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Biomedical subjects

D O Morgan

Publications and source records attributed to D O Morgan.

At least 91 records · Page 5Linked to original sources
PubMed1989

Foot-and-mouth disease virus-neutralizing antibodies induced in mice by anti-idiotypic antibodies.

A neutralizing monoclonal antibody (nmAb) to foot-and-mouth disease virus (FMDV) was used as antibody-1 (AB1) to induce anti-idiotypic antibodies (a-IdAb) in rabbits. The rabbit a-IdAb (AB2) were isolated on protein A-Sepharose, followed by cycles of separation on idiotype and isotype affinity columns. The specificity of the AB2 for the paratope of AB1 was determined by direct binding to AB1 in solid-phase radioimmunoassay (SP-RIA), and by competition RIA (C-RIA) with virus for binding to the AB1. The AB2, termed a-2PD11, was utilized to immunize six groups of female Swiss mice at weekly intervals with either one of three formulations, in doses of 50 micrograms or 5 micrograms, given in single subcutaneous (s.c.) spots. Anti-viral antibody (AB3) was first detected by RIA at the fifth week in the 50 micrograms/dose groups, and maximum levels were reached at the sixth week in the 50 and 5 micrograms/dose groups. The AB3 levels were at least three times higher for mice given 50 micrograms doses. In addition, the AB3 were also shown to neutralize FMDV infectivity in tissue culture and in a suckling mouse protection assay. Overall, mice exhibited variable responses to immunization with AB2. In a subsequent trial, mice received multispot s.c. and footpad injections of 50 micrograms of a-2PD11 coupled to keyhole limpet haemocyanin (KLH) on a weekly basis. In these mice, AB3 was detected earlier than in mice immunized with single s.c. injections. These results support the use of a-IdAb as potential surrogates of critical determinants for FMD vaccines.

Animals↗
PubMed1988

Neutralization sites of type O1 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants.

Monoclonal antibodies (MAbs) were derived from mice infected with foot-and-mouth disease virus type O1 Brugge (FMDV 01B) or immunized with inactivated virions (140 S) or viral subunits (12 S). A total of 19 neutralizing MAbs were characterized of which 17 recognized conformationally determined epitopes and two recognized amino acid sequences on isolated VP1. Neutralizing MAbs were used to select antigenic variants of FMDV O1B. Based on cross-neutralization and binding assays with MAbs the variants were divided into discrete groups demonstrating the presence of three unique neutralization sites on FMDV O1B. One site was present only on intact 140 S virions, a second was present on both 140 S virions and 12 S subunits, and the third was present on 140 S virions, 12 S subunits, and isolated VP1. Comparison of the deduced nucleic acid sequence of parental FMDV O1B with those of the O1B variants demonstrated that the epitope recognized by the VP1-reactive, neutralizing MAbs included amino acid residues 138, 144, and 148. Cross-neutralization assays demonstrated that these neutralization sites of FMDV O1B function on other type O1 strains of FMDV.

Animals↗
PubMed1987

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor.

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.

Carrier Proteins↗
PubMed1987

Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain.

Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.

Animals↗
PubMed1987

The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.

Antibodies, Monoclonal↗
PubMed1987

Acute insulin action requires insulin receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin.

The role of the insulin receptor tyrosine kinase (protein-tyrosine kinase, EC 2.7.1.112) in various rapid insulin effects was studied by injecting four different cell types (by osmotic lysis of pinocytotic vesicles) with a monoclonal antibody that specifically inhibits the kinase activity of the insulin receptor and the closely related receptor for insulin-like growth factor (IGF)-I. Injection of this inhibitory antibody resulted in a decreased ability of insulin to stimulate the uptake of 2-deoxyglucose in Chinese hamster ovary cells and freshly isolated rat adipocytes, ribosomal protein S6 phosphorylation in CHO cells, and glycogen synthesis in the human hepatoma cell line HepG2. The ability of insulin, IGF-I, and IGF-II to stimulate glucose uptake in TA1 mouse adipocytes was also inhibited. Studies with CHO cells demonstrated that these effects of the inhibitory antibody were specific, since there was no change in phorbol ester-stimulated glucose uptake and injection of a noninhibiting antibody to the kinase had no effect on insulin action. These studies indicate that the tyrosine kinase activity of the insulin receptor is important in mediating several rapid insulin effects in a variety of different cell types.

Adipose Tissue↗
PubMed1987

Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68gag-ros has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into alpha and hybrid beta subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine residues of the hybrid beta subunit in vivo and the phosphorylation of an exogenous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling.

Animals↗
PubMed1987

A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.

Animals↗
PubMed1986

Purification and characterization of the receptor for insulin-like growth factor I.

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.

Animals↗
PubMed1986

Identification of a monoclonal antibody which can distinguish between two distinct species of the type I receptor for insulin-like growth factor.

A monoclonal antibody was identified which equally inhibits 125I-labeled insulin and insulin-like growth factor I (IGF-I) binding to their respective receptors in human IM-9 lymphoid cells and solubilized placenta receptor preparations. In contrast, this monoclonal antibody inhibits insulin but not IGF-I binding to human hepatoma (HepG2) cells, fibroblasts and muscle cells. These results indicate that there are two distinct species of the type I insulin-like growth factor receptor (which we have named type IA and type IB) and suggest that this monoclonal antibody may be useful in determining whether different biological effects are mediated through these two receptors.

Antibodies, Monoclonal↗
PubMed1986

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.

Amino Acid Sequence↗
PubMed1986

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain.

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.

Adenosine Triphosphate↗
PubMed1986

Purification and characterization of the human brain insulin receptor.

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.

Amino Acid Sequence↗
PubMed1986

Antigenic comparison of foot-and-mouth disease virus serotypes with monoclonal antibodies.

The capsid structures of the 7 serotypes of foot-and-mouth disease virus have been compared utilizing a series of neutralizing monoclonal antibodies which were previously shown to recognize at least 4 distinct epitopes on type A12 virus. A radioimmune binding assay using radioactively labeled antigens and the monoclonal antibodies revealed that certain conformation dependent epitopes are conserved among A subtypes, while some continuous epitopes are conserved among A subtypes as well as other FMDV serotypes. On the basis of differential reactivity among other FMDV subtypes and serotypes two additional epitopes have been defined on the A12 particle. Binding and neutralization assays revealed that the presence and function of an epitope are not necessarily correlated.

Animals↗
PubMed1986

Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase.

Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor beta subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor beta subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

Animals↗
PubMed1986

Linking functional domains of the human insulin receptor with the bacterial aspartate receptor.

A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.

Amino Acid Sequence↗
PubMed1986

Analysis of foot-and-mouth disease virus type O1 Brugge neutralization epitopes using monoclonal antibodies.

Monoclonal antibodies (MAbs) were elicited with inactivated, purified foot-and-mouth disease virus (FMDV) type O1 strain Brugge (140S) and with 12S protein subunits. Each MAb was tested for its capacity to bind to FMDV O1 Brugge 140S virions, 12S subunits and purified VP1 by radioimmunoassay (RIA) and to neutralize viral infectivity in mouse protection assays. Those MAbs which reacted only with 12S subunits in RIA did not neutralize infectious virus. One MAb, 12FE9.2.1, reacted with 140S, 12S and purified VP1 of FMDV O1 Brugge and neutralized infectious virus. Reactions with different biosynthetic FMDV type O1 Campos VP1 polypeptides localized the binding site of 12FE9.2.1 between amino acid residues 135 and 172. Eight MAbs reacted with both 140S virions and 12S subunits and neutralized infectious virus. Monoclonal antibodies that reacted only with 12S protein subunits or with 140S, 12S and VP1 did not compete in RIA with 140S/12S-reactive neutralizing MAbs for 12S binding sites. The ability of 140S/12S-reactive MAbs to compete for the 12S binding site defined by MAb 10GA4.2.2 was directly related to their capacity to neutralize infectious virus, suggesting that these MAbs were all affecting a single neutralization site. In mouse protection and plaque reduction neutralization assays, 10GA4.2.2 and 12FE9.2.1 were equally effective in neutralizing the homologous FMDV type O1 Brugge, and in cross-neutralization assays both exhibited high titres against four additional strains of type O1 FMDV.

Animals↗