Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.     

Interference by the Catecholic Cephalosporin BRL-41897-A with Diagnostic Clinical Chemistry Assays

The catecholic cephalosporin BRL-41897-A, was investigated for in vitro interference with commonly used clinical chemistry assays. An important finding was a negative interference with the enzymatic cholesterol assay that incorporates the Trinder reaction, in which hydrogen peroxide produced by an oxidase drives the oxidative coupling of a phenol with 4-aminophenazone to produce a colored quinoneimine. The suspected interference by BRL-41897-A with the Trinder reaction was investigated using a uricase driven system in which the phenol reagent was substituted or combined with BRL-41897-A. The results indicate that BRL-41897-A competes with the phenol component to react preferentially with 4-aminophenazone to produce a quinoneimine of low absorptivity. Further investigations conducted with similar enzymatic assays for creatinine, cholesterol, triglycerides, and uric acid showed the interference varied substantially with analyte and was dependent on analyte concentration.     

Palladium complexes of bis(acyclic diaminocarbene) ligands with chiral N-substituents and 8-membered chelate rings

Reaction of cis-dichloridobis(p-trifluoromethylphenylisocyanide)palladium(II) with N,N′-bis[(R)-1-phenylethyl]-1,3-diaminopropane afforded an enantiomerically pure, C₁-symmetric bis(acyclic diaminocarbene)PdCl₂ complex in 41% yield. The X-ray crystal structure of the complex revealed that three of the four carbene nitrogens are twisted out of conjugation with the carbene units, apparently as a result of steric interactions between one phenyl group and the propylene backbone of the chelate. A similar reaction with N,N′-bis[(R)-1-(1-naphthyl)ethyl]-1,3-diaminopropane did not lead to an isolable bis(carbene) complex, instead forming significant amounts of bis(ammonium) salt as a decomposition product. However, reaction of the same palladium isocyanide precursor with a mixture of all diastereomers of N,N′-bis[1-(1-naphthyl)ethyl]-1,3-diaminopropane provided an achiral, C_s-symmetric palladium bis(acyclic diaminocarbene) complex derived exclusively from the (R,S) diamine in 20% yield. An X-ray structure showed that the (R,S) stereochemistry allows the bulky naphthyl groups to adopt an orientation that avoids steric interactions with the backbone that likely lead to the instability of the homochiral analogue. The two palladium carbene complexes catalyzed the aza-Claisen rearrangement of an allylic imidate to an allylic amide in 24–34% yield, with an enantiomeric excess of 8% ee for the [(R)-1-phenylethyl]-substituted complex.     

Gamma transitions between low-lying levels in 119Sn

We have carried out measurements on the decay of ¹¹⁹In isomers and the ¹¹⁸Sn(n, γ) reaction to supplement Coulomb excitation measurements on ¹¹⁹Sn. In addition to the 311.39 keV isomeric transition in ¹¹⁹In, we observed 13 γ-rays in ¹¹⁹Sn from the decay of the 2 min and 18 min ¹¹⁹In isomers. These γ-rays have been incorporated into a level scheme of ¹¹⁹Sn with levels at 0, 23.867, 89.54, 787.01, 920.5, 921.4, 1089.5, 1187.76, 1249.67, 1304.44 and 1354 keV. Conclusive evidence for the existence of a 920.5–921.4 keV, $\text{3}\text{2}{}^{\mathrm{+}}\text{-}\text{5}\text{2}{}^{\mathrm{+}}$ level doublet was obtained from capture γ-ray measurements of resonance energy neutrons.     

Geometric distortion in clinical MRI systems Part I: evaluation using a 3D phantom

Recently, a 3D phantom that can provide a comprehensive and accurate measurement of the geometric distortion in MRI has been developed. Using this phantom, a full assessment of the geometric distortion in a number of clinical MRI systems (GE and Siemens) has been carried out and detailed results are presented in this paper. As expected, the main source of geometric distortion in modern superconducting MRI systems arises from the gradient field nonlinearity. Significantly large distortions with maximum absolute geometric errors ranged between 10 and 25 mm within a volume of 240 x 240 x 240 mm(3) were observed when imaging with the new generation of gradient systems that employs shorter coils. By comparison, the geometric distortion was much less in the older-generation gradient systems. With the vendor's correction method, the geometric distortion measured was significantly reduced but only within the plane in which these 2D correction methods were applied. Distortion along the axis normal to the plane was, as expected, virtually unchanged. Two-dimensional correction methods are a convenient approach and in principle they are the only methods that can be applied to correct geometric distortion in a single slice or in multiple noncontiguous slices. However, these methods only provide an incomplete solution to the problem and their value can be significantly reduced if the distortion along the normal of the correction plane is not small.     

A study of atomic iodine and molecular thallium iodide by X-ray photoelectron and auger electron spectroscopy

The I3d5/2 binding energy has been measured in atomic iodine, thallium iodide and cesium iodide by high temperature gas-phase photoelectron spectrscopy using Al K (1486.6 eV) X-rays. The iodine M₅N_4,5N_4,5 (¹G₄) Auger energies for TlI and CsI have also been measured and combined with binding energies to yield extra-atomic relaxation energies of 0.5 and O.3 eV, respectively, after corrections are applied to the Auger parameter. Charges were calculated using the simple potential model, which was also used to obtain an estimate of the atomic T14f binding energy. Two other estimates of the atomic T14f7/2 binding energy have also been calculated, both based on Dirac-Fock ΔSCF binding energies. The results of the three methods suggest a value of 125.3 ± 0.2 eV for T14f7/2.