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K P Willey

Publications and source records attributed to K P Willey.

11 recordsLinked to original sources
PubMed1996

Epitope mapping of a recombinant human TSH receptor extracellular domain: identification of a predominant epitope using animal sera.

The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria. The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay. An epitope library was constructed and screened with affinity purified ovine and porcine antisera and detected a number of positive clones. Sequence analysis revealed that all of the epitopes contained sequences derived from the carboxyl terminus of the recombinant immunogen. One clone defined an epitope covering 16 amino acids from the carboxyl terminus and was the common epitope found in all of the other clones. Western blot screening of a large panel of Graves' sera with recombinant TSH receptor protein identified one patient sera that also recognized linear epitopes in the TSHR-561 protein. Experimentation demonstrated that the linear epitope recognized by this human sera was identical to the sequence recognised by the animal antisera. This sequence is unique to the TSH receptor and will be useful in further studies to analyze the TSH receptor protein.

Amino Acid Sequence↗
PubMed1996

The antibodies causing thyroid stimulating hormone-binding inhibition (TSH-BI) are not responsible for the specific inhibition of gonadal steroidogenesis by Graves' sera.

Graves' disease is attributed to the presence of autoantibodies with agonist activity which interact with the TSH receptor causing thyroid hyperstimulation and hyperthyroidism. The degree of TSH-binding inhibition (TSH-BI) caused by a Graves' serum in a TSH radioligand receptor assay is considered to be an index of the prevalence of anti-TSH receptor autoantibodies in that serum. We have previously shown that the specific inhibition by Graves' serum of hCG-stimulated steroidogenesis by Leydig cells was at a site distal to receptor binding and second messenger activation. In this report, we have investigated whether the effect of Graves' serum upon Leydig cells is a property of the constitutive antibodies. Immunoglobulin-enriched fractions were obtained from Graves' and normal sera using three increasingly rigorous procedures; ammonium sulphate precipitation, caprylic acid treatment and Protein A or G-affinity purification. The TSH-BI was determined for untreated and extracted sera in two radioreceptor assays developed for use with serum, one using human thyroid membranes and the other using HeLa cells transfected with the human TSH receptor, and the results were compared with effects in the Leydig cell steroidogenesis bioassay. The specific inhibition of hCG-stimulated Leydig cell steroidogenesis by Graves' sera was not retained in the antibody fraction causing TSH-BI. Thus, the inhibitory factor appears not to be an antibody and we are now attempting to purify and identify the responsible factor from Graves' serum.

Ammonium Sulfate↗
PubMed1993

Graves' autoimmune serum inhibits gonadal steroidogenesis: development of a Leydig cell bioassay to identify broad spectrum anti-endocrine autoantibodies.

In order to establish an assay for the detection of autoimmune sera with broad spectrum activity, we have investigated the effect of unselected normal and Graves' disease sera upon steroidogenesis by gonadal cells. Steroidogenesis was enhanced by the addition of normal serum in a 3-h primary Leydig cell bioassay, but was inhibited by the majority of Graves' sera. The inhibition was not related to clinical thyroid parameters, such as the severity of the TSH-binding inhibition index, and was not overcome by other agonists or second messenger supplements. Although pituitary TSH preparations bound to and stimulated Leydig cells, TSH receptor mRNA was not detectable and pure recombinant TSH failed to bind or stimulate, indicating contamination of pituitary TSH with LH. The binding of hCG to the Leydig cell luteinizing hormone receptor was not perturbed by the Graves' autoimmune sera, indicating that cross-reactive anti-TSH receptor antibodies were not responsible for the inhibition. By use of intermediates in the stimulatory pathway, the site of Graves' serum inhibition was identified to be distal to hormone receptor/adenylate cyclase coupled responses and proximal to supply of cholesterol for steroidogenesis.

Animals↗
PubMed1993

Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: evidence for a soluble form of the receptor in human thyroid.

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

Animals↗
PubMed1990

Histones inhibit human chorionic gonadotrophin-stimulated but not atrial peptide-stimulated testosterone production and cyclic nucleotide formation by isolated mouse Leydig cells.

Recently it has been reported that histone type H2A can inhibit gonadotrophin-stimulated cAMP formation and steroidogenesis by ovarian cells. In the present study we have investigated if similar antigonadotrophic effects of commercially available histones can also be demonstrated on testicular steroidogenic cells. Using percoll-purified mouse Leydig cells, we have demonstrated that several types of histones could almost completely inhibit hCG-stimulated testosterone production and cAMP formation. The inhibition was dose-dependent and could be reversed by the addition of excess of hCG. The most potent histone types were H2AS and H8S, both of which could inhibit hCG-stimulated cAMP formation half-maximally at concentrations of 4-5 micrograms/ml. Forskolin-stimulated cAMP formation was not affected by histones. When the cells were stimulated with either db-cAMP or rAP-II, histone H2AS and H8S failed to inhibit the testosterone production. In fact there was a marked increase in the amount of testosterone produced, the reason for which is not yet understood. The amount of cGMP accumulated in response to rAP-II was not affected by the presence of H2AS or H8S. In unstimulated cells, neither the cyclic nucleotide level nor the amount of steroid produced was affected by the histones. Based on the [125I]hCG binding data it is possible to conclude that histone H2AS inhibits the binding of hCG to its receptors on Leydig cells and thereby causes the inhibition of hCG-stimulated cAMP formation and steroidogenesis.

Animals↗
PubMed1989

Functionally distinct agonist and receptor-binding regions in human chorionic gonadotropin. Development of a tertiary structure model.

The histidine residues in human chorionic gonadotropin (hCG) were chemically modified using diethyl pyrocarbonate. Derivatives of hCG with an average of 0.5-3.5 histidines modified (maximum of 4 per hCG) had reduced receptor-binding and cell-stimulating activities. Acylation of hCG at progressively lower pH values (conditions in which 1 of the 2 absolutely conserved histidines alpha His-83 is not titratable, whereas alpha His-94 becomes increasingly protonated and resistant to modification) produced hCG derivatives with a greater retention of receptor-binding activity than cell-stimulating activity. The involvement of alpha His-94 as part of the receptor-binding region of the hormone and of alpha His-83 as a putative active site residue was inferred. Proteinaceous protease inhibitors were shown to neutralize the agonist activity of hCG and to reduce the binding of hCG to its receptor and also to specific antisera. It was presumed that an inhibitor-hormone complex was formed which was analogous to the complexing of inhibitor with the "substrate pocket" of a serine protease. The discovery of primary sequence analogies between hCG and the serine protease chymotrypsin enabled the prediction of hCG structure using the enzyme as a folding template. Solvent-exposed and buried core regions of the peptide chain were delineated using smoothed hydrophobicity profiles in combination with Chou-Fasman secondary structure predictions. Hypervariable hydrophobicity indices between residues 38 and 80 of the human beta subunits reflected different folding arrangements which presumably conferred the individual receptor specificities. When mapped to the putative structure these receptor-determinant loops were adjacent to an area of the alpha subunit analogous to the substrate pocket of serine proteases. Disulfide bond assignments and intersubunit contact regions were identifiable. The proposed tertiary structure for hCG manifests the topographical epitopes defined using monoclonal antibodies and satisfies the currently available data on specific modification and its effects upon hormonal structure and function. This paper is considered to be the first report of a differential effect upon the agonist and receptor binding abilities of a glycoprotein hormone after modification of the proteinaceous, as opposed to the glycosylated, moiety of the molecule.

Amino Acid Sequence↗
PubMed1985

Simple and complex antibody reactions in radioimmunoassay and the prediction of assay characteristics.

Radioimmunoassays are usually developed empirically since there have been few established rules which cover general RIA behaviour. Evidence is presented which enables the delineation of 2 types of RIA with distinct group characteristics. (1) Simple RIA, conforming to the law of mass-action kinetics, is a result of univalent interaction. Examples of simple RIA include hapten assays and those using a monoclonal antibody (McAb). (2) Complex RIA occurs when antigen reacts multivalently with a polyclonal antiserum (PcAs). The formation of multicomponent complexes between a large molecular weight antigen and a PcAs is demonstrated using gel exclusion chromatography. These complexes are resistant to dissociation and are responsible for higher affinity, greater sensitivity and slower equilibration times compared to simple RIA. The assignment of an assay to either the simple or complex RIA group is dependent upon either the molecular size of the antigen or the use of a McAb. The consequent predictability of RIA behaviour enables a more rational approach to optimal assay design than current theory allows. It is advocated that the inability of a McAb to form multicomponent complexes is a major disincentive to their indiscriminate adoption in RIA.

Antibodies, Monoclonal↗
PubMed1985

Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species.

The role of melatonin in animals which do not show marked seasonal changes in reproduction is disputed, in part because of the wide variation in reported concentrations. One reason for this may be the difficulties associated with the measurements of low molar concentrations of melatonin and the presence of a wide variety of potentially cross-reacting substances. The availability of a high affinity antiserum has allowed an assay, with low cross-reactivity and good sensitivity, to be established for the direct measurement of melatonin in a wide range of biological fluids, in particular serum, plasma and follicular fluid from man and rat. The high affinity of the antiserum enabled a tritium label of high specific activity to be used, removing the problems associated with the iodination of a small molecular weight compound. Melatonin concentrations in the assay were evaluated by four different methods: UV absorbance, gas chromatography, comparison of the immunoreactive concentrations of the label with the expected concentration by dilution and by comparison with a previously established assay which uses the same antiserum. Melatonin was measured in serum from twelve healthy women over two 24-h periods; eight women with normal menstrual cycles and four taking the contraceptive pill. Concentrations were found to range from 19.8 to 215 pmol/l during the day in both groups. In women with normal menstrual cycles peak concentrations of 513.2 +/- 54.1 (S.E.M.) pmol/l were recorded at 04.00 h, whereas higher concentrations were found in women taking the pill, reaching a peak of 849.12 +/- 21.8 (S.E.M.) pmol/l at 04.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Evidence concerning the mechanism of insulin-receptor interaction and the structure of the insulin receptor from biological properties of covalently linked insulin dimers.

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.

Adipose Tissue↗
PubMedDate not supplied

An elusive role for glycosylation in the structure and function of reproductive hormones.

The crescendo of events leading first to ovulation and subsequently to birth is orchestrated by a broad repertoire of hormones. The major hormones of the ovulatory cycle are representatives of four hormone classes: neurotransmitters, releasing factors, trophic hormones acting on target tissues, and steroid-like molecules released by the target tissues. The punctuate and staccato rhythm of the neurotransmitters and releasing hormones relentlessly drive the swelling and protracted wave of activity by the luteotrophic and steroid hormones. Carbohydrates alone are notably absent as hormones and the predominant role for glycosylation appears to be the conferment of increased solubility to endocrine molecules, either during their manufacture or by modulating circulatory half-life. Rarely considered examples of the importance of glycosylation in reproductive hormones include adenosine, important for spermatozoan activity, and the hormone-binding globulins, which ensure the aqueous transport of hydrophobic steroids. The archetype glycoprotein hormones, especially human chorionic gonadotrophin (HCG), are discussed more extensively, as the structural and functional roles of carbohydrate in these hormones have been studied exhaustively. Conversely, the direct involvement of HCG and the importance of its carbohydrate for autonomous growth, in both placental invasion and tumorigenesis, has received little attention in the literature.

Animals↗