Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Rehabilitation of Barbarine cull ewes using rosemary residues and linseed: effect on weight gain,carcass characteristics and meat quality

The cull ewes represent an important part of sheep flock. However, this category of animal is often submitted to under nutrition leading to poor BW and skeletal carcasses. Their rehabilitation using a high energy diet can be an alternative to improve their body condition. The objective of this experiment was to study the BW gain and carcass characteristics of Barbarine cull ewes using rosemary (Rosmarinus officinalis L.) distillation residues (RR) and extruded linseed. For this, 28 ewes above 6 years old and 33±0.5 kg of BW were divided into four groups: CCC was fed 500 g of barley-straw with concentrate, RCC received 300 g of straw and 200 g of RR as basal diet with concentrate; whereas two other groups received the experimental concentrate, containing 10% of linseed, with 500 g of straw for CLC and 300 g of straw plus 200 g of RR for RLC group. At the end of experiment (90 days), all animals were slaughtered. For all ewes, the daily concentrate intake averaged 700 g; the average daily gain was 131 g and the slaughter BW 43.4 kg without significant difference between groups. Neither basal diet nor concentrate type did affect the carcass’ weight, yield and composition. In addition, the organ’s proportions were similar for all groups. The RR intake slightly improved muscle’s protein content (P=0.03) and tended to decrease initial pH (P=0.06) and to increase meat redness (P=0.06), whereas linseed concentrate had no effect on meat color and its chemical composition. The subcutaneous fat color and firmness score relived a good quality trade for carcasses from all diets, in spite of higher yellowness and lower firmness recorded for linseed diet (P<0.05), which were moderately improved by rosemary combination with linseed. To conclude, the Barbarine cull ewes could gain up to 120 g/day in BW. The used diets permitted this BW gain without undesirable effects on carcass characteristics and meat quality. However, the study of meat fatty acid profile and antioxidant status should continue.     

Vocalisation sound pattern identification in young broiler chickens

In this study, we describe the monitoring of young broiler chicken vocalisation, with sound recorded and assessed at regular intervals throughout the life of the birds from day 1 to day 38, with a focus on the first week of life. We assess whether there are recognisable, and even predictable, vocalisation patterns based on frequency and sound spectrum analysis, which can be observed in birds at different ages and stages of growth within the relatively short life of the birds in commercial broiler production cycles. The experimental trials were carried out in a farm where the broiler where reared indoor, and audio recording procedures carried out over 38 days. The recordings were made using two microphones connected to a digital recorder, and the sonic data was collected in situations without disturbance of the animals beyond that created by the routine activities of the farmer. Digital files of 1 h duration were cut into short files of 10 min duration, and these sound recordings were analysed and labelled using audio analysis software. Analysis of these short sound files showed that the key vocalisation frequency and patterns changed in relation to increasing age and the weight of the broilers. Statistical analysis showed a significant correlation (P<0.001) between the frequency of vocalisation and the age of the birds. Based on the identification of specific frequencies of the sounds emitted, in relation to age and weight, it is proposed that there is potential for audio monitoring and comparison with ‘anticipated’ sound patterns to be used to evaluate the status of farmed broiler chicken.     

Effects of serial anesthesia using ketamine or ketamine/medetomidine on hematology and serum biochemistry values in rhesus macaques (Macaca Mulatta)

Background  This study aimed at determining the cumulative effect of daily anesthesia, using two drug regimens, over hematological and biochemical parameters.
Methods  Blood samples were obtained from rhesus monkeys 20 minutes after intramuscular administration of ketamine or ketamine/medetomidine combination for three consecutive days and results were evaluated to determine their effect on hematological and serum biochemistry values. Statistical significance of drug, day, and interaction of these two variables were evaluated.
Results  Drug effect resulted in a dramatic increase of aspartate aminotransferase and creatine kinase values. Day effect resulted in decreases of RBC, HCT, Hgb, and alkaline phosphatase but an increase of other biochemical parameters evaluated. The drug/day interaction effect was found to be –significant for RBC, platelets, aspartate aminotransferase, alanine aminotransferase, and creatine kinase values.
Conclusion  The results of our study suggest a cumulative effect of serial anesthesia and should be an important consideration when interpreting hematology and serum biochemistry in rhesus macaques.     

Multi-Sample Slippage Test for Ordered Observations

A multi-sample slippage test based on ordered observations has been given. The test statistic is based on the sum of ranks of the sample. The probability distribution of the test statistic has been worked out for small sample and it turns out to be chi-square distribution for large sample. The analytical procedure has been explained by a numerical example.     

Modela-r as a Froude and Strouhal dimensionless numbers combination for dynamic similarity in running

The aim of this study was to test the hypothesis that running at fixed fractions of Froude (Nfr) and Strouhal (Str) dimensionless numbers combinations induce dynamic similarity between humans of different sizes. Nineteen subjects ran in three experimental conditions, (i) constant speed, (ii) similar speed (Nfr) and (iii) similar speed and similar step frequency (Nfr and Str combination). In addition to anthropometric data, temporal, kinematic and kinetic parameters were assessed at each stage to measure dynamic similarity informed by dimensional scale factors and by the decrease of dimensionless mechanical parameter variability. Over a total of 54 dynamic parameters, dynamic similarity from scale factors was met for 16 (mean r=0.51), 32 (mean r=0.49) and 52 (mean r=0.60) parameters in the first, the second and the third experimental conditions, respectively. The variability of the dimensionless preceding parameters was lower in the third condition than in the others. This study shows that the combination of Nfr and Str, computed from the dimensionless energy ratio at the center of gravity (Modela-r) ensures dynamic similarity between different-sized subjects. The relevance of using similar experimental conditions to compare mechanical dimensionless parameters is also proved and will highlight the study of running techniques, or equipment, and will allow the identification of abnormal and pathogenic running patterns. Modela-r may be adapted to study other abilities requiring bounces in human or animal locomotion or to conduct investigations in comparative biomechanics.     

An Algorithm for Calculation of n-Dimensional Nonlinear Dependencies – Behaviour Tests on Rats

The evaluation of the data obtained during the behaviour tests always leads to the problem of multiple correlation, very often with non-linear dependencies on the target. All mathematical and statistical procedures that have been used so far are based on the assumption of an equation for the desired correlation for which parameters and related statistical equivalents are determined eventually. The MODAK system applied here (MODAK = algorithms of modelling for the calculation of multi-dimensional non-linear mathematical models) breaks down a complex correlation into individual dependencies in a mathematical and statistical way and selects suitable equations for each of them independently and determines the corresponding parameters. The numerical example evaluates data of behaviour tests on rats. First results obtained on the correlations of various behaviour tests indicate both the possibility of selecting suitable tests independent of each other and a better interpretation of the observed patterns of behaviour taking into account the interrelations between the tests. In addition, MODAK is a method which can be applied as a matter of course in a general way to all cases which call for the reduction and analysis of data occurring in process and system analysis and in the evaluation of test results requiring statistical modelling. So far, MODAK applications range from engineering sciences to medicine.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Effect of gibberellic acid on pod set in soybean

Field-grown soybean plants (Glycine max (L.) Merr. cv. Evans) were treated with gibberellic acid (GA₃; 10gl^–1) and/or (2-chloroethyl)-trimethylammonium chloride (CCC; 0.8gl^–1) in 1983 and 1984, and subsequent anthesis, pod set, seed size, seed number, and seed yield were determined at one node. The treatments were applied to five leaves in the center of each plant (typically leaves 7–11) and reproductive development at the node in the center of those leaves was monitored. Gibberellin A₃ applied Early (about 3d before anthesis of the first flower at the monitored node) had no effect on the number of flowers produced, but decreased the fraction of flowers that set pods in both experimental years (by 32% in 1983 and 76% in 1984). Seed size was slightly decreased by the GA₃ treatment in 1983 but not in 1984. The Middle GA₃ treatment (applied about 3 days after the Early treatment) slightly decreased the number of pods set; and Late treatments (9 days after) had no effect. None of the monitored parameters were affected by CCC.The Early experiments were repeated with two additional genotypes, Lincoln and T210. Genotype T210 is a single-gene, dwarf mutant of Lincoln whose stem elongation and leaf expansion are insensitive to GA₃. Gibberellin A₃ affected the reproductive parameters in Lincoln very similarly to Evans but those in T210 were unaffected. This indicates that GA₃ exerts its effect by increasing the mass of vegetative tissue and thus diverting assimilates away from the pods. However, since the mutation in T210 might affect a receptor that is in flowers as well as shoots, it is possible that GA₃ exerted its effect on the normal genotypes directly on the developing pods, rather than indirectly by diverting photoassimilates.     

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alphahelical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.