Phosphorylation of serine 212 confers novel activity to human estrogen receptor α

Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ERα a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ERα and mutants ERα S212A, ERα S212D, ERα S236A and ERα S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ERα. ERα S212D, but not ERα S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ERβ; the ERβ S176D mutant retained its trans-activation activity, but the ERβ S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ERα S212A or ERα S212D, revealed that phosphorylation of serine 212 enabled ERα to regulate a unique set of genes and cellular functions.     

Development and Validation of a New Questionnaire Assessing Quality of Life in Adults with Hypopituitarism: Adult Hypopituitarism Questionnaire (AHQ)

Objective

To develop and validate the Adult Hypopituitarism Questionnaire (AHQ) as a disease-specific, self-administered questionnaire for evaluation of quality of life (QOL) in adult patients with hypopituitarism.

Methods

We developed and validated this new questionnaire, using a standardized procedure which included item development, pilot-testing and psychometric validation. Of the patients who participated in psychometric validation, those whose clinical conditions were judged to be stable were asked to answer the survey questionnaire twice, in order to assess test-retest reliability.

Results

Content validity of the initial questionnaire was evaluated via two pilot tests. After these tests, we made minor revisions and finalized the initial version of the questionnaire. The questionnaire was constructed with two domains, one psycho-social and the other physical. For psychometric assessment, analyses were performed on the responses of 192 adult patients with various types of hypopituitarism. The intraclass correlations of the respective domains were 0.91 and 0.95, and the Cronbach’s alpha coefficients were 0.96 and 0.95, indicating adequate test-retest reliability and internal consistency for each domain. For known-group validity, patients with hypopituitarism due to hypothalamic disorder showed significantly lower scores in 11 out of 13 sub-domains compared to those who had hypopituitarism due to pituitary disorder. Regarding construct validity, the domain structure was found to be almost the same as that initially hypothesized. Exploratory factor analysis (n = 228) demonstrated that each domain consisted of six and seven sub-domains.

Conclusion

The AHQ showed good reliability and validity for evaluating QOL in adult patients with hypopituitarism.     

Bacteriome-associated endosymbionts of the green rice leafhopper <Emphasis Type="Italic">Nephotettix cincticeps</Emphasis> (Hemiptera: Cicadellidae)

The green rice leafhopper Nephotettix cincticeps (Uhler) is a commonly distributed pest of rice in East Asia. Early histological studies describe the presence of two bacteriome-associated symbionts and a rickettsial microorganism in N. cincticeps, but their microbiological affiliations have been elusive. We identified these bacterial symbionts using modern microbiological techniques. Cloning and sequencing of the 16S ribosomal RNA gene from dissected bacteriomes yielded two major and a minor bacterial sequences: a major sequence was placed in the Bacteroidetes clade of Sulcia muelleri, an ancient symbiont lineage associated with diverse hemipteran insects; another major sequence was allied to a β-proteobacterial sequence from a leafhopper Matsumuratettix hiroglyphicus; the minor sequence fell in the α-proteobacterial genus Rickettsia. In situ hybridization and transmission electron microscopy showed that the Sulcia symbiont and the β-proteobacterial symbiont are harbored within different types of bacteriocytes that constitute the outer and inner regions of the bacteriome, respectively. Oral administration of tetracycline to nymphal N. cincticeps resulted in retarded growth, high mortality rates, and failure in adult emergence, suggesting important biological roles of the symbionts for the host insect. The designation Candidatus Nasuia deltocephalinicola is proposed for the β-proteobacterial symbiont clade associated with N. cincticeps and allied leafhoppers of the subfamily Deltocephalinae.     

Genetic variants on chromosome 1q41 influence ocular axial length and high myopia

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = −0.16 mm per minor allele, P_meta = 2.69×10⁻¹⁰). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) = 0.75, 95% CI: 0.68–0.84, P_meta = 4.38×10⁻⁷) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.     

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

In efforts to define new targets for antithrombotic purposes, there is interest in utilizing antibodies targeting ligand binding domains of platelet receptors. To this end, we have recently shown that an antibody (designated C-EL2Ab), which targets the C-terminus of the 2nd extracellular loop (C-EL2) of the thromboxane A(2) receptor (TPR), selectively blocks TPR-mediated platelet aggregation, under both in vitro and ex vivo experimental conditions. In the current studies we sought to determine whether C-EL2Ab exhibits in vivo antithrombotic activity, by employing a carotid artery injury thrombosis model. It was found that mice treated with C-EL2Ab, exhibited a significant increase in time for occlusion, when compared to controls such as normal rabbit IgG, or an antibody which targets a region separate from the ligand binding site (i.e., EL1). We next examined the effect of C-EL2Ab on hemostasis, and found no increase in tail bleeding times in C-EL2Ab treated mice, compared to the aforementioned controls. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, and is devoid of increased bleeding risk. Moreover, the identification of a functionally active TPR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.     

TSBAb (TSH-stimulation blocking antibody) and TSAb (thyroid stimulating antibody) in TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism.

There are two types of TSH receptor antibodies (TRAb); thyroid stimulating antibody (TSAb) and TSH-stimulation blocking antibody (TSBAb). TSAb causes Graves' hyperthyroidism. TSBAb causes hypothyroidism. Both TSAb and TSBAb block TSH-binding to thyroid cells as TSH receptor antibodies (TRAb). TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism may have both TSBAb and TSAb. We studied TSBAb and TSAb in 43 TSBAb-positive patients with hypothyroidism and in 55 untreated Graves' patients with hyperthyroidism. TSBAb-activities were expressed as percentage inhibition of bovine (b) TSH-stimulated cAMP production by test IgG. Two formulas were used to calculate TSBAb-activities; TSBAb-A (%) = [1 - (c - b)/(a - b)] x 100 and TSBAb-B (%) = [1 - (c - d)/(a - b)] x 100, where a: cAMP generated in the presence of normal IgG and bTSH, b: cAMP generated in the presence of normal IgG, c: cAMP generated in the presence of test IgG and bTSH, and d: cAMP generated in the presence of test IgG. TSAb (%) = [d/b] x 100. All of the 43 TSBAb-positive patients with hypothyroidism had strongly positive TSBAb-A and -B. Some of them had weakly positive TSAb (<240%). All 55 untreated Graves' patients had positive TSAb (205-2509%). Some of them had both TSAb and TSBAb. TSBAb-positive patients with hypothyroidism had a limited distribution of TSBAb- and TSAb-activities (TSBAb-A + 75 - + 103%, TSBAb-B + 87 - + 106%, TSAb 92-240%), but Graves' patients with hyperthyroidsim had a wide distribution of TSAb- and TSBAb-activities (TSAb 205-2509%, TSBAb-A - 158 - + 43%, TSBAb-B - 14 - + 164%). TSBAb-A ignores TSAb activity in serum, and might give low TSBAb activity. However, TSBAb-A clearly differentiates TSBAb-positive patients with hypothyroidism from Graves' patients with hyperthyroidism; thus, we favor TSBAb-A over TSBAb-B. Some of TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism have both TSBAb and TSAb.     

Inhibition of the integrin signal constitutes a mouse iPS cell niche

Stem cells are regulated by their surrounding microenvironments, called niche, such as cell–cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS‐MEF‐Ng‐20D‐17, iPS‐MEF‐Ng‐178B‐5 and iPS‐MEF‐Fb/Ng‐440A‐3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder‐less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.