Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

不同胁迫预处理提高水稻幼苗抗寒性期间蛋白质的变化

水稻(Oryza sativa L.)幼苗经盐、热激和冷三种不同胁迫预处理均提高了幼苗的抗寒性。与未预处理苗相比,在处理后、低温伤害后和常温下恢复2d的三个时期,不同胁迫预处理苗的可溶性和热不稳定蛋白含量变化趋势甚为相似,但热稳定蛋白含量变化则各有异同。SDS-PAGE图谱分析显示,不同胁迫预处理提高水稻幼苗抗寒性时,其可溶性蛋白、热稳定和热不稳定蛋白组成变化亦各有异同。除诱导出共有的新多肽外,还各自诱导出特有的新多肽。结果表明,植物对不同胁迫的交叉适应存在一定的共同机理,但亦可看出植物对同一种环境胁迫似乎不是以同一的机理去适应。     

Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.     

Prospective validation of the EuroSCORE II risk model in a single Dutch cardiac surgery centre

Objective

The EuroSCORE I was one of the most frequently used pre-operative risk models in cardiac surgery. In 2011 it was replaced by its successor the EuroSCORE II. This study aims to validate the EuroSCORE II and to compare its performance with the EuroSCORE I in a Dutch hospital.

Methods

The EuroSCORE II was prospectively validated in 2,296 consecutive cardiac surgery patients between 1 April 2012 and 1 January 2014. Receiver operating characteristic curves on in-hospital mortality were plotted for EuroSCORE I and EuroSCORE II, and the area under the curve was calculated to assess discriminative power. Calibration was assessed by comparing observed versus expected mortality. Additionally, analyses were performed in which we stratified for type of surgery and for elective versus emergency surgery.

Results

The observed mortality was 2.4% (55 patients). The discriminative power of the EuroSCORE II surpassed that of the EuroSCORE I (area under the curve EuroSCORE II 0.871, 95% confidence interval (CI) 0.832–0.911; area under the curve additive EuroSCORE I 0.840, CI 0.798–0.882; area under the curve logistic EuroSCORE I 0.761, CI 0.695–0.828). Both the additive and the logistic EuroSCORE I overestimated mortality (predictive mortality additive EuroSCORE I median 5.0%, inter-quartile range 3.0–8.0%; logistic EuroSCORE I 10.7%, inter-quartile range 5.8–13.9), while the EuroSCORE II underestimated mortality (median 1.6%, inter-quartile range 1.0–3.5). In most stratified analyses the EuroSCORE II performed better.

Conclusion

Our results show that the EuroSCORE II produces a valid risk prediction and outperforms the EuroSCORE I in elective cardiac surgery patients.

     

The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.     

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l^?1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l^?1) was achieved in media containing 150 g l^?1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l^?1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.     

Identification of a pollen-specific gene,SlCRK1 (RFK2) in tomato

Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of development, hormone signaling, abiotic, and biotic stress responses. It has been suggested that cysteine-rich receptor-like kinases (CRKs), which are one of the largest RLK groups, is significant in pathogen defense and programmed cell death. The CRK1 gene is isolated and characterized from tomato (Solanum lycopersicum L.). The SlCRK1 has two C-X8-C-X2-C motifs: a trans-membrane region and a kinase domain similar to other CRKs. The semi-quantitative RT-PCR exhibits the specific expression of SlCRK1 in the flower, but not in the root, leaf, seed, and fruit of the tomato. In addition, SlCRK1 exhibits pollen-specific expression in the floral organ. SlCRK1 has pollen-specific cis-acting elements in the promoter region, and its promoter has pollen-specific activity in the homozygous transgenic plants of tomato and Arabidopsis as confirmed through histochemical GUS assays. Moreover, the expression of SlCRK1 is not detected via stress treatment or hormone treatment. In this study, SlCRK1 from tomato is characterized and its promoter can be useful in developing transgenic plants with foreign genes that should be expressed in pollens.     

2-O-substituted cyclodextrins as reversal agents for the neuromuscular blocker rocuronium bromide

A series of secondary face modified cyclodextrins (CDs) were synthesised with the aim of constructing host molecules capable of forming host-guest complexes with neuromuscular blockers, especially with rocuronium bromide. Perfacial 2-O-substitution of gamma-CD with 4-carboxybenzyl resulted in a CD host molecule 1 that forms a 1:1 binary complex with rocuronium bromide (K(a) 6.2 x 10(5) M(-1)). The biological activities of this compound and other derivatives as reversal agents of rocuronium bromide were examined in vitro (mouse hemi-diaphragm) and in vivo (anaesthetized guinea pigs). The host molecule 1 was found to exert potent reversal activity (ED(50) 0.21 micromol/kg, iv) against rocuronium-induced neuromuscular block, and thus proved the viability of using host molecules as antidotes of a biologically active compound.