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Background Osmotic demyelination syndrome commonly follows rapid correction of hyponatremia. Although pons is a common location, extrapontine locations, such as striatum and thalamus, have been reported. Case Description A 48-year-old woman presented with masked facies, shuffling gait, and pill-rolling tremors suggestive of acute-onset parkinsonism. Hyponatremia was diagnosed following a bout of diarrhea, which was corrected with hypertonic saline. Magnetic resonance imaging of the brain showed a giant pituitary adenoma. Hyperintensities on T2-weighted imaging were also seen at the level of pons and bilateral striatum. Central pontine myelinolysis and extrapontine myelinolysis were diagnosed. Hormonal assay showed hypocortisolism, secondary hypothyroidism, and hypogonadism. The patient was started on levodopa-carbidopa, steroids, and thyroxine. She underwent transnasal pituitary adenoma excision. At 6 months postoperatively, she had recovered completely with normal gait. Repeat imaging showed complete resolution of myelinolysis. At 36 months, she continued to have hypocortisolism and hypothyroidism requiring replacement. Conclusions Extrapontine myelinolysis with parkinsonism and asymptomatic central pontine myelinolysis is rare with few cases described in the literature. Our patient had a pituitary adenoma with hyponatremia requiring sodium correction, and we believe that hypopituitarism might have predisposed her to osmotic demyelination. We reviewed relevant literature on extrapontine myelinolysis in suprasellar tumors and the pathophysiology. Hypopituitarism is an underrecognized cause of hyponatremia. When treating a patient with hyponatremia, knowing the pituitary function status is a prerequisite for the physician to prevent osmotic demyelination syndrome. Highlights • ODS can occur with slow rates of sodium correction. • CPM and EPM can be morbid complications of sodium correction; rarely, EPM can manifest with acute-onset parkinsonism. • EPM associated with parkinsonism tends to have a better prognosis with levodopa-carbidopa therapy. • Hypopituitarism and hypoadrenalism can predispose to ODS in a patient with hyponatremia. • The treating physician needs to keep this predisposition in mind when treating patients with hyponatremia. [ABSTRACT FROM AUTHOR]

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Thyroid hormones (THs) are evolutionarily old hormones, having effects on metabolism in bacteria, invertebrates and vertebrates. THs bind specific distributor proteins (THDPs) to ensure their efficient distribution through the blood and cerebrospinal fluid in vertebrates. Albumin is a THDP in the blood of all studied species of vertebrates, so may be the original vertebrate THDP. However, albumin has weak affinity for THs. Transthyretin (TTR) has been identified in the blood across different lineages in adults vs juveniles. TTR has intermediate affinity for THs. Thyroxine-binding globulin has only been identified in mammals and has high affinity for THs. Of these THDPs, TTR is the only one known to be synthesised in the brain and is involved in moving THs from the blood into the cerebrospinal fluid. We analysed the rates of evolution of these three THDPs: TTR has been most highly conserved and albumin has had the highest rate of divergence. [ABSTRACT FROM AUTHOR]

Background/Aims: Congenital central hypothyroidism (CHC) can be detected on newborn screening (NBS) by programs using thyroxine (T4)-reflex thyroid-stimulating hormone (TSH) test approach. CH-C must be distinguished from T4-binding globulin (TBG) deficiency. We sought to determine whether thyroid function tests reliably separate CH-C from TBG deficiency. Methods: We analyzed NBS and serum free and total T4, T3 resin uptake (T3RU) or TBG, and TSH for infants in the Northwest Regional NBS Program (NWRSP) between the years 2008 and 2015 with either CH-C or TBG deficiency. Results: We discovered a significant overlap in T3RU and TBG levels amongst 21 cases of CH-C and 250 cases of TBG deficiency. Mean serum TBG levels were lower in CH-C cases (20.3 µg/mL, range 14.2-33.3) than what is reported in healthy infants (28.6 µg/mL, range 19.1-44.6). Serum free T4 was lower in CH-C cases than TBG deficiency but did not always differentiate between the two conditions. Conclusion: CH-C benefits from detection on NBS but must be distinguished from TBG deficiency. The diagnosis of CH-C rests solely on subnormal serum free T4, but is supported by the demonstration of other pituitary hormone deficiencies. As an overlap exists, serum TBG (or T3RU) levels do not play a role in the diagnosis of CH-C. [ABSTRACT FROM AUTHOR]

Rate-limiting dissociation of the tetrameric protein transthyretin (TTR), followed by monomer misfolding and misassembly, appears to cause degenerative diseases in humans known as the transthyretin amyloidoses, based on human genetic, biochemical and pharmacologic evidence. Small molecules that bind to the generally unoccupied thyroxine binding pockets in the native TTR tetramer kinetically stabilize the tetramer, slowing subunit dissociation proportional to the extent that the molecules stabilize the native state over the dissociative transition state—thereby inhibiting amyloidogenesis. Herein, we use previously reported structure-activity relationship data to develop two semi-quantitative algorithms for identifying the structures of potent and selective transthyretin kinetic stabilizers/amyloidogenesis inhibitors. The viability of these prediction algorithms, in particular the more robust in silico docking model, is perhaps best validated by the clinical success of tafamidis, the first-in-class drug approved in Europe, Japan, South America, and elsewhere for treating transthyretin aggregation-associated familial amyloid polyneuropathy. Tafamidis is also being evaluated in a fully-enrolled placebo-controlled clinical trial for its efficacy against TTR cardiomyopathy. These prediction algorithms will be useful for identifying second generation TTR kinetic stabilizers, should these be needed to ameliorate the central nervous system or ophthalmologic pathology caused by TTR aggregation in organs not accessed by oral tafamidis administration. [ABSTRACT FROM AUTHOR]

Measurement of total thyroxine (T4) is the first testing step in the work-up of thyroid disease in small animals. We evaluated an enzyme-linked fluorescent assay (ELFA) as an in-house method to measure T4 in cats and dogs. We compared the T4 concentration in sera of 122 cats and 176 dogs measured by the ELFA with an enzyme immunoassay (EIA) to assess the concordance of the 2 methods. Bias of the ELFA in cats was −11.4% and in dogs 1.4%. Using Bland–Altman plots, limits of agreement were −81.5 to 58.7% in cats and −71.4 to 74.4% in dogs. Imprecision was calculated for both methods. Intra- and interassay coefficients of variation (CVs) of the ELFA in feline sera were 0.7 and 3.4% and of the EIA 7.6 and 15.7%, respectively. Intra- and interassay CVs of both ELFA and EIA in canine sera were <9.5%. Reference intervals for the ELFA method were established and were 13.3–49.5 nmol/L for cats and 10.1–42.9 nmol/L for dogs. Accuracy of the EIA and ELFA was scored by assessing if the measured T4 value would identify the expected T4 range (low, normal, or elevated) of patients, based on history, clinical presentation, other diagnostic means, and response to therapy. This was possible for 75 cats and 50 dogs. Both methods yielded acceptable results, but the EIA was more accurate compared to the ELFA (percentage of true-positives in cats and dogs: EIA: 97% and 100%; ELFA: 92% and 94%). [ABSTRACT FROM AUTHOR]

Cortisol is transported in the blood by corticosteroid-binding globulin (CBG), a non-inhibitory member of the serpin family of serine protease inhibitors. Recent structural advances reveal how CBG acts as a releasing-agent as well as a carrier of cortisol. Taken together, the structures of the various forms of CBG and of the closely related thyroxine binding-globulin, show how the inherent conformational mechanism of the serpins has been adapted to modulate hormone release to the tissues by changes in binding affinities. A deduction from this, of the temperature dependence of hormone binding, is remarkably borne out with CBG, with a doubling in plasma free cortisol as the body temperature rises to 39 °C. Another insight, against a dogma in the corticosteroid field, is that the proteolytic cleavage of CBG in inflammation results in a partial and not a complete loss of cortisol binding. This becomes of medical importance in conjunction with recent evidence of a pool of the circulating cleaved-form of CBG. It is now evident that tissue levels of free cortisol are buffered by two responsive plasma pools, intact CBG with a high binding-affinity and, particularly in inflammation and sepsis, a further pool of cleaved-CBG with a ten-fold lower affinity. The new molecular understandings, as well as providing insights into the differential release of circulating hormones, also open prospects for therapeutic interventions and draw attention to the potential of CBG and TBG as vehicles for the targeted delivery of drugs. [ABSTRACT FROM AUTHOR]

Background: Emerging evidence suggests that mildly down-regulated thyroid function in older persons may protect and/or reflect maintained health.Methods: Using observational data collected between January 2006 and March 2014 on a volunteer sample of 602 men and women aged 68-97 years with normal thyroid function participating in the Baltimore Longitudinal Study of Aging, this study examines the concurrent relationship between reported walking ability, usual and rapid gait speed, endurance walk performance, fatigability, and reported energy level with respect to free thyroxine (FT4) within the normal range (0.76-1.50ng/dL) as a continuous variable and categorized as low (lower quartile), medium (interquartile), or high (upper quartile).Results: Adjusting for sex, age, race, height, weight, exercise and smoking, reported walking ability, usual and rapid gait speed, 400-m time, fatigability, and reported energy level were less favorable with increasing FT4 (p = .013 to <.001). In sex-strata, similar associations were observed except for walking ability in men and energy level in women. Categorical analyses revealed that persons with low FT4 exhibited better functional mobility, fitness, and reported energy than persons with intermediate or high levels (p < .05 for all). Persons with high-normal versus medium FT4 had slower usual and rapid gait speed (p < .05) only.Conclusion: Older adults with low-normal FT4 exhibit better mobility, fitness, and fatigue profiles. Mildly down-regulated thyroid function appears to align with better function in old age and may serve as a biomarker of healthy longevity. [ABSTRACT FROM AUTHOR]

The defect of low density lipoprotein receptor disturbs cholesterol metabolism and causes familial hypercholesterolaemia (FH). In this study, we directly delivered exogenous Ldlr gene into the liver of FH model mice ( Ldlr ) by lentiviral gene transfer system. The results showed that the Ldlr gene controlled by hepatocyte-specific human thyroxine-binding globulin (TBG) promoter successfully and exclusively expressed in livers. We found that, although, the content of high density lipoprotein in serum was not significantly affected by the Ldlr gene expression, the serum low density lipoprotein level was reduced by 46%, associated with a 30% and 28% decrease in triglyceride and total cholesterol, respectively, compared to uninjected Ldlr mice. Moreover, the TBG directed expression of Ldlr significantly decreased the lipid accumulation in liver and reduced plaque burden in aorta (32%). Our results indicated that the hepatocyte-specific expression of Ldlr gene strikingly lowered serum lipid levels and resulted in amelioration of hypercholesterolaemia. [ABSTRACT FROM AUTHOR]

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization. [ABSTRACT FROM AUTHOR]

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies. [ABSTRACT FROM AUTHOR]

Background: N-linked protein glycosylation plays an important role in various biological processes, including protein folding and trafficking, and cell adhesion and signaling. The acquisition of a novel N-glycosylation site may have significant effect on protein structure and function, and therefore, on the phenotype. Results: We analyzed the human glycoproteome data set (2,534 N-glycosylation sites in 1,027 proteins) and identified 112 novel N-glycosylation sites in 91 proteins that arose in the human lineage since the last common ancestor of Euarchonta (primates and treeshrews). Three of them, Asn-196 in adipocyte plasma membrane-associated protein (APMAP), Asn-91 in cluster of differentiation 166 (CD166/ALCAM), and Asn-76 in thyroglobulin, are human-specific. Molecular evolutionary analysis suggested that these sites were under positive selection during human evolution. Notably, the Asn-76 of thyroglobulin might be involved in the increased production of thyroid hormones in humans, especially thyroxine (T4), because the removal of the glycan moiety from this site was reported to result in a significant decrease in T4 production. Conclusions: We propose that the novel N-glycosylation sites described in this study may be useful candidates for functional analyses to identify innovative genetic modifications for beneficial phenotypes acquired in the human lineage. [ABSTRACT FROM AUTHOR]

The hormone thyroxine that regulates mammalian metabolism is carried and stored in the blood by thyroxine-binding globulin (TBG). We demonstrate here that the release of thyroxine from TBG occurs by a temperature-sensitive mechanism and show how this will provide a homoeostatic adjustment of the concentration of thyroxine to match metabolic needs, as with the hypothermia and torpor of small animals. In humans, a rise in temperature, as in infections, will trigger an accelerated release of thyroxine, resulting in a predictable 23% increase in the concentration of free thyroxine at 398C. The in vivo relevance of this fever-response is affirmed in an environmental adaptation in aboriginal Australians.We show how two mutations incorporated in their TBG interact in a way that will halve the surge in thyroxine release, and hence the boost in metabolic rate that would otherwise occur as body temperatures exceed 378C. The overall findings open insights into physiological changes that accompany variations in body temperature, as notably in fevers. [ABSTRACT FROM AUTHOR]

Partial thyroxine-binding globulin deficiency (TBG-PD) is an endocrine defect with a prevalence of 1:4 000 in newborns. Due to the presence of a single TBG gene on the X chromosome, most familial TBG defects follow an X-linked inheritance pattern. Abnormal T4 binding to T4-binding prealbumin (TTR) is a rare cause of euthyroid hyperthyroxinemia, which is transmitted by autosomal dominant inheritance. The purpose of the present study was to identify and characterize new mutations in the Serpina7 and TTR genes in a complete family with typical TBGPD. All patients underwent clinical and biochemical evaluation. Sequencing of DNA, population screening by (SSCP) analysis, and bioinformatics studies were performed. Molecular studies revealed a novel p.A64D mutation in the exon 1 of Serpina7 gene associated with the previously reported p.A109T mutation in the exon 4 of TTR gene. To our knowledge, this is the first report of a patient with a TBG-PD by a mutation in Serpina7 that was coincident with a mutation in TTR gene that increased affinity of TTR for T 4 . This work contributes to elucidate the molecular basis of the defects of thyroid hormone transport in serum and the improvement of the diagnosis avoiding unnecessary therapy. [ABSTRACT FROM AUTHOR]

Objectives: To assess the performance of thyroid-stimulating hormone (TSH), free thyroxine (FT4) and free triiodothyronine (FT3) determinations by luminescent oxygen channeling immunoassay (LOCI®) technology on the Dimension Vista® analyzer (Siemens Healthcare Diagnostics). Design & methods: We assessed 1) functional sensitivity for TSH (FSe-TSH), and intra- and inter-assay variations for TSH, FT4 and FT3 on Vista®; 2) comparisons of serum and heparin-treated plasma on Vista®; 3) comparisons of a) plasma TSH by Vista® versus electrochemiluminescence (ECLIA) on Roche Modular® analyzer, and b) plasma FT4 and FT3 by Vista® versus Immunotech®-Beckman radioimmunoassay (RIA); and 4) association of albumin and prealbumin levels with free thyroid hormone concentrations on Vista®. Results: 1) FSe-TSH concentration was below 0.005mIU/L. Maximum intra-assay CVs (2.1%, 1.4%, 5.2%) and inter-assay CVs (16.5%, 5.1%, 5.8%) were good for TSH, FT4 and FT3 respectively. 2) Heparin-treated plasma samples consistently gave slightly higher values than serum for TSH, FT4 and FT3. 3) Passing–Bablok regression gave: TSH: [LOCI]=0.91[ECLIA]−0.08 (concordance correlation coefficient ρ c =0.95); FT4: [LOCI]=1.05[RIA]−1.55 (ρ c =0.80); and FT3: [LOCI]=1.05[RIA]−0.06 (ρ c =0.81). 4) Both serum albumin and prealbumin concentrations were positively associated with FT3 levels and negatively associated with FT4 levels in patients. Conclusion: LOCI® is accurate for TSH, FT4 and FT3 analysis. Despite a slight significant bias compared to ECLIA, LOCI® is precise for TSH and fulfills the third-generation criteria. However, the poor concordance between LOCI® and RIA for FT4 and FT3, and the dependence of these hormones on binding proteins require further investigation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Thyroxine-binding globulin (TBG) is a non-inhibitory member of the serpin family of proteins whose main structural element is the reactive center loop (RCL), that, upon cleavage by proteases, is inserted into the protein core adopting a β-strand conformation (stressed to relaxed transition, S-to-R). After S-to-R transition thyroxine (T4) affinity decreases. However, crystallographic studies in the presence or absence of the hormone in different states are unable to show significant differences in the structure and interactions of the binding site. Experimental results also suggest the existence of several S states (differing in the number of inserted RCL residues), associated with a differential affinity. Methods: To shed light into the molecular basis that regulates T4 affinity according to the degree of RCL insertion in TBG, we performed extended molecular dynamics simulations combined with several thermodynamic analysis of the T4 binding to TBG in three different S states, and in the R state. Results: Our results show that, despite T4 binding in the protein by similar interactions in all states, a good correlation between the degree of RCL insertion and the binding affinity, driven by a change in TBG conformational entropy, was observed. Conclusion: TBG allosteric regulation is entropy driven. The presence of multiple S states may allow more efficient T4 release due to protease activity. General significance: The presented results are clear examples of how computer simulation methods can reveal the thermodynamic basis of allosteric effects, and provide a general framework for understanding serpin allosteric affinity regulation. [Copyright &y& Elsevier]

Transthyretin is the carrier protein of thyroxine and retinol in plasma and cerebrospinal fluid and has been described also as a neuroprotective molecule. 14-3-3 Proteins are very important in many cellular processes, being their absence related with deficits in memory and learning. The analysis of the relationship between these two proteins is the main objective of this work. We found that hippocampi of young TTR null mice presented lower levels of 14-3-3ζ protein, but no changes in gene expression when compared to TTR wild type littermates were noted. Cellular studies ascribed this finding to increased degradation of 14-3-3ζ in lysosomes in the absence of TTR, increasing autophagy. [ABSTRACT FROM AUTHOR]

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) was conceived of by structure-based design principles and was chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. Eleven of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore. [ABSTRACT FROM AUTHOR]

Objective To investigate the rate of thyroid testing during pregnancy. Design Population-based, retrospective record-linkage study. Setting Health care data on pregnant women in Tayside, Scotland. Participants All pregnant women who were 18 years and above and who delivered between 1 January 1993 and 31 March 2011 in Tayside were identified. Patients were included in the study if they have had at least three thyroxine prescriptions prior to pregnancy of which at least one prescription was within 6 months prior to pregnancy. Main outcome measures Number of thyroid-stimulating hormone ( TSH) assays performed during pregnancy and the changes in dosage of thyroxine prescribed during pregnancy. Results We identified 950 pregnancies that had thyroxine prescribed prior to pregnancy. Overall, 96·9% (95% CI: 96-98) of these pregnancies had at least one TSH assay performed during or just prior to pregnancy, with 81·2% (95% CI: 79-84) in the first trimester. The prescription of thyroxine was increased in 60·0% (95% CI: 57-63) at any time during pregnancy and in 34·0% (95% CI: 31-37) of pregnancies during the first trimester. Overall, 60% (95% CI: 57-63) of pregnancies had at least one elevated serum TSH during pregnancy with 55% (95% CI: 51-58) in the first trimester. Conclusion The TSH concentration is raised in many pregnancies in women taking long-term thyroxine. [ABSTRACT FROM AUTHOR]

Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. [ABSTRACT FROM AUTHOR]

Abstract: Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for TTRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities. [Copyright &y& Elsevier]

Abstract: The liver performs a vital role in metabolic process, which makes it an attractive target organ for gene therapy. To improve the effects of gene therapy in disorders caused by metabolic disturbance, we quantitatively evaluated six promoters, CMV, EF1α, PGK, apoE, thyroxine binding globulin (TBG), and cytochrome P450 2E1 (CYP2E1) by measuring the expression of α1-antitrypsin, which is controlled by these promoters and introduced via a lentivirus-mediated delivery system in the liver. The results showed that the TBG promoter presents as highly active though in general it is slightly lower than the ubiquitous CMV and EF1α. The expression of exogenous genes driven by the TBG promoter demonstrates to be much higher than by PGK, apoE, and CYP2E1 promoters, and the fragment of −435bp to −26bp from transcription start site (TSS) in the TBG promoter region is identified as the optimum region to direct transgene expression at a higher level. In addition, we further confirmed that the TBG promoter confers transgene persistent and specific expression within the liver up to several months after integration. The data suggests that the TBG promoter is a valuable tool and will greatly facilitate the optimization of vector design in hepatic gene therapy. [Copyright &y& Elsevier]

Abstract: Transthyretin (TTR) is one of the many proteins that are known to misfold and aggregate (i.e., undergo amyloidogenesis) in vivo. The process of TTR amyloidogenesis causes nervous system and/or heart pathology. While several of these maladies are associated with mutations that destabilize the native TTR quaternary and/or tertiary structure, wild-type TTR amyloidogenesis also leads to the degeneration of postmitotic tissue. Over the past 20 years, much has been learned about the factors that influence the propensity of TTR to aggregate. This biophysical information led to the development of a therapeutic strategy, termed “kinetic stabilization,” to prevent TTR amyloidogenesis. This strategy afforded the drug tafamidis which was recently approved by the European Medicines Agency for the treatment of TTR familial amyloid polyneuropathy, the most common familial TTR amyloid disease. Tafamidis is the first and currently the only medication approved to treat TTR familial amyloid polyneuropathy. Here we review the biophysical basis for the kinetic stabilization strategy and the structure-based drug design effort that led to this first-in-class pharmacologic agent. [Copyright &y& Elsevier]

Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period, which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. Adeno-associated virus (AAV) vector-based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose-limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC (mOTC) gene under a liver-specific thyroxine-binding globulin promoter and examined the therapeutic effects in a mouse model of OTCD, the spf ash mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC complementary DNA, which increased the OTC activity by five or twofold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy. [ABSTRACT FROM AUTHOR]

Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Ptenflox/flox with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T4) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background. [ABSTRACT FROM AUTHOR]

Objective: To determine the exact nature and timing of alterations in thyroid function throughout controlled ovarian hyperstimulation (COH). Design: Prospective cohort study. Setting: University fertility clinic. Patient(s): Fifty-seven women undergoing COH as part of planned in vitro fertilization. Intervention(s): None. Main Outcome Measure(s): Timing and magnitude of change in serum thyroid hormones, including TSH, total and free T4, E2, and thyroxine-binding globulin (TBG), measured at six time points from before stimulation to 2 weeks after serum pregnancy test. Result(s): Geometric mean serum TSH increased during stimulation, peaking 1 week after hCG administration compared with baseline (2.44 vs. 1.42 mIU/L), as did free T4 (1.52 vs. 1.38 ng/dL) and TBG (32.86 vs. 21.52 μg/mL). Estradiol levels increased, peaking at hCG administration (1743.21 vs. 71.37 pg/mL). Of 50 women with baseline TSH ≤2.5 mIU/L, 22 (44.0%) had a subsequent rise in TSH to >2.5 during or after COH. The pattern of change over time in TSH concentrations was significantly influenced by baseline hypothyroidism and whether pregnancy was achieved. Conclusion(s): COH led to significant elevations in TSH, often above pregnancy appropriate targets. These findings were particularly evident in women with preexisting hypothyroidism and may have important clinical implications for screening and thyroid hormone supplementation. [ABSTRACT FROM AUTHOR]

Transplacental delivery of maternal thyroid hormones to the fetus, in particular thyroxine (T4), is critical in ensuring normal fetal neurological development. The fetus relies on maternal T4 till around 16 weeks gestation, but mechanisms of placental T4 transport are not yet fully elucidated. Placenta produces, secretes and takes up the thyroid hormone-binding protein transthyretin (TTR). Many placental genes are regulated by oxygen levels, which are relatively low (1%) in the early first trimester, rising to 3% in the mid first trimester and 8% in the early second trimester and thereafter. We examined the expression and uptake of TTR in isolated primary human placental cytotrophoblast cells cultured under different oxygen concentrations (1, 3, 8, 21% O2 and 200 µM desferrioxamine (DFO)) for 24 h. We observed sevenfold higher expression of TTR mRNA and protein levels at 1% O2 than at 8 and 21% O2. Significant increases were observed after culture at 3% O2 and following DFO treatment. We observed significantly higher uptake of 125I-TTR and Alexa- 594-TTR when cells were cultured at 1 and 3% O2 and in the presence of 200 µM DFO than at 8 and 21% O2. When JEG-3 choriocarcinoma cells were transfected with TTR promoter reporter constructs, increased luciferase activity was measured in cells cultured at 1 and 3% O2 in comparison to 8 and 21% O2.We conclude that placental TTR expression and uptake is increased by the relative hypoxia observed in the first trimester of pregnancy, a time when materno-fetal T4 transfer is the sole source of fetal T4. [ABSTRACT FROM AUTHOR]

The prevalence of autoimmune thyroid disease (AITD) has progressively increased. Circulating autoantibodies associated with AITD may affect the result of laboratory analyses and cause incorrect conclusions in the assessment of thyroid hormone status. In this report, we present a patient with Hashimoto’s thyroiditis with high thyrotrophin and free thyroxine (T4) and triiodothyronine (T3) levels due to the presence of auto-antibodies to iodothyronines. Free T4 and free T3 levels measured by direct immunometric assays method were high, while those estimated by measurement of the serum T4-binding capacity were low. L-thyroxine replacement therapy was started. In patients with discrepant laboratory results, interference from autoantibodies should be considered. [ABSTRACT FROM AUTHOR]

Background: 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. Summary and Conclusions: 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. [ABSTRACT FROM AUTHOR]