T4-binding Proteins Research Articles

T3 levels and thyroid hormone signaling.

The clinical availability of tissue-specific biomarkers of thyroid hormone (TH) action constitutes a "holy grail" for the field. Scientists have investigated several TH-dependent markers, including the tissue content of triiodothyronine (T3)-the active form of TH. The study of animal models and humans indicates that the T3 content varies among different tissues, mostly due to the presence of low-affinity, high-capacity cytoplasmic T3 binding proteins. Nonetheless, given that T3 levels in the plasma and tissues are in equilibrium, T3 signaling is defined by the intracellular free T3 levels. The available techniques to assess tissue T3 are invasive and not clinically applicable. However, the tracer kinetic studies revealed that serum T3 levels can accurately predict tissue T3 content and T3 signaling in most tissues, except for the brain and pituitary gland. This is true not only for normal individuals but also for patients with hypo or hyperthyroidism-but not for patients with non-thyroidal illness syndrome. Given this direct relationship between serum and tissue T3 contents and T3 signaling in most tissues, clinicians managing patients with hypothyroidism could refocus attention on monitoring serum T3 levels. Future clinical trials should aim at correlating clinical outcomes with serum T3 levels.

Nicotine binds to the transthyretin-thyroxine complex and reduces its uptake by placental trophoblasts

BackgroundA supply of maternal thyroid hormone (thyroxine, T4) is essential for normal human fetal development. Human placental trophoblasts synthesize, secrete and take up the T4 binding protein transthyretin, providing a route for maternal T4 to enter the placenta. Transthyretin is also involved in T4 transport in other tissues such as the brain choroid plexus. Nicotine alters transthyretin synthesis and function in rat choroid plexus. If nicotine influences trophoblast turnover of transthyretin, then it may directly affect placental transfer of T4 to the developing fetus and contribute to the negative impacts of smoking on fetal growth, development and placental function. MethodsThe effect of nicotine on trophoblast uptake of Alexa-labelled transthyretin was measured using live cell imaging. The effect of nicotine on protein expression was measured by western blotting. Interactions between transthyretin, T4 and nicotine were investigated using chemical cross-linking techniques and molecular dynamic simulations. ResultsNicotine blocks uptake of transthyretin-T4 by human placental trophoblast cells. Nicotine reduces the expression of the trophoblast scavenger receptor class B type 1 (SR-B1) that plays a role in transthyretin-T4 uptake. Molecular dynamic modelling suggests that when T4 is bound to transthyretin, nicotine binding increases tetramer stability, reducing the ability of the transthyretin-T4 complex to enter trophoblast cells. ConclusionOur data suggest that nicotine exposure during pregnancy reduces transplacental transport of transthyretin and T4 to the placenta and developing fetus. This may contribute to the negative effects of smoking on fetal growth, development and pregnancy viability.

Profound Hypothyroidism 21 Days After Methimazole Initiation in a Patient With Graves’s Disease and Liver Cirrhosis

Introduction: Methimazole is the first line treatment of nonpregnant Grave’s disease (GD) patients with Grave’s ophthalmopathy. It usually results in euthyroidism within 4 to 12 weeks, and it is recommended to check FT4 every 4 to 6 weeks after initiation of therapy. We report a patient with GD who developed profound hypothyroidism 21 days after starting methimazole. Clinical Case: 51-year-old male with decompensated EtOH/NASH liver cirrhosis, Gilbert’s syndrome, and GD presented with severe hypothyroidism 21 days after initiating methimazole treatment. He initially presented to the emergency department (ED) with atrial fibrillation with a HR of 140 and found to have bilateral exophthalmos and lid lag. Labs: TSH <0.01 uIU/L (normal range (nl) 0.27-4.20), FT4 2.6 ng/dl (nl 0.7-1.7), TT4 6.2 ug/dl (nl 4.5-12), TT3 125 ng/dl (nl 71-180), TBG 14 ug/ml (nl 13-39), TSI 344 IU/l (nl 0-0.5), AST 55 IU/L (nl 14-44), ALT 27 IU/L (nl 9-57), Alk Phosphatase 497 IU/L (nl 45-129), Tbili 7.1 mg/dl (nl 0.2-.1), and albumin 2.1 g/dl (nl 3.4-5). He was started on methimazole 40 mg daily and propranolol 20 mg BID. Three days later, FT4 decreased to 1.3 ng/dl, so methimazole was decreased to 20 mg day. Ten days later, FT4 was 0.2 ng/dl and methimazole was reduced further to 10 mg daily. Eight days later, he presented to the ED with severe lethargy and found to have HR of 38 and oral temperature (T) of 94. TSH was 3.49 uIU/L and FT4 <0.1 ng/dl. Sixteen hours after 200 mcg IV levothyroxine, he had normal mental status, HR of 49, and T of 97.9. FT4 increased to 0.5 ng/dl within 48 hours of thyroxine initiation. Urine drug screen was only positive for tetrahydrocannabinol and other work up for severe lethargy and hypothermia was unremarkable. He was discharged on 100 mcg of PO levothyroxine. Conclusion: Only 21 days after starting methimazole, the patient developed severe lethargy associated with hypothermia, bradycardia, and undetectable FT4, with no alternative etiology and prompt response to IV levothyroxine, consistent with profound hypothyroidism. Although the half-life of T4 is shortened in hyperthyroidism, the particularly fast and profound effect of methimazole in this patient was unusual and likely due to a limited extra-thyroidal pool of thyroxine caused by low TBG level. As cirrhosis is usually associated with increased TBG level, he may have a contributing congenital etiology. Reduced levels of other T4 binding proteins, albumin and pre-albumin may also have contributed. Although methimazole clearance can be decreased in liver impairment, no dose adjustment is generally recommended, and even this would not have explained the observed rapid decrease in FT4 level. Methimazole should be used with caution and FT4 should be checked early and more frequently when treating hyperthyroid patients with liver disease and possible alteration in thyroid binding proteins.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

SummaryAdult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Thyroid disorders in pregnancy

There are significant changes that occur in the thyroid gland and its function during pregnancy, thus making the assessment of thyroid functions in pregnancy substantially important. Normal pregnancy is associated with an increase in renal iodine excretion, an increase in T4 binding proteins, an increase in thyroid hormone production, and thyroid stimulatory effects of human chorionic gonadotropin (hCG), and the treatment targets are different from women who are not pregnant. Both hypothyroidism and hyperthyroidism are associated with significant impact on the fetomaternal unit and pregnancy outcomes. Evidence appears to support an association between overt thyroid dysfunction and an increased risk of infertility, and there is strong evidence to recommend treatment for overt hypothyroidism in pregnancy. The recommended treatment of maternal hypothyroidism is the administration of oral LT4.

Thyroid Hormone Action: The p43 Mitochondrial Pathway.

The possibility that several pathways are involved in the multiplicity of thyroid hormone physiological influences led to searches for the occurrence of T3 extra nuclear receptors. The existence of a direct T3 mitochondrial pathway is now well established. The demonstration that TRα1 mRNA encodes not only a nuclear thyroid hormone receptor but also two proteins imported into mitochondria with molecular masses of 43 and 28kDa has provided new clues to understand the pleiotropic influence of iodinated hormones.The use of a T3 photo affinity label derivative (T3-PAL) allowed detecting two mitochondrial T3 binding proteins. In association with western blots using antibodies raised against the T3 nuclear receptor TRα1, mitochondrial T3 receptors were identified as truncated TRα1 forms. Import and in organello transcription experiments performed in isolated mitochondria led to the conclusion that p43 is a transcription factor of the mitochondrial genome, inducing changes in the mitochondrial/nuclear crosstalk. In vitro experiments indicated that this T3 mitochondrial pathway affects cell differentiation, apoptosis, and transformation. Generation of transgenic mice demonstrated the involvement of this mitochondrial pathway in the determination of muscle phenotype, glucose metabolism, and thermogenesis.

Characteristics of the brown hagfish Paramyxine atami transthyretin: Metal ion-dependent thyroid hormone binding

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33–50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3′,5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5–3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn2+ and Cu2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn2+ and Cu2+) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8μM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication.

Effects of thyroid hormone transporters MCT8 and MCT10 on nuclear activity of T3

Transport of thyroid hormone (TH) across the plasma membrane is necessary for the genomic action of T3 mediated by its nuclear T3 receptor. MCT8 and MCT10 have been identified as important TH transporters. Mutations in MCT8 result in severe psychomotor retardation. In addition to TH transport into the cell, MCT8 and MCT10 also facilitate TH efflux from cells. Therefore, the aim of this study was to examine if MCT8 and MCT10 increase the availability of T3 for its nuclear receptor rather than generate a rapid equilibrium between cellular and serum T3.T3 action was investigated in JEG3 cells co-transfected with TRβ1 and a T3 response element-driven luciferase construct, and T3 metabolism was analyzed in cells transfected with type 3 deiodinase (D3). In addition, cells were transfected with MCT8 or MCT10 and/or the cytoplasmic T3-binding protein mu-crystallin (CRYM). Luciferase signal was markedly stimulated by incubating cells for 24 h with 1 nM T3, but this response was not augmented by MCT8 or MCT10 expression. Limiting the time of T3 exposure to 1–6 h and co-transfection with CRYM allowed for a modest increase in luciferase response to T3. In contrast, T3 metabolism by D3 was potently stimulated by MCT8 or MCT10 expression, but it was not affected by expression of CRYM.These results suggest that MCT8 and MCT10 by virtue of their bidirectional T3 transport have less effect on steady-state nuclear T3 levels than on T3 levels at the cell periphery where D3 is located. CRYM alters the dynamics of cellular TH transport but its exact function in the cellular distribution of TH remains to be determined.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3′-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells

μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

Possible Roles of the AP-1 Site in the Cytosolic T3 Binding Protein Promoter and Insights into its Physiological Significance

Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to μ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on μ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in μ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of μ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased μ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of μ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on μ-crystallin mRNA expression.

Measurement of Total Rather Than Free Thyroxine in Pregnancy: The Diagnostic Implications

In light of several recent recommendations to use total thyroxine (T4) measurements in the diagnosis of thyroid function in pregnancy (in particular, "Clinical Practice Guidelines for Hypothyroidism in Adults," cosponsored by the American Thyroid Association and the American Association of Clinical Endocrinologists, which promote the use of T4 over free T4 [FT4 ]), we have examined the implications of employing T4 for diagnostic discrimination in both pregnant and nonpregnant patient panels. Use of T4 assays has significant drawbacks in this regard, and we believe that the suggestion is a retrograde step in thyroid function testing. Analysis of the interplay between the concentrations of T4 and thyroxine-binding globulin (TBG), typifying their respective reference ranges in either the nonpregnant or pregnant euthyroid state, shows that the effective T4 range is widened significantly by the accompanying hidden variation in TBG levels. Accordingly FT4 assays that fully compensate for serum T4-binding protein concentrations should discriminate dysfunctionality from normality more efficiently than total hormone measurements, whether in pregnant or nonpregnant states. The euthyroid FT4 reference ranges typical of late pregnancy should also be more compact than those for the total hormone, because of the increased dominance of higher, though equivalently variable TBG concentrations on T4 levels. Parallel effects on T4 from similarly variable, though lower concentrations of TBG are indicated in the nonpregnant group. While acknowledging the difficulties in FT4 measurement arising from inconsistent calibration of present-day commercial assays, this finding questions the recommendation that total hormone assays should supersede the former in pregnancy.

FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID–LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations

Serum free thyroxine (FT4) testing in pregnancy is known to be challenging for immunoassays (IAs). We verified the reliability of FT4 results by 3 commercial IAs throughout pregnancy, by comparison of the pattern to that obtained with an equilibrium dialysis isotope dilution-mass spectrometry (ED ID-MS) candidate reference measurement procedure. Pregnant females (107) and age-matched non-pregnant controls (26) were enrolled. The IAs tested were those performed on the Cobas 6000 (Roche Diagnostics), ARCHITECT i2000SR (Abbott Diagnostics) and Immulite 2000 (Siemens Healthcare) platforms. Compared to the controls (mean FT4: 18.2pmol/L), ED ID-MS gave in the late first trimester pregnancy an 8.8% lower (p<0.05) mean; in the second trimester it was 29.1% lower (p<0.001), to stabilize in the third trimester (p=0.99). Similar observations were made for the Cobas and Immulite IAs. The ARCHITECT IA showed no significant decrease in the late first trimester (mean 13.5pmol/L versus 13.6pmol/L in controls), but a significant, less pronounced, decrease in the second and third trimesters (15% and 14.4%, respectively). All IAs were susceptible towards alterations in T4 binding proteins during pregnancy. We proved that IAs may give a FT4 pattern during pregnancy similar to that obtained by ED ID-MS.

Unique alterations of thyroid function parameters after i.v. administration of alkylating drugs (cyclophosphamide and ifosfamide).

Alkylating drugs (cyclophosphamide and ifosfamide) have been in clinical use for the treatment of malignant diseases in the past. They are most useful anticancer agents and cyclophosphamide is also widely used for its immunosuppressive properties. However the effect of alkylating drugs on thyroid hormone parameters have not been evaluated so far. Three groups of patients were prospectively evaluated: Group I: 15 patients with Wegener's granulomatosis and 4 patients with severe scleritis received a single dose cyclophosphamide (15 mg/kg bw/day) and 250 mg prednisone i.v. Group II: 9 patients with malignant lymphomas were treated according to the IMVP 16-protocol. Patients received daily ifosfamide 1000 mg/m2 from day 0 to 4 and vepesid from day 0 to 2. Patients did not receive corticosteroids additionally. Group III: 6 patients with a relapse of malignant lymphomas received ifosfamide 1.500 mg/m2/day from day 0 to 4 i.v. and dexamethasone 40 mg/m2 as well as ara-c and etoposid. All patients received mesna to prevent hemorrhagic cystitis and odansetran or metoclopramide as antiemetic drugs. Alkylating drugs were given as a one hour infusion. Thyroid hormone parameters were determined before and on day 1, 2, 3, 4 after drug administration. We observed a significant increase in T4 and fT4 concentrations and a concomitant fall in TSH in either group one day after the administration of alkylating drugs. The effect was most pronounced in group III: T4 increased from 113 +/- 8 nmol/L to 175 +/- 8 (normal: 58-154) and fT4 from 14.0 +/- 0.8 to 24.8 +/- 2.5 pmol/L (normal 10-25). TSH dropped from 1.27 +/- 0.16 to 0.33 +/- 0.07 mU/L (normal 0.3-4). All changes were significant: p < 0.001. Two of the six patients displayed biochemical hyperthyroidism. Also reverse T3 increased significantly. Two days after drug administration a gradual normalization occurred. However, T3, Tg, TBG, Transthyretin and albumin levels did not change throughout the study period. One patient with coexisting hypothyroidism, who received his last thyroxine substitution therapy one day before the administration of cyclophosphamide (as in group I), also demonstrated an increase in T4, fT4 and rT3 and a fall in TSH concentrations. I.v. administrations of cyclophosphamide and ifosfamide induce a transient increase in T4 and fT4 concentrations and a concomitant fall of TSH in the presence of normal Tg, T3 and thyroid binding protein concentrations. These data suggest, that the changes are not due to a release of thyroid hormones from the thyroid itself, but is likewise related to a release of thyroxine from cellular pools such as the liver.

Identification of µ‐crystallin as an androgen‐regulated gene in human prostate cancer

Androgen receptor (AR) signaling is implicated in prostate cancer progression. Therefore, identification of AR downstream genes is potentially important for selection of novel markers and therapy targets in prostate cancer. Expression of a thyroid hormone T3-binding protein mu-crystallin (CRYM) mRNA and protein in cell lines was evaluated by real-time PCR and Western blot, respectively. CRYM expression in vivo was analyzed in patients' samples by immunohistochemistry. The effects of androgen and T3 on proliferation of MDA PCa 2b cells were assessed by (3)H-thymidine uptake assay. CRYM expression was detected in AR-positive LNCaP and MDA PCa 2b cells. In MDA PCA 2b cells, CRYM was regulated by androgens. Androgen-induced CRYM expression was diminished by antiandrogens or AR siRNA. Inhibition of transcription by alpha-amanitin caused a reduction in CRYM mRNA. The lack of CRYM expression was noted in LAPC-4 cells and in AR-negative prostate cancer cell lines PC-3 and DU-145. CRYM protein was increased in cancer tissue and decreased in samples from patients after hormonal therapy. In samples from patients with therapy-refractory cancer CRYM was not detectable. We also found that androgens and T3 have additive effects on stimulation of MDA PCa 2b cells proliferation. CRYM is a novel androgen-regulated gene whose expression is elevated in prostate cancer but down-regulated in castration therapy-resistant tumors.

Spiking Expression of μ-Crystallin mRNA during Treatment with Methimazole in Patients with Graves’ Hyperthyroidism

mu-Crystallin is an NADPH-dependent cytosolic T3-binding protein. A knockout study in mice showed that mu-crystallin has a physiological function as a reservoir of T3 in the cytoplasm in vivo. Patients with nonsyndromic deafness were reported to have point mutations in the mu-crystallin gene. The expression of mu-crystallin is regulated by multiple factors. The present study was performed to determine whether thyroid function is related to the expression of mu-crystallin mRNA in peripheral mononuclear cells. We examined 23 normal healthy male and female subjects and 15 patients with Graves' disease. mu-Crystallin protein expression was determined immunohistochemically in peripheral mononuclear cells. The expression of mu-crystallin mRNA was assessed by reverse transcription of total RNA from peripheral mononuclear cells followed by quantitative PCR. mu-Crystallin protein was detected in peripheral mononuclear cells. The mRNA expression was negatively correlated with age in normal female subjects. The values in female subjects were significantly higher than those in males. The values were positively correlated with serum TSH concentration. The values of the thyrotoxic patients with Graves' disease were lower than those in healthy subjects. A transient increase in mu-crystallin expression was observed within 14-42 days after the initial treatment with antithyroid medication. Thyroid hormone inversely relates to the expression of mu-crystallin mRNA in euthyroid mononuclear cells. Abrupt suppression of thyroid function leads to overexpression of mu-crystallin mRNA in thyrotoxic mononuclear cells. Thyroid hormone-regulated mu-crystallin expression may control thyroid hormone action via the intracytoplasmic T (3) capacity.

The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T4) was prepared by N-acylation of N-(3-aminopropyl) biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T4 conjugate with one or simultaneously with two binding proteins with affinity to Bt or T4 in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T4 was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T4. The Bt-T4 conjugate also formed a specific equimolar complex with T4-binding human globulin (TBG) by the same mechanism as that for T4. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T4 conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T4. The intact T4 competitively inhibited this interaction after its addition to the system. Bt-T4 also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu(3+)-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T4 immobilized on a solid phase. Free T4 inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T4 complex was actively bound to the T4-antibody, but had practically no interaction with serum T4-binding proteins, including TBG. Probably, nonspecific interactions of the T4 residue with streptavidin in its complex with Bt-T4, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T4-binding proteins of blood serum. The Bt-T4 stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.

Counterpoint: Legitimate and Illegitimate Tests of Free-Analyte Assay Function: We Need to Identify the Factors That Influence Free-Analyte Assay Results

There have been, and continue to be, reports of discordant free thyroxine (T4)1 measurements when different free-T4 methods are applied to the same serum samples (1)(2)(3)(4). Our laboratory has attempted to identify some of the factors that contribute to these disparities. There are 4 separate components of serum T4: free T4, albumin-bound T4, transthyretin-bound T4, and thyroxine-binding globulin (TBG)-bound T4. Free T4 is the one component that moves across semipermeable membranes during equilibrium dialysis and ultrafiltration. As Midgley and Christofides point out, the gold standard free-T4 methods are designed to measure this component. We applied analog-type methods and an equilibrium-dialysis/immunoassay gold standard method to carefully prepared solutions in which we varied these 4 components of serum T4. This equilibrium-dialysis method agrees closely with a liquid chromatography–tandem mass spectrometry method applied to serum ultrafiltrates (4). We also applied this equilibrium dialysis/immunoassay and a group of 7 analog-type free-T4 assays to solutions of T4 that did not include T4-binding proteins (5); that is, the only form of T4 was free T4. The analog-type assays required 28- to 588-fold higher concentrations of free T4 than …

Optical tweezers experiments resolve distinct modes of DNA‐protein binding

Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double-stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double-stranded structure. Proteins binding to both double- and single-stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single-stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double-stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding.

Spurious Conclusions on Analog Free Thyroxine Assay Performance

I read with misgivings the recent communication by Fritz et al. (1) alleging that because under some circumstances an analog free thyroxine (FT4) immunoassay correlates total T4 and FT4 values, it does not measure FT4 but something akin to T4. I have shown repeatedly through mass action analyses (2)(3)(4) that many experiments, including the one reported by Fritz et al. (1) and others described in earlier papers(5)(6), cannot meaningfully address the working of any of these assays. Allegations of shortcomings cannot be substantiated if they depend on experiments using artificial T4 solutions in which serum T4-binding proteins are either lacking or are present at concentrations insufficient to prevent overlarge T4 abstraction by the assay antibody (conditions strictly invalid for FT4 measurements). The amount of antibody that can be used in analog FT4 assays having a radioactive probe is limited (3). Constraints on the stability of molecules radiolabeled …