WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency.

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

Cloning of MYB gene and construction of greenfluorescent protein expression vector in Erigeron breviscapus / 中草药

Objective To study the role of MYB transcription factor in phenylpropane biosynthesis pathway and the response to biotic or abiotic stress, a full length sequence of putative MYB gene was cloned in Erigeron breviscapus. Methods Based on a partial sequence of putative MYB gene from transcriptome we have previous reported, the full length cDNA was cloned by RACE method in E. breviscapus. According to the sequence of cloning cDNA, the nucleotide sequence similarity, physicochemical properties, hydrophobicity, transmembrane structure, secondary structure and tertiary structure were predicted and analyzed by various softwares. Meanwhile, multiple sequence alignment of cloned MYB gene was performed, and the phylogenetic tree was constructed. In addition, the fusion expression vector of this gene and green fluorescent protein was constructed. Results The MYB gene was cloned and named eBMYB06. The open reading frame was 783 bp, encoding 260 amino acid residues with a relative molecular mass of 63.80 kDa and a theoretical pI of 5.18 which proved to be a stable protein. The secondary structure of the protein is mainly composed of irregular coil, alpha helix and beta fold. According to the result of phylogenetic tree alignment of E. breviscapus with R2R3MYBs from Arabidopsis thaliana, the cloned MYB gene was clustered to two subsets of R2R3-MYB genes from A. thaliana, which suggested that the cloned MYB gene would be similar with the two groups in structure and function, which would be involved in response to biotic and abiotic stress or phenylpropane biosynthesis pathway, respectively. Further experimental results show that the constructed expression can be used for the efficient transformation in E. breviscapus. Conclusion For the first time, a MYB gene was cloned may be involved in the phenylpropanoid metabolism or gene metabolism or gene regulation in response to the environment in E. breviscapus.

Spatial variations of storm runoff pollution and their correlation with land-use in a rapidly urbanizing catchment in China.

The composition of land use for a rapidly urbanizing catchment is usually heterogeneous, and this may result in significant spatial variations of storm runoff pollution and increase the difficulties of water quality management. The Shiyan Reservoir catchment, a typical rapidly urbanizing area in China, is chosen as a study area, and temporary monitoring sites were set at the downstream of its 6 sub-catchments to synchronously measure rainfall, runoff and water quality during 4 storm events in 2007 and 2009. Due to relatively low frequency monitoring, the IHACRES and exponential pollutant wash-off simulation models are used to interpolate the measured data to compensate for data insufficiency. Three indicators, event pollutant loads per unit area (EPL), event mean concentration (EMC) and pollutant loads transported by the first 50% of runoff volume (FF50), were used to describe the runoff pollution for different pollutants in each sub-catchment during the storm events, and the correlations between runoff pollution spatial variations and land-use patterns were tested by Spearman's rank correlation analysis. The results indicated that similar spatial variation trends were found for different pollutants (EPL or EMC) in light storm events, which strongly correlate with the proportion of residential land use; however, they have different trends in heavy storm events, which correlate with not only the residential land use, but also agricultural and bare land use. And some pairs of pollutants (such as COD/BOD, NH(3)-N/TN) might have the similar source because they have strong or moderate positive spatial correlation. Moreover, the first flush intensity (FF50) varies with impervious land areas and different interception ratio of initial storm runoff volume should be adopted in different sub-catchments.