Direct evidence for enzyme persulfide and disulfide intermediates during 4-thiouridine biosynthesis

Two proposed mechanisms for 4-thiouridine generation share key cysteine persulfide and disulfide intermediates, and indirect evidence of their existence has been previously reported; chemical trapping and mass spectrometry have now provided direct and definitive evidence of these key intermediates.     

Imaging of voltage-gated alamethicin pores in a reconstituted bilayer lipid membrane via scanning electrochemical microscopy

Voltage-gated biological ion channels were simulated by insertion of the peptaibol antibiotic alamethicin into reconstituted phosphatidylcholine bilayer lipid membranes (BLMs). Scanning electrochemical microscopy (SECM) was utilized to probe initial BLM resistivity, the insertion of alamethicin pores, and mass transport across the membrane. Acquired SECM images show the spatial location of inserted pore bundles, the verification of voltage control over the pore conformational state (open/closed), and variations in passive mass transport corresponding to different topographical areas of the BLM. SECM images were also used to evaluate overall BLM integrity prior to insertion as well as transport (flux in open state) and leakage (flux in closed state) currents following insertion.     

Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.     

Progress in understanding the intramolecular vibrational redistribution dynamics in the S(1) state of para-fluorotoluene

We employ zero-kinetic-energy (ZEKE) photoelectron spectroscopy with nanosecond laser pulses to study intramolecular vibrational redistribution (IVR) in S(1) para-fluorotoluene. The frequency resolution of the probe step is superior to that obtained in any studies on this molecule to date. We focus on the behavior of the 13(1) (C-CH(3) stretch) and 7a(1) (C-F stretch) vibrational states whose dynamics have previously received significant attention, but with contradictory results. We show conclusively that, under our experimental conditions, the 7a(1) vibrational state undergoes significantly more efficient IVR than does the 13(1) state. Indeed, under the experimental conditions used here, the 13(1) state undergoes very little IVR. These two states are especially interesting because their energies are only 36 cm(-1) apart, and the two vibrational modes have the same symmetry. We discuss the role of experimental conditions in observations of IVR in some detail, and thereby suggest explanations for the discrepancies reported to date.     

A forward model and conjugate gradient inversion technique for low-frequency ultrasonic imaging

Emerging methods of hyperthermia cancer treatment require noninvasive temperature monitoring, and ultrasonic techniques show promise in this regard. Various tomographic algorithms are available that reconstruct sound speed or contrast profiles, which can be related to temperature distribution. The requirement of a high enough frequency for adequate spatial resolution and a low enough frequency for adequate tissue penetration is a difficult compromise. In this study, the feasibility of using low frequency ultrasound for imaging and temperature monitoring was investigated. The transient probing wave field had a bandwidth spanning the frequency range 2.5-320.5 kHz. The results from a forward model which computed the propagation and scattering of low-frequency acoustic pressure and velocity wave fields were used to compare three imaging methods formulated within the Born approximation, representing two main types of reconstruction. The first uses Fourier techniques to reconstruct sound-speed profiles from projection or Radon data based on optical ray theory, seen as an asymptotical limit for comparison. The second uses backpropagation and conjugate gradient inversion methods based on acoustical wave theory. The results show that the accuracy in localization was 2.5 mm or better when using low frequencies and the conjugate gradient inversion scheme, which could be used for temperature monitoring.     

Impact of perforation impedance on the transmission loss of reactive and dissipative silencers

The effect of perforation impedance on the acoustic behavior of reactive and dissipative silencers is investigated using experimental and computational approaches. The boundary element method (BEM) is applied for the prediction of transmission loss of silencers with different perforation geometries. The variations are considered in the porosity (8.4 and 25.7%) and hole diameter (0.249 and 0.498 cm) of perforations for both reactive and dissipative silencers, as well as the fiber filling density (100 and 200 kg/m3) for the latter. The acoustic impedance for a number of perforations in contact with air alone and fibrous material has been incorporated into the predictions, which are then compared with the measured transmission loss using an impedance tube setup. The results demonstrate the significance of the accuracy of the perforation impedance in the predictions for both reactive and dissipative silencers.     

Single-mode low-loss chalcogenide glass waveguides for the mid-infrared

We demonstrate the design, fabrication, and characterization of single-mode low-loss waveguides for mid-infrared (MIR) wavelengths. Planar waveguide structures were fabricated from multilayer thin films of arsenic-based chalcogenide glasses followed by the creation of channel waveguides by using the photodarkening effect. Propagation losses as low as 0.5 dB/cm were measured for a quantum cascade laser end-fire coupled into the waveguides. This is a first step toward the design and fabrication of integrated optical components for MIR applications.     

Reflections on the disciplinary gulf between the natural and social sciences

The general purpose of this essay is to explore key features of the disciplinary gulf between the natural and social sciences, and, in particular, differences in fundamental assumptions concerning the nature and purpose of knowledge. The essay contrasts the claims of the natural sciences to objectivity and universality with those of the social sciences, especially the qualitative social sciences, to the historical and cultural contingency of knowledge. It examines the ways in which the use of two 'key words' - 'expertise' and 'responsibility'--serves to maintain the disciplinary gulf by reinforcing assumptions concerning the neutrality and technical nature of scientific knowledge and how those concepts marginalize social and ethical dimensions to create a politically influential hierarchy of knowledge claims.     

A 150 micron id packed column for the separation of soybean proteins by elution gradient mu-HPLC: simultaneous separation of soybean proteins from cereal and milk proteins

Mu-HPLC has previously been used to increase the resolution and sensitivity of protein separations but never for the analysis of soybean proteins. In this work, soybean proteins were, for the first time, separated using a capillary column with an internal diameter of 150 microm packed with a Genesis C18 stationary phase (4 microm, 300 angstroms) and UV detection. TFA and acetic acid were investigated as ion-pairing reagents in order to optimise water-ACN gradients to achieve this separation. The column showed good selectivity enabling the separation of soybean proteins from other vegetable proteins such as cereal (wheat, rice and corn) and also from milk proteins. The developed method was applied to the detection of soybean proteins in commercial products elaborated with mixtures of vegetable proteins.     

Absolute net charge and the biological activity of oligopeptides

Sequences of human proteins are frequently prepared as synthetic oligopeptides to assess their functional ability to act as compounds modulating pathways involving the parent protein. Our objective was to analyze a set of oligopeptides, to determine if their solubility or activity correlated with features of their primary sequence, or with features of properties inferred from three-dimensional structural models derived by conformational searches. We generated a conformational database for a set of 78 oligopeptides, derived from human proteins, and correlated their 3D structures with solubility and biological assay activity (as measured by platelet activation and inhibition). Parameters of these conformers (frequency of coil, frequency of turns, the degree of packing, and the energy) did not correlate with solubility, which was instead partly predicted by two measures obtained from primary sequence analysis, that is, the hydrophobic moment and the number of charges. The platelet activity of peptides was correlated with a parameter derived from the structural modeling; this was the second virial coefficient (a measure of the tendency for a structure to autoaggregate). This could be explained by an excess among the active peptides of those which had either a large number of positive charges or in some cases a large number of negative charges, with a corresponding deficit of peptides with a mixture of negative and positive charges. We subsequently determined that a panel of 523 commercially available (and biologically active) peptides shared this elevation of absolute net charge: there were significantly lower frequencies of peptides of mixed charges compared to expectations. We conclude that the design of biologically active peptides should consider favoring those with a higher absolute net charge.