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Biomedical subjects

A R Rees

Publications and source records attributed to A R Rees.

At least 55 records · Page 3Linked to original sources
PubMed1988

Engineering antibody affinity and specificity.

A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed.

Animals↗
PubMed1986

Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.

Animals↗
PubMed1986

Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells.

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

Antibodies, Monoclonal↗
PubMed1985

Growth factors in mammalian embryogenesis.

Malignant murine embryonal carcinoma (EC) cells have been studied as a means of characterizing the identity and action of growth regulatory molecules in the early postimplantation mouse embryo. The differentiation of EC cells in vitro is accompanied by significant changes in the control of cell proliferation, including the acquisition of dependence on specific exogenous growth factors for cell multiplication. This is at least partly controlled by the developmentally regulated expression of specific growth factor receptors and their intracellular response systems. The development of defined media has allowed the identification of the principal factors required for EC cell proliferation in vitro. These factors are synthesized in vivo by the extra-embryonic tissues of the developing embryo and by the differentiated progeny of EC cells in vitro. EC cells secrete a potent growth factor (embryonal carcinoma-derived growth factor, ECDGF) which has been purified and partly characterized. ECDGF induces proliferation of the differentiated progeny of EC cells and specific normal embryonic cell types in vitro, suggesting that ECDGF may act as an embryonic growth factor in vivo. Together these findings suggest that proliferation in the embryo may be controlled by reciprocal interaction between primitive ectoderm cells and their differentiated derivatives, mediated by the developmentally regulated expression of specific soluble growth factors.

Animals↗
PubMed1985

Isolation and characterization of an Fc receptor from neonatal rat small intestine.

Receptors for the Fc region of IgG from neonatal rat intestinal brush borders were solubilized using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate and purified by affinity chromatography. Analysis of IgG-binding material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions reveals two components with apparent Mr of 41 000-50 000 and 15 000. The larger component is glycosylated and may dimerize, giving a 100-110-kDa band on nonreduced gels. Both proteins are localized in the proximal small intestine, where IgG is specifically taken up during the first three weeks of neonatal life, and disappear when specific transport stops after weaning. Electron irradiation of brush borders shows that the functional unit for IgG binding has a molecular weight in situ of 110 kDa. These data suggest that a dimer of the 41-50-kDa protein together with the 15 kDa and other proteins may mediate intestinal transport of maternal IgG.

Animals↗
PubMed1985

Three distinct epitopes within the loop region of hen egg lysozyme defined with monoclonal antibodies.

Five monoclonal antibodies specific for the loop region of hen egg lysozyme were prepared by immunisation with a synthetic conjugate of a proteolytic fragment of lysozyme coupled to bovine serum albumin. Their fine specificities were investigated using a panel of variant lysozymes and peptide fragments of lysozyme in a quantitative radio-immunoassay procedure. Knowledge of the structure of hen lysozyme to high resolution and the use of computer graphics enables the localisation of the epitopes recognised by the antibodies with some precision. The antibodies were shown to define three distinct, overlapping epitopes within what was previously considered to be a single antigenic site. These results are discussed in relation to current ideas of the antigenic nature of proteins and other recent studies in which anti-protein antibodies have been elicited by immunisation with small peptides.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence.

Amino Acid Sequence↗
PubMed1985

Monoclonal antibody (EGR/G49) reactive with the epidermal growth factor receptor of A431 cells recognizes the blood group ALeb and ALey structures.

The carbohydrate specificity of the monoclonal antibody EGR/G49, raised against the epidermal growth factor (EGF) receptor of A431 cells, has been investigated by assessing its interactions with glycoproteins and erythrocytes derived from individuals of known blood group ABH, Lewis and secretor types, and by inhibition of binding assays using structurally defined oligosaccharides. The results indicate that this antibody reacts with the difucosylated blood group structures ALeb and ALey: (formula; see text) This antibody differs from the previously described anti-EGF receptor antibody. TL5, which is directed at the terminal blood group A trisaccharide structure and reacts poorly with the ALeb/Ley structures. Since both antibodies were selected for their reactivities with the receptor for EGF, their specificities provide evidence for the presence of both the mono- and difucosylated blood group A structures on the receptor glycoprotein. These antibodies will be invaluable in the studies of the distribution and the roles of blood group related carbohydrate structures in the organisation and function of the EGF and other receptor systems.

ABO Blood-Group System↗
PubMed1985

Proliferation of a human embryonal carcinoma-derived cell line in serum-free medium: inter-relationship between growth factor requirements and membrane receptor expression.

Substantial multiplication in vitro of cloned cells from a human embryonal carcinoma cell line, Tera 2, has been obtained in a basal medium (DMEM/Ham's F12,50:50, v/v) supplemented with 10 micrograms low density lipoprotein/ml, 100 micrograms high density lipoprotein/ml, 100 ng multiplication stimulating activity/ml, 100 ng insulin/ml and 1 microgram transferrin/ml. The growth rate appears to be similar to that obtained in 10% serum. Furthermore, studies on the expression of cell surface receptors revealed that cloned Tera 2 cells express high-affinity receptors for IGF-II but not for insulin. The cells also express receptors for Epidermal Growth Factor (EGF) even though the addition of EGF does not stimulate their proliferation in serum-free medium. These results suggest that the expression of specific growth factor receptors is not an absolute determinant of hormone responsiveness.

Cell Division↗
PubMed1984

Properties of a monoclonal antibody to epidermal growth factor receptor with implications for the mechanism of action of EGF.

A monoclonal antibody, EGR/ G49 , raised against the receptor for epidermal growth factor (EGF) present in A431 cells inhibits EGF binding by decreasing the affinity of the major population of low affinity receptors while leaving the minor high affinity population relatively unperturbed. The antibody, which binds to a carbohydrate determinant at a site distinct from the EGF binding site, induces clustering and internalisation of the receptor without stimulating the EGF receptor-kinase or affecting its ability to undergo stimulation by EGF. It is toxic to A431 cells and induces morphological changes similar to those seen when these cells are challenged with EGF in the concentration range 1-10 nM. These results suggest that high and low affinity EGF receptors can be distinguished and that they may serve different functions.

Animals↗
PubMed1984

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion.

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.

Carcinoma, Squamous Cell↗
PubMed1984

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells.

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type. (The term 'poly-lacto/neolacto' is used here to describe oligosaccharide backbone structures consisting of repeating Type 1, Gal beta 1-3GlcNAc (lacto) or Type 2, Gal beta 1-4GlcNAc (neolacto) sequences.) The glycosidic linkage of oligosaccharides to protein was investigated using Pronase digests of the receptor biosynthetically labelled with [3H]glucosamine or [3H]fucose. The oligosaccharides were alkali-resistant, consistent with N- rather than O-glycosidically linked chains. A proportion of [3H]fucose-labelled glycopeptides was susceptible to endo-beta-galactosidase, confirming the immunoblotting experiment using antibodies against the Lea and the difucosylated Type 2 antigenic determinants. Oligosaccharides were released from the [3H]fucose- and [3H]-glucosamine-labelled glycopeptides by hydrazinolysis. Chromatography of the oligosaccharides on Bio-Gel P6 and Concanavalin A columns indicated a spectrum of oligosaccharides which include those of high mannose type labelled with [3H]glucosamine, and a mixture of oligosaccharides labelled with [3H]fucose and [3H]glucosamine of bi- and multiantennary complex types of which a subpopulation is susceptible to digestion with endo-beta-galactosidase.

ABO Blood-Group System↗
PubMed1983

Properties of immunoglobulin G-Fc receptors from neonatal rat intestinal brush borders.

Newborn rats acquire immunity passively by receptor-mediated uptake of maternal immunoglobulin G (IgG) from the first milk. Specific IgG binding to brush borders and IgG transport across the gut increase concomitantly for 10-12 days after birth and then fall until closure at about 21 days. Cortisol acetate administration accelerates this decline. Two classes of binding site are resolved by their affinities (KA1 = 1.3 X 10(8) M; KA2 = 5.15 X 10(6) M). Persistence of the low-affinity site after closure precludes a transport role (see Rodewald et al, this volume). Target size analysis gives a preliminary Mr for the high-affinity site of 90 000-100 000. IgG recognition involves a small number of positively charged residues in the Fc region. An Fc binding activity is solubilized from intestinal brush borders by lithium 3,5-diiodosalicylate.

Animals↗
PubMed1981

The role of the arginine-B22 residue in insulin action.

We describe the modification of the side chain of the arginine-B22 residue of insulin by the N8N9-(1, 2-dihydroxycyclohex-1,2-ylene) group and by the adipoyl group. These are the first insulin derivatives described that contain a modified arginine residue in an otherwise unaltered molecule. When tested for their ability to lower blood sugar concentration, both modified insulins showed a specific activity indistinguishable from that of insulin. In view of the fact that the substituent groups involved are very bulky and in one case of opposite charge to that of the side chain, the retention of biological activity casts doubt on the idea, previously generally accepted, that the arginine-B22 residue is essential to the activity of the hormone.

Animals↗
PubMed1981

Epidermal growth factor receptors.

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.

Animals↗
PubMed1981

Appearance of functional insulin receptors during the differentiation of embryonal carcinoma cells.

The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.

Cell Differentiation↗