Using mass spectrometry to study copper-protein binding under native and non-native conditions: beta-2-microglobulin

A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of beta-2-microglobulin (beta2m). Recent studies have shown that beta2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native beta2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded beta2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of beta2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.     

Semiautomated high-throughput extraction and cleanup method for the measurement of polybrominated diphenyl ethers and polybrominated and polychlorinated biphenyls in breast milk

A semiautomated extraction and cleanup method has been developed to measure eight polybrominated diphenyl ethers (PBDEs), 2,2',4,4',5,5'-hexabromobiphenyl (BB-153), and 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153). The method employs solid-phase dispersion on diatomaceous earth in a solid-phase extraction cartridge followed by automated addition of internal standards ((13)C-labeled). Extraction is then performed using an automated modular solid-phase extraction system. The extraction procedure includes drying the sample on diatomaceous earth by pressurized nitrogen and eluting target analytes and lipids with dichloromethane. Lipid content is determined gravimetrically. Lipid determinations performed using this method are compared with other standard methods and with a certified reference material. A relative standard deviation of 7.9% was obtained for 130 determinations of the lipid content in a breast milk quality control sample. Final analytical determination of target analytes was performed by gas chromatography-isotope dilution high-resolution mass spectrometry. Relative standard deviations for the measurements of target analytes for which a labeled internal standard was available were below 10% for analytes at concentrations above 1 ng/g of lipid. Mean recoveries of the (13)C-labeled internal standards ranged from 60 to 89% for the eight PBDE congeners; 74 and 113% were recovered for BB-153 and CB-153, respectively.     

Analysis of organoselenium compounds in human urine using active carbon and chemically modified silica sol-gel surface-assisted laser desorption/ionization high-resolution time-of-flight mass spectrometry

Laser desorption/ionization time-of-flight mass spectra of three thermally labile low molecular weight organoselenium compounds (selenomethionine, selenoethionine, trimethylselenonium iodide) in human urine matrix have been obtained by using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Four active layers, active carbon, silica sol-gel, and silica sol-gel impregnated with crown ether and with active carbon, were deposited on three different support substrates: (i) microscope slide coverslips; (ii) Al foil; and (iii) Cu tapes. Primarily protonated molecular ions and alkali metal adducts were observed in the mass spectra. A mixed-silica sol-gel and active carbon substrate active layer gave the best signal response for all compounds. Sol-gel substrates offered very clean backgrounds compared to the pure active carbon layers; however, the mass spectral signal intensities acquired were substantially lower for the sol-gel surfaces than those of the pure active carbon under the same conditions. Cu tape and Al foil support materials gave strong mass spectra of molecular ions and alkali metal molecular ion adducts, with lower Na and K adducts found with the Cu tape than with Al foil. Glass coverslips gave no response under all experimental conditions examined. Aerosol deposition was used to prepare SALDI substrates to avoid the localized "sweet spot" phenomenon encountered in conventional SALDI substrate preparation. Various nebulizers were examined and found to be effective in producing SALDI films with controlled and reproducible thicknesses. We were able to obtain exact mass identification of all three selenium species by high-resolution TOF-MS. To the best of our knowledge, this is the first time low molecular weight organoselenium compounds have been identified by SALDI-high-resolution TOF-MS.     

Phase retrieval from subdivision of the focal plane with a lenslet array

A phase retrieval algorithm derived from subdivision of the complex field at the focal plane is proposed. This subdivision is achieved with a lenslet array at the focal plane in a manner similar to the pyramid wave-front sensor. The phase retrieval algorithm significantly improves the wave-front estimate that can be attained as a linear combination of the aperture images. This phase retrieval algorithm also avoids the twin-image stagnation problem inherent in phase retrieval and phase retrieval in conjunction with the Shack-Hartmann sensor.     

Mechanical characteristics of optical coatings prepared by various techniques: a comparative study

Good performance of optical coatings depends on the appropriate combination of optical and mechanical properties. Therefore, successful applications require good understanding of the relationship between optical microstructural and mechanical characteristics and film stability. In addition, there is a lack of standard mechanical tests that allow one to compare film properties measured in different laboratories. We give an overview of the methodology of mechanical measurements suitable for optical coatings; this includes depth-sensing indentation, scratch resistance, friction, abrasion and wear testing, and stress and adhesion evaluation. We used the techniques mentioned above in the same laboratory to systematically compare the mechanical behavior of frequently used high- and low-index materials, namely, TiO2, Ta2O5, and SiO2, prepared by different complementary techniques. They include ion-beam-assisted deposition by electron-beam evaporation, magnetron sputtering, dual-ion-beam sputtering, plasma-enhanced chemical-vapor deposition, and filtered cathodic arc deposition. The mechanical properties are correlated with the film microstructure that is inherently related to energetic conditions during film growth.     

Single-material inhomogeneous optical filters based on microstructural gradients in plasma-deposited silicon nitride

Transparent hydrogenated amorphous silicon nitride (SiNx:H) coatings were prepared by dual-mode microwave-radio-frequency plasma-enhanced chemical vapor deposition. By controlling the effects of plasma density and ion energy on the film growth, it was possible to modify the microstructure of the coatings and hence the refractive index n. Using this method, we were able to vary n from 1.6 to 2.0, at 550 nm, by adjusting the power levels of the radio-frequency and microwave components while keeping the gas composition (SiH4, N2) and pressure constant. An inhomogeneous bandpass filter with a controlled refractive-index depth profile was fabricated, and its optical performance was compared with that of its multilayer counterpart. Besides the attractive optical features of such single-material rugate filters, we found that the mechanical resistance of inhomogeneous films is superior to that of multilayer systems.     

Task-specific monitoring of nuclear medicine technologists' radiation exposure

Many studies have demonstrated that the exposure of nuclear medicine technologists arises primarily from radioactive patients rather than from preparation of radiopharmaceuticals. However, in order to devise strategies to reduce staff exposure, it is necessary to identify the specific tasks within each procedure that result in the highest radiation doses. An ESM Eberline FH41B-10 radiation dosemeter, which records the ambient dose equivalent rate, was used to monitor the radiation exposure of a technologist and to record the dose rate in microSv per hour every 32 s throughout a working day. The technologist recorded the procedures that were being performed so that the procedures that resulted in higher doses could be identified clearly. The measured doses clearly showed that the major contributions to the technologist's dose were the following: (1) transferring incapacitated patients from the imaging table to a hospital trolley; (2) difficult injections without syringe shields; and (3) setting up patients for gated myocardial scans. The average dose to the technologist from transferring patients after a bone scan was 0.54 microSv, 40% of the total dose of 1.3 microSv for the complete bone scan procedure. The average dose received injecting 900 MBq of 99Tcm-HDP using a tungsten syringe shield was 0.57microSv, but the highest dose was 1.6 microSv, in a patient in whom the injection was difficult. A 0.5 mm lead apron was found to reduce the dose when setting up a patient for a gated stress 99Tcm-sestamibi myocardial scan by approximately a factor of 2. The average dose per patient for this task was reduced from 1.1 to 0.6 microSv. It is recommended that staff waiting for assistance with patient transfers stand away from the patient, that tungsten syringe shields be used for all radiopharmaceutical injections and that a 0.5 mm lead apron be worn when attending patients containing high activities of 99Tcm radiopharmaceuticals, such as those having myocardial imaging.     

Schwa-Deletion in Hindi Text-to-Speech Synthesis

We describe the phenomenon of schwa-deletion in Hindi and how it is handled in the pronunciation component of a multilingual concatenative text-to-speech system. Each of the consonants in written Hindi is associated with an inherent schwa vowel which is not represented in the orthography. For instance, the Hindi word pronounced as [namak] (salt) is represented in the orthography using the consonantal characters for [n], [m], and [k]. Two main factors complicate the issue of schwa pronunciation in Hindi. First, not every schwa following a consonant is pronounced within the word. Second, in multimorphemic words, the presence of a morpheme boundary can block schwa deletion where it might otherwise occur. We propose a model for schwa-deletion which combines a general purpose schwa-deletion rule proposed in the linguistics literature (Ohala, 1983), with additional morphological analysis necessitated by the high frequency of compounds in our database. The system is implemented in the framework of finite-state transducer technology.     

FACTORS DESCRIPTIVE OF PSYCHOPATHOLOGY AND BEHAVIOR OF HOSPITALIZED PSYCHOTICS.

"A sample of 423 psychotic patients from five veterans hospitals was observed and rated on behavior in the interview and on the ward. The intercorrelations between 55 of the rating scales were subjected to a multiple group factor analysis. The 11 oblique first order factors were interpreted." (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The use of a physical symptom as a defense against psychosis.

It was hypothesized that, since anxiety is one of the main stress factors in producing psychotic symptoms, a psychotic's symptoms might decrease if his anxiety could be decreased. Using a single schizophrenic patient the authors attempted to relieve him of his psychotic symptoms by replacing them with a physical one. The symptom chosen was backache because the patient's history suggested that it would be more acceptable to him. A "conditioning" method, extending over a period of 3 months, was followed by all staff members having contact with the patient. Following this procedure, although no basic personality change occurred, the patient was able to make a satisfactory adjustment outside the hospital using his "back pain" as a defense. (PsycINFO Database Record (c) 2010 APA, all rights reserved)