Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

A Deployable In Vivo EPR Tooth Dosimeter for Triage After a Radiation Event Involving Large Populations

In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators.Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately five minutes, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care.     

Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]⁻ ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C_oxLRRASLG) [M – H + O₃]⁻ ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d _i fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a _i ions followed by the elimination of the side chain to form d _i ions).     

Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids

Locked nucleic acids (LNAs) are novel base modifications containing a methylene bridge uniting the 2'-oxygen and the 4'-carbon. In this study, LNA-modified single-stranded molecules directed the repair of single base mutations in a yeast chromosomal gene. Using a genetic assay involving a mutant hygromycin-resistance gene, correction of point and frameshift mutations was facilitated by vectors containing an LNA residue on each terminus. Increasing the number of LNA bases on each terminus reduced the correction frequency progressively. When the LNA vector is used in combination with a phosphorothioate-modified vector (74-mer), however, a high level of gene-repair activity occurs; hence, short LNA-based vectors can augment the activity of other types of targeting vectors. These data suggest that oligonucleotides containing locked nucleic acid residues can be used to direct single nucleotide exchange reactions in vivo.     

Experimental Procedures for Sensitive and Reproducible In Situ EPR Tooth Dosimetry

In vivo electron paramagnetic resonance (EPR) tooth dosimetry provides a means for non-invasive retrospective assessment of personal radiation exposure. While there is a clear need for such capabilities following radiation accidents, the most pressing need for the development of this technology is the heightened likelihood of terrorist events or nuclear conflicts. This technique will enable such measurements to be made at the site of an incident, while the subject is present, to assist emergency personnel as they perform triage for the affected population. At Dartmouth Medical School this development is currently being tested with normal volunteers with irradiated teeth placed in their mouths and with patients who have undergone radiation therapy. Here we describe progress in practical procedures to provide accurate and reproducible in vivo dose estimates.     

Periodic Fluctuations of Flow and Porosity in Spouted Beds

The results of experimental investigations of bed flow hydrodynamics in spouted beds are compared with CFD simulations (Eulerian–Eulerian approach) for two different column geometries. The experimental results of bed porosity and fluctuation frequency of mass flow rate of grain in the fountain region are compared with the corresponding results of simulations. The simulation results confirmed the observations of Muir et al. (1990, Chem. Eng. Comm. 88: 153–171) and Yang and Keairns (1978, AlchE Symp. Ser. No. 176 74: 218) that fluctuations of bed flow in DTSB are caused by particle cluster formation in the loading region at the bottom of column. The solids cross into the jet and cover the column inlet and are carried upward periodically through a draft tube. Subsequent figures obtained from simulations, which show stages of particle cluster formation at the entrance of column, exactly match visual observations. The frequency of fluctuations of grain mass flow rate predicted in simulations (~5–6 Hz) is in the range of that experimentally determined. The fluctuating inflow of solids results in slug formation and explains the vertical variations of height and porosity of the fountain.     

Intercalation of Toluidines into α-Zirconium Hydrogenphosphate

Intercalates of o-, m-, and p-toluidine into α-Zr(HPO₄)₂ · H₂O were prepared and characterized by powder X-ray diffraction, thermogravimetric analysis and infrared spectroscopy. As follows from IR, toludine molecules are protonated in the interlayer space. Toluidine molecules are arranged in a bimolecular way in the intercalates containing more than 1.5 toluidine molecules per Zr atom. On the other hand, a monolayer of the toluidine molecules is supposed in the intercalates with less than one toluidine molecule per Zr atom.     

Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

Background  

Huntington's Disease (HD) is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt) is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.     

In Vivo EPR For Dosimetry

As a result of terrorism, accident, or war, populations potentially can be exposed to doses of ionizing radiation that could cause direct clinical effects within days or weeks. There is a critical need to determine the magnitude of the exposure to individuals so that those with significant risk have appropriate procedures initiated immediately, while those without a significant probability of acute effects can be reassured and removed from the need for further consideration in the medical/emergency system. In many of the plausible scenarios there is an urgent need to make the determination very soon after the event and while the subject is still present. In vivo EPR measurements of radiation-induced changes in the enamel of teeth is a method, perhaps the only such method, which can differentiate among doses sufficiently for classifying individuals into categories for treatment with sufficient accuracy to facilitate decisions on medical treatment. In its current state, the in vivo EPR dosimeter can provide estimates of absorbed dose with an error approximately +/- 50 cGy over the range of interest for acute biological effects of radiation, assuming repeated measurements of the tooth in the mouth of the subject. The time required for acquisition, the lower limit, and the precision are expected to improve, with improvements in the resonator and the algorithm for acquiring and calculating the dose. The magnet system that is currently used, while potentially deployable, is somewhat large and heavy, requiring that it be mounted on a small truck or trailer. Several smaller magnets, including an intraoral magnet are under development, which would extend the ease of use of this technique.     

APPLICATION OF HIGH RESOLUTION GEOMETRIC MOIRÉ METHOD TO FRACTURE PROBLEMS

A high density geometric moiré technique has been developed and successfully applied to determine fracture parameters, namely the opening mode stress intensity factor, in both transparent and opaque engineering materials. With the development of this technique the displacement resolution capability of geometric moiré has been increased at least twofold, from 0.025 mm to 0.0125 mm. Further development of gratings with line densities higher than 80 line pairs/mm will further bridge the gap which currently exists between the displacement measuring capabilities of geometric and interferometric moiré.