Surfactant induced symmetric and thermally stable interfaces in Cu/Co multilayers.

In this work we studied Ag surfactant induced growth of Cu/Co multilayers. The Cu/Co multilayers were deposited using Ag surfactant by the ion beam sputtering technique. It was found that Ag surfactant balances the asymmetry between the surface free energies of Cu and Co. As a result, the Co-on-Cu and Cu-on-Co interfaces become sharp and symmetric and thereby improve the thermal stability of the multilayer. On the basis of obtained results, a mechanism leading to symmetric and stable interfaces in Cu/Co multilayers is discussed.

Gene expression profiling in ethnic Malays with type 2 diabetes mellitus, with and without diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) among type 2 diabetes mellitus patients (DM) in Malaysia. This study used microarray analysis to determine the gene expression profiling in ethnic Malay patients with type 2 DM. METHODS: A total of 312 patients were recruited; 25 were on dialysis due to ESRD, 128 were classified as normoalbuminuric, 93 as microalbuminuric and 66 as macroalbuminuric, based on urine albumin to creatinine ratio of <3.5, between 3.5 and 35 and =35 mg/mmol, respectively. RESULTS: Microalbuminuria was associated with up- and down-regulation of 2,694 and 2,538 genes, respectively, while macroalbuminuria was associated with up-regulation of 2,520 genes and down-regulation of 2,920 genes. There was significant up-regulation of 1,135 genes and down-regulation of 908 genes in the ESRD samples. Thirty-seven significantly up-regulated genes and 40 down-regulated genes were commonly expressed in all 3 groups of patients with worsening of renal functions. Up-regulated genes included major histocompatibility complex (HLA-C), complement component 3a receptor 1 (C3AR1), solute carrier family 16, member 3 (SLC16A3) and solute carrier family 9 (sodium/hydrogen exchanger) (SLC9A8). Consistently down-regulated genes included were bone morphogenetic phosphatase kinase (BMP2K), solute carrier family 12, member 1 (SLC12A1), solute carrier family 7 (SLC7A2), paternally expressed 10 (PEG10) and protein phosphatase 1 regulatory (inhibitor unit) (PPP1R1C). CONCLUSION: This study has identified several genes of interest, such as HLA-C, SLC16A3, SLC9A8, SLC12A1 and SLC7A2, that require verification of their roles as susceptibility genes for diabetic nephropathy in ethnic Malays with type 2 DM.

Variation in the thyrotropic activity of human chorionic gonadotropin in Chinese hamster ovary cells arises from differential expression of the human thyrotropin receptor and microheterogeneity of the hormone.

The role of hCG as a stimulator of the human thyroid has been a subject of controversy, because discrepant results have been obtained in different in vitro assays. In an attempt to explain the variation observed in the thyroid response to hCG, we investigated the ability of hCG and that of its isoforms and glycosylation variants to inhibit [125I]bovine (b) TSH binding and stimulate adenylate cyclase in two clones, JP09 and JP26, of Chinese hamster ovary cells stably transfected with the human TSH receptor (hTSHr). The two clones differed with respect to the number of hTSHr expressed per cell (34,000 in JP09 and 2,000 in JP26 cells). Both responded extremely well to bTSH; the cAMP response to 0.001 IU/L bTSH was distinguishable from basal values. Interestingly, JP09 cells were readily stimulated by hCG (20-100 mg/L; 0.52-2.6 x 10(-6) mol/L) to release cAMP, whereas JP26 cells showed little if any response. Also, cAMP stimulation produced by asialo-hCG was 12-fold in JP09 cells and only 4-fold in JP26 cells compared to 45- and 67-fold stimulations by bTSH, respectively. Stimulation by asialo-hCG was approximately 30% that of bTSH in JP09 cells, but less than 6% in JP26 cells. When assessing the thyrotropic activity of the microheterogeneous isoforms of hCG, more alkaline pI forms were found to be more active than those of a more acidic pI regardless of whether they were derived from normal or molar pregnancy urine. Further studies with hCG, asialo-hCG, asialoagalacto-hCG, and deglycosylated hCG revealed that removal of sialic acid caused a marked increase in both its affinity for hTSHr and its cAMP-releasing potency, whereas removal of further carbohydrate, although it slightly enhanced receptor binding, was detrimental to adenylate cyclase activation. In conclusion, differences in hTSHr expression may cause a variation in the cAMP response to hCG or its glycosylation variants, as does the microheterogeneity of the hormone itself. These mechanisms may be responsible at least in part for the divergent responses of different cell types to hCG and render interpretation of the physiological meaning of the data obtained in recombinant receptor systems difficult.

Role of subunit sialic acid in hepatic binding, plasma survival rate, and in vivo thyrotropic activity of human chorionic gonadotropin.

Previous studies have shown that desialylation of human chorionic gonadotropin (hCG) results in a sharp enhancement of its affinity for thyroid thyroid-stimulating hormone (TSH) receptors, transforming it from a weak to a potent antagonist of adenylate cyclase activity in vitro. Because most of the information on the structure-function relation of hCG as a thyroid stimulator has been derived from in vitro experiments, the present studies were undertaken to assess the role of its sialic acid residues in the expression of its thyrotropic activity in vivo. hCG and its various desialylated forms, viz., intact-alpha-asialo-beta, asialo-alpha-intact-beta, and asialo-hCG (ashCG), were initially characterized in terms of their immunoreactivities and receptor-binding abilities as assessed in the rat testis assay. In neither assay did hCG or its variants exhibit a major discordance in activity. In the mouse bioassay, intact hCG (150 micrograms) proved to be a thyroid stimulator of considerable potency, exceeding the response induced by 0.2 mIU bovine TSH (bTSH), as measured by 125I release into the blood after 2- and 8-h intervals. Remarkably, both asialo-alpha-intact-beta and ashCG significantly stimulated the mouse thyroid in this assay, though to a lesser degree than hCG itself. However, in the same assay intact-alpha-asialo-beta was inactive. Studies of the survival of hCG and its variants in the circulation of the mouse, as assessed by radioimmunoassay (RIA) in multiple serum samples drawn over 30 min, showed hCG to have a long half-life, whereas ashCG was cleared very rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate modifications transform human chorionic gonadotropin into a potent stimulator of adenosine 3',5'-monophosphate and growth responses in FRTL-5 thyroid cells.

To delineate the role of carbohydrate moiety in the expression of in vitro thyrotropic activity of hCG, its variants that lacked sialic acid residues or the entire carbohydrate moiety on one or both subunits were prepared. They along with intact hCG were then tested for the abilities to bind to TSH receptor and stimulate cAMP production and growth responses in FRTL-5 cells. The removal of sialic acid from either one or both subunits sharply increased the [125I]bovine TSH binding-inhibiting activity of hCG in the receptor assay. Among the variants tested, desialylated hCG (as-hCG) was the most potent inhibitor, followed by alpha-as-beta, as-alpha-beta, and hCG in that order. With respect to their abilities to stimulate cAMP generation in the cells, the activities of all desialylated hCG variants were markedly higher than that of hCG itself, and as in the receptor assay, as-hCG was the most potent stimulator tested. At the same concentration (100 micrograms/ml), as-alpha-beta, alpha-as-beta, and hCG were approximately only 57%, 46%, and 27% as active as as-hCG in activating cAMP production. The findings in the growth assay were entirely consistent with those noted in the cAMP response assay. Among the deglycosylated hybrids examined, alpha-dg-beta was the most effective in activating cAMP release. An equivalent dose (200 micrograms/ml) was about 1.7, 2.7, and 3.8 times as active as dg-hCG dg-alpha-beta, and hCG, respectively. The deglycosylated variants of hCG showed a similar pattern of activity in growth response and cAMP accumulation assays. In both cases, alpha-dg-beta was the most potent stimulator, while hCG was the least active. No significant difference between the potencies of dg-alpha-beta and alpha-dg-beta was discerned in the receptor assay; however, deglycosylated hCG (dg-hCG) was sharply more active. Results of these studies strongly suggest that the optimal expression of in vitro thyrotropic activity of hCG variants in FRTL-5 cells may require an alpha-subunit, with intact carbohydrate, in combination with the deglycosylated beta-subunit. Further, they demonstrate that modification of the hCG carbohydrate moiety alone can transform it from a weak agonist to a potent stimulator of in vitro thyrotropic bioactivity. These results also provide further evidence to support the notion that the degree of expression of thyrotropic activity is strongly affected by the species in which the studies are performed.

The charge characterization of native and deglycosylated thyrotropin.

Chromatofocusing was used to characterize the charge microheterogeneity of crude pituitary, highly purified native bovine (b) and deglycosylated (dg) thyrotropin (TSH) preparations. Greater than 90% of crude pituitary TSH and native bTSH-I-1 bound to concanavalin-A (conA) columns while dgbTSH was excluded from the column. Extracts of ovine (o) pituitaries contained at least nine species (isohormones) of immunoreactive TSH when chromatofocused on pH 7.5-4 gradients. Highly purified native bTSH-I-1 displayed a similar elution profile. In contrast, dgbTSH eluted as a single homogeneous species with an apparent pI greater than 7.5. When subjected to chromatofocusing on a pH 10.5-7 gradient, 68% of crude pituitary oTSH and 96% of native bTSH-I-1 was bound to the column but could be eluted with NaCl indicating acidic species, while at least three peaks of dgbTSH could be resolved having apparent pI's of 9.12, 9.03 and 8.98. These data suggest that although removal of the carbohydrate moieties markedly alters the isohormone pattern of TSH, chemical deglycosylation does not completely eliminate the charge microheterogeneity of bTSH.

Synthesis and characterization of some chromogenic crown ethers as potential optical sensors for potassium ions.

The complexing abilities of a series of chromogenic crown ethers for potassium and sodium ions have been investigated, by spectrophotometry for the reactions in solution, and by diffuse reflectance spectroscopy when the crown ethers were immobilized. The binding coefficients of the reagents increased with increasing negative charge in the cation-binding site and with increasing extent of chelation. Centimolar K(+) concentrations could be determined with the immobilized reagents, with a K(+)/Na(+) selectivity ratio of approximately 10.

Design of a long-lived thyrotropin antagonist from derivatives of human chorionic gonadotropin.

Previous studies have revealed that desialylated forms of hCG have a high potency to inhibit both the binding of bovine TSH (bTSH) to human thyroid membranes and the bTSH-stimulated adenylate cyclase activity therein. The biological activities of these desialylated forms, however, are greatly reduced in vivo due to the high affinity of asialo-glycoproteins for hepatic receptors and their consequent rapid clearance from the circulation. Intact purified hCG (hCGp), on the other hand, has little affinity for hepatic receptors and has a relatively long half-life in vivo, but displays only negligible inhibitory potency in the human thyroid. In the present studies, we have sought to obtain one or more compounds derived from hCG that are able to inhibit binding to TSH to the high affinity receptor in human thyroid membranes, and, consequently, could inhibit the stimulatory effect of TSH and Graves' immunoglobulin G and which also display a relatively low affinity for the hepatic asialoglycoprotein receptor and, as a consequence, have a reasonable survival in the circulation. Two hCG forms isolated by sequential chromatography of crude hCG on DEAE-52 and Sephadex G-100 were of interest, since they displayed some degree of selectivity in binding to thyroid and liver receptors. The first form (hCGv), whose isolation we described previously, was comprised of partially desialylated variant forms of intact hCG. The second material, as judged from its immunoreactivity, elution behavior on Bio-Gel P-100, and migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained a fragment of the beta-subunit (BF), similar to the beta-core fragment described by others. However, BF differed from the beta-core fragment in having a higher sialic acid content. Interestingly, we found that BF as well as the enzymatically desialylated form of BF displayed a much lower affinity for mouse liver receptors than did asialo-hCGp or the free asialo-beta- and asialo-alpha-subunits. Further, the activity of BF to inhibit the binding of [125I]bTSH to human thyroid membranes exceeded that of the desialylated subunits of hCGp as well as that of intact hCGp, but was only exerted at the low affinity binding site and was not accompanied by inhibition of TSH-stimulated adenylate cyclase. In an attempt to shift the locus of BF action from the low to the high affinity TSH receptor, we recombined BF with either an intact alpha-subunit of hCG or an asialo-alpha-subunit. This led to the creation of two novel forms of hCG with properties of the type that we were seeking.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that partially desialylated variants of human chorionic gonadotropin (hCG) are the factors in crude hCG that inhibit the response to thyrotropin in human thyroid membranes.

Previous studies have revealed that pure unmodified hCG (hCGp) has little potency to inhibit the binding of bovine TSH (bTSH) to human thyroid membranes and to either stimulate adenylate cyclase or inhibit TSH-stimulated adenylate cyclase therein. On the other hand, preparations of crude hCG (hCGc) as well as enzymatically desialylated hCGp (asialo-hCGp) are relatively potent inhibitors of bTSH binding (TBI activity) and bTSH-stimulated adenylate cyclase activity in human thyroid membrane preparations. In the present studies we have sought to identify and characterize the inhibitory moieties in crude hCG that are responsible for these inhibitory activities and to elucidate the properties of their interaction with the TSH receptor in human thyroid membranes. Preparations of hCGc were processed by DEAE-52 chromatography; this separated components of interest, which were not adsorbed, from intact hCGp, which was adsorbed to the column. The former were then subjected to gel filtration on Sephadex G-100, and the earliest eluting fractions, which proved to have the greatest TBI activity, were pooled, rechromatographed, and designated as variant hCG (hCGv). Three different preparations of hCGv were studied. All displayed a lower sialic acid content, by about half, than that in hCGp. Though their potencies varied somewhat, all had significant TBI activity, which was less than that of asialo-hCGp, but more than that of hCGc. Saturation studies revealed that the TBI activities of hCGv and asialo-hCGp were due to a competitive inhibition of bTSH binding at both the high and low affinity bTSH-binding sites, whereas the inhibitory activity of hCGc was exerted primarily at the low affinity binding site. Preparations of hCGv were also capable of inhibiting the adenylate cyclase response to TSH in human thyroid membranes, and Lineweaver-Burk analysis revealed this inhibition to be competitive in nature. As with its TBI activity, the potency of hCGv to inhibit the adenylate cyclase response was intermediate between that of asialo-hCGp and hCGc. Among the three batches of hCGv, their inhibitory effects on bTSH binding and adenylate cyclase activation appeared to vary inversely with with their sialic acid content. Enzymatic desialylation of hCGv increased its potency to that of asialo-hCGp. Several lines of evidence, as follows, indicate that the moieties that comprise hCGv are modified forms of hCGp itself. 1) On a unit weight basis, hCGv was at least as potent as hCGp in its ability to inhibit the binding of [125I]hCG to rat testicular membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Stabilization, partial purification, and characterization of thyrotropin receptors in solubilized guinea pig fat cell membranes.

Specimens obtained during the course of efforts to purify the TSH receptor in guinea pig fat cell membranes solubilized with Triton X-100 displayed extensive loss of TSH binding activity, analogous to that seen by others during the purification of the TSH receptor in thyroid membranes. Therefore, as a preliminary to efforts to purify the TSH receptor in solubilized fat cell membranes (SFCM), experiments were undertaken to ascertain the factors responsible for this loss of binding activity and to find means for its prevention. Temperature proved to be the most important variable. SFCM stored at -70 C retained their activity with respect to the binding of bovine TSH (bTSH) for months, but binding activity was rapidly lost during storage of SFCM at 4 C, a loss due to a decrease in receptor number rather than binding affinity. Loss of binding activity during storage of SFCM at 4 C was unaffected by the addition of 1 mM cystine and was only slightly and temporarily retarded by a mixture of three protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and leupeptin). These results indicated that loss of TSH receptors during storage at 4 C is not due to reduction of disulfide bonds or to proteolytic degradation. On the other hand, activity of the receptor was largely or entirely preserved during at least a week of storage at 4 C by the formation of a TSH-TSH receptor complex, by the addition of 40-50% glycerol either during solubilization or immediately thereafter, or by lyophilization immediately after solubilization. Receptors preserved by these three measures retained their original affinity for bTSH and their response to the TSH binding inhibitory activity of Graves'-immunoglobulin G. In the light of these results, SFCM were prepared in 40% glycerol and then subjected to TSH-affinity gel chromatography. The resulting materials contained at least 29.3% of the original binding activity, and their specific TSH binding activity was increased 227-fold. Saturation analysis of [125I]bTSH binding to this preparation revealed an association constant (Ka) (2.2 x 10(9) M-1), very similar to that in the original SFCM preparation. Binding of [125I]bTSH was inhibited in a dose-dependent manner by Graves'-immunoglobulin G, and in multiple preparations of the latter, TSH binding inhibitory activity measured in the affinity-purified receptor preparation was closely correlated with that measured in SFCM.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of subunit sialic acid in the thyrotropic and gonadotropic activities of human chorionic gonadotropin.

The present studies were undertaken to delineate the role of sialic acid residues in the two subunits of hCG in relation to its hormonal activity. Sialic acid was removed by treatment of the individual subunits or intact hCG with insolubilized neuraminidase. Desialylated variants of hCG were obtained by combining an asialo subunit (as alpha or as beta) with its complementary intact or desialylated subunit. When tested in the hCG receptor assay using a rat testis fraction, none of the hCG variants exhibited any loss of activity compared with that of intact purified hCG. In in vitro bioassays that employed cAMP and testosterone generation in rat Leydig cells as indices of response, intact hCG and as alpha in combination with intact beta (as alpha-beta) were equipotent. In contrast, intact alpha-subunit combined with as beta (alpha-as beta) and desialylated intact hCG (asialo-hCG) showed activity that at the highest concentration of each tested (20 ng/ml) was no more than half of that evoked by intact hCG. In a TSH receptor assay in human thyroid membranes, loss of sialic acid from either one or both hCG subunits resulted in enhancement of binding affinity; the rank order of decreasing potency was asialo-hCG, alpha-as beta, as alpha-beta. However, despite their enhanced binding affinity, and like intact hCG itself, none of the variants elicited a cAMP response in either human thyroid membranes or cultured human thyroid cells. Rather, asialo-hCG and alpha-as beta, but not intact hCG and as alpha-beta, were effective antagonists of the stimulatory response induced by bovine TSH in thyroid cells. These findings indicate that desialylation of hCG enhances its binding affinity for hCG receptors in testis and, much more so, for TSH receptors in human thyroid. Desialylation also changes hCG from a full agonist to a partial agonist in testis and from a nonagonist to an antagonist in human thyroid. In all cases, sialic acid in the beta-subunit of hCG appears to have a predominant role in these effects.

The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for receptor recognition.

TSH is a glycoprotein hormone whose carbohydrate content varies among different species. Although recent studies suggest that variants of TSH deficient in carbohydrate occur naturally, the significance of the carbohydrate moiety of TSH in respect to its thyrotropic function is unclear. The present studies were undertaken, therefore, to examine this question. A highly purified preparation of bovine TSH (bTSH) was deglycosylated by treatment with anhydrous hydrogen fluoride. Amino acid and carbohydrate analyses of the original and deglycosylated preparations indicated that approximately 85% of the carbohydrate originally present had been removed and that the protein moiety was unaltered. As judged from TSH radioreceptor assays, bTSH and deglycosylated bTSH (dg-bTSH) bound to human thyroid membranes with equal affinity, since both caused a half-maximal inhibition of [125I]bTSH binding at approximately equal concentrations. Nonetheless, dg-bTSH at optimal concentration displayed only about one third the activity of intact TSH in stimulating adenylate cyclase activity in human thyroid membranes. dg-bTSH also antagonized the adenylate cyclase-stimulating activity of intact bTSH in this system, but only weakly, since abolition of the bTSH effect required an approximately 40-fold higher concentration of dg-bTSH. In cultures of FRTL5 cells, a cloned line of follicular cells derived from normal rat thyroid, both intact and dg-bTSH enhanced cell growth, as measured by [3H]thymidine incorporation and stimulated cAMP release in the medium, but the response elicited by dg-bTSH was much less than that caused by equal concentrations of the intact hormone. In accord with the findings in the in vitro assays, dg-bTSH evoked a much smaller response than bTSH did in the in vivo mouse assay. It is concluded that although not required for receptor recognition, the carbohydrate moiety of bTSH is essential for the full expression of its biological activity.

Binding of bovine thyrotropin to specific sites in thyroid tissue from control and hemithyroidectomized rats.

The binding of 125I-bovine thyrotropin to thyroid particulate fractions from sham-operated (control) and hemithyroidectomized rats was compared to determine if a change in either the number of bovine thyroid-stimulating hormone (bTSH) binding sites or their affinity for bTSH occurs in physiological situations that evoke changes in the intensity of thyroid stimulation. Following hemithyroidectomy serum TSH levels increase and the remnant thyroid lobe enlarges. Because of compensatory thyroid hypertrophy the concentration of TSH binding sites in the thyroid glands from hemithyroidectomized and control rats was related to particulate protein concentration, to the degree of thyroid cellularity as indicated by DNA concentration, and to the concentration of the plasma membrane markers, 5'-nucleotidase and magnesium-dependent ATPase. In each of four experiments, saturation studies revealed that the maximum specific binding of TSH per unit particulate protein and per thyroid lobe was greater in particulates from remnant than from control thyroid lobes. When related to DNA concentration, the concentration of TSH binding sites in remnant lobes was approximately twice that in control lobes. Because of an increase in plasma membrane markers per lobe after hemithyroidectomy, however, there was no difference in the number of TSH binding sites when related to the concentrations of the membrane marker enzymes in the particulate fractions. As judged from Scatchard analysis, the affinity of TSH binding was lower in remnant than in control lobes. This was partially but not completely due to the increased concentration of particulate protein in the remnant thyroid. These experiments demonstrate that the increase in serum TSH levels after hemithyroidectomy in the rat is associated with alterations in TSH receptor capacity and affinity.

Polyphloretin phosphate, an antagonist of thyrotropin action: evidence for an interaction with the hormone rather than the receptor.

Polyphloretin phosphate (PPP) is known to be an inhibitor of bovine TSH (bTSH)-induced stimulation of the thyroid in both in vivo and in vitro assays. The present studies were undertaken to delineate the mechanism of these effects. A high molecular weight PPP preparation strongly inhibited both the binding of 125I-labeled bTSH [( 125I]bTSH) to human thyroid membranes and the stimulation of adenylate cyclase evoked by bTSH therein. Inhibition of bTSH-induced adenylate cyclase activity by PPP was evident both in the absence and the presence of NaCl (150 mM) in the incubation medium. Incubation of membranes with PPP, followed by its removal, did not affect subsequent binding of [125I]bTSH, indicating that PPP did not bind firmly to or damage the TSH receptor. Gel chromatography on Sephadex G-100 revealed that [125I]bTSH incubated with PPP eluted earlier than [125I]bTSH alone, indicating that PPP had formed a higher molecular weight complex with [125I]bTSH. This effect could be prevented by the addition of an excess of unlabeled bTSH to the incubation mixture. Binding of [125I] bTSH in the higher molecular weight peak generated by incubation with PPP was less than half that in control specimens of [125I]bTSH. Studies with PPP were also conducted in a highly sensitive assay that employs cultured porcine thyroid cells and measures the cAMP response induced by bTSH. The inhibitory effect of PPP on bTSH-induced cAMP accumulation was also evident in this assay. However, the presence of divalent cations Ca++ and Mg++ in the assay medium greatly diminished the inhibitory effect of PPP. Similarly, addition of Ca++ and Mg++ to the incubation medium greatly reduced or abolished the inhibitory effect of PPP on [125I]bTSH binding. Both effects of these salts to lessen the inhibitory response to PPP were overcome by increasing the PPP concentration. Gel chromatographic studies revealed that Ca++ and Mg++ acted by inhibiting the formation of the high molecular weight complex of bTSH and PPP. From these findings, we conclude that PPP exerts its inhibitory effect on TSH-induced stimulatory responses in the thyroid, in vivo as well as in vitro, by forming a complex with the hormone. The complex either does not bind to TSH receptors or does so with much lower affinity.

The active principles of plant extracts with antithyrotropic activity: oxidation products of derivatives of 3,4-dihydroxycinnamic acid.

We have recently reported that freeze-dried extracts (FDE) of certain plants form high molecular weight adducts with bovine TSH (bTSH), preventing it from binding to and stimulating adenylate cyclase in human thyroid membranes. We have now studied 34 pure compounds identical or structurally related to compounds present in FDE from Lycopus or Lithospermum, 2 of the 3 species of active plants studied previously. In studies conducted at 4 C in 20 mM Tris-HCl-0.5% BSA buffer, pH 7.45, eight 3,4-dihydroxylated compounds, all structurally related to cinnamic acid, inhibited the binding of [125I] bTSH to human thyroid membranes. Of these, 4 (caffeic, rosmarinic, chlorogenic, and ellagic acids) are present in the plants, and 4 (3,4-dihydroxyphenylacetic acid, deoxyepinephrine, adenochrome, and nordihydroguaretic acid) are structurally related thereto. These compounds were inactive when tested directly but became active when allowed to undergo auto-oxidation. With all 8 compounds, half-maximum inhibition of [125I]bTSH binding required quantities of oxidized product equivalent to 20-80 micrograms/ml (60-195 microM) of the original compound. Half-maximum inhibitory concentrations of oxidized caffeic and ellagic acids were increased 2- to 4-fold when experiments were performed at 37 C in medium containing 50 mM NaCl. Preincubation of membranes with active oxidation products in concentrations up to 100 micrograms/ml, followed by washing, had no effect on the subsequent binding of [125I]bTSH. As has been shown in the case of FDE, when [125I]bTSH was preincubated with oxidation products of caffeic and ellagic acids and was then chromatographed on Sephadex G-100, its elution pattern was advanced from an apparent mol wt of 30,000 to the void volume, and [125I]bTSH in the early eluting fractions displayed greatly reduced binding to thyroid membrane preparations. Addition of a large excess of unlabeled bTSH during preincubation prevented the shift in the elution pattern of [125I]bTSH produced by these oxidation products. To ascertain whether FDE and active compounds interact with the protein or carbohydrate moieties of bTSH, studies of their effects on the binding and chromatographic behavior of 125I-deglycosylated-bTSH (dg-bTSH) were also performed. Effects were similar to those observed for intact bTSH, suggesting that they do not interact with the carbohydrate moiety of TSH. Preincubation of both bTSH and dg-bTSH with either active FDE or oxidation products of caffeic or rosmarinic acid also greatly decreased their activity in the McKenzie mouse assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracts and auto-oxidized constituents of certain plants inhibit the receptor-binding and the biological activity of Graves' immunoglobulins.

Freeze-dried extracts (FDE) of the plants Lycopus virginicus, Lycopus europaeus, Melissa officinalis, and Lithospermum officinale, as well as products of the oxidation of certain of their constituents, have been shown to exert antithyrotropic activity by virtue of their ability to form adducts with TSH that bind weakly, if at all, to the TSH receptor. The thyroid-stimulating immunoglobulin G (IgG) found in the blood of patients with Graves' disease (Graves'-IgG) resemble TSH in their ability to bind to the thyroid plasma membrane, probably at the TSH receptor, and to activate the gland. In view of this similarity, and since some of the aforementioned FDE have been used in the treatment of hyperthyroidism in Graves' disease, we undertook studies of the effect of these FDE and their constituents on the binding and biological action of Graves'-IgG. In all samples of Graves'-IgG tested, incubation with antithyrotropic FDE or their antithyrotropic auto-oxidized constituents decreased their TSH-binding inhibitory activity in a dose-dependent manner. FDE from L. officinale also inhibited in a dose-dependent manner the direct binding to human thyroid membranes of a 125I-labeled preparation of receptor-purified Graves'-IgG. As judged from both stimulation of adenylate cyclase activity in vitro (thyroid-stimulating Ig activity) and enhancement of thyroid iodine release in the McKenzie assay system (LATS activity), antithyrotropic FDE and their auto-oxidized constituents also inhibited the biological responses to Graves'-IgG. FDE and constituents lacking antithyrotropic activity had little or no effect on the TSH-binding inhibitory activity, thyroid-stimulating Ig activity, or LATS activities of Graves'-IgG. Evidence of some degree of specificity of the inhibitory effects of the active compounds on Graves'-IgG was obtained in the demonstration that they failed to inhibit both the direct binding of [125I]insulin to its receptors in human lymphoblastoid IM-9 cells and the ability of IgG preparations containing antiinsulin receptor antibodies to inhibit the binding of labeled insulin. These observations suggest that the active principles in those FDE and their oxidized constituents with antithyrotropic activity may interact with the pathogenically important components of Graves'-IgG to inhibit their ability to bind to the TSH receptor and activate the thyroid, as they do with TSH. Our findings provide a possible rationale for the empirical, though poorly documented, use of FDE in the treatment of Graves' disease and some support for the suggestion that Graves'-specific IgG may have structural similarities to TSH itself.

Heterogeneity of thyroxine binding by serum albumins in normal subjects and patients with familial dysalbuminemic hyperthyroxinemia.

The nature and properties of the T4-binding albumins in the sera of normal subjects and patients with the syndrome of familial dysalbuminemic hyperthyroxinemia (FDH) were investigated by means of isoelectric focusing in polyacrylamide gels. Albumins isolated from normal sera and sera of patients with FDH displayed multiple protein bands, with isoelectric points between 4.65 and 5.75, and protein patterns were the same in the two groups. In normal albumin, [125I]T4 was consistently localized in two bands, termed B1 and B2, whose isoelectric points (pIs) were 5.44 +/- 0.03 and 5.31 +/- 0.02 (mean +/- SE), respectively. Occasionally, binding of far smaller proportions of [125I]T4 was seen in two additional bands of lower pI (5.22 +/- 0.03 and 4.97 +/- 0.07), termed B3 and B4, respectively. In FDH albumin, [125I]T4 was also localized in four bands whose PIs were almost identical to those of B1-B4 in normal albumin. In FDH albumin, however, bands corresponding to B1 and B2 bound only a minor fraction of [125I]T4, the great majority being bound by bands corresponding to B3 and B4, especially the latter. Identity of the four T4-binding albumins in normal and FDH albumin was suggested by their virtually identical pIs in both types of albumin, by the emergence or intensification of the B3 band in both after extraction of endogenous T4, and by the finding that an 125I-labeled derivative of T4 used in a commercial one-step assay for serum free T4 was bound by the B4 band in both normal and FDH albumin, though more intensely in the latter. Treatment of FDH albumin with increasing concentrations of dithiothreitol (DTT; 0.1-5.0 mM) caused a progressive loss of [125I]T4 binding by B3 and B4, leaving [125I]T4 bound by B1 or B2, and had little effect on the binding of [125I]T4 by B1 and B2 in normal albumin. These effects of DTT appeared to correlate well with earlier dialysis studies at physiological pH which revealed that the same concentrations of DTT decreased or abolished the high affinity binding of T4 in FDH albumin but had little effect on the residual lower affinity binding of T4 in FDH albumin or that characteristic of normal albumin. These findings suggest that T4 binding patterns evident during isoelectric focusing of albumin at low pH have relevance to binding of T4 by albumins at physiological pH.(ABSTRACT TRUNCATED AT 400 WORDS)

Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro.

hCG is a known stimulator of mouse thyroid in vivo. Studies were therefore performed to ascertain whether the thyroid-stimulating activity of hCG in the mouse could also be demonstrated by the in vitro techniques that had failed to show any activity of hCG in the human thyroid. When labeled with 125I and incubated at 22 degrees C in 20 mM Tris-0.5% bovine serum albumin (Tris-BSA), pH 7.45, with increasing concentrations (70-300 micrograms protein/ml) of a mouse thyroid fraction, a purified hCG preparation [( 125I]hCG) showed 5-12% specific binding. In contrast, its binding to a human thyroid particulate fraction, over the same range of protein concentrations, did not exceed 1%. When similar studies were performed at 37 degrees C in 10 mM Tris-50 mM NaCl-0.5% BSA, pH 7.45, [125I]hCG showed no detectable binding either to the human or the mouse thyroid fractions. At concentrations ranging from 1 to 20 mIU/ml (0.9-18 X 10(-9) M), bTSH stimulated cAMP release from human thyroid slices into the medium in a dose-dependent manner. In contrast, hCG concentrations from 10(3) to 10(4) IU/ml (2-20 X 10(-6) M) were without effect on cAMP release. bTSH, at concentrations of 4.5 and 9.0 mIU/ml (4 and 8 X 10(-9)M), stimulated cAMP release from the mouse thyroid, producing in the medium approximately 11- and 28-fold increases in cAMP concentration. hCG also stimulated cAMP release from the mouse thyroid, the increases being approximately 2.3- and 1.8-fold, in the presence of 2270 and 4540 IU/ml (4.5 and 9.0 X 10(-6) M), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Human chorionic gonadotropin and thyroid function in patients with hydatidiform mole.

In view of the controversy regarding the role of human chorionic gonadotropin as the stimulator of thyroid function in patients with trophoblastic tumors, especially hydatidiform mole, we conducted studies to explore whether a correlation between serum human chorionic gonadotropin levels and thyroid function was demonstrable in such patients. Among 47 patients studied, only one was clinically hyperthyroid, although 10 had serum total thyroxine values exceeding those found in normal pregnancy (8 to 17 micrograms/dl). Among 34 patients in whom free thyroxine indices could be calculated, 18 had elevated values for the free thyroxine index (greater than 10.6), and nine had elevated values for both total thyroxine and free thyroxine index. Serum total 3,5,3'-triiodothyronine concentrations were also measured in 17 patients, and only one of them had a value (400 ng/dl) above the normal limit for pregnancy (greater than 350 ng/dl). Among the 13 patients for whom free 3,5,3'-triiodothyronine indices were calculated, three had values above the normal range (greater than 215). A weakly positive correlation (r = 0.35, p less than 0.05, n = 47) between the serum human chorionic gonadotropin levels and serum total thyroxine concentrations was observed in these patients. However, no correlation was found between serum human chorionic gonadotropin levels and free thyroxine index values (r = 0.32, p greater than 0.05, n = 34). Also there was no correlation between serum human chorionic gonadotropin levels and either serum total 3,5,3'-triiodothyronine concentrations (r = 0.32, p greater than 0.1, n = 17) or free 3,5,3'-triiodothyronine index values (r = 0.27, p greater than 0.1, n = 13). chi 2 Analysis revealed no significant relationship between elevations of serum human chorionic gonadotropin concentration and abnormally high values of the free thyroxine index. These studies do not support the premise that human chorionic gonadotropin per se is the thyroid stimulator of molar pregnancy and suggest that a substance or substances, distinct from human chorionic gonadotropin and elaborated by the gestational trophoblastic tissue, are responsible for thyrotoxicosis observed in patients with trophoblastic tumors.