Reaction pathway and free energy profiles for butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine

The catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations on both acylation and deacylation of BChE. Additional quantum mechanical (QM) calculations have been carried out, along with the QM/MM-FE calculations, to understand the known substrate activation effect on the enzymatic hydrolysis of ATCh. It has been shown that the acylation of BChE with ATCh consists of two reaction steps including the nucleophilic attack on the carbonyl carbon of ATCh and the dissociation of thiocholine ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain. QM/MM-FE calculation results reveal that the acylation of BChE is rate-determining. It has also been demonstrated that an additional substrate molecule binding to the peripheral anionic site (PAS) of BChE is responsible for the substrate activation effect. In the presence of this additional substrate molecule at PAS, the calculated free energy barrier for the acylation stage (rate-determining step) is decreased by ~1.7 kcal/mol. All of our computational predictions are consistent with available experimental kinetic data. The overall free energy barriers calculated for BChE-catalyzed hydrolysis of ATCh at regular hydrolysis phase and substrate activation phase are ~13.6 and ~11.9 kcal/mol, respectively, which are in reasonable agreement with the corresponding experimentally derived activation free energies of 14.0 kcal/mol (for regular hydrolysis phase) and 13.5 kcal/mol (for substrate activation phase).     

Kinetics of adsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosic suspensions

This study conducted quantitative kinetic modeling and in situ and temporally resolved measurements of adsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosic suspensions. The study defined a cellulase adsorption and desorption competition parameter, a pseudo rate of binding and desorption, binding and desorption capacity, as well as cellulase-binding reversibility (a thermodynamic property) and recyclability (a engineering parameter). The results indicate that both substrate chemical and physical structures play important roles in cellulase binding and desorption. Binding of a commercial cellulase onto a cellulosic substrate was reversible. Bindings to two different lignocellulosic substrates were almost irreversible. While lignin and its structure positively affect binding capacity to substrate, they negatively affect cellulase recyclability. Collapsing of substrate pores reduced cellulose accessibility and cellulase-binding capacity and increased reversibility and recyclability. Increasing temperature and pH increase cellulase desorption and increased binding reversibility and capacity. This study lays the foundation for developing effective cellulase recycling strategies.     

HSF4 is involved in DNA damage repair through regulation of Rad51

Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.     

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.     

Synthesis, drug release and anti-HIV activity of a series of PEGylated zidovudine conjugates

A series of methoxy poly(ethylene glycol)-succinyl-5'-O-zidovudine conjugates (mPEG-succinyl-AZT) with different molecular weight (M(w): 750 Da, 2, 5 or 10 kDa) of mPEG were synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy, (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy, and matrix-assisted laser desorption/ionization time of flight mass (MALDI TOF MS) spectrometry analysis. All conjugates showed good stability in vitro release experiments, and good anti-HIV activity and low cytotoxicity in MT-4 cells, in which, mPEG(750)-succinyl-AZT exhibited good inhibition to wild-type viruses (strains IIIB and ROD) with EC(50) values of 0.11 and 0.090 μmol/L, respectively, and it showed no cytotoxicity up to 110 μmol/L. Oral pharmacokinetic study in rats showed the half-life time (T(1/2)) of all conjugates are prolonged compared to free AZT. Especially, mPEG(750)-succinyl-AZT displayed a ~2.3-fold prolonged half-life and approximately 224% increased bioavailability of AZT.     

Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen

The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.     

PDGFRβ triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRβ (platelet-derived growth factor receptor β) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRβ mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRβ, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRβ mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRβ/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.     

基于本体论的植物遗传资源数据整合研究

随着现代生物学的发展,全球范围内建立了大量的生物学数据共享中心,同时,在生物学发展的带动下,植物遗传资源数据变得更为复杂、异构化和海量。本文在分析国内外几大著名的数据整合共享中心的基础上,简要介绍了本体论的概念及其在生物学领域中的研究现状,提出了基于生物本体论将植物遗传数据、数据挖掘工具、科技文献和科技交流进行整合的设想,并对数据整合需要考虑的几个问题进行了讨论。