Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.     

Repeatability of 3D gait kinematics obtained from an electromagnetic tracking system during treadmill locomotion

The purpose of this paper was to describe a technique that enables three-dimensional (3D) gait kinematics to be obtained using an electromagnetic tracking system, and to report the intra-trial, intra-day/inter-tester and inter-day/intra-tester repeatability of kinematic gait data obtained using this technique. Ten able-bodied adults underwent four gait assessments; the same two testers tested each subject independently on two different days. Gait assessments were conducted on a custom-built long-bed treadmill with no metal components between the rollers. Each gait assessment involved familiarisation to treadmill walking, subject anatomical and functional calibration, and a period of steady-state treadmill walking at a self-selected speed. Following data collection, 3D joint kinematics were calculated using the joint coordinate system approach. 3D joint angle waveforms for 10 left and right strides were extracted and temporally normalised for each trial. Intra-trial, intra-day/inter-tester and inter-day/intra-tester repeatability of the temporally normalised kinematic waveforms were quantified using the coefficient of multiple determination (CMD). CMDs for joint kinematics averaged 0.942 intra-trial, 0.849 intra-day/inter-tester and 0.773 inter-day/intra-tester. In general, sagittal plane kinematics were more repeatable than frontal or transverse plane kinematics, and kinematics at the hip were more repeatable than at the knee or ankle. The level of repeatability of kinematic gait data obtained during treadmill walking using this protocol was equal or superior to that reported previously for overground walking using image-based protocols.     

Characterisation of the main drivers of intra- and inter- breed variability in the plasma metabolome of dogs

Introduction

Dog breeds are a consequence of artificial selection for specific attributes. These closed genetic populations have metabolic and physiological characteristics that may be revealed by metabolomic analysis.

Objectives

To identify and characterise the drivers of metabolic differences in the fasted plasma metabolome and then determine metabolites differentiating breeds.

Methods

Fasted plasma samples were collected from dogs maintained under two environmental conditions (controlled and client-owned at home). The former (n = 33) consisted of three breeds (Labrador Retriever, Cocker Spaniel and Miniature Schnauzer) fed a single diet batch, the latter (n = 96), client-owned dogs consisted of 9 breeds (Beagle, Chihuahua, Cocker Spaniel, Dachshund, Golden Retriever, Greyhound, German Shepherd, Labrador Retriever and Maltese) consuming various diets under differing feeding regimens. Triplicate samples were taken from Beagle (n = 10) and Labrador Retriever (n = 9) over 3 months. Non-targeted metabolite fingerprinting was performed using flow infusion electrospray-ionization mass spectrometry which was coupled with multivariate data analysis. Metadata factors including age, gender, sexual status, weight, diet and breed were investigated.

Results

Breed differences were identified in the plasma metabolome of dogs housed in a controlled environment. Triplicate samples from two breeds identified intra-individual variability, yet breed separation was still observed. The main drivers of variance in dogs maintained in the home environment were associated with breed and gender. Furthermore, metabolite signals were identified that discriminated between Labrador Retriever and Cocker Spaniels in both environments.

Conclusion

Metabolite fingerprinting of plasma samples can be used to investigate breed differences in client-owned dogs, despite added variance of diet, sexual status and environment.

     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

Fe(III) Oxide Reduction by a Gram-positive Thermophile: Physiological Mechanisms for Dissimilatory Reduction of Poorly Crystalline Fe(III) Oxide by a Thermophilic Gram-positive Bacterium Carboxydothermus ferrireducens

Physiological strategies driving the reduction of poorly crystalline Fe(III) oxide by the thermophilic Gram-positive dissimilatory Fe(III)-reducing bacterium C. ferrireducens were evaluated. Direct cell-to-mineral contact appears to be the major physiological strategy for ferrihydrite reduction. This strategy is promoted by cell surface-associated c-type cytochromes, and the extracellular electron transfer to ferrihydrite is linked to energy generation via a membrane-bound electron transport chain. The involvement of pili-like appendages in ferrihydrite reduction has been detected for the first time in a thermophilic microorganism. A supplementary strategy for the utilization of a siderophore (DFO) in dissimilatory ferrihydrite reduction has also been characterized.     

Heritability estimates for carcass traits of cattle: a review

We present estimates of heritability for carcass traits of cattle published in the scientific literature. Seventy-two papers published from 1962 to 2004, which reported estimates of heritability for carcass traits, were reviewed. The unweighted means of estimates of heritability for 14 carcass traits by slaughter end point (age, weight, and fat depth) were calculated. Among the three end points, carcass weight, backfat thickness, longissimus muscle area, and marbling score were the carcass traits with the most estimates of heritability (56 相似文献   

Comparative fitness of irradiated light brown apple moths (Lepidoptera: Tortricidae) in a wind tunnel, hedgerow, and vineyard

Light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is the target of the sterile insect technique, but reduced moth fitness from irradiation lowers the effective overflooding ratio of sterile to wild moths. New measures of insect quality are being sought to improve field performance of irradiated insects, thus improving the cost effectiveness of this technique. Male pupae were irradiated at intervals between 0 and 300 Gy, and adult flight success was assessed in a wind tunnel equipped with flight track recording software. A dose response was evident with reduced successful search behaviors at higher irradiation doses. Irradiation at 250 Gy reduced arrival success to 49% of untreated controls, during 2-min assays. Mark-release-recapture of males irradiated at 250 Gy indicated reduced male moth recapture in hedgerows (75% of control values of 7.22% +/- 1.20 [SEM] males recaptured) and in vineyards (78% of control values 10.5% +/- 1.66% [SEM] recaptured). Males dispersed similar distances in both habitats, and overflooding ratios dropped off rapidly from the release point in both landscapes. Transects of traps with central releases proved to be an efficient method for measuring the quality of released males. Relative field performance of moths was greater than suggested by wind tunnel performance, which could be due to time differences between the two assays, two-minute wind tunnel tests compared with days in the field treatments. Release strategies involving ground releases should consider the effect of limited postrelease dispersal. Aerial release could solve this problem and warrants investigation.     

Identification of residues important for DNA binding in the full-length human Rad52 protein

Human Rad52 (HsRad52) is a DNA-binding protein (418 residues) that promotes the catalysis of DNA double strand break repair by the Rad51 recombinase. HsRad52 self-associates to form ring-shaped oligomers as well as higher order complexes of these rings. Analysis of the structural and functional organization of protein domains suggests that many of the determinants of DNA binding lie within the N-terminal 85 residues. Crystal structures of two truncation mutants, HsRad52(1-212) and HsRad52(1-209) support the idea that this region makes up an important part of the DNA binding domain. Here, we report the results of saturating alanine scanning mutagenesis of the N-terminal domain of full-length HsRad52 in which we identify residues that are likely involved in direct contact with single-stranded DNA (ssDNA). Our results largely agree with the position of side-chains seen in the crystal structures but also suggest that certain DNA binding and cross-subunit interactions differ between the 11 subunit ring in the crystal structures of the truncation mutant proteins versus the seven subunit ring formed by full-length HsRad52.