Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.
Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.
A cell-free system that actively synthesizes collagen was prepared from L-929 fibroblasts. Chromatographic and electrophoretic techniques were used to demonstrate that the only collagenous products are pro alpha(1) and pro alpha(2) chains. The collagen synthesized by the cell-free system was also compared to the collagen extracted from the cells. The cellular collagen was composed of aggregates of pro-alpha chains, while no alpha chains were found. Procollagen peptidase activity could not be detected in the cells, and the activity present in the medium was low, comparable to that in dermatosparaxic cell cultures. These properties indicate that L-929 cells may be a model system for dermatosparaxis.
Three patients with a form of the Ehlers-Danlos syndrome, a generalized disorder of connective tissue, have detectable amounts of procollagen in extracts of their skin and tendon. The activity of procollagen peptidase, the enzyme that converts procollagen to collagen, is reduced in cultures of fibroblasts. The clinical manifestations of this syndrome may be related to impaired enzymatic conversion of procollagen to collagen. Cultures of skin fibroblasts from these patients have an increased rate of synthesis of collagenous protein (collagen and procollagen), possibly related to the inability of these cells to convert procollagen to collagen.
Thyroglobulin (TG), the primary synthetic product of the thyroid, is the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and degradation control thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (TSHR), increases thyroid hormone levels by up-regulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of several thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex class I gene expression, which also is regulated by TTF-1 and Pax-8 in the thyroid, is decreased simultaneously. This helps maintain self-tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. In this report we show that follicular TG counter-regulates TSH-increased, thyroid-specific gene transcription by suppressing expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS, and TSHR genes, but increases class I expression. TG acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the 5' flanking region of TTF-1. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS, and/or TSHR gene expression. The inhibitory effect of TG on gene expression is not duplicated by thyroid hormones or iodide and may be mediated by a TG-binding protein on the apical membrane. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism that limits follicular function and contributes to follicular heterogeneity.
Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.
Accumulation of the permeant lipophilic cation [ 3 H]tetraphenylphosphonium (TPP + ) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K + and by veratridine. A further 1:10 to 1:15 decrease in TPP + uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (ΔΨ, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K + diffusion gradient (K + in →K + out ). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP + concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP + uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP + uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP + uptake is not evident in the presence of 193 mM K + or veratridine but remains under conditions known to abolish the mitochondrial ΔΨ. Moreover, tetanus toxin has no effect on TPP + uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.
Thyroid cell membranes contain a multiplicity of gangliosides, some of which inhibit thyrotropin binding to thyroid membranes. The most potent inhibitor is a ganglioside which is present in only trace amounts and appears to have a novel structure. Thyroid gangliosides may play a role in relaying the hormonal message to the thyroid cell.
The major collagenous products synthesized in a cell-free polysome preparation are pro-alpha1 and pro-alpha2, formed in a ratio of 2:1. They are the precursor forms of alpha1 and alpha2 chains of normal connective-tissue collagen that are also formed in small amounts. A procollagen peptidase activity has been demonstrated in the supernatant fraction that can account for the formation of alpha1 and alpha2 chains from their precursors. The polysomal system is activated by a salt extract of reticulocyte ribosomes and is inhibited by aurintricarboxylic acid, suggesting that the polysomes are able to initiate protein synthesis.
Pyruvic carboxylase activated by acetyl coenzyme A is highly active in the mitochondria of rodent liver, and its activity is increased in fasting and alloxan diabetes. In conjunction with acyl carboxylase activated by di- and tricarboxylic acid, it forms a reciprocating control network. Analog models of similar networks tend to correct for perturbations, stabilizing the overall system.
