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A R Rees

Publications and source records attributed to A R Rees.

66 records · Page 4Linked to original sources
PubMed1981

A behavioural and biochemical study in rats of 5-hydroxytryptamine receptor agonists and antagonists, with observations on structure-activity requirements for the agonists.

1 The effect of the putative 5-hydroxytryptamine (5-HT) receptor antagonists, methysergide, methergoline, mianserin, cyproheptadine, cinanserin (all at 10 mg/kg), methiothepin (5 mg/kg) and (-)-propranolol (20 mg/kg) on the behavioural responses to tranylcypromine (10 mg/kg) followed 30 min later by L-tryptophan (100 mg/kg) was examined.2 Methysergide, methergoline, methiothepin and (-)-propranolol inhibited head weaving, forepaw treading and hind-limb abduction. Methysergide and methergoline increased reactivity. In contrast, cypropheptadine, cinanserin and mianserin had no effects on the behaviour.3 Similar findings were obtained when the behaviours were elicited by administration of tranylcypromine (10 mg/kg) followed by the putative 5-HT receptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) (2 mg/kg).4 When the behaviours were elicited by the putative 5-HT receptor agonist, quipazine (50 mg/kg), all the drugs effectively inhibited head weaving and forepaw treading.5 When the dose of cypropheptadine was doubled to 20 mg/kg an inhibition of the tranylcypromine/L-tryptophan induced behaviours was seen.6 Methiothepin produced a marked inhibition of apomorphine-induced locomotor activity whilst all the others enhanced this response, suggesting that only methiothepin inhibits the 5-HT behaviours by dopamine antagonism and that the increased reactivity seen following tranylcypromine/L-tryptophan after pretreatment with methysergide or methergoline might be due to enhanced dopamine function.7 Pretreatment with p-chlorophenylalanine resulted in enhanced behavioural responses to both 5-MeODMT and quipazine.8 Both methergoline and methiothepin decreased the rate of 5-HT synthesis in whole brain but not spinal cord and methergoline decreased spinal cord 5-HIAA concentration. None of the other drugs had any significant effects on the concentration of 5-HT, 5-HIAA or 5-HT synthesis rate in brain or spinal cord.9 Experiments with compounds structurally related to quipazine and with molecular models suggested that quipazine produces behavioural changes probably by stimulating the 5-HT receptor in a similar way to 5-HT but that it would bind weakly, in agreement with ligand-receptor binding studies.10 It is suggested, therefore, that cyproheptadine, cinanserin and mianserin fail to inhibit 5-HT and 5-MeODMT-induced behaviours because they are weak antagonists whilst they are able to inhibit the same behaviours induced by quipazine because it is a weak agonist.11 These data indicate that extreme care should be taken in accepting or rejecting 5-HT as a mediator of behaviours or of other responses unless several antagonists or agonists have been examined.

Animals↗
PubMed1980

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.

Animals↗
PubMed1979

Epidermal growth factor receptors increase during the differentiation of embryonal carcinoma cells.

Mouse teratocarcinoma stem cells (embryonal carcinoma, or EC cells) bind very small amounts of mouse epidermal growth factor (EGF) and the latter hormone seems to have no stimulatory effect on the growth of two cloned lines of EC cells. However, when EC cells are induced to differentiate into large flat endodern-like cells (END cells), EGF receptors increase in number reaching a plateau in 6 to 8 days. At 8 to 10 days after induction, END cells multiply very slowly, but when EGF is added (3 x 10(-10) M) to the medium, cell division is stimulated and a further change in morphology occurs. This letter describes the binding characteristics and numbers of the EGF receptors on EC and END cells and shows that exogenous retinoic acid increases the numbers of EGF receptors on END cells. We were unable to find endogenous competing factors produced by EC cells. Such factors could account for the lack of detectable binding of EGF on these cells. As EC cells differentiate to END cells, so the ability of the cells to form tumours is reduced. Since this change is accompanied by an increase in the number of EGF receptors there may be a relationship between these two events.

Animals↗
PubMed1976

The preparation of protected fragments of lysozyme for semisynthesis.

This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain...

Acylation↗
PubMed1976

The semisynthesis of portions of hen's-egg lysozyme by fragment condensation.

1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion.

Amino Acid Sequence↗
PubMed1972

X-ray crystallography and the amino acid sequence of lysozyme.

Certain parts of the amino acid sequence of hen's-egg lysozyme were reinvestigated. The parts in question are those in which X-ray-crystallographic data were used to decide between the two reported versions of the sequence that were based on chemical data. We conclude that the X-ray evidence, in the main, led to the correct choice between the sequences.

Amino Acid Sequence↗
PubMedDate not supplied

Antibody-combining sites. Extending the natural limits.

The antibody repertoire is very large with at least 10(9) different antibody specificities, yet there are currently only 800 variable-region sequences known and < 23 Fab structures deposited with the Brookhaven Protein Data Bank. To engineer the antibody-combining site rationally, we need to define the rules that govern antibody structure. To understand the process of antibody-antigen recognition, we need not only to predict complementary determining regions accurately, but to simulate accurately the interaction of antibody with antigen. We have made progress in the modeling of antibody-combining sites and in the simulation of antibody complex formation. The combination of these approaches will allow us to extend the natural limits of antibody-combining sites in a more rational manner.

Algorithms↗
PubMedDate not supplied

Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering.

A detailed description of the interactions between an antibody and its epitope is necessary to allow an understanding of the way in which antibodies bind to antigenic surfaces presented by foreign molecules. Ideally this should be done by analysis of crystal structures of antibody-antigen complexes, but so far only two of these are available. An alternative strategy combines molecular modelling with site-directed mutagenesis (SDM) and using this we have generated a preliminary model of the complex between Gloop2, an antibody raised against a peptide containing the 'loop' determinant of hen egg-white lysozyme (HEL) which also binds the native protein, and its epitope on the protein surface. The main predictions from our model were; (1) that the surface of interaction between the antibody and the antigen is large (20 A X 15 A) and involves all the complementarity-determining regions (CDRs), (2) that electrostatic interactions were important in the formation of the complex, and (3) that conformational changes in either the loop or in the CDRs may occur during the formation of the complex. Here we report SDM studies which test some of these predictions; removal of two charged residues at the periphery of the combining site increases the affinity of the antibody for its antigen over 8-fold and decreases its ability to cross-react with closely-related antigens. This result is at variance with our original prediction but can be accommodated within our newly refined model; the role of electrostatics in antigen-antibody interactions is now questionable.

Animals↗
PubMedDate not supplied

The cloning and expression of an anti-peptide antibody: a system for rapid analysis of the binding properties of engineered antibodies.

The cloning of the light and heavy chain genes of an antilysozyme monoclonal antibody is described and the application of site-directed mutagenesis for reconstruction of complete cDNAs from incomplete cDNA clones is discussed. The behaviour of the RNA transcripts produced from these cDNAs after their subcloning into an appropriate Salmonella typhimurium, SP6, vector and subsequent injection into Xenopus oocytes has been analysed. The antibody secreted by oocytes after RNA injection has been quantitated and its antigen-binding properties compared with wild-type hybridoma antibody. The results show that this route for expression of antibodies, though limited in its capacity to produce more than a few micrograms of protein, is potentially a means for rapid evaluation of the antigen-binding properties of engineered antibodies.

Animals↗