Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

Evaluation of Corn/Soybeans Separability Using Thematic Mapper And Thematic Mapper Simulator Data

Multitemporal Thematic Mapper, Thematic Mapper Simulator, and detailed ground truth data were collected for a 9-by 1-km sample segment in Webster County, Iowa, in the summer of 1982. Three dates were acquired each with Thematic Mapper Simulator (June 7, June 23, and July 31) and Thematic Mapper (August 2, September 3, and October 21). The Thematic Mapper Simulator data were converted to equivalent TM count values using TM and TMS calibration data and model based estimates of atmospheric effects. The July 31, TMS image was compared to the August 2, TM image to verify the conversion process. A quantitative measure of proportion estimation variance (Fisher information) was used to evaluate the corn/soybeans separability for each TM band as a function of time during the growing season. The additional bands in the middle infrared allowed corn and soybeans to be separated much earlier than was possible with the visible and near-infrared bands alone. Using the TM and TMS data, temporal profiles of the TM principal components were developed. The greenness and brightness exhibited behavior similar to MSS greenness and brightness for corn and soybeans.     

Compound screening for the presence of the primary N-oxide functionality via ion-molecule reactions in a mass spectrometer

A mass spectrometry method is presented for the identification of compounds that contain the primary N-oxide functional group. This method utilizes a gas-phase ion-molecule reaction with dimethyl disulfide that rapidly and selectively derivatizes the protonated primary N-oxide functional group in a mass spectrometer to yield an ionic reaction product (with 31 Da higher mass than that of the protonated molecule) that is diagnostic for the presence of a primary N-oxide functionality. A variety of protonated analytes containing different functional groups were tested in Fourier transform ion-cyclotron resonance and triple quadrupole mass spectrometers to probe the selectivity of the reaction. Only molecules containing the protonated primary N-oxide functional group yielded the diagnostic reaction product; all other protonated molecules gave protonated dimethyl disulfide or no reaction products. The feasibility of this method for compound screening was tested by examining six analytes with the same molecular formula but different atom connectivity. The one analyte that contained the primary N-oxide functional group was readily differentiated from the other analytes.     

Resonant laser ablation of metals detected by atomic emission in a microwave plasma and by inductively coupled plasma mass spectrometry

It has been shown that an increase in sensitivity and selectivity of detection of an analyte can be achieved by tuning the ablation laser wavelength to match that of a resonant gas-phase transition of that analyte. This has been termed resonant laser ablation (RLA). For a pulsed tunable nanosecond laser, the data presented here illustrate the resonant enhancement effect in pure copper and aluminum samples, chromium oxide thin films, and for trace molybdenum in stainless steel samples, and indicate two main characteristics of the RLA phenomenon. The first is that there is an increase in the number of atoms ablated from the surface. The second is that the bandwidth of the wavelength dependence of the ablation is on the order of 1 nm. The effect was found to be virtually identical whether the atoms were detected by use of a microwave-induced plasma with atomic emission detection, by an inductively coupled plasma with mass spectrometric detection, or by observation of the number of laser pulses required to penetrate through thin films. The data indicate that a distinct ablation laser wavelength dependence exists, probably initiated via resonant radiation trapping, and accompanied by collisional broadening. Desorption contributions through radiation trapping are substantiated by changes in crater morphology as a function of wavelength and by the relatively broad linewidth of the ablation laser wavelength scans, compared to gas-phase excitation spectra. Also, other experiments with thin films demonstrate the existence of a distinct laser-material interaction and suggest that a combination of desorption induced by electronic transition (DIET) with resonant radiation trapping could assist in the enhancement of desorption yields. These results were obtained by a detailed inspection of the effect of the wavelength of the ablation laser over a narrow range of energy densities that lie between the threshold of laser-induced desorption of species and the usual analytical ablation regime. Normal ablation employs high-power lasers in an attempt to create a vapor plume without selective vaporization, and with a stoichiometry that accurately represents the stoichiometry of species in the solid sample. RLA, as a method of selective vaporization, appears to provide an opportunity to exploit selective vaporization in new ways.     

A comparison of sample weight and culture methods for the detection of Salmonella in pig feces

Five protocols were compared to determine the combined effects of different sample weights and culture methods for the recovery of Salmonella from 310 pig cecal samples taken in abattoirs as part of the Canadian Integrated Program for Anti-microbial Resistance Surveillance. Sample weights evaluated were 1 and 10 g. Culture methods used with each sample weight were modified semisolid Rappaport-Vassiliadis agar (MSRV) and brilliant green agar with sulfa and novobiocin (BGSN) and xylose-lysine-tergitol-4 agar (XLT4). A preliminary sample preparation step in saline was also evaluated using a 10-g sample and MSRV. The Salmonella recovery rate varied from 20% for the saline MSRV 10-g protocol to 32% for the MSRV 10-g and the BGSN-XLT4 10-g protocols. A good agreement (K > 0.8) was observed between pairs of protocols except whenever the saline MSRV 10-g and the MSRV 1-g protocols were compared. Larger samples (10 g) yielded higher detection of Salmonella than 1-g samples for the MSRV protocol (32 versus 25%), whereas the differences were not statistically significant for the BGSN-XLT4 protocols. Protocols using the BGSN-XLT4 agar yielded higher detection rates of Salmonella compared with MSRV with 1-g samples (30 versus 25%), whereas it was equivalent with 10-g samples. Considering a greater recovery rate, the ease of use, and a better time and resource efficiency, the MSRV 10-g protocol was therefore adopted by the Canadian Integrated Program for Antimicrobial Resistance Surveillance.     

Education for the optometric educator

Optometric education is faced with the need for new thinking in developing and training educators and new methods of financing their education. Establishing of research faculty and the instituting of research in present schools of optometry must be given a high priority in the future of optometric education.     

A queueing model for an ATM rate control scheme

Many applications in telecommunications engineering lead to highly degenerate partitioned Markov chains of QBD type. In this paper, we study a traffic shaping scheme which is based on a generalization of the bucket method. The arrival process is modeled by a discrete Markovian arrival process. For this model, a detailed mathematical analysis leads to special algorithms involving matrices of lower order. The characteristics of the model are discussed from this viewpoint. Some performance measurements are derived and numerical examples are shown to demonstrate the effectiveness of the rate control scheme.This research was supported in part by Grant No. DDM-8915235 from the National Science Foundation.     

Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons

Adrenalectomy (ADX) is known to block the acquisition of intravenous cocaine self-administration. A previous study therefore examined whether ADX decreases sensitivity of the 'brain reward system' in general, or its response to cocaine in particular, by measuring thresholds for intracranial self-stimulation with and without concurrent cocaine administration. ADX had no effect on thresholds for lateral hypothalamic self-stimulation (LHSS) and did not alter the cocaine dose-response curve for lowering the LHSS threshold. This result suggested that ADX does not affect sensitivity of the brain reward system. However, medial prefrontal cortex (MPFC) appears to be an important site in the mediation of cocaine reinforcing effects, and MPFC self-stimulation (MPFCSS) is mediated by a neural substrate that is largely independent of that which mediates LHSS. The present study therefore assessed whether ADX diminishes cocaine facilitation of MPFCSS. It was found that the threshold-lowering effect of cocaine (5.0, 10.0 and 20.0 mg/kg, i.p. ) did not differ between ADX rats maintained on 0.7% saline, ADX rats maintained on corticosterone (50 microg/ml) in 0.7% saline, and sham-operated controls. However, there was a trend toward desensitization of MPFCSS, itself, following ADX in the group that did not receive corticosterone supplementation. Based on this observation, and the similar responses of MPFCSS and cocaine self-administration to noncontingent priming stimulation, stress, and NMDA receptor antagonism, it is speculated that acquisition of MPFCSS and cocaine self-administration may be dependent upon a common sensitization process that is regulated by corticosterone.