A new assay for the determination of phosphoribosylamine.

This report describes a new assay for glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) (EC 2.4.2.14) based on the formation of a stable compound between the ribose-5-phosphate moiety of phosphoribosylamine and [³⁵S]cysteine. The assay is comparable in sensitivity to the [¹⁴C]glutamine assay, and its potential in the evaluation of NH₃ utilization by this enzyme is documented. The stoichiometry of the enzymatic reaction and the analysis of the ribose-5-phosphate/cysteine compound demonstrate that 1 mol of phosphoribosylamine (or ribose-5-phosphate) combines with 1 mol of [³⁵S] cysteine to form this compound. Preliminary studies with a number of carbohydrates indicate that an aldopentose phosphate is most reactive with cysteine. The substitution of a phosphate, pyrophosphate, or purine ring at C-1 of the pentose markedly reduces its reactivity with cysteine.     

A rapid boiling method for the preparation of bacterial plasmids

Compounds separated on polyamide thin-layers and located in an appropriate manner can be introduced directly into the ion source of the mass spectrometer together with the chromatographic polyamide adsorbent. In spite of the large excess of polyamide, excellent mass spectra are obtained exhibiting low backgrounds. In this way, organic compounds can be rationally identified on a nanogram scale not requiring reference substances. The application of the procedure is described for selected compounds of different classes of substances, e.g., phenols, steroids, nucleosides, biogenic amines, and amino acids.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Design and development of a series of borocycles as selective,covalent kallikrein 5 inhibitors

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.     

Cannot see the random forest for the decision trees: selecting predictive models for restoration ecology

Improving predictions of restoration outcomes is increasingly important to resource managers for accountability and adaptive management, yet there is limited guidance for selecting a predictive model from the multitude available. The goal of this article was to identify an optimal predictive framework for restoration ecology using 11 modeling frameworks (including machine learning, inferential, and ensemble approaches) and three data groups (field data, geographic data [GIS], and a combination thereof). We test this approach with a dataset from a large postfire sagebrush reestablishment project in the Great Basin, U.S.A. Predictive power varied among models and data groups, ranging from 58% to 79% accuracy. Finer‐scale field data generally had the greatest predictive power, although GIS data were present in the best models overall. An ensemble prediction computed from the 10 models parameterized to field data was well above average for accuracy but was outperformed by others that prioritized model parsimony by selecting predictor variables based on rankings of their importance among all candidate models. The variation in predictive power among a suite of modeling frameworks underscores the importance of a model comparison and refinement approach that evaluates multiple models and data groups, and selects variables based on their contribution to predictive power. The enhanced understanding of factors influencing restoration outcomes accomplished by this framework has the potential to aid the adaptive management process for improving future restoration outcomes.     

Metabonomic studies on the physiological effects of acute and chronic psychological stress in Sprague-Dawley rats

The biochemical effects of acute and chronic psychological stress have been investigated in male Sprague-Dawley rats using a combination of 1H NMR spectral analysis of plasma and conventional hematological analyses. Animals were subjected to 35 consecutive days of 6-h sessions of stress, and following a 9 day break, were stressed for a further 6-h period. Plasma samples were collected at 0, 1, 3, and 6 h on days 1, 9, 21, 35, and 44, measured using 600 MHz 1H NMR spectroscopy, and analyzed by Principal Components Analysis. Time-dependent biochemical effects of psychological stress on a range of endogenous metabolites were evident and were correlated with the intensity of the stress response as defined by corticosterone and hematological parameters. Following acute stress, increases in the levels of glucose and ketone bodies, and decreases in the levels of acetate, alanine, isoleucine, lactate, leucine, valine, and lipoproteins, were observed. Chronic stress-induced increases in plasma levels of alanine, lactate (day 9), and leucine, valine, and choline (day 44) and decreases in acetate (day 9) and lipoprotein concentrations were observed. Positive correlations between plasma corticosterone level and glucose and glycerol, and between plasma lipoprotein concentrations and hemoglobin levels, were established using Projection to Latent Structures (PLS) analysis. This study indicates the potential of using NMR-based metabonomic strategies for the characterization of endogenous metabolic perturbations induced by psychological stressors and lifestyle choices.     

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae

The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, ADDelta. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K(m) value and the V(max) value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.