Evaluation of chemiluminescence immunoassays for detecting thyroglobulin (Tg) and thyroid peroxidase (TPO) autoantibodies using the IMMULITE 2000 system.

The chemiluminescence assays for detection of autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg) implemented on the IMMULITE 2000 system (Diagnostic Products Corporation) were evaluated. These were immunometric assays with antigen-coated beads and monoclonal murine anti-IgG antibodies conjugated with alkaline phosphatase. Precision was satisfactory with an intraassay precision of 5.3-5.5% for anti-Tg and 4.8-5.3% for anti-TPO and an interassay precision of 5.7-7.3% for anti-TPO and 5.2-7.5% for anti-Tg. The lower detection limit was determined as 5 IU/ml for anti-TPO and 2.2 IU/ml for anti-Tg. The average dilution linearities of 102% for anti-TPO and 100% for anti-Tg and the average recovery of 80-127% for anti-TPO and 93-112% for anti-Tg were acceptable. The findings of the tests were compared with the systems from Pharmacia & Upjohn, ORGenTec, Roche Diagnostics, Byk Sangtec Diagnostica and BRAHMS Diagnostica. Taking the respective cutoff value into account, concordance was 87-96% for anti-Tg and 87-97% for anti-TPO. Summarizing all results from the different methods revealed a clinical agreement of 95% for anti-TPO and 93% for anti-Tg. A good agreement was found with the IMMULITE anti-TPO and anti-Tg assays, which are closely related as regards method and biochemistry. Regression analysis gave the following results: anti-TPO IMMULITE 2000 vs anti-TPO IMMULITE: anti-TPO IMMULITE 2000 = 0.99 x IMMULITE anti-TPO - 1.43 IU/ml (r = 0.99, n = 144). anti-Tg IMMULITE 2000 vs anti-Tg IMMULITE: anti-Tg IMMULITE 2000 = 0.98 x IMMULITE anti-Tg + 1.63 IU/ml (r = 0.99, n = 86). Further age-dependent normal ranges were evaluated. A higher prevalence of elevated autoantibody titers was found for patients older than 50 years. The rate of elevated antibody titer can be reduced by using an age-dependent reference range: < or = 50 years anti-TPO < 35 IU/ml, anti-Tg < 40 IU/ml and > 50 years anti-TPO < 100 IU/ml, anti-Tg < 80 IU/ml. Further samples from clinically diagnosed Hashimoto's thyroiditis and Graves' disease were investigated. The levels of positive anti-Tg values and anti-TPO values accorded with those stated in the literature and were comparable to those measured with a reference assay. In the tested INSTAND e. V. interlaboratory samples there was high-level accordance with the expected clinical results.

Gcn4 activator targets Gcn5 histone acetyltransferase to specific promoters independently of transcription.

Histone acetylation correlates well with transcriptional activity, and histone acetyltransferases (HATs) selectively regulate subsets of target genes by mechanisms that remain unclear. Here, we provide in vivo evidence that the yeast transcriptional activator Gcn4 recruits Gcn5 HAT complexes to selective promoters positioned in natural or ectopic locations, thereby creating local domains of histone H3 hyperacetylation and subsequent transcriptional activation. A significant portion of the Gcn4-targeted histone acetylation by Gcn5 is independent of transcriptional activity. These observations provide strong evidence for promoter-selective, targeted histone acetylation by Gcn5 that facilitates transcription in a causal fashion. In addition, Gcn5 also functions in an untargeted manner to acetylate H3 on a genome-wide scale.

Two distinct nuclear receptor interaction domains in NSD1, a novel SET protein that exhibits characteristics of both corepressors and coactivators.

NSD1, a novel 2588 amino acid mouse nuclear protein that interacts directly with the ligand-binding domain (LBD) of several nuclear receptors (NRs), has been identified and characterized. NSD1 contains a SET domain and multiple PHD fingers. In addition to these conserved domains found in both positive and negative Drosophila chromosomal regulators, NSD1 contains two distinct NR interaction domains, NID-L and NID+L, that exhibit binding properties of NIDs found in NR corepressors and coactivators, respectively. NID-L, but not NID+L, interacts with the unliganded LBDs of retinoic acid receptors (RAR) and thyroid hormone receptors (TR), and this interaction is severely impaired by mutations in the LBD alpha-helix 1 that prevent binding of corepressors and transcriptional silencing by apo-NRs. NID+L, but not NID-L, interacts with the liganded LBDs of RAR, TR, retinoid X receptor (RXR), and estrogen receptor (ER), and this interaction is abrogated by mutations in the LBD alpha-helix 12 that prevent binding of coactivators of the ligand-induced transcriptional activation function AF-2. A novel variant (FxxLL) of the NR box motif (LxxLL) is present in NID+L and is required for the binding of NSD1 to holo-LBDs. Interestingly, NSD1 contains separate repression and activation domains. Thus, NSD1 may define a novel class of bifunctional transcriptional intermediary factors playing distinct roles in both the presence and absence of ligand.

The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors.

Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution. Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors. This protein is identical to Ada3, a component of the yeast Ada coactivator complex. We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells. Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD.

Ligand-dependent interaction of nuclear receptors with potential transcriptional intermediary factors (mediators).

The activity of the ligand-inducible activation function 2 (AF-2) contained in the ligand binding domain (LBD) of nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs). We have recently reported the isolation and characterization of two novel mouse proteins, designated TIF1 and mSUG1, that interact in a ligand-dependent fashion with the LBD (region E) of several NRs in vivo as well as in vitro. Remarkably, these interactions require the conserved core motif of the AF-2 activating domain (AF-2 AD) and can be blocked by AF-2 antagonists. TIF1 and mSUG1 might therefore represent TIFs/mediators for the ligand-dependent AF-2 of NRs. By comparing the interaction properties of these two putative TIFs with different NRs including the oestrogen (ER), thyroid hormone (TR), vitamin D3 (VDR), retinoic acid (RAR alpha) and retinoid X (RXR) receptors, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with both TIF1 and mSUG1; (ii) the amphipathic alpha helix core of AF-2 AD is differentially involved in the interactions of RAR alpha with TIF1 and mSUG1; and (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus the interaction interfaces between the various NRs and either TIF1 or mSUG1 may vary depending on the nature of both the receptor and the putative mediator of its AF-2 function. We discuss the possible roles of TIF1 and mSUG1 as mediators of the transcriptional activity of the AF-2 of NRs.

Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms.