Crystal structural studies of changes in the native dinuclear iron center of ribonucleotide reductase protein R2 from mouse

Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms. The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical. Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions. In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron. The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1. There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies. Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study. Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible. Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2.     

Capillary high-performance liquid chromatographic determination of lutein and zeaxanthin in aqueous humor from a single mouse eye

To protect the eye from ultraviolet phototoxicity caused by free radicals, ocular components such as the aqueous humor accumulate antioxidants, such as the carotenoids. Lutein and zeaxanthin are the only carotenoids known to be present in the aqueous humor. Due to the small sample volume, pooling of samples from an undesirable large number of animals is often required for sufficient sensitivity and statistically significant differences to be achieved. In this paper we present a rapid, sensitive and robust packed capillary high-performance liquid chromatographic visible detection method for the quantification of lutein and zeaxanthin in the aqueous humor of single mouse eyes.     

Discrepancy between cardiorespiratory system and skeletal muscle in elite cyclists after hypoxic training

BACKGROUND: The purpose of this study was to determine the effects of hypoxic training on the cardiorespiratory system and skeletal muscle among well-trained endurance athletes in a randomized cross-over design. METHODS: Eight junior national level competitive cyclists were separated into two groups; Group A trained under normoxic condition (21% O2) for 2 hours/day, 3 days/week for 3 weeks while Group B used the same training protocol under hypoxic condition (15% O2). After 3 weeks of each initial training condition, five weeks of self-training under usual field conditions intervened before the training condition was switched from NT to HT in Group A, from HT to NT in Group B. The subjects were tested at sea level before and after each training period. O2 uptake (O2), blood samples, and muscle deoxygenation were measured during bicycle exercise test. RESULTS AND DISCUSSION: No changes in maximal workload, arterial O2 content, O2 at lactate threshold and O2max were observed before or after each training period. In contrast, deoxygenation change during submaximal exercise in the vastus lateralis was significantly higher at HT than NT (p < 0.01). In addition, half time of oxygenation recovery was significantly faster after HT (13.2 PlusMinus; 2.6 sec) than NT (18.8 PlusMinus; 2.7 sec) (p < 0.001). CONCLUSIONS: Three weeks of HT may not give an additional performance benefit at sea level for elite competitive cyclists, even though HT may induce some physiological adaptations on muscle tissue level.     

Cobalt substitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state: spectroscopic and structural evidence for a ferromagnetically coupled dinuclear cobalt cluster

The R2 dimer of mouse ribonucleotide reductase contains a dinuclear iron-oxygen cluster and tyrosyl radical/subunit. The dinuclear diferrous form reacts with dioxygen to generate the tyrosyl radical essential for the catalytic reaction that occurs at the R1 dimer. It is important to understand how the reactivity toward oxygen is related to the crystal structure of the dinuclear cluster. For the mouse R2 protein, no structure has been available with a fully occupied dinuclear metal ion site. A cobalt substitution of mouse R2 was performed to produce a good model for the very air-sensitive diferrous form of the enzyme. X-band EPR and light absorption studies (epsilon(550 nm) = 100 mm(-1) cm(-1)/Co(II)) revealed a strong cooperative binding of cobalt to the dinuclear site. In perpendicular mode EPR, the axial signal from mouse R2 incubated with Co(II) showed a typical S = 3/2 Co(II) signal, and its low intensity indicated that the majority of the Co(II) bound to R2 is magnetically coupled. In parallel mode EPR, a typical integer spin signal (M(s) = +/-3) with g approximately 12 is observed at 3.6 K and 10 K, showing that the two Co(II) ions (S = 3/2) in the dinuclear site are ferromagnetically coupled. We have solved the 2.4 A crystal structure of the Co(II)-substituted R2 with a fully occupied dinuclear cluster. The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2. This might be important for proper O(2) activation of the more exposed native diferrous site in mouse R2 compared with E. coli R2.     

Fine mapping of a quantitative trait locus for twinning rate using combined linkage and linkage disequilibrium mapping

A novel and robust method for the fine-scale mapping of genes affecting complex traits, which combines linkage and linkage-disequilibrium information, is proposed. Linkage information refers to recombinations within the marker-genotyped generations and linkage disequilibrium to historical recombinations before genotyping started. The identity-by-descent (IBD) probabilities at the quantitative trait locus (QTL) between first generation haplotypes were obtained from the similarity of the marker alleles surrounding the QTL, whereas IBD probabilities at the QTL between later generation haplotypes were obtained by using the markers to trace the inheritance of the QTL. The variance explained by the QTL is estimated by residual maximum likelihood using the correlation structure defined by the IBD probabilities. Unlinked background genes were accounted for by fitting a polygenic variance component. The method was used to fine map a QTL for twinning rate in cattle, previously mapped on chromosome 5 by linkage analysis. The data consisted of large half-sib families, but the method could also handle more complex pedigrees. The likelihood of the putative QTL was very small along most of the chromosome, except for a sharp likelihood peak in the ninth marker bracket, which positioned the QTL within a region <1 cM in the middle part of bovine chromosome 5. The method was expected to be robust against multiple genes affecting the trait, multiple mutations at the QTL, and relatively low marker density.     

A vegetation-based method to map climatic variation in the arctic–boreal transition area of Finnmark, north-easternmost Norway

Aim To develop a new method for bioclimate mapping where the vegetation layer is the main source of climate information. Location The study area includes four subareas, all situated on the Varangerhalvøya peninsula in Finnmark, north‐easternmost Norway (70–71° N). The four subareas were chosen to represent most of the climatic, topographic, geomorphologic and botanic diversity along the arctic–boreal gradient in the area. The four meteorological stations in the area show a climatic gradient with mean July temperature ranging from 10.1 to 12.3 °C. Methods The new vegetation‐based method is based on the fact that most plant species and plant communities both in the Arctic and adjacent areas have a distribution pattern limited by temperature to some extent. The vegetation is mapped using Landsat TM data and a contextual correction process in a geographic information system. The mapped vegetation units are defined as temperature indicators based on their total distribution patterns and the temperature indicator value of their high frequency and dominant species. The indicator value and degree of cover of all thermophilous vegetation units, within each 500 × 500 m study unit, are combined in a Vegetation‐based Index of Thermophily, VI_tm. This new vegetation‐based method is based on the same basic idea as a recently published floristic‐based method for calculating a Floristic‐based Index of Thermophily, FI_tm. The VI_tm values are tested by comparison with the FI_tm values, and temperature data collected in the field during two growing seasons, and the differences are interpreted ecologically. Results Twenty‐one of the mapped vegetation units were defined as thermophilous and categorized in five groups of temperature indicators. The VI_tm values showed a strong positive linear relationship with the temperatures measured during the years 2001 and 2002, with r² values of 0.79 and 0.85, respectively. The VI_tm values show a high linear relationship (r² = 0.76) with the 71 study units where the FI_tm values were calculated. As interpreted from the relationship with temperature measurements and FI_tm values, the vegetation‐based method seems to work at a broad range of ecological conditions, with very dry, acidic sites being the most important exception. The VI_tm values are related to growing degree‐days of a normal year, and the four subareas are mapped, showing a diversity of 13 bioclimatic classes. The birch forest line is estimated to occur at about 980 °C‐days. The results show climatic gradients with temperatures increasing from the cold coast towards the interior, from wind‐exposed convex hills towards wind‐protected valleys, and from mountain plateaux towards south‐facing lowlands. The north‐easternmost study site at the coast is positioned within the arctic shrub tundra zone. Main conclusions The vegetation‐based method shows a strong positive correlation both with measured temperatures and the floristic‐based method within a broad range of different ecological conditions. The vegetation‐based method has the potential for bioclimatic mapping of large areas in a cost‐effective way. The floristic‐based method has higher accuracy and is more flexible than the vegetation‐based method, and the two methods seem to complement each other.     

High-throughput analysis of vitamin C in human plasma with the use of HPLC with monolithic column and UV-detection

Vitamin C plays a central role in the body. One of its important functions is its role as an antioxidant, and accurate measurements are important for interpretations of this role. However, its reactive nature and instability complicates the assessment, especially in biological samples. A high-throughput chromatographic method using monolithic column and UV-detection was developed for the assessment of plasma ascorbic acid and total ascorbic acid. The method showed excellent analytical sensitivity, specificity, precision, recovery and linearity during the validation study. The method was used for the assessment of ascorbic acid and total ascorbic acid during several clinical studies.     

Geometrical E/Z isomers of (6R)- and (6S)-neoxanthin and biological implications

9'Z-(3S,5R,6R,3'S,5'R,6'S)-Neoxanthin reisolated from spinach (Spinacea oleracea) and characterized by HPLC, VIS, MS, and 2D (1)H NMR, has been submitted to photoinduced stereomutation in the presence of iodine or diphenyl diselenide at conditions not involving isomerization of the allenic bond. The six individual geometrical isomers, all-E,9Z,9'Z,13Z,13'Z,15Z and three minor di-Z-isomers, presumably 9,9'-di-Z,9',13-di-Z and 9',13'-di-Z, present in the equilibrium mixture have been characterized by HPLC, VIS data, 1H NMR and reversibility tests. Judged by the quantitative composition of the equilibrium mixture the naturally occurring 9'Z-isomer is thermodynamically less stable than the all-E-isomer. The availability of these isomers facilitates future search in natural sources. 9'Z-(6R90% of total neoxanthin in spinach and broccoli (Brassica oleracea var. italica), consistent with previous findings of its abundance in chloroplasts. all-E90% of total violaxanthin in the same sources. It is postulated that a neoxanthin Delta9'-isomerase is present and involved in the biosynthesis of abscisic acid in higher plants. Allenic S-isomers are of interest as postulated biosynthetic precursors of R-allenes. All-E-(6S)- and 9'Z-(6S)-neoxanthin were available as semi-synthetic model compounds. The allenic (6S)-diastereomers could not be detected in spinach or broccoli.     

Risk taking during parental care: a test of the harm-to-offspring hypothesis

Amount of risk taking during parental care is often explainedin relation to the reproductive value of the offspring. The"harm-to-offspring hypothesis" focuses on the relative harma period of no parental care can do to the offspring. Accordingto this hypothesis, parents should take greater risks for offspringin poor condition than for offspring in good condition. We manipulatedoffspring condition in the pied flycatcher (Ficedula hypoleuca)and tested the harm-to-offspring hypothesis by exposing parentsto a predator model (a sparrowhawk, Accipiter nisus). Time elapseduntil a parent first entered the nest-box was used as a risk-takingmeasure. Parents spent significantly shorter time until first nestvisit for offspring in poor condition than for offspring ingood condition. Hence, the harm-to-offspring hypothesis wassupported.     

Low frequency hearing in cephalopods

Summary Classical conditioning was employed to test the sensitivity of cephalopods to vibrations between 1 and 100 Hz generated in a standing wave acoustic tube. The animals were trained to associate sound stimuli with a weak electric shock, and the recorded conditioned responses were changes in breathing and jetting activity. Five specimens of Sepia officinalis were tested, and all responded to these low frequency sounds. The relevant stimulus parameter was particle motion rather than sound pressure. The threshold values (measured as particle acceleration) decreased towards lower frequencies in the tested range, reaching values below 4 × 10^-3 m/s². The thresholds in the most sensitive range may have been masked by the considerable background noise at the experimental site (Naples). Two individuals of Octopus vulgaris and one Loligo vulgaris were also tested, and showed a similar sensitivity to low frequency sound.