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1.
Int J Biol Macromol ; 261(Pt 2): 129645, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38296143

RESUMO

Thyroxine-binding globulin (TBG) plays a vital role in regulating metabolism, growth, organ differentiation, and energy homeostasis, exerting significant effects in various key metabolic pathways. Halogenated thiophenols (HTPs) exhibit high toxicity and harmfulness to organisms, and numerous studies have demonstrated their thyroid-disrupting effects. To understand the mechanism of action of HTPs on TBG, a combination of competitive binding experiments, multiple fluorescence spectroscopy techniques, molecular docking, and molecular simulations was employed to investigate the binding mechanism and identify the binding site. The competition binding assay between HTPs and ANS confirmed the competition of HTPs with thyroid hormone T4 for the active site of TBG, resulting in changes in the TBG microenvironment upon the binding of HTPs to the active site. Key amino acid residues involved in the binding process of HTPs and TBG were further investigated through residue energy decomposition. The distribution of high-energy contributing residues was determined. Analysis of root-mean-square deviation (RMSD) demonstrated the stability of the HTPs-TBG complex. These findings confirm the toxic mechanism of HTPs in thyroid disruption, providing a fundamental reference for accurately assessing the ecological risk of pollutants and human health. Providing mechanistic insights into how HTPS causes thyroid diseases.


Assuntos
Fenóis , Compostos de Sulfidrila , Globulina de Ligação a Tiroxina , Tiroxina , Humanos , Globulina de Ligação a Tiroxina/metabolismo , Tiroxina/farmacologia , Proteínas de Ligação a Tiroxina/metabolismo , Simulação de Acoplamento Molecular
2.
Endocrinol Metab (Seoul) ; 37(6): 870-878, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36475360

RESUMO

BACKGRUOUND: Thyroxine-binding globulin (TBG) is a major transporter protein for thyroid hormones. The serpin family A member 7 (SERPINA7) gene codes for TBG, and mutations of the SERPINA7 gene result in TBG deficiency. Although more than 40 mutations have been reported in several countries, only a few studies of TBG deficiency and SERPINA7 gene mutation have been performed in Korea. The aim of this study is to review the clinical presentations and laboratory findings of patients with TBG deficiency and to investigate the types of SERPINA7 gene mutation. METHODS: Five unrelated Korean adults with TBG deficiency attending endocrinology clinic underwent SERPINA7 gene sequencing. Four patients harbored a SERPINA7 gene mutation. Serum thyroid hormones, anti-microsomal antibodies, and TBG were measured. Genomic DNA was extracted from whole blood. All exons and intron-exon boundaries of the TBG gene were amplified and sequencing was performed. RESULTS: Two patients were heterozygous females, and the other two were hemizygous males. One heterozygous female had coexisting hypothyroidism. The other heterozygous female was erroneously prescribed levothyroxine at a local clinic. One hemizygous male harbored a novel mutation, p.Phe269Cysfs*18, which caused TBG partial deficiency. Three patients had the p.Leu372Phefs*23 mutation, which is known as TBG-complete deficiency Japan (TBG-CDJ) and was also presented in previous mutation analyses in Korea. CONCLUSION: This study presents four patients diagnosed with TBG deficiency and provides the results of SERPINA7 gene sequencing. One novel mutation, p.Phe269Cysfs*18, causing TBD-partial deficiency and three cases of TBG-CDJ were demonstrated. It is necessary to identify TBG deficiency to prevent improper treatment. Also, sequencing of the SERPINA7 gene would provide valuable information about the TBG variants in Korea.


Assuntos
Globulina de Ligação a Tiroxina , Proteínas de Ligação a Tiroxina , Adulto , Humanos , Masculino , Feminino , Globulina de Ligação a Tiroxina/genética , Globulina de Ligação a Tiroxina/metabolismo , Proteínas de Ligação a Tiroxina/genética , Proteínas de Ligação a Tiroxina/metabolismo , Mutação , República da Coreia/epidemiologia
3.
Hormones (Athens) ; 20(1): 101-110, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32996026

RESUMO

PURPOSE: The potential benefits of treating subclinical hypothyroidism (SCH) are unclear and still controversial. Thus, we surgically induced SCH in rats and evaluated the effects of thyroxine (T4) replacement on the gene expression levels of deiodinases and thyroid hormone (TH) transporters in different tissues. METHODS: SCH was induced by hemithyroid electrocauterization. The control animals underwent the same surgical procedure but were not subjected to electrocauterization (sham). After 14 days, half of the SCH animals were treated with T4 (SCH + T4). At the end of the experimental protocol, all of the rats were euthanized, serum hormone concentrations were measured, and RNA analyses were performed on different tissues and organs. RESULTS: Consistent with previous studies, we observed increased TSH levels, normal TH levels, and reduced hypothalamic TRH expression in the SCH group. Additionally, Dio2 mRNA expression was downregulated in the hippocampus and pituitary, and Dio1 was upregulated in the kidney and pituitary of the SCH animals. The changes in Dio3 expression were tissue-specific. Concerning TH transporters, Mct10 expression was upregulated in the pituitary, kidney, hypothalamus, and hippocampus, and Mct8 expression was downregulated in the kidney of the SCH group. Crym expression was upregulated in the kidney and pituitary. Notably, T4 replacement significantly attenuated serum TSH levels and reverted Dio1, Dio2, Mct10, and Crym expression in the pituitary, hippocampus, and kidney to levels that were similar to the sham group. Tissue-specific responses were also observed in the liver and hypothalamus. CONCLUSION: Our results indicate that treatment of SCH should be considered before the appearance of clinical symptoms of hypothyroidism.


Assuntos
Hipotireoidismo/tratamento farmacológico , Iodeto Peroxidase/metabolismo , Proteínas de Ligação a Tiroxina/metabolismo , Tiroxina/uso terapêutico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/fisiologia , Hipotireoidismo/etiologia , Iodeto Peroxidase/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas de Ligação a Tiroxina/genética , Cristalinas mu
4.
J Biomol Struct Dyn ; 37(6): 1402-1413, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29620440

RESUMO

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are a class of toxic environmental pollutants that are persistent, bioaccumulative, and difficult to degrade. Their structure is very similar to the thyroid hormone (T4) and uses the body's thyroid transporter (TTR) binding to interfere with the endocrine balance, disrupting the body's normal physiological activity. According to Fourier transform infrared spectroscopy and dynamics simulation of do_dssp module analysis, there are three kinds of OH-PBDEs that can induce TTR secondary structural changes. Fluorescence spectra and UV-Vis spectra show that for the three kinds of OH-PBDEs for TTR, the main methods of quenching are static quenching and non-radiative energy transfer. According to thermodynamic analysis, ΔG < 0, ΔH > 0, and ΔS > 0 combine to show that the hydrophobic interaction is the main driving force of the combination. From the molecular docking analysis, it was found that 4'-hydroxy-2,2',4,5'- tetrabromodiphenyl ether (4'-OH-BDE49) and 4 hydroxy-2,2',3,4',5,6,6'- heptabromodiphenyl ether (4-OH-BDE188) had a cationic-π interaction with TTR, whereas 4 hydroxy-2,2',3,4,5,5',6- heptabromodiphenyl ether (4-OH-BDE187) was bonded to TTR by hydrogen bonds to form stable complexes. In this paper, we highlight the consistency of spectroscopic experiments and computer simulations so as to provide a reliable analytical method for the toxicological properties of small molecule contaminants.


Assuntos
Éteres Difenil Halogenados/química , Modelos Moleculares , Proteínas de Ligação a Tiroxina/química , Sítios de Ligação , Éteres Difenil Halogenados/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Termodinâmica , Proteínas de Ligação a Tiroxina/metabolismo
5.
Front Biosci (Schol Ed) ; 10(2): 326-336, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29293436

RESUMO

We hypothesized that a thyroid hormone (TH)-binding consensus sequence, which is shared by human and animal TH plasma carriers (THPC), might also be shared by cell surface TH transporters (CMTTH) and TH nuclear receptors (THR). We generated the consensus for CMTTH or THR from 8,691 or 624 sequences. In the 49 position-long THPC consensus, eight positions were occupied by very highly conserved (>50% of sequences) and 11 by highly conserved (>33-50% of sequences) groups of residues. Matches between very highly conserved residues of the same group were seven, five or nine when comparing CMTTH vs THPC, THR vs THPC or THR vs CMTTH. Matches between highly conserved residues of the same group were found at one position when comparing CMTTH vs THPC. Noteworthy, the 5-residue TH-binding motif (Y,L/I/M,X,X,V/L/I) originally detected in a few THPC and then confirmed in the 426 THPC (Y/F/W,L/V/I/M,L/V/M/I,l/v/i/m,V/L/I/M) at positions 22-26, was also confirmed in the total 9,741 sequences (W/F/Y,L/I/V/M,I/V/M/L,P,L/V/I/M). In conclusion, proteins so genetically and functionally diverse share TH binding because they share a homologous region that remains conserved phylogenetically.


Assuntos
Proteínas Sanguíneas/química , Hormônios Tireóideos/química , Sequência de Aminoácidos , Animais , Sequência Consenso , Sequência Conservada , Humanos , Proteínas de Ligação a Tiroxina/química
6.
J Toxicol Sci ; 42(6): 663-669, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29142165

RESUMO

We have previously reported that decrease in level of serum thyroxine T4 by Kanechor 500 (KC500) in rats would occur through the increase in hepatic T4 accumulation rather than the increase in hepatic T4-glucuronyl transferase activity. In the present study, to understand the mechanism underlying the KC500-mediated increase in hepatic T4 accumulation, we examined the relationship between the KC500-mediated changes in hepatic T4 accumulation and the expression levels of mRNAs of hepatic transporters including T4 transporters. [125I]T4 was intravenously injected into KC500-pretreated and control (KC500-untreated) Wistar rats, and [125I]T4 uptake levels of liver parenchymal cells were comparatively examined. The amount of [125I]T4 uptake by hepatic cells increased in a time-dependent manner up to 96 hr after KC500 treatment. Following KC500 treatment, a time-dependent increase in the mRNA level of hepatic T4 influx transporter LAT1 was observed up to 96 hr later, while a significant increase in hepatic T4 influx transporter Oatp2 mRNA occurred only at 96 hr later. No KC500-mediated increases in the mRNAs of other hepatic transporters (Oatp1, Oatp3, Oatp4, Ntcp, LAT2, and Mrp2) were observed at any timepoints, although the mRNA expression of the T4 conjugate(s) efflux transporter Mrp3 significantly increased in a time-dependent manner 24-96 hr following KC500 treatment. The present findings suggest that KC500-mediated increase in hepatic T4 accumulation occurs, at least in part, through the increase in the expression of hepatic T4-transporters, such as LAT1 and Oatp2.


Assuntos
Fígado/metabolismo , Bifenilos Policlorados/efeitos adversos , Tiroxina/metabolismo , Animais , Expressão Gênica/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos Wistar , Proteínas de Ligação a Tiroxina/genética , Proteínas de Ligação a Tiroxina/metabolismo , Fatores de Tempo , Distribuição Tecidual
7.
Best Pract Res Clin Endocrinol Metab ; 29(5): 735-47, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26522458

RESUMO

Thyroid hormones (TH) are bound to three major serum transport proteins, thyroxine-binding globulin (TBG), transthyretin (TTR) and human serum albumin (HSA). TBG has the strongest affinity for TH, whereas HSA is the most abundant protein in plasma. Individuals harboring genetic variations in TH transport proteins present with altered thyroid function tests, but are clinically euthyroid and do not require treatment. Clinical awareness and early recognition of these conditions are important to prevent unnecessary therapy with possible untoward effects. This review summarizes the gene, molecular structure and properties of these TH transport proteins and provides an overview of their inherited abnormalities, clinical presentation, genetic background and pathophysiologic mechanisms.


Assuntos
Hipertireoxinemia Disalbuminêmica Familiar/genética , Proteínas de Ligação a Tiroxina/genética , Humanos , Hipertireoxinemia Disalbuminêmica Familiar/diagnóstico , Mutação , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Tiroxina/química , Proteínas de Ligação a Tiroxina/metabolismo
8.
Ned Tijdschr Geneeskd ; 158: A6564, 2014.
Artigo em Holandês | MEDLINE | ID: mdl-25248730

RESUMO

OBJECTIVE: To describe the Dutch neonatal screening programme for congenital hypothyroidism (CH). DESIGN: Descriptive study. METHOD: Data on neonatal screening for CH in the period 1 January 1981 through 31 December 2011 were obtained from the Department for Vaccine Supply and Prevention Programmes of the Dutch National Institute for Public Health and the Environment (RIVM), laboratories and paediatricians to whom babies with abnormal screening results were referred. The screening procedure has been amended several times. In the period 1981-1994, only T4 and TSH were measured in heel prick blood, for example. From 1995, thyroxine-binding globulin (TBG) was added to the screening protocol. RESULTS: The participation rate was 99.7%. Before 1995 the sensitivity, specificity and positive predictive value were 94%, 99.51% and 6%, respectively. From 1995 these percentages were 98%, 99.85% and 21%, respectively. The total prevalence of CH was 1:2670 (prevalence of CH of thyroidal origin was 1:3100 and CH of central origin was 1:21,600). The percentages of patients with severe CH treated before day 15 in the periods 1981-1990, 1991-2000 and 2001-2011 were 24% (63/263), 63% (170/269) and 96% (176/184), respectively. CONCLUSION: The sensitivity and specificity of the screening procedure has considerably increased since 1995 compared with the period before 1995. In recent years patients with severe CH were treated considerably earlier than in the first years of the screening. Neonatal screening for CH may be considered as an important success for public health care.


Assuntos
Hipotireoidismo Congênito/diagnóstico , Triagem Neonatal/métodos , Hipotireoidismo Congênito/sangue , Feminino , Humanos , Recém-Nascido , Masculino , Triagem Neonatal/normas , Países Baixos/epidemiologia , Prevalência , Sensibilidade e Especificidade , Tireotropina/sangue , Tiroxina/sangue , Proteínas de Ligação a Tiroxina/análise
9.
Eur J Endocrinol ; 169(6): 835-44, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24086088

RESUMO

OBJECTIVE: Variation in thyroid hormone (TH) concentrations between subjects is greater than in a single subject over a prolonged period of time, suggesting an individual set point for thyroid function. We have previously shown that TH levels within normal range are associated with clinical indices such as bone mass, BMI, and heart rate. The aim of this study on young men was therefore to gain insight into the determinants of variation in TH levels among healthy subjects. METHODS: Healthy male siblings (n=941, 25-45 years) were recruited in a cross-sectional, population-based study; a history or treatment of thyroid disease and thyroid auto-immunity were exclusion criteria. A complete assessment of TH status was performed (TSH, free thyroxine (FT4), free triiodothyronine (FT3), thyroperoxidase, and thyroglobulin antibodies, reverse T3 (rT3), thyroid-binding globulin (TBG), and urinary iodine levels). Genotyping was performed by TaqMan and KASP (KBiosciences) genotyping assays. RESULTS: (F)T4, rT3, and TBG had heritability estimates between 80 and 90%. Estimates were lower for (F)T3 (60%) and lowest for TSH (49%). Significant associations were observed between different single-nucleotide polymorphisms (SNPs) in the thyroid pathway and TSH, FT4, ratio FT3:FT4, and rT3. Nevertheless, these SNPs only explain a limited part of the heredity. As to age and lifestyle-related factors, (F)T3 was negatively related to age and education level, positively to smoking and BMI (all P<0.0001) but not substantially to urinary iodine concentrations. Smoking was also negatively related to TSH and positively to FT4. CONCLUSION: Both genetic and lifestyle-related factors play a role in determining between-subject variation in TH levels in euthyroid young men, although genetic factors seem most important.


Assuntos
Hereditariedade , Estilo de Vida , Glândula Tireoide/metabolismo , Hormônios Tireóideos/sangue , Adulto , Fatores Etários , Bélgica , Estudos Transversais , Escolaridade , Genótipo , Humanos , Iodeto Peroxidase/sangue , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fumar/sangue , Tireoglobulina/sangue , Testes de Função Tireóidea , Tireotropina/sangue , Tiroxina/sangue , Proteínas de Ligação a Tiroxina/metabolismo , Tri-Iodotironina/sangue , Tri-Iodotironina Reversa/sangue
10.
Horm Mol Biol Clin Investig ; 13(2): 13-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25436710

RESUMO

This article discusses the conditions that may lead to a phenomenon called dysthyronemia. Here, the thyroid gland has concentration of thyrotropin in circulation within the reference range, but the concentrations of free or total fractions of thyroid hormones are outside the reference range. Normal values of thyrotropin (TSH) and increased values of THs are referred to as hyperthyroxinemia, while normal values of thyrotropin and decreased values of thyroid hormone are hypothyroxinemia. As shown by our observations, it is a relatively frequent situation in the parallel determinations of TSH and free thyroxine, when results verging on hyperthyroxinemia were found in 7% of cases (6.74%, n=259,590), and also in the parallel sets of TSH and total triiodothyronine when hypotriiodothyroninemia reached 8.5% (8.48%, n=73,143). We are assuming that the main cause of hyperthyroxinemia in the free thyroxine and TSH system is the presence of autoantibodies against thyroxine in patients with autoimmune thyroid disease. The reason of hypotriiodothyroninemia in the system of triiodothyronine and TSH is a decreased concentration of thyroid binding globulin in postmenopausal women. Manufacturers of immunoanalytical kits should take into account the potential adverse effects of autoantibodies against thyroid hormones when measuring the results of immunoassay determination of the free fraction of these hormones.


Assuntos
Hipertireoxinemia/sangue , Hormônios Tireóideos/sangue , Tireotropina/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hipertireoxinemia/diagnóstico , Masculino , Pessoa de Meia-Idade , Valores de Referência , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tiroxina/sangue , Proteínas de Ligação a Tiroxina/metabolismo , Tri-Iodotironina/sangue , Adulto Jovem
11.
Eur J Endocrinol ; 167(5): 719-26, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22956557

RESUMO

OBJECTIVE: Thyroid disorders affect metabolism and body composition. Existing literature has been conflicting on whether this is also the case for thyroid hormone levels within the euthyroid range. Therefore, we have investigated the relationship between thyroid hormone concentrations and body composition together with metabolic parameters in a population of healthy euthyroid men. METHODS: Healthy male siblings (n=941, 25-45 years, median BMI 24.6) were recruited in a cross-sectional, population-based study; a history or treatment of thyroid disease and thyroid autoimmunity were exclusion criteria. Body composition and muscle cross-sectional area were assessed by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Total (triiodothyronine (T(3); TT(3)) thyroxine and (T(4); TT(4))) and free thyroid hormones (FT(3) and FT(4)), TSH, and reverse T(3) (rT(3)) and thyroid-binding globulin (TBG) were determined using immunoassays. RESULTS: BMI was positively associated with (F)T(3) (P<0.0001). Whole body fat mass displayed positive associations with TT(3) and with (F)T(4) and TBG (P≤0.0006). Positive associations were further observed between leptin and (F)T(3), TT(4), and TBG (P≤0.0002). Inverse associations between lean mass and muscle cross-sectional area and (F)T(3), (F)T(4), and TBG were observed (P≤0.0003). Higher levels of (F)T(3) and TBG were associated with lower insulin sensitivity, assessed by homeostatic model assessment of insulin resistance (IR; P≤0.0001). No associations between TSH and body composition or metabolic parameters were seen. CONCLUSION: We show that a less favorable body composition (with higher fat and lower muscle mass and accompanying higher leptin concentrations) and IR are associated with higher thyroid hormone levels in healthy young men with well characterized euthyroidism.


Assuntos
Tecido Adiposo , Composição Corporal , Índice de Massa Corporal , Resistência à Insulina , Leptina/sangue , Hormônios Tireóideos/sangue , Adulto , Biomarcadores/sangue , Proteínas de Transporte/sangue , Fatores de Confusão Epidemiológicos , Estudos Transversais , Estradiol/sangue , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/sangue , Tireoglobulina/sangue , Tireotropina/sangue , Tiroxina/sangue , Proteínas de Ligação a Tiroxina/metabolismo , Tri-Iodotironina/sangue
12.
Ann Clin Biochem ; 48(Pt 6): 550-7, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21885471

RESUMO

BACKGROUND: Valid assays measuring free thyroxine (FT4) must perform without bias despite large variations in the concentrations and affinities of serum thyroxine-binding proteins in the population. We developed a new, rapid one-step labelled-antibody time-resolved fluoroimmunoassay (TRFIA) for FT4. METHODS: Based on the heterologous combination of anti-T4 monoclonal antibody and triiodothyronine-immunoglobulin G conjugate, a one-step TRFIA for FT4 detection was established and compared with the two-step DELFIA(®) Free Thyroxine Assay. Matrix interference caused by endogenous binders and exogenous non-esterified fatty acids (NEFA) was also accessed in the proposed assay. RESULTS: The developed method generally took only one hour, had a detection limit of 0.6 pmol/L and a large linear range of 2.5-120 pmol/L. The inter- and intra-assay coefficients of variation were 3.5-6.6% and 4.4-9.8%, respectively. Results from 110 specimens showed apparent agreement with that from the DELFIA(®) FT4 Assay with the square of the correlation coefficient of 0.975. This assay indicated that there was no significant dependence on endogenous binders and displayed potential interference by exogenous NEFA up to 5 mmol/L. CONCLUSIONS: The proposed one-step heterologous TRFIA FT4 assay possesses simplicity, accuracy, high sensitivity and exhibits great potential for FT4 measurement. The combination of heterologous immunoassay with TRFIA may be advantageous for FT4 immunoassay development.


Assuntos
Tiroxina/sangue , Anticorpos Imobilizados , Calibragem , Estudos de Casos e Controles , Síndromes do Eutireóideo Doente/sangue , Síndromes do Eutireóideo Doente/diagnóstico , Ácidos Graxos não Esterificados/química , Técnica Direta de Fluorescência para Anticorpo/métodos , Técnica Direta de Fluorescência para Anticorpo/normas , Humanos , Hipertireoidismo/sangue , Hipertireoidismo/diagnóstico , Hipotireoidismo/sangue , Hipotireoidismo/diagnóstico , Imunoglobulina G/química , Limite de Detecção , Padrões de Referência , Proteínas de Ligação a Tiroxina/química , Tri-Iodotironina/química
13.
J Neuroendocrinol ; 23(12): 1194-203, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21910767

RESUMO

Transporters are essential in thyroid hormone metabolism. Thyroxine (T4) is transported by solute carrier organic anion transporter 1c1 (SLCO1C1, OATP14) into the adult brain, where T4 is converted to 3,5,3'-triiodothyronine (T3). In adults, SLCO1C1 expression is found in two brain barrier structures: the blood-brain barrier (BBB) and choroid plexus. However, little is known about how T4 is transported in the developing brain, when the BBB is not yet completely formed. We employed bacterial artificial chromosome recombineering to generate transgenic mice carrying Cre recombinase in the Slco1c1 locus (Slco1c1-Cre mice). In Slco1c1-Cre mice Cre was expressed at the sites that have been previously reported for SLCO1C1 in adults. To trace Cre expression during development, we crossed Slco1c1-Cre transgenic mice with Rosa26 reporter mice. ß-galactosidase staining showed Cre activity in neurones of various brain structures, such as cortical layer 2/3 and the hippocampus, suggesting transient Slco1c1 expression during brain development. At embryonic day15, SLCO1C1 was expressed at the same site as TBR2, a marker of neuronal progenitors. Neurones that express SLCO1C1 during their development could be T4 sensitive. In support of this hypothesis, hypothyroxinaemia induced by propylthiouracil treatment of dams decreased the number of ß-galactosidase-positive neurones in cortical layer 2/3 of newborn Slco1c1-Cre/Rosa26 mice. In conclusion, by generating Slco1c1-Cre transgenic mice, we demonstrated that SLCO1C1 is expressed in the neuronal cell lineage during brain development. Expression of SLCO1C1 may underlie the extraordinary sensitivity of specific neuronal populations to hypothyroxinaemia.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Feminino , Hipotireoidismo/genética , Hipotireoidismo/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas de Ligação a Tiroxina/genética , Proteínas de Ligação a Tiroxina/metabolismo , Distribuição Tecidual
14.
Environ Toxicol Chem ; 30(11): 2431-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21842493

RESUMO

Molecular docking and three-dimensional quantitative structure-activity relationships (3D-QSAR) were used to develop models to predict binding affinity of polybrominated diphenyl ether (PBDE) compounds to the human transthyretin (TTR). Based on the molecular conformations derived from the molecular docking, predictive comparative molecular similarity indices analysis (CoMSIA) models were developed. The results of CoMSIA models were as follows: leave-one-out (LOO) cross-validated squared coefficient q² (LOO) = 0.827 (full model, for all 28 compounds); q² (LOO) = 0.752 (split model, for 22 compounds in the training set); leave-many-out (LMO) cross-validated squared coefficient q² (LMO, two groups) = 0.723 ± 0.100 (full model, for all 28 compounds); q² (LMO, five groups) = 0.795 ± 0.030 (full model, for all 28 compounds); and the predictive squared correlation coefficient r²(pred) = 0.928 (for six compounds in the test set). The developed CoMSIA models can be used to infer the activities of compounds with similar structural characteristics. In addition, the interaction mechanism between hydroxylated polybrominated diphenyl ethers (HO-PBDEs) and the TTR was explored. Hydrogen bonding with amino acid residues Asp74, Ala29, and Asn27 may be an important determinant for HO-PBDEs binding to TTR. Among them, forming hydrogen bonds with amino acid residues Asp74 might exert a more important function.


Assuntos
Éteres Difenil Halogenados/metabolismo , Modelos Químicos , Relação Quantitativa Estrutura-Atividade , Proteínas de Ligação a Tiroxina/metabolismo , Éteres Difenil Halogenados/química , Humanos , Ligação de Hidrogênio , Conformação Molecular , Ligação Proteica , Proteínas de Ligação a Tiroxina/química
15.
Environ Sci Technol ; 45(18): 7936-44, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21842831

RESUMO

A sample preparation method combining solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was developed to be used in Effect-Directed Analysis (EDA) of blood plasma. Until now such a method was not available. It can be used for extraction of a broad range of thyroid hormone (TH)-disruptors from plasma with high recoveries. Validation of the method using spiked cow plasma showed good recoveries for hydroxylated polybrominated diphenyl ethers (OH-PBDEs; 93.8 ± 19.5%), hydroxylated polychlorinated biphenyls (OH-PCBs; 93.8 ± 15.5%), other halogenated phenols (OHPs; 107 ± 8.1%), and for short-chain (<8 C-atoms) perfluoroalkyl substances (PFASs; 85.2 ± 24.6%). In the same extracts, the potency of the compound classes spiked to the cow plasma to competitively bind to transthyretin (TTR) was recovered by 84.9 ± 8.8%. Furthermore, the SPE-LLE method efficiently removed endogenous THs from the extracts, thereby eliminating their possible contribution to the binding assay response. The SPE-LLE method was applied to polar bear plasma samples to investigate its applicability in future EDA studies focusing on TH-disrupting compounds in this top predator species that is exposed to relatively high levels of bioaccumulating pollutants. A first screening revealed TTR-binding potency in the polar bear plasma extracts, which could be explained for 60-85% by the presence of OH-PCBs.


Assuntos
Disruptores Endócrinos/metabolismo , Plasma/química , Proteínas de Ligação a Tiroxina/metabolismo , Animais , Ligação Competitiva , Bovinos/sangue , Disruptores Endócrinos/análise , Feminino , Fluorocarbonos/análise , Fluorocarbonos/metabolismo , Éteres Difenil Halogenados/análise , Éteres Difenil Halogenados/metabolismo , Fenóis/análise , Fenóis/metabolismo , Bifenilos Policlorados/análise , Bifenilos Policlorados/metabolismo , Reprodutibilidade dos Testes , Extração em Fase Sólida , Tiroxina/metabolismo , Ursidae/sangue
18.
Placenta ; 32(2): 128-33, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21146209

RESUMO

Maternal thyroid hormone is provided to the fetus before the onset of fetal thyroid function (at about 16 weeks) and is essential for normal neurologic development. Mechanisms of transport are uncertain but transthyretin (TTR), a thyroxine binding protein produced by the placenta may be involved. Placental oxygen concentrations in early pregnancy are low, about 1% early in the first trimester and rising to 8% over the next 12 weeks. This study investigated the regulation of TTR expression, secretion and uptake in JEG-3 placental cells cultured at different oxygen concentrations. TTR mRNA and protein expression and (125)I-TTR and Alexa-Fluor594-TTR uptake were significantly higher in cells cultured at 1% and 3% O(2), than at 8% O(2). This suggests that increased carrier mediated T(4) transport by placental TTR may be induced by the low oxygen environment of early pregnancy, a time when the fetus has its highest requirement for transport of maternal T(4).


Assuntos
Coriocarcinoma/metabolismo , Oxigênio/administração & dosagem , Pré-Albumina/metabolismo , Proteínas de Ligação a Tiroxina/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Humanos , Oxigênio/farmacologia , Placenta/metabolismo , Pré-Albumina/biossíntese , Gravidez , Primeiro Trimestre da Gravidez , RNA Mensageiro , Neoplasias Uterinas
19.
Toxicology ; 277(1-3): 20-8, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-20804816

RESUMO

Polybrominated diphenyl ethers (PBDEs) have been shown to alter thyroid hormone level in experimental animals. One of the possible mechanisms for hormone disruption is the competitive binding of hydroxylated PBDEs (OH-PBDEs) with hormone transport proteins. In this study, binding interaction of 14 diversely structured OH-PBDEs with two thyroxine transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking. Binding affinity of the 14 OH-PBDEs with transthyretin (TTR) and thyroxine-binding globulin (TBG) was measured by competitive fluorescence displacement assay. The binding constant was found to fall in the range of 1.4 x 107 M and 6.9 x 108 M⁻¹ for TTR, and between 6.5 x 106 M⁻¹ and 2.2 x 108 M⁻¹ for TBG. Binding affinity increased significantly with bromination number from 1 to 4, whereas 5- and 6-brominated diphenyl ethers did not show any further increase. Protein secondary structural change of TTR and TBG upon binding with 5-OH-BDE-047 was investigated by circular dichroism. The spectral change displayed a pattern similar to the one with thyroxine, suggesting that the environmental chemical binds to the two proteins at the same sites as the hormone. In molecular docking analysis, a ligand-binding channel in TTR was revealed for OH-PBDEs binding, which appeared to be mostly hydrophobic inside but guarded by positively charged residue Lys15 at the entrance. Binding affinity of the 14 OH-PBDEs with TTR could be rationalized reasonably well by their pocket binding mode and hydrophobic characteristics. Based on the binding constant obtained in this work, possibility of in vitro competitive displacement of thyroid hormones from the transport proteins by OH-PBDEs was evaluated.


Assuntos
Éteres Difenil Halogenados/química , Éteres Difenil Halogenados/metabolismo , Proteínas de Ligação a Tiroxina/metabolismo , Ligação Competitiva/fisiologia , Cristalografia por Raios X , Humanos , Hidroxilação/fisiologia , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Hormônios Tireóideos/metabolismo
20.
Endocr Regul ; 44(2): 43-7, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20429632

RESUMO

OBJECTIVE: Thyroxine binding globulin-Chicago (TBG-Chicago), a variant of TBG with enhanced heat stability, was isolated at the University of Chicago from a 22 year old subject of an African American lineage. High thermodynamic stability in serine proteinase inhibitors (serpin) is the hallmark of reactive center loop (RCL) inserted conformation and this aspect has not been explored in TBG-Chicago, a serpin molecule. It is hypothesised that the high heat stability of TBG-Chicago is due its loop inserted state. METHODS: Recombinant (r) TBG-Chicago and normal (r) TBG were expressed in baculovirus system. Stability of the freshly made proteins was assessed on Native- PAGE by: 1. heating at 65 degrees C/30 min; 2. incubating at 37 degrees C/24 h. Susceptibility of the RCL of both the TBG's to endoproteinase cleavage and the ability to accept synthetic RCL mimetic peptide were assessed before and after incubation at 37 degrees C /24 h (SDS-PAGE/ Native PAGE). RESULTS: It was found that rTBG-Chicago aggregates at 65 degrees C, accepts RCL mimetic peptide and is cleaved by endoproteinase. In contrast, rTBG-Chicago that had been incubated at 37 degrees C/24 h showed enhanced heat stability at 65 degrees C, reduced ability to accept synthetic peptide and decreased susceptibility to endoproteinase cleavage (besides having changed mobility on Native gel). CONCLUSION: The results support the conclusion that freshly isolated TBG-Chicago exists in loop expelled conformation. However at 37 oC, the protein readily converts to a more stable loop inserted conformation and could explain why plasma TBG-Chicago was found to have enhanced heat stability in the 22 year old subject.


Assuntos
Temperatura Alta , Proteínas de Ligação a Tiroxina/química , Baculoviridae/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Metaloendopeptidases/química , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas de Ligação a Tiroxina/genética , Adulto Jovem
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