Binding parameters for σ₁ receptors in the absence and presence of dextromethorphan.

<p>Saturation binding assays in brain homogenates for σ₁ receptors were conducted using [³H](+)-pentazocine as the radioligand. The assays were performed in the absence or presence of dextromethorphan (400 nM). The K_d and B_max were determined using nonlinear regression. Dextromethorphan produced a significant decrease in K_d and B_max. Data shown are expressed as mean ± S.E.M. ^a<i>P</i><0.05, compared with [³H](+)-pentazocine alone; paired t-test.</p

Antidepressant-like effects of imipramine and dextromethorphan in the forced swim test in mice.

<p>Imipramine (0–20 mg/kg, i.p.) significantly decreased immobility time (A), but had no significant effects on locomotor activity (B). Dextromethorphan (0–30 mg/kg, i.p.) significantly decreased immobility time (C), and significantly increased locomotor activity (D). However, there was no correlation between dextromethorphan (30 mg/kg)-induced locomotor stimulatory effects and decreased immobility times (E). Data shown are expressed as mean ± S.E.M. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, compared with the saline-treated group; one-way ANOVA followed by <i>post-hoc</i> Dunnett's tests. Pearson's r correlation test for correlation analysis. IM, imipramine. DM, dextromethorphan.</p

Potentiation of the antidepressant-like effects of dextromethorphan by quinidine.

<p>A single dose of the CYP2D6 inhibitor quinidine (30 mg/kg, i.p.) administered concomitantly with dextromethorphan (0–30 mg/kg, i.p.) significantly potentiated the decrease in immobility time for dextromethorphan at 10 mg/kg (A). In contrast, in the locomotor study, dextromethorphan in combination with quinidine had no stimulatory effects (B). Data shown are expressed as mean ± S.E.M. ***<i>P</i><0.001, compared with the saline-treated group; one-way ANOVA followed by <i>post-hoc</i> Tukey's tests. QND, quinidine. DM, dextromethorphan.</p

Attenuation of the antidepressant-like effects of dextromethorphan, but not imipramine, by σ₁ receptor antagonism.

<p>Pretreatment with the σ₁ receptor antagonist BD1063 (10 mg/kg, i.p.) prevented the dextromethorphan (30 mg/kg, i.p.)-induced decrease in immobility time (A). BD1047 (10 mg/kg, i.p.) pretreatment also produced a noticeable, albeit not statistically significant, trend toward the prevention of the decreased immobility time induced by dextromethorphan (B). In contrast, the antidepressant-like effect of imipramine (20 mg/kg, i.p.) in the forced swim test was not significantly prevented by BD1063 pretreatment (C). Data shown are expressed as mean ± S.E.M. *<i>P</i><0.05, ***<i>P</i><0.001, compared with the saline-treated group; #<i>P</i><0.05, compared with the dextromethorphan-treated group; one-way ANOVA followed by <i>post-hoc</i> Tukey's tests. IM, imipramine. DM, dextromethorphan.</p

The Ubiquitin-Conjugating Enzyme, UbcM2, Is Restricted to Monoubiquitylation by a Two-Fold Mechanism That Involves Backside Residues of E2 and Lys48 of Ubiquitin

Proteins can be modified on lysines (K) with a single ubiquitin (Ub) or with polymers of Ub (polyUb). These different configurations and their respective topologies are primary factors for determining whether substrates are targeted to the proteasome for degradation or directed to nonproteolytic outcomes. We report here on the intrinsic ubiquitylation properties of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked to cell proliferation, development, and the cellular antioxidant defense system. Using a fully recombinant ubiquitylation assay, we show that UbcM2 is severely limited in its ability to synthesize polyUb chains with wild-type Ub. Restriction to monoubiquitylation is governed by multiple residues on the backside of the enzyme, far removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated backside residues can synthesize K63-linked polyUb chains and to a lesser extent K6- and K48-linked chains. Additionally, we identified a single residue on the backside of the enzyme that promotes monoubiquitylation. Together, these findings reveal that a combination of noncatalytic residues within the Ubc catalytic core domain of UbcM2 as well as a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate its cognate targets despite having the latent capacity to construct polyUb chains. The two-fold mechanism for restricting activity to monoubiquitylation provides added insurance that UbcM2 will not build polyUb chains on its substrates, even under conditions of high local Ub concentrations

Additional file 1: of Perceptions of patients and providers on myocardial perfusion imaging for asymptomatic patients, choosing wisely, and professional liability

Patient Survey. Description: Full version of questionnaire given to patients. (DOCX 25 kb

Additional file 2: of Perceptions of patients and providers on myocardial perfusion imaging for asymptomatic patients, choosing wisely, and professional liability

Provider Survey. Description: Full version of questionnaire given to providers. (DOCX 27 kb

The Ubiquitin-Conjugating Enzyme, UbcM2, Is Restricted to Monoubiquitylation by a Two-Fold Mechanism That Involves Backside Residues of E2 and Lys48 of Ubiquitin

Proteins can be modified on lysines (K) with a single ubiquitin (Ub) or with polymers of Ub (polyUb). These different configurations and their respective topologies are primary factors for determining whether substrates are targeted to the proteasome for degradation or directed to nonproteolytic outcomes. We report here on the intrinsic ubiquitylation properties of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked to cell proliferation, development, and the cellular antioxidant defense system. Using a fully recombinant ubiquitylation assay, we show that UbcM2 is severely limited in its ability to synthesize polyUb chains with wild-type Ub. Restriction to monoubiquitylation is governed by multiple residues on the backside of the enzyme, far removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated backside residues can synthesize K63-linked polyUb chains and to a lesser extent K6- and K48-linked chains. Additionally, we identified a single residue on the backside of the enzyme that promotes monoubiquitylation. Together, these findings reveal that a combination of noncatalytic residues within the Ubc catalytic core domain of UbcM2 as well as a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate its cognate targets despite having the latent capacity to construct polyUb chains. The two-fold mechanism for restricting activity to monoubiquitylation provides added insurance that UbcM2 will not build polyUb chains on its substrates, even under conditions of high local Ub concentrations

A Structural Element That Facilitates Proton-Coupled Electron Transfer in Oxalate Decarboxylase

The conformational properties of an active-site loop segment, defined by residues Ser¹⁶¹-Glu¹⁶²-Asn¹⁶³-Ser¹⁶⁴, have been shown to be important for modulating the intrinsic reactivity of Mn­(II) in the active site of <i>Bacillus subtilis</i> oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen-bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step <i>prior</i> to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments [Imaram et al. (2011) <i>Free Radicals Biol. Med. 50</i>, 1009–1015]. This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center

Flowchart for the GLUMIT-DG study.

<p>This figure shows the Flowchart for patient recruitment for the GLUMIT-DG study, which follows a non-randomized trial design. A TREND checklist accompanies this Flowchart as the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194759#pone.0194759.s001" target="_blank">S1 File</a> in the Supplemental Information section.</p