Enzyme deactivation during cellulose hydrolysis

Hydrolysis of cellulose by Trichoderma viride cellulase reached a plateau after some 25 hr. If the initial enzyme-to-substrate ratio was low, resuspension of substrate in fresh enzyme or addition of enzyme resulted in further high rate hydrolysis. This did not occur if the initial ratio was high. Over 75% hydrolysis might be achieved in the former case, while less than 60% in the latter. A model postulating inactivation of adsorbed enzyme–substrate complex which blocked further hydrolysis was proposed, and it was found to fit the data well. The proposed model had five parameters, four of which could be checked by graphical methods, and all of which had physical meanings. The parameters were estimated by a nonlinear least-squares minimization FORTRAN computer program, using numerical integration and optimization of the parameters. The model was used to predict the resuspension data, powdered enzyme addition data, cellobiose addition data, and cellulose addition data; the deviations from the model are discussed. It was found that average values could be used for four out of the five parameters, while the fifth (initial enzyme concentration) did not correlate with independent measurements such as the filter paper activity or protein concentration.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

Oxylipin Pathway in Rice and Arabidopsis

Plants have evolved complex signaling pathways to coordinate responses to developmental and environmental Information. The oxylipin pathway Is one pivotal lipid-based signaling network, composed of several competing branch pathways, that determines the plant＇s ability to adapt to various stimuli. Activation of the oxyllpln pathway Induces the de novo synthesis of biologically active metabolltes called ＂oxyllplns＂. The relative levels of these metabolltes are a distinct indicator of each plant species and determine the ability of plants to adapt to different stimuli. The two major branches of the oxyllpln pathway, allene oxide synthase （AOS） and hydroperoxlde lyase （HPL） are responsible for production of the signaling compounds, jasmonates and aldehydes respectively. Here, we compare and contrast the regulation of AOS and HPL branch pathways In rice and Arabidopsis as model monocotyledonous and dicotyledonous systems. These analyses provide new Insights Into the evolution of JAs and aldehydes signaling pathways, and the complex network of processes responsible for stress adaptations In monocots and dicots.     

The root zone dynamics of water uptake by a mature apple tree

We report the results from a field experiment in which we examined the spatial and temporal patterns of water uptake by a mature apple tree (Malus domestica Borkh., ‘Splendour’) in an orchard. Time Domain Reflectometry (TDR) was used to measure changes in the soil's volumetric water content, and heat-pulse was used to monitor locally the rates of sap flow in the trunk and roots of the tree. We also measured the tree's distribution of root-length density and obtained supporting data to characterize the soil's hydraulic properties. The experimental data were used to examine the output of the WAVE-model (Vanclooster et al, 1995; Ecol. Model. 81, 183–185) in which soil water transport is predicted using Richards' equation, and where root uptake is represented by a distributed macroscopic sink term. When the surface soil layers were uniformly wet, 70% of the trees water uptake occurred in the top 0.4 m of the root zone, in which approximately 70% of the tree's fine roots were located. When a partial irrigation was applied to just one side of the root zone, the apple tree quickly shifted its pattern of water uptake with an almost two-fold increase in uptake from the wetter soil parts and a corresponding reduction in uptake from the drier parts. The response of root-sap flow to irrigation was almost immediate (i.e. root flow increased within hours of the irrigation). Following subsequent irrigations over the whole soil surface, TDR measurements revealed a surface-ward shift in the pattern of water extraction, and root flow measurements revealed a recovery in the uptake function of seemingly inactive roots located in the previously-dry soil. Via our root sap flow measurements, we observed two roots on the same tree locally responding quite differently to similar events of soil wetting. This observation suggests that there may be considerable functional variability across the apple root system. Our measurement-model calculations yielded similar results and stress the prime role played by the plant in modifying the root zone balance of water. Following an irrigation or rainfall event, root uptake by apple appears to be more dependent upon the near-surface availability of water than it is related to the distribution of fine roots.     

Re-Examining the Association between Vitamin D and Childhood Caries

Background

Previous studies have reported an inverse association between vitamin D and childhood dental caries, but whether this is causal is unclear.

Objective

To determine the causal effect of circulating 25-hydroxyvitamin D concentration on dental caries experience, early caries onset and the requirement for a dental general anesthetic.

Design

A Mendelian randomization study was undertaken, using genetic variants known to be associated with circulating 25-hydroxyvitamin D concentrations in 5,545 European origin children from the South West of England. Data on caries and related characteristics were obtained from parental and child completed questionnaires between 38 and 91 months and clinical assessments in a random 10% sample at 31, 44 and 61 months.

Results

In multivariable confounder adjusted analyses no strong evidence for an association of 25-hydroxyvitamin D with caries experience or severity was found but there was evidence for an association with early caries onset, or having a general anesthetic for dental problems. In Mendelian randomization analysis the odds ratio for caries experience per 10 nmol/L increase in 25-hydroxyvitamin D was 0.93 (95% confidence interval: 0.83, 1.05; P = 0.26) and the odds ratio for dental general anaesthetic per 10 nmol/L increase in 25-hydroxyvitamin D was 0.96 (95% confidence interval: 0.75, 1.22; P = 0.72).

Conclusions

This Mendelian randomization study provides little evidence to support an inverse causal effect of 25-hydroxyvitamin D on dental caries. However, the estimates are imprecise and a larger study is required to refine these analyses.     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.     

Native faunal communities depend on habitat from non-native plants in novel but not in natural ecosystems

Invasive non-native plants are a major driver of native biodiversity loss, yet native biodiversity can sometimes benefit from non-native species. Depending on habitat context, even the same non-native species can have positive and negative effects on biodiversity. Blackberry (Rubus fruticosus aggregate) is a useful model organism to better understand a non-native plant with conflicting impacts on biodiversity. We used a replicated capture-mark-recapture study across 11 consecutive seasons to examine the response of small mammal diversity and abundance to vegetation structure and density associated with non-native blackberry (R. anglocandicans) in native, hybrid and blackberry-dominated novel ecosystems in Australia. Across the three habitat types, increasing blackberry dominance had a positive influence on mammal diversity, while the strength and direction of this influence varied for abundance. At a microhabitat scale within hybrid and native habitat there were no significant differences in diversity, or the abundance of most species, between microhabitats where blackberry was absent versus dominant. In contrast, in novel ecosystems diversity and abundances were very low without blackberry, yet high (comparable to native ecosystems) within blackberry as it provided functionally-analogous vegetation structure and density to the lost native understory. Our results indicate the ecological functions of non-native plant species vary depending on habitat and need to be considered for management. Comparative studies such as ours that apply a standardized approach across a broad range of conditions at the landscape and habitat scale are crucial for guiding land managers on control options for non-native species (remove, reduce or retain and contain) that are context-sensitive and scale-dependent.     

Autoshaping in the rat: conditioned licking response to a stimulus that signals sucrose reinforcement

The present experiments were designed to determine if repeated presentations of an empty sipper tube (the conditioned stimulus or CS) with the response-independent delivery of a sucrose solution (the unconditioned stimulus or US) from a second spout results in the development of Pavlovian conditioned responding. In Experiment 1, rats in the experimental condition received paired CS-US presentations whereas subjects in the control condition were exposed to random presentations of CS and US. In Experiment 2, an additional control condition (CS alone) was included and, to encourage generalized responding between the US and CS, the CS tube was filled with water for all groups. The results of both experiments indicate that the CS-directed responding in the paired CS-US condition was Pavlovian in nature. Thus, the present procedure serves as an autoshaping task in which conditioned licking is generated.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.