Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): insights into the evolution of thyrotropin receptor specificity.

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH-TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T(4)-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone-receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11-20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin ß subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T(4), thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.

A rational design strategy for protein hormone superagonists.

By combining evolutionary considerations, sequence comparisons and homology modeling we have designed recombinant human thyroid-stimulating hormone (hTSH) analogs with increased receptor binding and activity. The introduction of seven basic residues into the peripheral loops of hTSH resulted in up to a 50,000-fold increase in receptor binding affinity and 1300-fold increase in intrinsic activity. Such analogs are not only of potential clinical interest but can be tools to explore molecular aspects of conventional as well as nonclassical actions of glycoprotein hormones. These design strategies should be applicable to the development of novel analogs of other related hormones and growth factors with a variety of therapeutic and basic science applications, particularly for proteins that have undergone evolutionary decrease in bioactivity.

Engineering human glycoprotein hormone superactive analogues.

We report the generation of superactive analogues of human glycoprotein hormones, with potential applications in thyroid and reproductive disorders. Current biological and structural data were used to rationalize mutagenesis. The 11-20 region in the alpha-subunit with a cluster of lysine residues forms a previously unrecognized domain critical for receptor binding and signal transduction, as well as an important motif in the evolution of glycoprotein hormone activities. The gradual elimination of basic residues in the alpha-subunit coincided with the evolutionary divergence of the hominids from the Old World monkeys. By selective reconstitution of certain critical residues present in homologous nonhuman hormones we have developed human thyroid stimulating hormone and chorionic gonadotropin analogues with substantial increases in receptor binding affinity and bioactivity, thus providing a paradigm for the design of novel therapeutic protein analogues.

Structure-function studies of human TSH: new advances in design of glycoprotein hormone analogs.

Recent progress in structure-function studies of glycoprotein hormones has provided new insights into the molecular mechanisms of action of these hormones and has further supported the concept that physiological modulation of assembly, bioactivity, and clearance of these hormones is dependent on specific structural components. This review emphasizes current advances in the structure-function relationships of human TSH, which have contributed to further elucidation of common and hormone specific features within the glycoprotein hormones family. Novel strategies are now being applied to investigate the role of individual structural elements. The principks discovered in such studies are essential to understand the physiological regulation of hormone bioactivity and allow for the rational design of novel analogs with potential therapeutic applications.

Role of the carboxy-terminal residues of the alpha-subunit in the expression and bioactivity of human thyroid-stimulating hormone.

The glycoprotein hormones TSH, CG, LH, and FSH are heterodimers consisting of a hormone-specific beta-subunit and a common alpha-subunit. The aim of the present study was to investigate the role of the carboxy terminus of the common alpha-subunit (amino acids Tyr89-His90-Lys91-Ser92), which has been shown to be important for human (h) CG and hFSH, for the activity of hTSH. Successive truncations of the alpha-carboxy terminus by site-directed mutagenesis revealed a stepwise reduction of bioactivity occurring at residues alpha Ser92 and alpha His90 to 64% and 13%, respectively. This contrasts with previous findings for hCG and hFSH, where loss of bioactivity occurred in a single step with the deletion of alpha Lys91 but alpha Ser92 was not important. The decreased bioactivities of the hTSH alpha-truncation mutants were reflected by concomitant reductions of cAMP production, thyroid hormone synthesis and cell growth and were accompanied by a loss of receptor binding. Substitution of residues alpha Lys91 or alpha His90 with either a hydrophobic or a bulkier residues resulted in a reduction of receptor binding and signal transduction, indicating that the alpha-carboxy terminus of hTSH may interact with the TSH receptor in a tight contact area. Conversely, substitution of alpha His90 with smaller residues enhanced bioactivity. In addition, the integrity of the alpha-carboxy terminus was essential for hTSH expression. Thus, we showed common and different roles of the alpha-carboxy-terminal residues for the glycoprotein hormones. The unique role of alpha Ser92 in hTSH activity explains the evolutionary constraint to preserve the alpha-carboxy-terminal Ser92 in all glycoprotein hormones.

Site-directed mutagenesis of amino acids 33-44 of the common alpha-subunit reveals different structural requirements for heterodimer expression among the glycoprotein hormones and suggests that cyclic adenosine 3',5'-monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin.

Amino acid residues 33-44 of the common alpha-subunit of the glycoprotein hormones have been implicated in heterodimerization as well as high affinity receptor binding of human (h) CG. In the present study, we compared the role of specific amino acids within this region for glycoprotein hormone heterodimer formation, using a transient transfection system to coexpress different mutant alpha-subunit constructs with the beta-subunit of either hTSH, hCG, or hFSH. Our results identified a crucial role for alpha Pro38 in the heterodimer expression of hTSH as well as hFSH, similiar to what had been described for hCG. In contrast, alpha Ala38, which had been critical for hCG, was not essential for hTSH heterodimer expression and less important for hFSH, whereas alpha Phe33 and alpha Arg35 appeared uniquely important for hFSH. Furthermore, we assessed the role of these residues for bioactivity and receptor binding of hTSH. Mutation of the surface-exposed residues alpha Arg42-Ser43-Lys44, which form part of a unique alpha-helical structure, to Ala42-Ala43-Ala44, decreased TSH receptor binding using porcine thyroid membranes as well as rat FRTL-5 cells. Residues alpha Phe33 and alpha Arg35, in contrast, were not important for high affinity binding of hTSH. In the signal transduction of hTSH, alpha Ala36 was necessary for efficient growth induction in FRTL-5 cells but not for cAMP production in either FRTL-5 cells or Chinese hamster ovary cells expressing the human TSH receptor (JP09). Similarly, residues alpha Arg42-Ser43-Lys44 were more important for hTSH-mediated induction of cell growth than cAMP production. Mutating alpha Arg35 to Ala reduced cAMP induction but not receptor binding of hTSH. In summary, using site-directed mutagenesis, we identified a domain, residues 33-44 of the common alpha-subunit, important in heterodimer expression, receptor binding, and activation of hTSH. The comparison of the relative roles of specific amino acids within this region in hTSH with hCG and hFSH highlights previously unrecognized differences in the structural requirements for heterodimer expression among the members of the glycoprotein hormone family. Moreover, our findings revealed a novel role for residues alpha 33-44 in triggering different postreceptor events, suggesting that cAMP production and growth promotion may, at least in part, be dissociable functions of hTSH.

The role of the oligosaccharide chains of thyrotropin alpha- and beta-subunits in hormone action.

TSH, a dimeric glycoprotein hormone, has two N-linked complex-type oligosaccharide chains on the alpha-subunit and one on the beta-subunit. The oligosaccharide chains of TSH are important for the expression of hormonal activity, but the contribution of those on each of the subunits to the activity is not clear. In the current study we have determined the relative importance of the oligosaccharide chains of TSH subunits using a recently reported method of enzymatic deglycosylation. The alpha- and beta-subunits of bovine TSH were deglycosylated with endoglycosidase-F and recombined to obtain differentially deglycosylated TSH. The derivatives showed no differences in their receptor-binding activities. The in vitro bioactivity of these derivatives was assessed by measuring adenylyl cyclase activity in bovine thyroid membranes and stimulation of cAMP production and growth in FRTL-5 cells. In the adenylyl cyclase assay, deglycosylation of the alpha-subunit alone had a more profound effect on the activity [maximal stimulatory activity (Vmax), 13% that of alpha.beta) than when the beta-subunit alone was deglycosylated (Vmax, 50% that of alpha.beta). In the FRTL-5 assays, removal of carbohydrate from TSH alpha, but not the beta-subunit, caused a 2- to 3-fold increase in the concentration required for half-maximal stimulation, with minimal change in the apparent Vmax. The adenylyl cyclase assay in bovine membranes was more sensitive to carbohydrate removal than the assays of rat FRTL-5 cells, in which the derivatives with lower activity were able to stimulate cAMP and growth to near-maximal levels, albeit at 3-fold higher concentrations. These results indicate that the carbohydrate chains in both subunits of TSH, particularly those in the alpha-subunit, are important in hormone action. In contrast to previous reports, our study shows that the beta-subunit plays a role in signal transduction.

The relationship between prorenin levels in follicular fluid and follicular atresia in bovine ovaries.

Bovine follicles having a higher concentration of progesterone than estradiol in the follicular fluid can be considered as atretic. Since we observed previously that there was an inverse relationship between the follicular fluid estradiol to progesterone (E/P) ratio and the prorenin level, we have proposed that a high prorenin level may be associated with follicular atresia. The aim of the present study was to corroborate this hypothesis by including additional indices to distinguish unambiguously between atretic and nonatretic follicles and to compare the prorenin levels in these two groups of follicles. The present study included examination of more than 200 follicles in the follicular fluid of which we have measured steroid and prorenin levels. The results obtained show a highly significant negative correlation between the prorenin level on the one hand and the E/P ratio, estrogen to total androgen ratio, or estradiol concentration on the other hand. As a further criterion for atresia, we have examined the histological characteristics of the follicles by light and electron microscopy and have found that 90% of histologically characterized atretic follicles had an E/P ratio less than 1 and an average prorenin level four to five times higher than nonatretic follicles. Finally, when we determined the FSH-stimulated cAMP response and the aromatase activity, in terms of the ability to convert exogenous androgen to estrogen in granulosa cells isolated from individual follicles, we observed a markedly higher prorenin level in the fluid of follicles whose granulosa cells responded poorly to FSH and showed a low aromatase activity, compared to follicles whose granulosa cells responded strongly to FSH and contained high aromatase activity. In summary, follicles that were classified as atretic on the basis of a number of biochemical and histological parameters contained significantly higher prorenin levels in their follicular fluid than nonatretic ones. Thus, a high follicular fluid prorenin level is a valid indicator for follicular atresia in bovine ovaries. However, the reason for this increase in follicular fluid prorenin level and whether this increase is a cause or a consequence of atresia remains to be determined.

Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance.

Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity.

Asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin.

The recombinant human TSH (rhTSH) with highly sialylated oligosaccharide chains showed higher in vivo bioactivity and a lower MCR than the predominantly sulfated pituitary human TSH (phTSH). The aim of the present study was to investigate the role of terminal carbohydrate residues in organ distribution and metabolic clearance of TSH using an in vivo rat model. The different 125I-labeled TSH preparations with distinct carbohydrate composition were injected i.v. At various time points (5-180 min) after bolus TSH injection, blood, liver, kidney, spleen, lung, heart, and thyroid samples were collected. TSH uptake was determined by trichloroacetic acid precipitation of [125I]TSH in the organ homogenates. The rhTSH (solely sialylated) was distributed predominantly to the kidneys 5, 15, and 30 min after injection. In contrast, phTSH (sulfated/sialylated) and bovine TSH (bTSH; solely sulfated) were cleared predominantly by the liver (at 5 min), with a later renal phase of clearance (at 30 min). Asialo-rhTSH was cleared by the liver with only minor involvement of other organs. The early liver uptake (at 5 min) was proportionally highest for the asialo-rhTSH and bTSH preparations and lowest for rhTSH, which correlated inversely with the serum levels and the degree of sialylation. Blockade of the N-acetylgalactosamine (GalNAc) sulfate receptors by injection of bovine LH resulted in a significant decrease in liver uptake of phTSH. Similarly, liver uptake of asialo-rhTSH was significantly inhibited by injection of asialo-fetuin. Thus, phTSH and bTSH preparations containing sulfated oligosaccharide chains are cleared at least in part by the GalNAc sulfate-specific receptors in the liver. In contrast, rhTSH with highly sialylated oligosaccharides in both subunits accumulates predominantly in the kidneys, even at the early phase of clearance, indicating that sialylated glycoprotein hormones escape from specific receptor-mediated clearance mechanisms in the liver. These data indicate that terminal sialic acid and GalNAc sulfate residues, each to a different extent, determine glycoprotein hormone distribution and thereby plasma level, which as we have shown previously is a major factor in determining the in vivo potency of TSH.

Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity.

The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity. We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes. In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones. We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor. The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis. rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values. Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions. phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions. The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions. In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms. These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation. The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.

The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design.

Among glycoprotein hormone receptors the TSH receptor (TSHR) is the most susceptible to constitutive activation by mutations in various regions of the molecule, including mutations in the extracellular domain (ECD) and extracellular loops of the transmembrane domain (TMD). To understand the role of the ECD in TSHR activation we have tested several TSHR constructs with major deletions of the ECD. Previous studies reported very low expression of such truncated glycoprotein hormone receptors, which prevented reliable assessment of their ligand-binding and basal constitutive activities. We have eliminated this problem using TSHR tagged at its N-terminus with a hemagglutinin tag (HA) recognized by the HA-specific monoclonal antibody. Based on such quantitation the TSHR deletion mutant missing 386 N-terminal amino acid residues, constituting 98% of the entire ECD, showed 4-7 fold higher normalized basal activity compared to activity of the corresponding wild-type (WT) TSHR construct. This increase in basal activity was significantly inhibited by linking the common alpha-subunit of glycoprotein hormones at the N-terminus of the truncated TSH receptor. The role of a hypothetical activating fragment (409-418) in TSHR activation was further studied using peptides and mutagenesis of charged residues. This study provides important evidence supporting the "two-state" model of TSHR activation and the potential role of proteolytic cleavage for receptor activation. Accordingly, the mechanism of hormone-induced receptor activation is dependent, at least in part, on the elimination of inhibitory interactions within the receptor. Such intra-molecular inhibition of TSHR may include electrostatic interactions between the ECD and extracellular loops of TMD. Moreover, the truncated, constitutively active receptors described herein provide new insights valuable in the design of TSHR antagonists.

Expression of biologically active human thyrotropin (hTSH) in a baculovirus system: effect of insect cell glycosylation on hTSH activity in vitro and in vivo.

To obtain large amounts of hTSH and to study the role of the N-linked oligosaccharides for its biological activity, hTSH was produced using recombinant baculovirus containing the human alpha-subunit and a hTSH beta-minigene, respectively, both under the control of the polyhedrin promoter. Expression in insect cells was 800-1000 ng/ml, 30-fold higher than in our optimized mammalian transient transfection system using Chinese hamster ovary (CHO) cells (20-50 ng/ml). The in vitro activity of insect-cell expressed hTSH (IC-hTSH) was increased 5-fold compared with CHO-hTSH, judged by the ability to induce cAMP production in CHO cells stably transfected with the hTSH receptor (JP09) and the rat thyroid cell line FRTL-5, as well as growth promotion in FRTL-5 cells. Lectin binding and enzymatic desialylation studies suggested that in contrast to CHO-hTSH, IC-hTSH lacked complex-type oligosaccharides terminating with sialic acid but contained predominantly high mannose-type oligosaccharides. The in vitro activity of CHO-hTSH also increased 5- to 6-fold upon treatment of the hTSH-producing cells with the oligosaccharide processing inhibitors swainsonine and castanospermine, which inhibit formation of complex, terminally sialylated oligosaccharides, and upon enzymatic desialylation. In contrast, insect cell-expression or treatment with processing inhibitors did not affect TSH receptor binding. Despite the higher in vitro activity, IC-hTSH had a much lower in vivo activity than CHO-hTSH, due to rapid clearance from the circulation. In summary, this study shows for the first time that relatively high levels of recombinant hTSH with high in vitro bioactivity can be produced in a baculovirus system. Cell-dependent glycosylation is a major factor that determines the final in vivo biopotency of recombinant glycoproteins, a finding that should be of general relevance for all insect cell-produced glycosylated proteins. Although not suitable for clinical use, highly bioactive recombinant hTSH derived from high expression in insect cells should be useful in defining structure-function relations of hormone analogs.

Effects of omeprazole on the number of immunoreactive gastrin- and somatostatin-cells in the rat gastric mucosa.

The effects of omeprazole--an inhibitor of gastric acid secretion--on gastrin (G)- and somatostatin (D)-cell density in the gastric antral mucosa epithelium in rats were examined, following a 5-day treatment. It was found that omeprazole increased the density of G-cells, whereas it decreased the density of D-cells. That effect was probably independent of hypergastrinaemia, since it could not be blocked by a simultaneous treatment with proglumide--a gastrin receptor blocker. It is concluded that the observed phenomenon is a direct result of a lower gastric acidity, as a consequence of omeprazole treatment.

Influence of somatostatin and epidermal growth factor (EGF) on the proliferation of thyroid follicular cells in organ culture.

The aim of the present study has been to examine the effects of various concentrations of somatostatin (SS), epidermal growth factor (EGF), as well as of interactions among SS, EGF and thyrotropin (TSH) in their influence upon the mitotic activity of thyroid follicular cells (TFC) in organ culture. The stathmokinetic method was employed. It was shown that: (1) SS, at the concentration of 10(-7) M, suppressed the mitogenic effect of TSH, as well as of TSH and EGF employed together, on TFC; (2) EGF, at the concentration of 10 and 100 ng/ml, increased the mean mitotic activity rate of TFC; (3) TSH and EGF revealed an additive action on TFC proliferation. The obtained results evidently suggest an antiproliferative effect of SS and mitogenic action of EGF on TFC in organ culture.

Suppressing effect of cysteamine on the TSH-stimulated mitotic activity of the rat thyroid follicular cells in vivo.

We have examined the effect of the thiol agent-cysteamine, known to deplete the hypothalamus, the pancreas, the gut of somatostatin--on the basal and TSH-stimulated mitotic activity of the thyroid follicular cells (TFC) in adult male rats in vivo. The animals of different groups were administered cysteamine alone (300 mg/kg BW, s.c.), TSH alone (2IU, i.p.), or cysteamine with TSH jointly, the former agent being injected 2 hrs prior to the latter. In order to evaluate the thyroid mitotic activity, the metaphase-arrest technique was used in the study. It was shown that: 1. TSH significantly increased the mitotic activity of TFC, as compared to controls, 2. cysteamine alone had no effect on the TFC proliferation, 3. unexpectedly, cysteamine, when administered 2 hrs prior to TSH, suppressed the stimulatory effect of TSH on the mitotic activity of TFC.

Development and in vitro characterization of human recombinant thyrotropin.

We have gained insight into the molecular mechanism of human thyrotropin (hTSH) action through cloning of the human TSHbeta subunit gene, development of recombinant TSH and novel analogues and chimeras produced by site-directed as well as cassette mutagenesis. A variety of loss of function mutations have shown several key domains in both the alpha- and beta-subunits that are important for high-affinity ligand interaction with the receptor. In contrast the specificity of receptor interaction was shown to be determined primarily by areas within the hTSH-beta "seat-belt" region. We have also designed various gain of function mutants (superagonists) using evolutionary considerations, homology modeling, and sequence comparisons within the cystine knot growth factor superfamily. Such superagonists resulted from increasing the positive charge by introduction of lysine or arginine residues or neutralization of negatively charged residues of the peripheral hairpin loops of each subunit in various combinations. Certain superagonists increased receptor binding, in vitro and in vivo bioactivity 100- to 1000-fold, more than that achieved previously for any other known protein ligand. In vivo metabolic clearance and biologic activity could be separately modulated by alteration of TSH carbohydrate structure including production of chimeras that added sites of O-glycosylation and/or covalently linked the alpha- and beta-subunits. These data suggest that electrostatic interactions resulting from net positive charge in TSH and net negative charge in its receptor play an important role in high-affinity TSH receptor binding and signal transduction. Insights gained from the design of such novel recombinant TSH analogues and chimeras should have many diagnostic and therapeutic applications. These include the design of improved in vitro assays for thyrotropic factors as well as the design of second generation recombinant TSH analogues for the detection and treatment of thyroid cancer.

A novel, nonradioactive in vivo bioassay of thyrotropin (TSH).

A new and simple in vivo bioassay suitable for routine testing of pituitary and recombinant TSH preparations was developed. Male Albino Swiss CF-1 mice were given T3 in their drinking water to suppress endogenous TSH. T3, 3.0 micrograms/mL, given to mice for a period of 4 days decreased plasma total T4 (TT4) level to less than 10% of the nonsuppressed (control) level. Various doses of exogenous pituitary and recombinant TSH preparations were injected intraperitoneally and blood samples were obtained from the orbital sinus 6 h later. The TT4 level, measured by radioimmunoassay, served as the assay end-point. The assay required injection of approximately 3.0 micrograms of pituitary human TSH (phTSH), 1.0 microgram recombinant human TSH (rhTSH), 0.2 microgram bovine TSH (bTSH), and 0.1 microgram rat TSH (rTSH) to attain half-maximal response. The maximal level of TT4 after TSH stimulation was similar to that observed in normal, nonsuppressed mice. The procedure developed is relatively easy to perform, economical, and, unlike earlier TSH bioassays, does not require the use of radionuclides. This bioassay showed acceptable sensitivity and reliability in structure-function studies of pituitary TSH from different species as well as rhTSH.

A proposed mechanism of anticancer activity of indomethacin.

The exact mechanism of anticancer activity of indomethacin still remains unclear. In view of the gathered observations and ideas, I hypothesize that the effect of indomethacin may be related--at least in certain cases--to its antagonism to gastrin/cholecystokinin family of peptides and/or to its agonistic activity to thromboxane A2 and melatonin. Future experiments in this direction are required.

Glutamine derivatives as novel anticancer agents acting via gastrin binding sites.

With regard to the structural analogies between the C-terminal tetrapeptide of gastrin, gastrin receptor antagonists and anticancer derivatives of glutamine, it is plausible to propose that the mechanism of the anticancer activity may be related to interaction with the gastrin receptor. Dietary glutamine intake and glutamine metabolites may also influence the activity of gastrin receptors.