A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies

We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.     

High-throughput method for the production and analysis of large natural product libraries for drug discovery

High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.     

Extreme-ultraviolet calibration of thin-film Zr filters for the cosmic hot interstellar plasma spectrometer

The design and calibration of thin-film Zr transmission filters for the Cosmic Hot Interstellar Plasma Spectrometer (CHIPS) spaceborne astronomical telescope is presented. The transmission of a 1,000-A-thick Zr filter is measured for wavelengths from 45 to 1,304 A as well as its variability with respect to expected space-flight enviromental extremes of temperature, vibration, and atomic O bombardment. For the CHIPS spectral band of 90-260 A, the transmission of the Zr fiter is deemed useful between 90 and 200 A and is stable with respect to expected temperature and vibrational changes. The maximum atomic O bombardment decreases the filter transmission between 90 and 130 A by approximately 20% and does not appreciably affect the rest of the CHIPS bandpass. These results make Zr an acceptable filter candidate for CHIPS.     

Direct deflection method for determining refractive-index profiles of polymer optical fiber preforms

We present a method for determining the refractive-index profile of polymer optical fiber preforms through a direct-deflection measurement. The method is simple to use, compact, and has good resolution. The profile is obtained from the deflection data by numerically integrating the differential-ray equation for a radial refractive-index gradient. Corrections for topographical deviations are also discussed. Results for both graded-index and step-index fibers are presented.     

Preliminary results on a dedicated silicon diode detector for proton dosimetry

The present work reports preliminary measurements on the behaviour of a new p-type stereotactic silicon diode. Hi-pSi, produced by Scanditronix and dedicated to proton dosimetry. Diode response was investigated in low-energy proton beams (26.7 MeV and 12 MeV nominal energy), mainly with attention to stability, linearity, dose rate and energy dependence of the detector response. Three different Hi-pSi diodes of the same type were investigated. The diode response was linear with dose and the standard deviation of repeated readings was less than 2.5%. A marked dependence on dose rate was observed for one of the diodes (a response increase of 47% in the 0.7-11 Gy x min(-1) range). After the dose rate and water to silicon mass collision stopping power ratio correction of the diode response in the depth dose measurements, the difference, at the Bragg peak, with respect to the reference chamber was about 4%, ascribed to poor knowledge of the materials in front of the sensitive volume. The diode response was also nearly independent of linear energy transfer (LET) in the 9.6-21.5 MeV effective energy range.     

QSAR studies of the pyrethroid insecticides. Part 3. A putative pharmacophore derived using methodology based on molecular dynamics and hierarchical cluster analysis

Previous studies of the conformational behaviour of a group of synthetic pyrethroid insecticides have been extended to a more structurally diverse set. This includes compounds with different backbones and differing stereochemistry, with both Types I and II biological activity. These compounds also encompass a large range of biological activities. A parameterisation of the CHARMM force field for these compounds has been performed and the extra parameters are reported. Conformational sampling, using molecular dynamics (MD), has been performed for each of the 41 active structures. The accessible conformations of each have been characterised by the values of the common torsion angles using hierarchichal cluster analysis (HCA). A further CA, based on the centroids derived from the conformational sampling, identified a conformation common to at least 39 of the 41 structures. The critical torsion angles of this conformation lie at the centre of the molecule about the ester linkage and are defining an extended conformation, which differs from the minimum energy conformation of deltamethrin used previously. This may represent a putative pharmacophore for kill. The methods used here improve significantly on those used previously. The CHARMM force field was parameterised for the compounds and an improved method of conformational sampling, based on centroid clustering, has also been used.     

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations,activation and development of mouse oocytes

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.     

Fast, all-optical Rayleigh wave microscope: imaging on isotropic and anisotropic materials

A fast, non-contact Rayleigh wave scanning microscope is demonstrated, which is capable of scan rates of up to a maximum of 1000 measurements/s with typical speeds of up to 250 measurements/s on real samples. The system uses a mode-locked, Q-switched Nd:YAG laser operating at a mode-locked frequency of 82 MHz and a Q-switch frequency of 1 kHz. The Q-switch frequency determines the upper limit of the scanning rate. The generating laser illumination is delivered and controlled by a computer-generated hologram (CGH). The generating laser produces around 30 pulses at 82 MHz and additional harmonics at 164 and 246 MHz and above. The microscope can operate at these harmonics provided the spatial bandwidth of the optics and the temporal bandwidth of the electronics are suitable. The ultrasound is detected with a specialized knife-edge detector. The microscope has been developed for imaging on isotropic materials. Despite this, the system can be used on anisotropic materials, but imaging and interpreting images can be difficult. The anisotropy and grain structure of the material can distort the Rayleigh wavefront, leading to signal loss. A model has been developed to simulate polycrystalline-anisotropic materials; this is discussed along with possible solutions that would overcome the problems associated with anisotropy. Rayleigh wave amplitude images are demonstrated on silicon nitride at 82 and 164 MHz and on polycrystalline aluminium at 82 MHz.     

An improvement in the hygienic instruction of workers in dairy industry enterprises

The article presents the experience of the work on the sanitary education of the workers of the milk industry plants in Moscow and Moscow Province. The Institute's specialists had worked out basing on the studied experience the program of the full-time and external differential sanitary education of the workers of the milk industry which was approved by the Department of the State Sanitary Control of the Ministry of Health of Russia the April 22, 1998. "An exercise book for the sanitary education of the workers of the milk industry plants" was also prepared and published in 1999.     

Modeling the customer in electronic commerce

This paper reviews interface design of web pages for e-commerce. Different tasks in e-commerce are contrasted. A systems model is used to illustrate the information flow between three subsystems in e-commerce: store environment, customer, and web technology. A customer makes several decisions: to enter the store, to navigate, to purchase, to pay, and to keep the merchandize. This artificial environment must be designed so that it can support customer decision-making. To retain customers it must be pleasing and fun, and create a task with natural flow. Customers have different needs, competence and motivation, which affect decision-making. It may therefore be important to customize the design of the e-store environment. Future ergonomics research will have to investigate perceptual aspects, such as presentation of merchandize, and cognitive issues, such as product search and navigation, as well as decision making while considering various economic parameters. Five theories on e-commerce research are presented.