Purification and characterization of recombinant human liver glycolate oxidase

Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.     

The microneme proteins EtMIC4 and EtMIC5 of Eimeria tenella form a novel, ultra-high molecular mass protein complex that binds target host cells

Eimeria tenella, in common with other parasitic protozoa of the phylum Apicomplexa, invades host cells using an actinomyosin-powered "glideosome" complex and requires the secretion of adhesive proteins from the microneme organelles onto the parasite surface. Microneme proteins of E. tenella include EtMIC4, a transmembrane protein that has multiple thrombospondin type I domains and calcium-binding epidermal growth factor-like domains in its extracellular domain, and EtMIC5, a soluble protein composed of 11 tandemly repeated domains that belong to the plasminogen-apple-nematode superfamily. We show here that EtMIC4 and EtMIC5 interact to form an oligomeric, ultrahigh molecular mass protein complex. The complex was purified from lysed parasites by non-denaturing techniques, and the stoichiometry was shown to be [EtMIC4](2):[EtMIC5](1), with an octamer of EtMIC4 bound non-covalently to a tetramer of EtMIC5. The complex is formed within the parasite secretory pathway and is maintained after secretion onto the surface of the parasite. The purified complex binds to a number of epithelial cell lines in culture. Identification and characterization of this complex contributes to an overall understanding of the role of multimolecular protein complexes in specific interactions between pathogens and their hosts during infection.     

G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes

In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.     

Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping

High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine-hamster whole-genome radiation hybrid panel (WGRH3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine whole-genome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.     

Context matters: female aggression and testosterone in a year-round territorial neotropical songbird (Thryothorus leucotis)

Testosterone promotes aggressive behaviour in male vertebrates during the breeding season, but the importance of testosterone in female aggression remains unclear. Testosterone has both beneficial and detrimental effects on behaviour and physiology, prompting the hypothesis that selection favours an association between aggression and testosterone only in certain contexts in which intense or persistent aggression may be beneficial. We tested this hypothesis in a year-round territorial female buff-breasted wrens (Thryothorus leucotis), by exposing free-living females to experimental intrusions in different social (either single female or male, or paired decoys) and seasonal (pre-breeding or breeding) contexts. Females responded more aggressively to intrusions by females and pairs than to males. However, female intrusions elicited stronger responses during pre-breeding, whereas responses to pair intrusions were more intense during breeding. Territorial females had elevated testosterone levels after female intrusions and intermediate levels after pair intrusions during pre-breeding, but the levels of testosterone remained low after these intrusions during breeding. These results demonstrate seasonal differences in circulating testosterone following territorial aggression in female buff-breasted wrens and are suggestive of differences according to social context as well. Context-dependent elevation of testosterone implies that selection acts directly on female vertebrates to shape patterns of testosterone secretion.     

Fructose-induced hypertension in Wistar-Kyoto rats: interaction with moderately high dietary salt

We investigated the effects of 4% fructose plus moderately high salt (MHS) (4% NaCl) treatment on tissue aldehyde conjugates, platelet cytosolic free calcium ([Ca2+]i), renal morphology, and systolic blood pressure (SBP) in Wistar-Kyoto rats, and whether these effects were reversible (R) after withdrawal of treatment. At age 7 weeks, rats were divided into 4 groups: NS group, given normal salt (NS) diet (0.7% NaCl) for 18 weeks; NS+F(R) group, NS diet and fructose in water for 14 weeks, then 4 weeks fructose withdrawal; MHS+F group, NS diet and fructose for 6 weeks, then MHS diet and fructose for 12 weeks; and MHS+F(R) group, NS diet and fructose for 6 weeks, then MHS diet and fructose for 8 weeks, then MHS and fructose withdrawal for 4 weeks. SBP in the NS+F(R) group increased during fructose treatment, but normalized within 1 week of withdrawal. Tissue aldehyde conjugates and platelet [Ca2+]i were normal at completion. Adverse renal vascular changes did not reverse to normal and were similar to those of the salt plus fructose-treated groups. This may have implications for future development of hypertension. MHS did not cause any additional increase in SBP or associated tissue alterations when added to fructose treatment. However, the SBP and tissue changes persisted even after discontinuation of treatment. The fructose and salt combination may result in long-lasting vascular alterations leading to hypertension.     

Response of lead-induced oxidative stress and alterations in biogenic amines in different rat brain regions to combined administration of DMSA and MiADMSA

The present study was planned to investigate if combined administration of meso-2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) could achieve better recovery in the altered biochemical parameters suggestive of brain oxidative stress and depletion of lead from blood and brain following acute lead exposure. Male Wistar rats were exposed to lead nitrate (50 mg/kg, i.p., once daily for 5 days) followed by treatment with the above chelating agents using two different doses of 25 or 50 mg/kg (orally) either alone and in combination once daily for five consecutive days. Lead exposure resulted in the significant inhibition of δ-aminolevulinic acid dehydratase activity and depletion of glutathione (GSH) in blood. These changes were accompanied by significant reduction in blood hemoglobin, RBC levels and superoxide dismutase and catalase activities. Significant increase in blood reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) levels were noted. We observed marked increase in brain ROS level while GSH/oxidized glutathione ratio showed significant decrease accompanied by a significant increase in blood and brain lead concentration. The levels of norepinephrine, dopamine and serotonin in different brain regions were also altered on lead exposure. Co-administration of DMSA and MiADMSA particularly at the lower dose was most effective in the recovery of lead-induced changes in the hematological variables and oxidative stress and resulted in more pronounced depletion of lead from blood and brain compared to monotherapy with these chelators. On the other hand, combined administration of MiADMSA (50 mg/kg) in combination with DMSA (25 mg/kg each) had additional beneficial effect over the individual effect of chelating agent in the recovery of altered levels of brain biogenic amines. The study suggests that administration of MiADMSA is generally a better lead chelator than DMSA while combined administration of DMSA and MiADMSA might be a better treatment option compared to monotherapy at least in the removal of lead from the target tissues.     

The effect of contrast water therapy on symptoms of delayed onset muscle soreness

This study examined the effect of contrast water therapy (CWT) on the physiological and functional symptoms of delayed onset muscle soreness (DOMS) following DOMS-inducing leg press exercise. Thirteen recreational athletes performed 2 experimental trials separated by 6 weeks in a randomized crossover design. On each occasion, subjects performed a DOMS-inducing leg press protocol consisting of 5 x 10 eccentric contractions (180 seconds recovery between sets) at 140% of 1 repetition maximum (1RM). This was followed by a 15-minute recovery period incorporating either CWT or no intervention, passive recovery (PAS). Creatine kinase concentration (CK), perceived pain, thigh volume, isometric squat strength, and weighted jump squat performance were measured prior to the eccentric exercise, immediately post recovery, and 24, 48, and 72 hours post recovery. Isometric force production was not reduced below baseline measures throughout the 72-hour data collection period following CWT ( approximately 4-10%). However, following PAS, isometric force production (mean +/- SD) was 14.8 +/- 11.4% below baseline immediately post recovery (p < 0.05), 20.8 +/- 15.6% 24 hours post recovery (p < 0.05), and 22.5 +/- 12.3% 48 hours post recovery (p < 0.05). Peak power produced during the jump squat was significantly reduced (p < 0.05) following both PAS (20.9 +/- 13.4%) and CWT (12.8 +/- 8.0%), with the mean reduction in power for PAS being marginally (not significantly) greater than for CWT (effect size = 0.76). Thigh volume measured immediately following CWT was significantly less than PAS. No significant differences in the changes in CK were found; in addition, there were no significant (p > 0.01) differences in perceived pain between treatments. Contrast water therapy was associated with a smaller reduction, and faster restoration, of strength and power measured by isometric force and jump squat production following DOMS-inducing leg press exercise when compared to PAS. Therefore, CWT seems to be effective in reducing and improving the recovery of functional deficiencies that result from DOMS, as opposed to passive recovery.     

Why do earlier‐arriving migratory birds have better breeding success?

In migratory birds, early arrival on breeding sites is typically associated with greater breeding success, but the mechanisms driving these benefits are rarely known. One mechanism through which greater breeding success among early arrivers can potentially be achieved is the increased time available for replacement clutches following nest loss. However, the contribution of replacement clutches to breeding success will depend on seasonal variation in nest survival rates, and the consequences for juvenile recruitment of hatching at different times in the season. In particular, lower recruitment rates of late‐hatched chicks could offset the benefits to early arrivers of being able to lay replacement clutches, which would reduce the likelihood of replacement clutch opportunities influencing selection on migratory timings. Using a simulation model of time‐constrained capacity for replacement clutches, paramaterized with empirically‐derived estimates from avian migratory systems, we show that greater reproductive success among early‐arriving individuals can arise solely through the greater time capacity for replacement clutches among early arrivers, even when later renesting attempts contribute fewer recruits to the population. However, these relationships vary depending on the seasonal pattern of nest survival. The benefits of early arrival are greatest when nest survival rates are constant or decline seasonally, and early arrival is least beneficial when nest success rates increase over the breeding season, although replacement clutches can mitigate this effect. The time benefits of early arrival facilitating replacement clutches following nest loss may therefore be an important but overlooked source of selection on migratory timings. Empirical measures of seasonal variation in nest survival, renesting, and juvenile recruitment rates are therefore needed in order to identify the costs and benefits associated with individual migration phenology, the selection pressures influencing migratory timings, and the implications for ongoing shifts in migration and breeding phenology.