A mammalian genetic system to screen for small molecules capable of disrupting protein-protein interactions

A mammalian two-hybrid system was developed for high-throughput screening of compounds that disrupt specific protein-protein interactions. The existing mammalian systems are unsatisfactory for drug screening due to nonregulated expression of interacting proteins. To construct a tightly regulated system, the tetracycline repressor was fused with the inhibitory KRAB domain as a suppressor. The binding of the suppressor to the tet operator entirely blocked expression of two interacting proteins. When both the inducer doxycycline and drugs were added to the culture, the reporter gene was either activated by interaction of the paired proteins with ineffective drugs or remained silent due to disruption of the protein interactions by the effective drugs. We demonstrate that interactions of the type I receptor for TGFbeta with FKBP12 and the epidermal growth factor receptor (EGFR) with p85 are effectively disrupted by FK506 and EGFR kinase inhibitor AG1478, respectively. The power of this system for drug screening was further demonstrated by rapid identification of inhibitors from a druglike library for the receptor kinases.     

Impact of nano- and mesoscale particles on the performance of microcantilever-based sensors

Microcantilever-based sensors comprise an emerging class of chemomechanical sensors. The crucial challenge for every new and promising sensing platform lies in its performance in complex mixtures. Since most biofluids are rich in particulates, we assessed the impact of particles in the liquid stream on the performance of microcantilever sensors operated in both deflection and resonance modes. For both detection modes, sensor response depends on the particle diameter, concentration, and velocity as well as the composition of a thin-film coating. The presence of particles in the fluid stream produce substantial scattering of the laser beam used to measure cantilever deflection. Thus, prior removal of particulate matter from biofluids is required for optimal performance of microcantilever-based biosensors.     

High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.     

Sellmeier's equation and the expression of the thermal refractive-index coefficient for a Nd0.007Gd0.993VO4 crystal

The principal refractive indices of a Nd0.007Gd0.993VO4 crystal for wavelengths of 0.488, 0.6328, 1.064, 1.0795, and 1.3414 microm under the temperature range of 20-170 degrees C are measured by the autocollimation method, and the Sellmeier's equation and the expression of the thermal refractive-index coefficient are then obtained. The reliability of these results is checked by comparing the calculated values with the measured values for the measured wavelengths and the temperature region, as well as with the published values by Studenikin et al. [Quantum Electron. 25, 1162-1165 (1995)] for wavelengths of 0.491, 0.546, 0.578, 0.632, 0.808, and 1.062 microm at 20 degrees C. In addition, the birefringence and the thermal coefficient of the birefringence were obtained by measured results. The results show that the birefringence is larger than that of a YVO4 crystal and that the thermal coefficient of birefringence is less than that of a YVO4 crystal. Therefore it can be expected that the GdVO4 crystal is not only an excellent laser crystal but also an excellent birefringent crystal used in a modern optical transmission system as a passive fiber-optic component.     

Chemical approaches to reversible protein phosphorylation

Protein phosphorylation catalyzed by protein kinases plays a critical role in cellular signaling. Here we review several chemical approaches to understanding protein kinases and the consequences of protein phosphorylation. We discuss the design of bisubstrate analogue inhibitors based on a dissociative transition state, the development of reagents for cross-linking protein kinases with their substrates, the chemical rescue of mutant protein tyrosine kinases, and the application of expressed protein ligation to understanding protein phosphorylation.     

Excitation saturation in two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.     

Optical sensor based on Fabry-Perot resonance modes

An oscillating wave sensor based on Fabry-Perot resonance modes has been developed. Different from the surface plasmon resonance sensors and the waveguide mode sensors in which the sample is located in the evanescent field region, the proposed device contains the sample in the core region that supports the oscillating field. Owing to the strong concentration of the electromagnetic field in the sensing medium, the proposed device exhibits unusual sensitivity enhancement, which has never been exploited in any other devices.     

Pulse-inversion-based fundamental imaging for contrast detection

Pulse-inversion-based fundamental imaging was experimentally investigated for the enhancement of contrast detection. The pulse-inversion technique involves two firings with inverted waveforms. When the returning echoes from the two firings are summed, the residue signal is limited to even-order harmonics for tissue. However, when the returning echoes are from microbubbles, the fundamental signal is not completely cancelled because the reaction of the bubbles under compression is different from that under rarefaction. Thus, with the application of pulse-inversion technique, the fundamental signal can be used to enhance the contrast-to-tissue ratio. In this paper, B-mode, pulse-inversion-based fundamental images were constructed with various transmit waveforms. Motion artifacts also were studied. The results indicate that the contrast-to-tissue ratio was significantly enhanced compared to that obtained using either conventional, fundamental imaging or second-harmonic imaging. Longer transmit pulses resulted in a better signal-to-noise ratio, but did not noticeably affect the nonlinear response of the bubbles. In addition, the optimal ratio of the magnitude of the positive pulse to that of the negative pulse was unity, in terms of avoiding the uncancelled, third-order response in the fundamental frequency range. It also was found that the pulse-inversion fundamental technique is highly sensitive to tissue motion because the fundamental tissue signal is not cancelled when motion is present.     

Preparation and characterization of novel silica-butyrylchitosan hybrid biomaterials*

Novel silica-butyrylchitosan hybrid biomaterials were produced by a sol–gel technique, using butyrylchitosan as the organic species incorporated into the silicon alkoxide (TEOS) based network. 3-acryloxypropyl trimethoxysilane (MPTMS) was used effectively to combine the organic and inorganic species to form uniform hybrid biomaterials. All the samples made were in the form of thin, flexible films with transparent clarity. The blood-clotting and platelet adhesion assay confirmed that these hybrid biomaterials displayed potential good blood compatibility.     

Comparison of soluble and immobilized acetate for removing Pb from contaminated soil

Five lead (Pb) contaminated soils were used in a laboratory and modeling study to examine the effects of soluble and immobilized acetate on Pb removal from a contaminated soil as a function of pH. Soluble acetate was added as sodium acetate; immobilized acetate was added in the form of a cation exchange resin. For comparative purposes, Pb adsorption with no acetate also was measured as a function of pH. A surface complexation modeling framework was used to interpret experimental data. Experimental results showed the cation exchange resin was much more effective than soluble acetate in removing Pb from soils due to a strong affinity of the resin for Pb. In addition, concentrations of soluble Pb in resin/soil slurry were very low, minimizing the pollution threat if discharged. As deduced from modeling studies, soluble acetate performed poorly compared to the resin, in part, due to adsorption of the soluble PbAc(+) complex. The effectiveness of both soluble and immobilized acetate was diminished below pH 4 as a result of competition by H(+) for acetate. Modeling results based on resin affinity for Pb compare well with experimental data for resin/soil mixtures, suggesting that Pb partitioning in resin/soil mixtures may be predicted reasonably well if soil/Pb and resin/Pb partitioning are known. Thus, the modeling approach may be used as a screening tool to determine the performance of alternative resins.