Goiter in a 6-year-old patient with novel thyroglobulin gene variant (Gly145Glu) causing intracellular thyroglobulin transport disorder: Correlation between goiter size and the free T3 to free T4 ratio.

Thyroglobulin gene abnormalities cause thyroid dyshormonogenesis. A 6-yr-old boy of consanguineous parents presented with a large goiter and mild hypothyroidism (thyroid-stimulating hormone [TSH] 7.2 µIU/mL, free T3 [FT3] 3.4 pg/mL, free T4 [FT4] 0.6 ng/dL). Despite levothyroxine (LT4) administration and normal TSH levels, the goiter progressed slowly and increased rapidly in size at the onset of puberty. Thyroid scintigraphy revealed a remarkably high 123I uptake of 75.2%, with a serum thyroglobulin level of 13 ng/ml, which was disproportionately low for the goiter size. DNA sequencing revealed a novel homozygous missense variant, c.434G>A [p.Gly145Glu], in the thyroglobulin gene. Goiter growth was suppressed by increasing the LT4 dose. Thyroidectomy was performed at 17-yr-of-age. Thyroglobulin analysis of the thyroid tissue detected mutant thyroglobulin present in the endoplasmic reticulum, demonstrating that thyroglobulin transport from the endoplasmic reticulum to the Golgi apparatus was impaired by the Gly145Glu variant. During the clinical course, an elevated FT3/FT4 ratio was observed along with thyroid enlargement. A high FT3/FT4 ratio and goiter seemed to be compensatory responses to impaired hormone synthesis. Thyroglobulin defects with goiter should be treated with LT4, even if TSH levels are normal.

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

BACKGROUND: We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. METHODS: We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. RESULTS: A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. CONCLUSIONS: Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis.

Development of an ultra-sensitive enzyme immunoassay for human insulin autoantibodies.

OBJECTIVES: We developed an ultrasensitive enzyme immunoassay (ICT-EIA) for insulin autoantibody (IAA) measurements to better understand the pathophysiology of diabetes. DESIGN AND METHODS: We developed ICT-EIA for IAA and measured IAA in 24 patients with type 1 diabetes, 30 patients with type 2 diabetes, 30 patients with methimazole-treated Graves' disease, 20 patients with Hashimoto's disease, 9 patients with hyperinsulinemia, and 73 healthy control subjects. RESULTS: The conventional ELISA identified 3 patients with type 1 diabetes and 2 patients with type 2 diabetes as IAA positive, whereas 15 patients with type 1 diabetes, 7 patients with type 2 diabetes, and 4 patients with methimazole-treated Graves' disease were identified as IAA positive using ICT-EIA. CONCLUSIONS: The ICT-EIA is an ultrasensitive and specific assay for IAA, and its use may provide a better understanding of the role of IAA in diabetes onset and progression.

Site specific phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) for evaluating clinical relevancy in fetal growth restriction.

Fetal growth restriction (FGR) is a leading cause of fetal and neonatal morbidity and mortality. Insulin-like growth factor binding protein-1 (IGFBP-1) is one of the major insulin-like growth factor (IGF) binding proteins involved in fetal growth and development. Our recent data shows that phosphorylation of IGFBP-1 carries both functional and biological relevance in FGR. Considering that IGFBP-1 phosphorylation can be valuable in diagnostics, we examined strategies to enrich IGFBP-1 so that its phosphorylation sites could be assessed by mass spectrometry (MS). Using <1 mL of human amniotic fluid, widely employed immunoprecipitation with IGFBP-1 monoclonal antibody (Mab 6303) coenriched IgGs that interfered with MS. Covalent coupling of Mab 6303 with Seize immunoprecipitation resin (Pierce) mitigated this drawback. However, LC-MS/MS analysis with the titanium dioxide (TiO(2)) enriched IGFBP-1 phosphopeptides in the immunoprecipitated samples revealed pSer101 and pSer119, but not pSer169 nor pSer98 of the previously identified phosphorylation sites. The alternative, ZOOM isoelectric focusing (IEF) (Invitrogen) rendered low-IGFBP-1 recovery with overlapping albumin. Subsequently, depletion of albumin using Affi-GelBlue gel (Bio-Rad) maximized IGFBP-1 yield. ELISA estimation showed approximately 8.5% residual albumin (3.73 x 10(5) +/- 2.35 x 10(5) ng/mL), whereas up to approximately 68% IGFBP-1 was recovered (1.36 x 10(3) +/- 0.174 x 10(3) microg/L, IEMA). LC-MS/MS analysis with the albumin depleted samples detected all four expected phosphorylation sites. Additionally, LC-MS analysis semiquantitatively indicated much reduced phosphopeptide peak intensities, approximately 20-fold with pSer169 and approximately 10-fold lower with pSer98 sites as compared to pSer101. With the use of our depletion strategy, this study offers a novel simple proteomic approach to enrich IGFBP-1 for identification of site-specific changes in IGFBP-1 phosphorylation. This strategy will be vital in performing differential IGFBP-1 phosphorylation profiling clinically, to help establish its link with FGR and develop diagnostic assays, as well as elucidating novel mechanisms potentially involved in regulation of fetal growth.

A photoelectronic switching device using a mixed self-assembled monolayer.

A cathodic-anodic biway photoelectronic device has been successfully constructed using a self-assembled monolayer (SAM). The SAM consists of two kinds of photofunctional thiol derivatives, a ruthenium complex-viologen linked compound (RuVS) and a phthalocyanine derivative (PcS), on a gold electrode. Structural characterization of the SAM has been carried out by absorption spectroscopy, cyclic voltammetry, and differential pulse voltammetry. Photocurrent responses were measured in the presence of methyl viologen (MV2+) and oxygen as electron acceptors and triethanolamine (TEOA) as a sacrificial reagent. For the SAM of RuVS alone, intramolecular electron transfer (ET) was superior to intermolecular ET, resulting in anodic photocurrents even in the presence of MV2+ and oxygen at 0 V vs Ag/AgCl. On the contrary, only cathodic photocurrents were observed at 0 V for the SAM of PcS alone. Photocurrents from the mixed SAM of RuVS and PcS were roughly the sum of individual photocurrents from RuVS and PcS. In fact, photocurrents from the mixed SAM of RuVS and PcS were observed in the anodic direction below approximately 550 nm, and in the cathodic direction above approximately 550 nm at 0 V vs Ag/AgCl. In the case of the mixed SAM of RuS (ruthenium complex disulfide) and PcS, only cathodic photocurrents were observed at 0 V vs Ag/AgCl, due to the lack of an intramolecular ET pathway. The results indicate that in the mixed SAM of RuVS and PcS both dyes can individually function for opposite photocurrent generation. We have also applied the mixed SAM as a photoelectronic logic device by using two LEDs (470 and 640 nm). The system clearly operated as an XOR logic device.

Bi-directional photocurrent generation dependent on the wavelength of irradiation of a mixed monolayer assembly.

A mixed self-assembled monolayer consisting of a ruthenium tris-bipyridine complex linked to viologen and a palladium phthalocyanine derivative was fabricated on a gold electrode by means of pendant thiol groups. The direction of the photocurrent which is induced in the electrode when it is irradiated depends on the wavelength of the light used.

An alternative synthetic route of [3(5)](1,2,3,4,5)cyclophane, and structural properties of multibridged [3(n)]cyclophanes and their charge-transfer complexes in the solid state.

To develop an improved synthetic route to [3(6)](1,2,3,4,5,6)cyclophane (CP) 2, a more practical synthetic route to [3(5)](1,2,3,4,5)CP 3 than the original one was developed, which started from [3(2)](1,3)CP 7 via [3(4)](1,2,4,5)CP 5. The fundamental structural parameters of [3(n)]CPs (n = 3-6) in the solid state were elucidated, and the observed structures were in good agreement with the most stable conformers in solution and those predicted by the theoretical calculations. In the case of [3(6)]CP 2, the most stable C(6)(h) structure was observed in the crystal structure of the 2-TCNQ-F(4) (1:1) complex, whereas the highly strained structure with a D(6)(h) symmetry was observed in the crystal structure of 2 and the 2:TCNQ:benzene (1:1:1) complex because of a severe disorder problem. [3(n)]CPs (n > 3) showed reversible redox processes, and 2 (+0.39 V vs F(c)/F(c)(+), Cl(2)CHCHCl(2)) showed the lowest first half-wave oxidation potential [E(1/2) (I)] in [3(n)]CPs. The E(1/2) (I) data support the strong donating ability of 2 and its lower homologues. This is attributed to their molecular structures where effective hyperconjugation between the benzyl hydrogens and benzene ring is possible. By taking advantage of the strong electron-donating ability of [3(n)]CPs, their CT complexes with TCNE, TCNQ, and TCNQ-F(4) were prepared, and their crystal structural properties were examined. The single-crystal conductivity data of the CT complexes indicated that the TCNQ-F(4) complexes showed higher conductivities than the corresponding TCNQ complexes mainly due to a larger charge separation. Among the [3(n)]CP-TCNQ complexes, the [3(3)](1,3,5)CP 6-TCNQ-F(4) (1:1) complex showed the highest conductivity (10(-)(4) S cm(-)(1)), and this was ascribed to the formation of an infinite column of partially overlapped acceptors with a short acceptor-acceptor distance, while the formation of such a column was not observed in the 2-TCNQ-F(4) complex. Although the conductivities of the cyclophane-CT complexes are much lower than those of the TTF related complexes, this study successfully provides the basic knowledge for understanding the CT interactions in the solid state.

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.