Iron regulatory protein 2 (IRP2) is a central regulator of cellular iron homeostasis due to its regulation of specific mRNAs encoding proteins of iron uptake and storage. Iron regulates IRP2 by mediating its rapid proteasomal degradation, where hypoxia and the hypoxia mimetics CoCl 2 and desferrioxamine (DFO) stabilize it. Previous studies showed that iron-mediated degradation of IRP2 requires the presence of critical cysteines that reside within a 73-amino acid unique region. Here we show that a mutant IRP2 protein lacking this 73-amino acid region degraded at a rate similar to that of wild-type IRP2. In addition, DFO and hypoxia blocked the degradation of both the wild-type and mutant IRP2 proteins. Recently, members of the 2-oxoglutarate (2-OG)-dependent dioxygenase family have been shown to hydroxylate hypoxia-inducible factor-1␣ (HIF-1␣), a modification required for its ubiquitination and proteasomal degradation. Since 2-OG-dependent dioxygenases require iron and oxygen, in addition to 2-OG, for substrate hydroxylation, we hypothesized that this activity may be involved in the regulation of IRP2 stability. To test this we used the 2-OG-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG) and showed that it blocked iron-mediated IRP2 degradation. In addition, hypoxia, DFO and DMOG blocked IRP2 ubiquitination. These data indicate that the region of IRP2 that is involved in IRP2 iron-mediated degradation lies outside of the 73-amino acid unique region and suggest a model whereby 2-OG-dependent dioxygenase activity may be involved in the oxygen and iron regulation of IRP2 protein stability.Iron regulatory proteins 1 and 2 (IRP1 and IRP2) 1 are cytosolic RNA-binding proteins that have a central role in iron homeostasis (reviewed in Refs. 1 and 2). IRP1 and IRP2 bind to stem-loop structures, known as iron-responsive elements, that are found in the untranslated regions of mRNAs encoding proteins important in iron homeostasis. The interaction of IRPs with iron-responsive elements represses translation of ferritin mRNA while increasing the stability of the transferrin receptor mRNA. Since IRP1 and IRP2 RNA binding activities are regulated by intracellular iron concentration, these proteins couple cellular iron with the regulation of expression of proteins important in iron homeostasis. In iron-replete cells, IRP1 RNA binding activity decreases as the protein assembles a [4Fe-4S] cluster having cytosolic aconitase activity, whereas in irondepleted cells apo-IRP1 binds RNA (3). In the case of IRP2, iron controls its RNA binding activity through the regulation of protein stability. This is achieved through iron stimulation of its ubiquitination and proteasomal degradation (4 -7). Thus, the amount of IRP2 is inversely related to cellular iron concentration. The mechanism by which iron accelerates IRP2 degradation has been reported to involve iron-catalyzed oxidation of the protein within a unique 73-amino acid region termed the "degradation domain" (6, 7). It has been recently shown that in vitro iron can oxidiz...

Background: Thyroid-stimulating hormone (TSH) is used to detect primary hypo-and hyperthyroidism. Current guidelines for TSH assays recommend a functional sensitivity of <0.02 mIU/L. The protocol for determining the functional sensitivity of TSH assays specifies analyses of serum samples with two reagent lots over a 6-to 8-week period.

Measurement of circulating B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) can identify patients with heart failure and guide therapy. The limit of detection, linearity, imprecision, method comparison, analytic concordance, and reference intervals of the Access 2 BNP (Biosite, San Diego, CA), ADVIA Centaur BNP (Bayer Diagnostics, Tarrytown, NY), AxSYM BNP (Abbott Diagnostics, Abbott Park, IL), and E170 NT-proBNP (Roche Diagnostics, Indianapolis, IN) methods were evaluated. The Triage meter BNP assay (Biosite) was the comparison method. Imprecision testing showed total coefficients of variation of 4.1%, 4.4%, 5.5%, and 0.8% for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. Relative to the Triage meter, method comparison revealed a slope of 0.96 and r = 0.95, a slope of 0.77 and r = 0.92, a slope of 1.13 and r = 0.94, and a slope of 8.8 and r = 0.80 for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. Overall analytic concordance values with the Triage meter were 95.9%, 92.9%, 92.4%, and 84.3% for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. All automated natriuretic peptide methods showed acceptable analytic performance.

While the Newmarket EIA was a generally more specific assay, it was insufficiently sensitive relative to the IFA and the Cellabs EIA for screening purposes for malaria antibodies. The Cellabs EIA demonstrated the best overall sensitivity and is a reasonable choice as a serodiagnostic tool for malaria. It may also be useful as an adjunct to Giemsa-stained smear examination, to aid in cases of low parasitemia in previously nonimmune individuals.

Measurement of circulating B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) can identify patients with heart failure and guide therapy. The limit of detection, linearity, imprecision, method comparison, analytic concordance, and reference intervals of the Access 2 BNP (Biosite, San Diego, CA), ADVIA Centaur BNP (Bayer Diagnostics, Tarrytown, NY), AxSYM BNP (Abbott Diagnostics, Abbott Park, IL), and E170 NT-proBNP (Roche Diagnostics, Indianapolis, IN) methods were evaluated. The Triage meter BNP assay (Biosite) was the comparison method. Imprecision testing showed total coefficients of variation of 4.1%, 4.4%, 5.5%, and 0.8% for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. Relative to the Triage meter, method comparison revealed a slope of 0.96 and r = 0.95, a slope of 0.77 and r = 0.92, a slope of 1.13 and r = 0.94, and a slope of 8.8 and r = 0.80 for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. Overall analytic concordance values with the Triage meter were 95.9%, 92.9%, 92.4%, and 84.3% for the Access 2, ADVIA Centaur, AxSYM, and E170, respectively. All automated natriuretic peptide methods showed acceptable analytic performance.