Neurotrophic factors mediate their signal by binding to specific cell surface receptors of the trk family. The binding sites of neurotrophin-3 (NT-3) and nerve growth factor (NGF) to their preferred receptors trkC and trkA, respectively, were previously determined by mutational analyses. These and other studies showed that trkA can discriminate between NGF and NT-3 primarily by recognition of their N-terminal residues. The mechanism of trkC discrimination, however, remained unclear especially since the most important residue in NT-3 involved in binding to trkC, R103, is conserved in all neurotrophins. In this study residues that are part of the central beta-strand bundle of NT-3 and are not conserved among the neurotrophins were grafted onto NGF and tested for recruitment of trkC affinity. Exchange of NGF residues at positions 18, 20, 23, 29, 84, and 86 by their NT-3 counterparts resulted in NGF variants that bound to trkC, while maintaining their affinity to trkA, and were able to induce autophosphorylation and differentiation of PC12 cells expressing trkC. These variants show that the amino acid at position 23 (glycine in NGF, threonine in NT-3) is critical for trkC recognition while other residues fine tune the specificity of NT-3 for trkC. The results demonstrate the importance of nonconserved residues of the central beta-strand bundle region for the interaction of NT-3 with trkC and emphasize the different mechanism of specificity determination that is employed in the NT-3/trkC and NGF/trkA ligand/receptor pairs.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.

Numerous clinical studies show that direct interference with the IgE response leads to a decrease or elimination of allergic symptoms. The aim of these studies was to design a therapy aimed at decreasing IgE levels in order to ameliorate atopic disease. To this end, a murine monoclonal antibody, MAE11, directed against IgE was identified, which had all the properties necessary to interfere with IgE responses, but lacked the harmful side effects of inducing receptor cross-linking. The antibody was selected on the basis of its ability to bind circulating IgE at the same site as the high-affinity receptor, thus blocking the binding of IgE to mast cells and basophils. To allow for possible chronic administration and to avoid the problems of antigenicity, MAE11 as humanized. The best of several humanized variants, version 25 (rhumAb-E25) was selected since it possessed binding affinity and biological activity comparable to MAE11. Clinical studies are underway to determine the safety and efficacy of this treatment for allergic rhinitis and asthma.

Immunoglobulin (Ig) E antibodies mediate allergic responses by binding to specific high affinity receptors, Fc epsilon RI, on mast cells and basophils. Previous studies have shown that the principal Fc epsilon RI binding site is located on the third constant domain, Fc epsilon 3, of IgE. Based on a model of the IgE Fc epsilon 3 (which is homologous to the second constant domain of IgG), homology scanning mutagenesis and replacement of individual residues were used to determine the specific amino acids of human IgE involved in binding to human Fc epsilon RI. The amino acids are localized in three loops, which form a putative ridge on the most exposed side of the Fc epsilon 3 domain of IgE and include Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469. The preponderance of charged residues suggests that IgE-Fc epsilon RI binding is mediated primarily by electrostatic interaction. Furthermore, it is possible to confer Fc epsilon RI binding to an IgG molecule by introducing these three IgE loops into the IgG C gamma 2 domain.

The X-ray crystal structure of relaxin at 1.5 A resolution is reported for the physiologically active form of the human hormone. Relaxin is a small, two-chain polypeptide that is a member of the protein hormone family that also includes insulin and the insulin-like growth factors IGF-I and IGF-II. These hormones have biologically diverse activities but are structurally similar, sharing a distinctive pattern of cysteine and glycine residues. The predicted structural homology of relaxin to insulin is confirmed by this structural analysis; however, there are significant differences in the terminal regions of the b-chain. Although relaxin, like insulin, crystallizes as a dimer, the orientation of the molecules in the respective dimers is completely different. The region of the relaxin molecule proposed to be involved in receptor binding is part of the dimer interface, suggesting that some of the other residues contained in the dimer contact surface might be receptor binding determinants as well. The proposed receptor binding determinants for insulin likewise include residues at its dimer interface. However, because the dimer contacts of relaxin and insulin are quite different, it appears that these two structurally related hormones have evolved somewhat dissimilar mechanisms for receptor binding.

Inorderto evaluate the relation between gastric emptying and gastric motility, we developed acomputerizedtechniqueto measure concurrently these two parameters in a primate model.Three strain gauges were sutured to the fundus, wrpus, and antrum of five rhesus monkeys undergeneralanesthesiaandasepticwnditions.Afteratenday recovery period,we used radionucliie imaging to measure gastric emptying of liquids and solkfs in conscious animals, while ECG electrodes were placed on the skin of the abdomen and on the legs to obtain an electrogastrogram (EGG).Gastric mechanical signals and the EGG were concurrently recorded, taped, and aubaequentlydigitized,filtered, and analyzed using a MNC/DECLAB-23 computer.For all signals,we determined the amplitude, frequency, motility index (frequency x ampltiude; MI), and activity (area underthecurve)of eachwave.Duringfasiing,m'grating motorcomplexes (Phase III)were observedevery60-80minongaatriomechanicalsignals.Following a rnlxed liquid and solid meal, gastric mechanical activity was also significantly (~~0.05)iiireased in the fundus, corpus, and antrum compared to fasting.In addition, fasting and postcibal mechahical activities were significantly greater in the corpus and anlrum than in the fundus (~~0.05).The radioprotectant and hypocalcemic agent WR-2721 (150 m@kg, IV) abolished gastrii emptying of liquids and solids, and decreased gasIfs nwtor activity by 65% in the corpus and antturn, but only by 10% in the fundus.Similarly, cutaneous activity was decreased by 50%, suggesting that the EGG refleots an average of gastric motiliy in the fundus,corpus and anlrum.Furthermore, EGG Mlwas significantly oorrelatedwiih m%hanicalMIinthefundus (Rt0.43;p~O.Ol),corpus (R-0.60;~0.001) and antrum (R.0.38; pcO.01).These observations indicate that gastric emptying of a mixed liquid and solid meal is relatedtogaatrii mechanical activity.In addition, although the EGG and the gastric mechanical aoii\riiies aresignificantly ccrrblated,they are sufficiently different to suggest that the EGG may reflect intraabdominal activities unrelated to stomach motility.

Total body irradiation is followed within minutes by nausea and vomiting. In dogs, the authors found that gastric emptying was suppressed for at least 3 hours after exposure to 8 Gy Cobalt-60; in addition, pretreatment with domperidone (D) could prevent vomiting without improving gastric emptying. In the present studies, the authors used a primate model to further investigate the possibility of treating radiation induced vomiting and delayed gastric emptying. Gastric emptying was measured using either (1) radionuclide imaging after intragastric administration of chicken liver tagged in vivo with 1 mCi Tc-99m sulfur colloid and water containing 0.2 mCi In-111-DTPA; or (2) a Tc-99 m-DPTA dilution technique. Chair-adapted rhesus monkeys were studied in the basal state receiving either saline (S; 1.0 ml), D (0.1 mg/kg) or metoclopramide (M; 0.15 mg/kg). All monkeys were then exposed to total body irradiation (8 Gy, Cobalt-60) after receiving either S, D or M. Vomiting was observed in 5 of 6 monkeys receiving S or D but in only one of 5 animals pretreated with M. Gastric emptying was unaffected by any drug in the basal state (S: 5.54 +- 0.86; D: 6.21 +- 1.01; P: 6.52 +- 0.95%/min; means +- SE). After irradiation, emptying wasmore » completely abolished in animals treated with S (0.2 +- 0.2%/min) or D (0.1 +- 0.1%/min; in contrast, pretreatment with M improved gastric emptying significantly (1.81 +- 0.52%/min. Thus, gastric emptying is suppressed in monkeys who vomit after exposure to total body irradiation. In addition, vomiting is prevented and suppression of gastric emptying is improved by M, a dopamine antagonist that acts centrally but not by D, a peripherally acting dopamine antagonist.« less