Biophysical Characterisation of Neuroglobin of the Icefish, a Natural Knockout for Hemoglobin and Myoglobin. Comparison with Human Neuroglobin

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe²⁺ form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.     

Acyl coenzyme A synthetase long-chain 1 (ACSL1) gene polymorphism (rs6552828) and elite endurance athletic status: a replication study

The aim of this study was to determine the association between the rs6552828 polymorphism in acyl coenzyme A synthetase (ACSL1) and elite endurance athletic status. We studied 82 Caucasian (Spanish) World/Olympic-class endurance male athletes, and a group of sex and ethnically matched healthy young adults (controls, n=197). The analyses were replicated in a cohort of a different ethnic origin (Chinese of the Han ethnic group), composed of elite endurance athletes (runners) [cases, n=241 (128 male)] and healthy sedentary adults [controls, n=504 (267 male)]. In the Spanish cohort, genotype (P=0.591) and minor allele (A) frequencies were similar in cases and controls (P=0.978). In the Chinese cohort, genotype (P=0.973) and minor allele (G) frequencies were comparable in female endurance athletes and sedentary controls (P=0.881), whereas in males the frequency of the G allele was higher in endurance athletes (0.40) compared with their controls (0.32, P=0.040). The odds ratio (95%CI) for an elite endurance Chinese athlete to carry the G allele compared with ethnically matched controls was 1.381 (1.015-1.880) (P-value=0.04). Our findings suggest that the ACSL1 gene polymorphism rs6552828 is not associated with elite endurance athletic status in Caucasians, yet a marginal association seems to exist for the Chinese (Han) male population.     

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Background

Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.

Methodology/Principal Findings

We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.

Conclusions/Significance

These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.     

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (b_KCl = 0.15 mol/kg) and high (b_KCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.     

A peach linkage map integrating RFLPs, SSRs, RAPDs, and morphological markers.

A linkage map was obtained using a BC1 progeny (Prunus persica x (P. persica x P ferganensis)). The map is composed of 109 loci (74 RFLPs, 17 SSRs, 16 RAPDs, and two morphological traits) distributed in 10 linkage groups. Loci, segregating in five different ratios, were integrated in the map with JoinMap 2.0 software. The map covers 521 cM of the peach genome. The average distance between adjacent loci is 4.8 cM. Two monogenic traits, flesh adhesion (F/f) and leaf glands (E/e), were placed on the map. Thirty-two loci in common with a saturated linkage map of Prunus allowed a comparative analysis to be made between the two maps. Homologies were found among the respective linkage groups. No relevant differences were observed in the linear order of the common loci.     

Plasma progesterone levels during the estrous cycle of Holstein and Brahman cows, Carora type and cross-bred heifers

Daily plasma progesterone (P(4)) was determined during one estrous cycle of 19 cows and 18 heifers of four different breeds: Holstein (H), Brahman (B), Carora-type (C) and crossbred (CB) females. Estrus detection was made by visual observation and using a teaser bull with a chin-ball marker. The P(4) profiles showed no differences among groups. In Group 1 (H), P(4) levels ranged from 0.5 ng/ml plasma on the day of estrus (Day 0) to 5.1 ng/ml at the luteal phase peak (Day 13). In Group 2 (B), P(4) levels ranged from 0.5 ng/ml on Day 0 to 9.2 ng/ml on Day 13. In Groups 3 (C) and 4 (CB), P(4) levels ranged from 0.5 ng/ml, on Day 0, to 13.7 ng/ml on Day 12 and 8.8 ng/ml on Day 13. These last two groups were moved to the same location and then compared. It was found that P(4) concentrations were significantly higher (P < 0.025) in Group 3 between Days 7 and 14 of the estrous cycle. In all groups, P(4) levels were lower than 1 ng/ml one day before the next estrus, and levels of 0.4, 0.5, 0.4 and 0.4 ng/ml were obtained the day of estrus in Groups 1 to 4, respectively. Results indicated that the pattern of P(4) for each one of the groups was similar to those reported by other investigators.     

On growth and form: control of cell morphogenesis in fission yeast

In the past year, we have gained considerable insight into the process of cell morphogenesis and the establishment of positional information in fission yeast. The highlights include a better understanding of the role of the microtubule cytoskeleton in the control of cell shape, as well as the identification of novel genes essential for the establishment of cell polarity and for the positioning of the site of cell division.     

Effect of restricted suckling on pregnancy rates and calf performance in Brahman cows

Seventy-six Brahman cows and first-calf heifers were assigned to one of two groups: 1) normal suckling (34 cows) or 2) twice-daily suckling (45 minutes of suckling each time; 42 cows). Twice-daily suckling was carried out from 30 days postcalving until weaning (seven months). All animals were maintained under artificial insemination for a four-month breeding period. Mean pregnancy rate was 63.06 +/- 0.06% and was influenced by suckling group (P<0.01) and number of parturitions (P<0.05). The pregnancy rates were 33% higher in twice-daily suckled cows. Forty-four percent of the first calf heifers in the twice-daily suckling group became pregnant compared to 9% in the normal suckling group (P<0.01). Twice-daily suckling improved pregnancy rate without depressing preweaning calf performance.     

Lattice defects in microtubules: protofilament numbers vary within individual microtubules

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.