Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

No evidence of inbreeding depression in fast declining herds of migratory caribou

Identifying inbreeding depression early in small and declining populations is essential for management and conservation decisions. Correlations between heterozygosity and fitness (HFCs) provide a way to identify inbreeding depression without prior knowledge of kinship among individuals. In Northern Quebec and Labrador, the size of two herds of migratory caribou (Rivière‐George, RG and Rivière‐aux‐Feuilles, RAF) has declined by one to two orders of magnitude in the last three decades. This raises the question of a possible increase in inbreeding depression originating from, and possibly contributing to, the demographic decline in those populations. Here, we tested for the association of genomic inbreeding indices (estimated with 22,073 SNPs) with body mass and survival in 400 caribou sampled in RG and RAF herds between 1996 and 2016. We found no association of individual heterozygosity or inbreeding coefficient with body mass or annual survival. Furthermore, those genomic inbreeding indices remained stable over the period monitored. These results suggest that the rapid and intense demographic decline of the herds did not cause inbreeding depression in those populations. Although we found no evidence for HFCs, if demographic decline continues, it is possible that such inbreeding depression would be triggered.     

Crystallographic analysis of the pH-dependent binding of iminobiotin by streptavidin.

Streptavidin binds 2'-iminobiotin in a pH-dependent fashion--affinity decreases as the pH is lowered. This property makes the purification of compounds conjugated to streptavidin or immobiotin possible under mild conditions by affinity chromatography. In order to understand the molecular details of this pH-dependent binding, we analyzed the crystal structures of the complex of core streptavidin with 2'-iminobiotin at pH values 4.0 and 7.5. The two structures are very similar to each other even at their binding sites. Although the relative abundance of the protonated species of the ligand is increased more than 3,000-fold on going from pH 7.5 to pH 4.0, both structures contain only the nonprotonated from of the ligand. Streptavidin selects the nonprotonated form, which, at pH 4.0, is one part in 7.9 x 10(7).     

利用肠激酶加工融合蛋白的方法制备重组人甲状旁腺素（1—34）肽

采用全基因分段合成和补丁连接相结合的方法构建了人甲状旁腺素 (hPTH) (1～ 34)肽基因 ,并利用GST融合表达系统 ,在大肠杆菌中克隆和可溶性表达了hPTH(1～ 34)肽基因。融合蛋白表达量占菌体总蛋白质的 2 5 %以上 ,经高密度发酵、亲和纯化后 ,在每升发酵液中得到了 10 g融合蛋白。融合蛋白经肠激酶一步加工并亲合纯化和反相脱盐后 ,最终可以得到约 0 .6g /L人甲状旁腺素 (1～ 34)肽纯品 ,样品的理论回收值达到了4 8.75 %。产物的纯度和性质经HPLC、毛细管电泳、质谱分析、N端测序等得到证明 ,并与化学合成产物的结果相吻合。兔肾皮质细胞测活表明 ,重组产物与化学合成人甲状旁腺素 (1～ 34)肽具有相近的腺苷酸环化酶激活活性。     

Sperm quality, secondary sexual characters and parasitism in roach (Rutilus rutilus L.)

According to sperm competition models, a male spawning in a disfavoured role should have spermatozoa with higher velocity but shorter longevity compared with a male spawning in a favoured role. Moreover, immunosuppressive androgens are needed to produce both secondary sexual characters and sperm cells. The 'sperm protection' hypothesis suggests that the immunosuppressive action of androgens has evolved to protect haploid spermatozoa, which are antigenic, from autoimmune attacks. Therefore, a male with high sexual ornamentation may be more susceptible to diseases but may possess better quality ejaculate than his less ornamented rival. We studied sexual ornamentation (breeding tubercles), ejaculation quality (sperm concentration, longevity and spermatozoal velocity) and intensity of parasitism in the cyprinid, Rutilus rutilus . Sperm longevity and spermatozoal velocity were positively correlated. Males having elaborated sexual ornamentation had longer-lived sperm and more Myxobolus mülleri parasites in the liver compared with males with low ornamentation. However, no difference was found between males with different degrees of ornamentation with respect to sperm concentration, spermatozoal velocity or other parasites. Since the highly ornamented males had higher sperm longevity, the present results are partly consistent with the predictions of the sperm competition models and the 'sperm protection' hypothesis.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 111–117.     

Habitat-specific clutch size and cost of incubation in eiders reconsidered

The energetic incubation constraint hypothesis (EICH) for clutch size states that birds breeding in poor habitat may free up resources for future reproduction by laying a smaller clutch. The eider (Somateria mollissima) is considered a candidate for supporting this hypothesis. Clutch size is smaller in exposed nests, presumably because of faster heat loss and higher incubation cost, and, hence, smaller optimal clutch size. However, an alternative explanation is partial predation: the first egg(s) are left unattended and vulnerable to predation, which may disproportionately affect exposed nests, so clutch size may be underestimated. We experimentally investigated whether predation on first-laid eggs in eiders depends on nest cover. We then re-evaluated how nesting habitat affects clutch size and incubation costs based on long-term data, accounting for confounding effects between habitat and individual quality. We also experimentally assessed adult survival costs of nesting in sheltered nests. The risk of egg predation in experimental nests decreased with cover. Confounding between individual and habitat quality is unlikely, as clutch size was also smaller in open nests within individuals, and early and late breeders had similar nest cover characteristics. A trade-off between clutch and female safety may explain nest cover variation, as the risk of female capture by us, mimicking predation on adults, increased with nest cover. Nest habitat had no effect on female hatching weight or weight loss, while lower temperature during incubation had an unanticipated positive relationship with hatching weight. There were no indications of elevated costs of incubating larger clutches, while clutch size and colony size were positively correlated, a pattern not predicted by the ‘energetic incubation constraint’ hypothesis. Differential partial clutch predation thus offers the more parsimonious explanation for clutch size variation among habitats in eiders, highlighting the need for caution when analysing fecundity and associated life-history parameters when habitat-specific rates of clutch predation occur.     

Increased performance of Cirsium arvense from the invasive range

The Evolution of Increased Competitive Ability (EICA) hypothesis suggests that plants from the invasive range should perform better than plants of the same species from the native range. To properly test this, we need to compare growth of plants from the two ranges in a common environment. Ideally, all the natural enemies should be excluded, to make sure that the differences are not due to different response of plants from the two ranges to the natural enemies. We used the above design to examine the difference in growth and reproduction in Cirsium arvense plants from the invasive (North America) and native range (Europe). To account for possible differences within the ranges, we used plants from two regions, separated by at least 1000 km, in each range. Because the higher performance of species from the invasive range can be caused by their higher ability to acquire resources we compared growth of the plants in two different nutrient levels. The results indicate that plants from the invasive range are larger in most size parameters as well as parameters more closely related to fitness. For aboveground biomass, the response of plants from the invasive range to nutrient addition was weaker than that of plants from the native range and the difference between the ranges was stronger in the nutrient poor substrate. The results are in agreement with the EICA hypothesis and suggest that plants from the invasive range have higher ability to use resources and are thus able to perform well also in nutrient poor conditions.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

You are what your mother eats: evidence for maternal preconception diet influencing foetal sex in humans

Facultative adjustment of sex ratios by mothers occurs in some animals, and has been linked to resource availability. In mammals, the search for consistent patterns is complicated by variations in mating systems, social hierarchies and litter sizes. Humans have low fecundity, high maternal investment and a potentially high differential between the numbers of offspring produced by sons and daughters: these conditions should favour the evolution of facultative sex ratio variation. Yet little is known of natural mechanisms of sex allocation in humans. Here, using data from 740 British women who were unaware of their foetus's gender, we show that foetal sex is associated with maternal diet at conception. Fifty six per cent of women in the highest third of preconceptional energy intake bore boys, compared with 45% in the lowest third. Intakes during pregnancy were not associated with sex, suggesting that the foetus does not manipulate maternal diet. Our results support hypotheses predicting investment in costly male offspring when resources are plentiful. Dietary changes may therefore explain the falling proportion of male births in industrialized countries. The results are relevant to the current debate about the artificial selection of offspring sex in fertility treatment and commercial 'gender clinics'.