animals↗
PubMed1984

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.

Animals↗
PubMed1983

Identification of amino acid and nucleotide sequence of the foot-and-mouth disease virus RNA polymerase.

Foot-and-mouth disease virus (FMDV) RNA polymerase was purified from the polyethylene glycol (PEG)-treated supernatant of infected cell media by a combination of ion-exchange chromatography, membrane molecular filtration, and affinity chromatography. The purified RNA polymerase which migrated as a single band of 56,000 molecular weight on a polyacrylamide gel was subjected to automated Edman degradation and the sequence of the first 30 amino acid residues established. On the basis of previous evidence, which indicated that the RNA polymerase was the most 3'-translated polypeptide, plasmids containing cDNA mapping at the 3' end of the genome were characterized by restriction enzyme analysis and nucleotide sequencing. These investigations definitively established the derived amino acid sequence by confirmation of 28 of the amino terminal residues determined by amino acid sequence analysis; the location of the FMDV RNA polymerase coding region at the extreme 3' end of the genome, 96 nucleotides from the poly(A) tail; and the N-terminal cleavage point of the RNA polymerase from its precursor P100 was found to be a glutamic acid-glycine bond.

Amino Acid Sequence↗
PubMed1983

Immunogenicity of foot-and-mouth disease virus type O1 replicated in either monolayer or suspended BHK cell system.

The efficacy of vaccines formulated from the 10th passage of foot-and-mouth disease virus (FMDV) type O1 in monolayer baby hamster kidney (BHK) cells and the 8th passage in suspension BHK cells was compared in steers. The vaccines were inactivated with ethylenimine, contained an equal amount of antigen and were emulsified in oil-adjuvant. Six animals were vaccinated with each vaccine. During the challenge of immunity (91 days post-vaccination, DPV), one out of the six steers from the monolayer vaccine group became infected with the challenge virus while none of the six steers from the suspension vaccine group contracted the disease during the test period. The neutralizing antibody titers (means) of the serum samples taken at different DPV also did not suggest significant variation between these vaccines. In addition exposure to FMDV infected animals demonstrated that both vaccines elicited an immune state in the vaccinates.

Animals↗
PubMed1982

Foot-and-mouth disease virus: immunogenicity and structure of fragments derived from capsid protein VP and of virus containing cleaved VP.

Peptide fragments were obtained from the immunogenic capsid protein VP3, ca. 24 kilodaltons (kd), of foot-and-mouth disease virus type A12 119ab by three procedures: (1) spontaneous proteolysis of in virion VP3 in tissue cultures to produce a 15 kd peptide, designated S fragment; (2) trypsin treatment of purified virus to produce a 16 kg peptide, designated T fragment; and (3) cyanogen bromide cleavage of purified VP3 to produce a 13 kd fragment. Following isolation and purification by gel electrophoresis, VP3 and each of the three fragments were immunogenic for livestock. Lyophilization appeared to impair the immunogenicity of VP3. In addition, viruses containing VP3 fragments produced either by the spontaneous- or trypsin-induced proteolysis were as immunogenic as virus with its VP3 intact. Amino acid sequencing of N-terminal regions revealed that the S fragment was homologous with the N-terminus of VP3, whereas the 13 kd fragment possessed a unique N-terminus. Thus, putative common immunogenic amino acid sequences would appear to reside within an overlap region of the 15 kd S and 13 kd fragments. Sequencing of cDNA prepared to viral genome RNA provided three kinds of information: it (1) placed the above overlap region in the second and third quarters of VP3; (2) demonstrated that the codons for the C-terminus of VP1 and N-terminus of VP3 are contiguous; and (3) supported earlier evidence that these same codons program a chain reversal where VP1 and VP3 are joined in the precursor polyprotein.

Amino Acid Sequence↗
PubMed1981

Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine.

A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

Amino Acid Sequence↗
PubMed1980

Oxytocin action: lack of correlation between receptor number and tissue responsiveness.

Brattleboro rats exhibit diabetes insipidus (DI) because of a genetic autosomal recessive defect in the synthesis of vasopressin; oxytocin is synthesized normally. Preliminary work suggests that elevated circulating oxytocin levels may compensate for the absence of vasopressin. To evaluate the consequences of presumed elevations of oxytocin levels, oxytocin binding and tissue responsiveness have been measured in the uterus and epididymal fat cells of homozygous-DI (HoDI) and heterozygous-DI (HeDI) animals and Sprague-Dawley and Long-Evans controls. Surprisingly, whereas membranes from HoDI rat uteri exhibited an 85% reduction in oxytocin binding, the biological response (contraction) to oxytocin was indistinguishable from the uteri of HeDI or Sprague-Dawley animals. The uterine response to carbachol was also normal in HoDI rats. In contrast, in adipocytes from HoDI animals, the biological response to oxytocin (glucose oxidation) was abolished, whereas the binding of oxytocin was normal; insulin-stimulated glucose oxidation was, however, normal. These results indicate that receptor binding, while critical to hormone action, is not the sole determining factor. With oxytocin action, postreceptor mechanisms are most important in determining oxytocin responsiveness.

Adipose Tissue↗