Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence

The effects of guanine nucleotides on the intrinsic and extrinsic fluorescence properties of dynamin were assessed. The intrinsic Trp (tryptophan) fluorescence spectra of purified recombinant dynamin-1 and -2 were very similar, with a maximum at 332 nm. Collisional quenching by KI was weak (∼30%), suggesting that the majority of Trp residues are buried. Binding of guanine nucleotides decreased intrinsic fluorescence by 15–20%. Titration of the effects showed that GTP and GDP bound to a single class of non-interacting sites in dynamin tetramers with apparent dissociation constants (K(d)) values of 5.4 and 7.4 μM (dynamin-1) and 13.2 and 7.1 μM (dynamin-2) respectively. Similar dissociation constant values for both nucleotides were obtained by titrating the quenching of IAEDANS [N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine]-labelled dynamin-2. Despite the similar binding affinities, GTP and GDP result in different conformations of the protein, as revealed by sensitivity to proteinase K fragmentation. Dynamins contain five Trp residues, of which four are in the PH domain (pleckstrin homology domain) and one is in the C-terminal PRD (proline/arginine-rich domain). Guanine nucleotides quenched fluorescence emission from a truncated (ΔPRD) mutant dynamin-1 to the same extent as in the full-length protein, suggesting conformational coupling between the G (groove)-domain and the PH domain. Efficient resonance energy transfer from PH domain Trp residues to bound mant-GTP [where mant stands for 2′-(3′)-O-(N-methylanthraniloyl)] suggests that the G-domain and PH domain are in close proximity (5–6 nm). Promotion of dynamin-2 oligomerization, by reduction in ionic strength or increasing protein concentration, had little effect on intrinsic dynamin fluorescence. However, fluorescence emission from IAEDANS·dynamin-2 showed a significant spectral shift on oligomerization. In addition, energy transfer was observed when oligomerization was promoted in mixtures of IAEDANS·dynamin-2 and 4-(4-dimethylaminophenylazo)benzoic acid-coupled dynamin-2, an effect that was counteracted by GTP but not GDP

Triage vital signs predict in-hospital mortality among emergency department patients with acute poisoning: a case control study

<p>Abstract</p> <p>Background</p> <p>To document the relationship between triage vital signs and in-hospital mortality among emergency department (ED) patients with acute poisoning.</p> <p>Methods</p> <p>Poisoning patients who admitted to our emergency department during the study period were enrolled. Patient’s demographic data were collected and odds ratios (OR) of triage vital signs to in-hospital mortality were assessed. Receiver operating characteristic curve was used to determine the proper cut-off value of vital signs that predict in-hospital mortality. Logistic regression analysis was performed to test the association of in-hospital mortality and vital signs after adjusting for different variables.</p> <p>Results</p> <p>997 acute poisoning patients were enrolled, with 70 fatal cases (6.7%). A J-shaped relationship was found between triage vital signs and in-hospital mortality. ED triage vital signs exceed cut-off values independently predict in-hospital mortality after adjusting for variables were as follow: body temperature <36 or >37°C, <it>p</it> < 0.01, OR = 2.8; systolic blood pressure <100 or >150 mmHg, <it>p</it> < 0.01, OR: 2.5; heart rate <35 or >120 bpm, <it>p</it> < 0.01, OR: 3.1; respiratory rate <16 or >20 per minute, <it>p</it> = 0.38, OR: 1.4.</p> <p>Conclusions</p> <p>Triage vital signs could predict in-hospital mortality among ED patients with acute poisoning. A J-curve relationship was found between triage vital signs and in-hospital mortality. ED physicians should take note of the extreme initial vital signs in these patients.</p