In situ polymerization of conducting polymers around living neural cells: Cellular effect study.

Conducting polymer has been directly polymerized around living neural cells or in the cortex with the aim of creating an intimate contact between implantable electrical devices and electrogenetic cells. The long term cellular effect after conductive polymer coating, a critical issue for practical applications, has not been reported. In this study, poly(3,4-ethylenedioxythiophene) PEDOT was directly polymerized around the living primary neural and PC12 cells under varying current densities, potentials and charge-balanced current pulses. The cell morphology, nuclei evolution, and cell viability post PEDOT polymerization were studied at different time points. The aim of this study was to investigate the immediate and long-term cellular response towards in-situ polymerization of conductive polymers and to provide experimental information on the feasibility of this technique in practical applications.

Large air pressure changes triggered by P-SV ground motion in a cave in northern Taiwan.

Acoustic-gravity waves are generally considered to be one of the major factors that drive changes of the total electron content in the ionosphere. However, causal mechanisms of couplings between sources in the lithosphere and responses in the atmosphere and the ionosphere are not fully understood, yet. A barometer in the cave of the SBCB station records an unusual phenomenon of larger amplitudes in air pressure changes inside than those at the Xinwu station (outside). Accordingly, the comparison between the recorded data at the SBCB and Xinwu station can drive investigations of potential sources of the unusual phenomenon. Analytical results of phase angle differences reveal that the air pressure outside the cave at the Xinwu station often leads air pressure changes inside at the SBCB station at relatively low frequency bands. In contrast, the larger pressure changes at frequencies > ~ 5 × 10-4 Hz inside the cave at the SBCB station lead smaller changes outside at the Xinwu station. To expose causal mechanisms of the unusual phenomenon, continuous seismic waveforms are further conducted for examination. When the horizontal and vertical ground velocities of ground vibrations yield a difference in the phase angle close to 90°, coherence values between the air pressure changes and ground vibrations become large. This suggests that the pressure-shear vertical ground vibrations can drive air pressure changes. Meanwhile, the results shed light on investigating the existence of acoustic waves near the Earth's surface using a partially confined space underground due to that the assumptions of the waves can propagate upward into the atmosphere driving changes in the ionosphere.

Structure of a Thyrotropin Receptor Monoclonal Antibody Variable Region Provides Insight into Potential Mechanisms for its Inverse Agonist Activity.

BACKGROUND: The high constitutive, or ligand-independent, activity of the thyrotropin receptor (TSHR) is of clinical importance in some thyroid conditions, particularly well-differentiated thyroid carcinoma remnants following incomplete ablative therapy (surgery and radioiodine). Under these conditions, even total suppression of TSH by thyroid hormone administration does not fully reduce TSHR activity, a driver of thyrocyte growth. METHODS: CS-17 is a murine monoclonal antibody that has inverse agonist activity in that it suppresses TSHR constitutive activity. This study crystallized the CS-17 Fab and determined its atomic structure at a resolution of 3.4 Å. RESULTS: In silico docking of this structure to that of the TSHR extracellular domain was accomplished by targeting to TSHR residue tyrosine 195 (Y195) known to contribute to the CS-17 epitope. High affinity interaction between these two molecules, primarily by the CS-17 immunoglobulin heavy chain, was validated by energetic analysis (KD of 8.7 × 10-11 M), as well as by previously obtained data on a number of individual TSHR amino acids in three regions whose mutagenesis reduced CS-17 binding as detected by flow cytometry. CONCLUSIONS: Structural insight at atomic resolution of a TSHR antibody with inverse agonist activity opens the way for the development of a molecule with therapeutic potential, particularly in thyroid carcinoma. For this purpose, CS-17 will require "humanization" by substitution of its constant region (Fc component). In addition, with its epitope defined, the CS-17 affinity can be increased further by mutagenesis of selected amino acids in its heavy- and light-chain complementarity determining regions.

Screening of different sample types associated with sheep and cattle for the presence of nematophagous fungi in China.

A total of 1502 samples, including feces of sheep (793) and cattle (348), pasture soil (118), dung compost (147) and barn soil (96), were examined between October 2012 and August 2014 to discover potential strains of nematophagous fungi for the biological control of livestock-parasitic nematodes. These samples were collected from 87 sites located in 48 counties of 20 provinces (autonomous regions/municipalities) of China. Fungi were identified down to a species level. Four hundred and seventy-seven isolates, which were distributed in 8 genera and 28 taxa, were identified as nematophagous fungi. Nematode-trapping fungi included 17 species and one unidentified species of Arthrobotrys, two of Dactylella, Drechslerella dactyloides, and Duddingtonia flagrans. Five identified species and two unidentified species of endoparasitic fungi were isolated. The predominant species from all regions were Arthrobotrys oligospora, followed by Arthrobotrys musiformis, Arthrobotrys (Monacrosporium) thaumasiun, and Arthrobotrys (Monacrosporium) microscaphoides. Species with adhesive networks were the most frequently isolated. Among the endoparasitic fungi, Podocrella harposporifera (Harposporium anguillulae) was the most common species, followed by Harposporium lilliputanum and Harposporium arcuatum. Based on Shannon diversity index, the diversity levels of nematophagous fungi were relatively higher in samples associated with cattle, barn soil, and subtropical monsoon climate zone. Three species isolated from this study, namely, Duddingtonia flagrans, Arthrobotrys salina (Monacrosporium salinum), and Arthrobotrys oligospora var. sarmatica, are newly recorded in China, and 20 species (including one unidentified species) are newly recorded in sheep and cattle barn soils worldwide.

A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor.

Abs that stimulate the thyrotropin receptor (TSHR), the cause of Graves' hyperthyroidism, only develop in humans. TSHR Abs can be induced in mice by immunization, but studying pathogenesis and therapeutic intervention requires a model without immunization. Spontaneous, iodine-accelerated, thyroid autoimmunity develops in NOD.H2(h4) mice associated with thyroglobulin and thyroid-peroxidase, but not TSHR, Abs. We hypothesized that transferring the human TSHR A-subunit to NOD.H2(h4) mice would result in loss of tolerance to this protein. BALB/c human TSHR A-subunit mice were bred to NOD.H2(h4) mice, and transgenic offspring were repeatedly backcrossed to NOD.H2(h4) mice. All offspring developed Abs to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2(h4) mice (TSHR/NOD.H2(h4)) developed pathogenic TSHR Abs as detected using clinical Graves' disease assays. As in humans, TSHR/NOD.H2(h4) female mice were more prone than male mice to developing pathogenic TSHR Abs. Fortunately, in view of the confounding effect of excess thyroid hormone on immune responses, spontaneously arising pathogenic human TSHR Abs cross-react poorly with the mouse TSHR and do not cause thyrotoxicosis. In summary, the TSHR/NOD.H2(h4) mouse strain develops spontaneous, iodine-accelerated, pathogenic TSHR Abs in female mice, providing a unique model to investigate disease pathogenesis and test novel TSHR Ag-specific immunotherapies aimed at curing Graves' disease in humans.

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells.

The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody.

Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease.

CONTEXT: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. OBJECTIVE: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. DESIGN: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. RESULTS: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. CONCLUSIONS: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

Isolation, identification, and characterization of the nematophagous fungus Monacrosporium salinum from China.

Nematophagous fungi are considered to have the best potential as biological agents for the control of gastrointestinal nematodes in domestic animals. However, relatively few studies have been conducted with the genus Monacrosporium, especially with strains native to China. In the present study, we isolated and identified nematophagous fungi from fresh sheep feces. A pure fungal strain was molecularly characterized, and its nematophagous activity was evaluated. The morphological plasticity of the isolated strain, as well as its interaction with the nematode targets, was observed by scanning electron microscopy of the infected Trichostrongylus colubriformis L3 and the free-living nematode Caenorhabditis elegans. Three isolated fungal strains from the 30 fresh fecal samples of sheep from Inner Mongolia, China exhibited predatory activity; however, only a single strain was successfully purified (SF 0459). The SF 0459 strain was characterized by morphological analysis of its conidia and sequencing of its ITS1-5.8S rDNA-ITS2 region. This strain was identified to be Monacrosporium salinum (GenBank ID: KP036623). Nematophagous fungus helper bacteria were found at the interaction points between fungi and nematodes. The percentage of live T. colubriformis L3 was reduced by 83.79-88.69% based on the in vitro assay.

Crystal structure of a TSH receptor monoclonal antibody: insight into Graves' disease pathogenesis.

The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis. The 3-dimensional structure of the active A-subunit (in complex with a human TSAb Fab, M22) is known, but the structural difference with inactive A-subunits is unknown. We solved the 3BD10 Fab 3-dimensional crystal structure. Guided by prior knowledge of a portion of its epitope, 3BD10 docked in silico with the known active TSHR-289 monomeric structure. Because both TSAb and 3BD10 recognize the active TSHR A-subunit monomer, this form of the molecule can be excluded as the basis for the active-inactive dichotomy, suggesting, instead a role for A-subunit quaternary structure. Indeed, in silico analysis revealed that M22, but not 3BD10, bound to a TSHR-289 trimer. In contrast, 3BD10, but not M22, bound to a TSHR-289 dimer. The validity of these models is supported experimentally by the temperature-dependent balance between active and inactive TSHR-289. In summary, we provide evidence for a structural basis to explain the conformational heterogeneity of TSHR A-subunits (TSHR-289). The pathophysiologic importance of these findings is that affinity maturation of pathogenic TSAb in Graves' disease is likely to involve a trimer of the shed TSHR A-subunit.

Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms.

Despite elucidation of the crystal structure of M22, a human thyroid-stimulating autoantibody (TSAb) bound to the TSH receptor (TSHR) leucine-rich repeat domain (LRD), the mechanism by which TSAs activate the TSHR and cause Graves' disease remains unknown. A nonstimulatory murine monoclonal antibody, 3BD10, and TSAb interact with the LRD N-terminal cysteine cluster and reciprocally distinguish between two different LRD conformational forms. To study this remarkable phenomenon, we investigated properties of 3BD10, which has a linear epitopic component. By synthetic peptide ELISA, we identified 3BD10 binding to TSHR amino acids E34, E35, and D36 within TSHR cysteine-bonded loop 2 (C31-C41), which includes R38, the most N-terminal contact residue of TSAb M22. On flow cytometry, despite not contributing to the 3BD10 and M22 epitopes, chimeric substitution (but not deletion) of TSHR cysteine-bonded loop 1 (C24-C29) eliminated 3BD10 binding to the TSHR ectodomain (ECD) expressed on the cell surface, as found previously for TSAb including M22. Furthermore, 3BD10 did not recognize all cell surface TSHR ECDs, consistent with recognition of only one conformational receptor form. Reversion to wild-type of small components of the loop 1 chimeric substitution partially restored 3BD10 binding to the TSHR-ECD but not to synthetic peptides tested by ELISA. Molecular modeling supports the concept that modification of TSHR C-bonded loop 1 influences loop 2 conformation as well as LRD residues further downstream. In conclusion, the present study with mouse monoclonal antibody 3BD10 confirms TSHR conformational heterogeneity and suggests that the N-terminal cysteine cluster may contribute to this structural variability.

Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.

The thyrotropin receptor hinge region as a surrogate ligand: identification of loci contributing to the coupling of thyrotropin binding and receptor activation.

The TSH receptor (TSHR) hinge region, the least well understood component, bridges the leucine-rich repeat and transmembrane domains. We report data on clusters of hinge charged residues the mutation of which to Ala is compatible with cell surface expression and normal, or near normal, TSH binding affinity yet with a relative reduction in receptor activation. Mutation to Ala of E409 at the junction with the transmembrane domain was the most potent in uncoupling TSH binding and signal transduction (~22-fold less sensitive than the wild-type TSHR) and was unique among the residues studied in reducing both the amplitude and the sensitivity of the ligand-induced signal. Unexpectedly, a dual E409A/D410A mutation partially corrected the major suppressive effect of TSHR-E409A. The combined Ala substitution of a cluster of positively charged hinge residues (K287, K290, K291, R293; termed "K3R1") synergistically reduced sensitivity to TSH stimulation approximately 21-fold without altering the TSH binding affinity. Simultaneous Ala substitutions of a cluster of acidic hinge residues D392, E394, and D395 (termed "DE392-5A") partially uncoupled TSH binding from signal transduction (4.4-fold reduction in sensitivity), less than for E409A and K3R1A. Remarkably, the combination of the K3R1A and DE392-5A mutations was not additive but ameliorated the major uncoupling effect of K3R1A. This lack of additivity suggests that these two clusters contribute to a common signaling pathway. In summary, we identify several TSHR hinge residues involved in signal transmission. Our data support the concept that the hinge regions of the TSHR (and other glycoprotein hormone receptors) act as surrogate ligands for receptor activation.

Role of self-tolerance and chronic stimulation in the long-term persistence of adenovirus-induced thyrotropin receptor antibodies in wild-type and transgenic mice.

BACKGROUND: Graves'-like disease, reflected by thyrotropin receptor (TSHR) antibodies and hyperthyroidism in some mouse strains, can be induced by immunization with adenovirus-expressing DNA for the human TSHR or its A-subunit. The conventional approach involves two or three adenovirus injections at 3-week intervals and euthanasia 10 weeks after the first injection. To investigate TSHR antibody persistence in mice with differing degrees of self-tolerance to the TSHR A-subunit, we studied the effect of delaying euthanasia until 20 weeks after the initial immunization. METHODS: Wild-type (WT) mice and transgenic (tg) mice expressing low intrathyroidal levels of the human TSHR A-subunit were immunized with A-subunit-adenovirus on two occasions; a second group of mice was immunized on three occasions. Sera obtained 4, 10, and 20 weeks (euthanasia) after the initial immunization were tested for thyrotropin (TSH) binding inhibition (TBI), antibody binding to TSHR A-subunit protein-coated enzyme-linked immunosorbent assay (ELISA) plates, and thyroid stimulating antibody activity (TSAb; cyclic adenosine monophosphate [cAMP] generation). Serum thyroxine (T4) and thyroid histology were studied at euthanasia. RESULTS: THE majority of WT mice retained high TSHR antibody levels measured by TBI or ELISA at euthanasia but only about 50% were TSAb positive. Low-expressor tgs exhibited self-tolerance, with fewer mice positive by TBI or ELISA and antibody levels were lower than in WT littermates. In WT mice, antibody persistence was similar after two or three immunizations; for tgs, only mice immunized three times had detectable TSAb at 20 weeks. Unlike our previous observations of hyperthyroidism in WT mice examined 4 or 10 weeks after immunization, all mice were euthyroid at 20 weeks. CONCLUSIONS: Our findings for induced TSHR antibodies in mice, similar to data for human thyroid autoantibodies, indicate that the parameters that contribute to the concentration of the antibody and thereby play a critical role in long-term persistence of TSHR antibodies are the degree of self-tolerance to the TSHR and chronic stimulation.

Rescue of a pathogenic mutant human glucagon receptor by pharmacological chaperones.

We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.

Long term persistence of thyrotropin receptor antibodies in wild-type and transgenic mice in a Graves' disease model.

Background: Graves'-like disease, reflected by TSHR antibodies and hyperthyroidism in some mouse strains, can be induced by immunization with adenovirus expressing DNA for the human thyrotropin receptor (TSHR) or it's A-subunit. The conventional approach involves two or three adenovirus injections at three-weekly intervals and euthanasia 10 weeks after the first injection. In order to investigate TSHR antibody persistence in mice with differing degrees of self-tolerance to the TSHR A-subunit, we studied the effect of delaying euthanasia until 20 weeks after the initial immunization. Methods: Wild-type mice and transgenic mice expressing low intrathyroidal levels of the human TSHR A-subunit were immunized with A-subunit-adenovirus on two occasions; a second group of mice was immunized on three occasions. Sera obtained 4, 10 and 20 weeks (euthanasia) after the initial immunization were tested for TSH binding inhibition (TBI), antibody binding to TSHR A-subunit protein-coated ELISA plates and thyroid stimulating antibody activity (TSAb; cAMP generation). Serum thyroxine and thyroid histology were studied at euthanasia. Results: The majority of wild-type mice retained high TSHR antibody levels measured by TBI or ELISA at euthanasia but only about 50% were TSAb positive. Low expressor transgenics exhibited self-tolerance, with fewer mice positive by TBI or ELISA and antibody levels were lower than in wild-type littermates. In wild-type mice, antibody persistence was similar after two or three immunizations; for transgenics, only mice immunized three times had detectable TSAb at 20 weeks. Unlike our previous observations of hyperthyroidism in wild-type mice examined 4 or 10 weeks after immunization, all mice were euthyroid at 20 weeks. Conclusions: Our findings for induced TSHR antibodies in mice, similar to data for human thyroid autoantibodies, indicate that the parameters that contribute to the concentration of the antibody and thereby play a critical role in long term persistence of TSHR antibodies are the degree of self-tolerance to the TSHR and chronic stimulation.

Structure of Co-2 × 2 nanoislands grown on Ag/Ge(111)-√3 × âˆš3 surface studied by scanning tunneling microscopy.

We have found that Co-2 × 2 islands grown on an Ag/Ge(111)-√3 × âˆš3 surface have hcp structure with the (11-20) orientation. The island evolution involves transformation of the unit cell shape from parallelogram into rectangular, which is accompanied by the island shape transformation from hexagonal into stripe-like. Identified are two crystallographic directions for the island growth, the pseudo-[0001] and the pseudo-[1-100]. We have observed the occurrence of a lateral shift between the topmost and the underlying bilayers in the case of the island growth along the pseudo-[0001] direction. In contrast, the topmost and the underlying bilayers are unshifted for the growth along the pseudo-[1-100] direction.

Novel information on the epitope of an inverse agonist monoclonal antibody provides insight into the structure of the TSH receptor.

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.

Insight into thyroid-stimulating autoantibody interaction with the thyrotropin receptor N-terminus based on mutagenesis and re-evaluation of ambiguity in this region of the receptor crystal structure.

BACKGROUND: Thyroid-stimulating autoantibodies (TSAb) bind to the thyrotropin receptor (TSHR) extracellular domain, or ectodomain (ECD), comprising a leucine-rich repeat domain (LRD) linked by a hinge region to the transmembrane domain (TMD). The LRD (residues 22-260; signal peptide 1-21) contains two disulfide-bonded loops at its N-terminus. In the crystal structure of the isolated LRD complexed with human TSAb monoclonal antibody (mAb) M22, N-terminal disulfide loop 1 (residues 22-30) could not be determined because of crystal disorder. Nevertheless, present crystal structure data are interpreted to exclude a role for the LRD N-terminal disulfide loops in the TSAb epitope(s), contradicting prior functional evidence of a role for these loops in TSAb function. MATERIALS AND METHODS: To re-examine this issue we studied two cell types expressing the TSHR with the extreme N-terminal loop 1 (residues 22-30) deleted: the TSHR ECD lacking the TMD and tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and the TSH holoreceptor containing the TMD. Because TSAb including M22 "see" the holoreceptor poorly relative to the TSHR ECD-GPI, we used the latter to examine the effect of deleting residues 22-30 on M22 binding by flow cytometry and the holoreceptor to test the effect of this deletion on the functional response to M22. RESULTS: Deletion of TSHR N-terminal loop 1 (residues 22-30) reduced the number of TSHR-ECD-GPI recognized by M22 relative to two TSHR mAb with epitopes far downstream of the LRD N-terminal loops. Relative to control mAb 2C11, M22 recognized only 60.4% of cell surface receptors (p = 0.02). In contrast to M22 binding to TSHR-ECD-GPI, in functional studies with the TSH holoreceptor, M22 stimulation of cAMP generation was unaltered by the loop 1 deletion. CONCLUSIONS: Our data support the concept that TSAb interact with the cysteine-rich N-terminus of the TSHR. Comparison of crystal structures of the same TSHR LRD in complex with TSAb M22 or blocking antibody K1-70 helps reconcile contradictory viewpoints. A difference between M22 interaction with the identical TSHR N-terminus expressed on the TSHR-ECD-GPI and holoreceptor suggests that crystallization of the TSHR LRD-M22 complex may not provide a complete understanding of the functional TSAb epitope(s) in Graves' disease.

Exceptional hyperthyroidism and a role for both major histocompatibility class I and class II genes in a murine model of Graves' disease.

Autoimmune hyperthyroidism, Graves' disease, can be induced by immunizing susceptible strains of mice with adenovirus encoding the human thyrotropin receptor (TSHR) or its A-subunit. Studies in two small families of recombinant inbred strains showed that susceptibility to developing TSHR antibodies (measured by TSH binding inhibition, TBI) was linked to the MHC region whereas genes on different chromosomes contributed to hyperthyroidism. We have now investigated TSHR antibody production and hyperthyroidism induced by TSHR A-subunit adenovirus immunization of a larger family of strains (26 of the AXB and BXA strains). Analysis of the combined AXB and BXA families provided unexpected insight into several aspects of Graves' disease. First, extreme thyroid hyperplasia and hyperthyroidism in one remarkable strain, BXA13, reflected an inability to generate non-functional TSHR antibodies measured by ELISA. Although neutral TSHR antibodies have been detected in Graves' sera, pathogenic, functional TSHR antibodies in Graves' patients are undetectable by ELISA. Therefore, this strain immunized with A-subunit-adenovirus that generates only functional TSHR antibodies may provide an improved model for studies of induced Graves' disease. Second, our combined analysis of linkage data from this and previous work strengthens the evidence that gene variants in the immunoglobulin heavy chain V region contribute to generating thyroid stimulating antibodies. Third, a broad region that encompasses the MHC region on mouse chromosome 17 is linked to the development of TSHR antibodies (measured by TBI). Most importantly, unlike other strains, TBI linkage in the AXB and BXA families to MHC class I and class II genes provides an explanation for the unresolved class I/class II difference in humans.

An attempt to induce "Graves' disease of the gonads" by immunizing mice with the luteinizing hormone receptor provides insight into breaking tolerance to self-antigens.

BACKGROUND: Gonadotropin receptors, unlike the thyrotropin receptor (TSHR), are not cleaved into disulfide-linked A- and B-subunits, nor do they shed A-subunits. Heavily glycosylated TSHR A-subunits initiate or amplify responses leading to stimulating TSHR-autoantibodies and Graves' hyperthyroidism. METHODS: To investigate the possibility that mice immunized with luteinizing hormone receptor (LHR) would develop functional antibodies, we constructed adenoviruses expressing the rat-LH holoreceptor (LHR-Ad) and an LHR A-subunit equivalent (LHR-289-Ad). Female BALB/c mice were immunized with high doses (10(11) particles) of LHR-Ad, LHR-289-Ad, or control (Con)-Ad. Sera were tested using LHR-expressing eukaryotic cells for antibody binding by flow cytometry and for bioactivity by measuring cyclic adenosine monophosphate (cAMP) stimulation. RESULTS: Elevated serum binding to LHR cells in some LHR-Ad and LHR-289-Ad immunized mice was not specific for LHR-expressing cells. Moreover, sera lacked bioactivity, consistent with unchanged serum estradiol and ovary histology. The difference between rat and mouse LHR-ectodomains is relatively small (3% at the amino-acid level). In contrast, despite amino-acid identity, immunization of mice with adenovirus expressing membrane-bound mouse thyroid peroxidase (TPO), but not soluble mouse TPO ectodomain, elicited strong TPO-specific antibodies. CONCLUSIONS: Our investigations provide insight into antibody responses to self-antigens. First, antibodies are induced to large self-antigens like mouse-TPO when membrane bound. Second, lesser amino acid homology between the immunogen and mouse protein (91% vs. 97% for the human-TSHR and rat-LHR, respectively) favors antibody induction. Finally, from previous studies demonstrating the immunogenicity of the highly glycosylated human TSHR A-subunit versus our present data for the nonimmunogenic less glycosylated rat LHR, we suggest that the extent of glycosylation contributes to breaking self-tolerance.