Disordered eating behaviors and reward sensitivity.

This research evaluated the extent to which reward sensitivity demonstrated associations with binge and purge behavior frequency. A verbal operant conditioning task designed to assess conditionability to reward cues was administered to a sample of 34 women who exhibited disordered eating patterns for at least I month prior to study participation. Reward sensitivity significantly correlated with the average weekly frequency of purge (r = 0.44) but not binge behaviors. These findings suggest that reward sensitivity has some associations with aspects of disordered eating and therefore may have relevance for theories on the maintenance of some forms of eating disorder-related behavior.

The subpopulations of circulating white blood cells in inflammatory bowel disease.

A detailed analysis of the species of lymphocytes was carried out in 58 patients with inflammatory bowel disease (IBD). These individuals were further divided into 31 with Crohn's disease (CD) and 27 with ulcerative colitis (UC). There were 13 CD patients with only small bowel involvement called "regional enteritis" and 18 who had some degree of colonic involvement called "ileocolitis". Similarly, the UC group was subdivided into 9 patients with disease confined to the rectosigmoid area called "proctosigmoiditis" and 18 with more extensive involvement called "universal colitis". We also studied 13 patients who had undergone previous colectomy and ileostomy and 78 healthy age- and sex-matched controls. Although there was no increase in the absolute number of lymphocytes in patients with ileocolitis and universal colitis, the percentage of these cells was decreased because of an increase in both polymorphonuclear leukocytes and monocytes. In IBD and its subgroups, mean T lymphocytes, determined by the sheep red blood cell rosette technique, were not significantly different from the controls either in percentage or absolute number. Furthermore, no difference was noted between UC and CD. However, there seems to be a subpopulation of patients with UC or CD whose T cells are reduced below 1 SD of the mean. There was also no difference in the number of immunoglobulin-bearing B cells in both diseases; however, when the B cells were enumerated by their ability to rosette with antibody-complement-coated sheep cells (EAC), we found a marked decrease in percentage (P less than 0.001) and absolute number (P less than 0.0005) relative to the control population. The decrease bore a direct relation to the severity of the disease process and, although more marked in patients with UC, was present in CD also.

Effects of sulfasalazine on selected lymphocyte subpopulations in vivo and in vitro.

Sulfasalazine has proven to be an effective agent in the therapy of inflammatory bowel disease (IBD). Despite long and widespread usage, the mechanism of action of this drug is still not understood. Several investigators have suggested that the drug might act as an immunosuppressant. To examine this possibility, an in vivo study was undertaken to ascertain any quantitative change in the circulating T cells, Ig-bearing B cells, and complement receptor-bearing lymphocytes (CRL) of patients before and during therapy with sulfaslazine. Concomitant responses to skin test antigens were also evaluated. In vitro studies with control cells were performed to determine the influence of sulfasalazine and its components (sulfapyridine or 5-aminosalicylic acid) on the extent of antibody-dependent cellular cytotoxicity (ADCC), as well as on the number of T cells and CRL. Results indicate that neither sulfasalazine nor either of its components quantitatively alters those subpopulations of circulating mononuclear cells studied in vivo or in vitro--nor are these compounds responsible for any functional inhibition of ADCC.

Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity.

Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.

Hybrid and atypical insulin/insulin-like growth factor I receptors.

The insulin receptor and type 1 insulin-like growth factor (IGF) receptor as classically described are each the product of a single gene. Various receptor subtypes have been described, however, with distinct structures or binding properties. Two of these subtypes have been studied, namely hybrid and atypical IGF-I receptors. Hybrid receptors contain alpha beta halves of both the insulin and the IGF receptor. They are identifiable as a high-affinity IGF-I-binding species reacting with both IGF-receptor-specific and insulin-receptor-specific monoclonal antibodies, and account for a substantial fraction of IGF receptor in many mammalian tissues. Hybrid receptors purified from human placenta bind IGF-I with approximately 25-fold higher affinity than insulin, the affinity for insulin being 10-fold less than that of the classical insulin receptor. It is therefore likely that hybrids will respond more readily to IGF-I than to insulin in vivo. Atypical IGF receptors are characterized by an ability to bind insulin as well as IGFs with relatively high affinity, but are immunologically indistinguishable from classical IGF receptor and do not react with insulin receptor-specific antibodies. The structural basis of atypical binding behaviour is unknown, though the effect is mimicked by binding of certain anti-IGF receptor monoclonal antibodies, which dramatically increase the affinity of the IGF receptor for insulin. Specific physiological roles have not been demonstrated for hybrid or atypical receptors, but the available information concerning their distribution and properties suggests that these receptor subtypes may have an important influence on the specificity of action of insulin and IGFs in vivo.

Skeletal maturation of Hong Kong Chinese children in the first five years of life.

This report describes the skeletal maturity during the first five years of life of 492 Chinese children in Hong Kong in a longitudinal study. Hand-wrist radiographs taken half-yearly were assessed by the Tanner-Whitehouse method and rated according to the TW1 20-bone self-weighting maturity score. Skeletal age was in advance of chronological age in both sexes, but significantly more so in females, especially between 18 months and three years. The relationship of the skeletal maturation of these children to various socioeconomic factors is discussed, including the effect of preferential child rearing in favour of males.

Skin test reactivity in inflammatory bowel disease in the United States and Czechoslovakia.

Inflammatory bowel disease (IBD) patients in the United States of America (USA) and Czechoslovakia (CSSR) were categorized by clinical, pathological, and radiological criteria as having Crohn's disease (CD) or ulcerative colitis (UC) and were tested with five skin test antigens [Candida, mumps, purified protein derivative (PPD), streptokinase-streptodornase (SK-SD), and trichophytin] at two different dilutions in an attempt to elicit some evidence of anergy. No significant differences were encountered between the USA and CSSR populations or between any patient group and its controls.

A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors.

A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.

High throughput direct end sequencing of BAC clones.

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.