Resolution of microbial mixtures by free flow electrophoresis

Summary The resolution of bacterial mixtures by free flow electrophoresis (FFE) was not affected by the position of the microbes on the growth curve and approximately 70% of the individual cells applied were recovered as viable cells. The dependence of bacterial electrophoretic mobility on the pH, salt concentration, and viscosity of the electrolyte was determined. Suspending media and running electrolyte were developed which allowed collection of samples of>99% purity within two minutes of introduction of a mixture of Escherichia coli and Staphylococcus aureus. Most bacterial strains migrated in a single band, although some migrated in more than one band. Escherichia coli was resolved from each of 10 different species. The considerable variation in mobility found in 21 different E. coli strains, however, appears to preclude use of FFE as a method of species identification.     

Structural characterization and bioactivity evaluation of an acidic proteoglycan extract from Ganoderma lucidum fruiting bodies for PTP1B inhibition and anti‐diabetes

A water‐soluble PTP1B inhibitor, named FYGL‐a, was fractionated for structure investigation and bioactivity evaluation. FYGL‐a is an ingredient of a reported antihyperglycemia extract from Ganoderma Lucidum fruiting bodies. Composition analysis indicated that FYGL‐a was a 100.2 kDa acidic proteoglycan, consisting of 85 ± 2% heteropolysaccharide chain with rhamnose, galactose, glucose, and glucuronic acid residues in a mole ratio of 1.0:3.7:3.9:2.0, and the 15 ± 2% protein moiety of FYGL‐a was covalently bonded to the polysaccharide chain in O‐linkage type via threonine residues. The complete sequence of FYGL‐a was characterized systematically by periodate oxidation, Smith degradation, methylation analysis, ¹H & ¹³C 1D NMR, and 2D NMR (HSQC, HMBC, NOESY, COSY, & TOCSY). The chemical structure of FYGL‐a was determined as following, which may play special role in the competitive inhibition of PTP1B and antihyperglycemia potency. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 613–623, 2014.

     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.     

Response of CH4 and N2O Emissions and Wheat Yields to Tillage Method Changes in the North China Plain

The objective of this study was to quantify soil methane (CH₄) and nitrous oxide (N₂O) emissions when converting from minimum and no-tillage systems to subsoiling (tilled soil to a depth of 40 cm to 45 cm) in the North China Plain. The relationships between CH₄ and N₂O flux and soil temperature, moisture, NH₄ ⁺-N, organic carbon (SOC) and pH were investigated over 18 months using a split-plot design. The soil absorption of CH₄ appeared to increase after conversion from no-tillage (NT) to subsoiling (NTS), from harrow tillage (HT) to subsoiling (HTS) and from rotary tillage (RT) to subsoiling (RTS). N₂O emissions also increased after conversion. Furthermore, after conversion to subsoiling, the combined global warming potential (GWP) of CH₄ and N₂O increased by approximately 0.05 kg CO₂ ha⁻¹ for HTS, 0.02 kg CO₂ ha⁻¹ for RTS and 0.23 kg CO₂ ha⁻¹ for NTS. Soil temperature, moisture, SOC, NH₄ ⁺-N and pH also changed after conversion to subsoiling. These changes were correlated with CH₄ uptake and N₂O emissions. However, there was no significant correlation between N₂O emissions and soil temperature in this study. The grain yields of wheat improved after conversion to subsoiling. Under HTS, RTS and NTS, the average grain yield was elevated by approximately 42.5%, 27.8% and 60.3% respectively. Our findings indicate that RTS and HTS would be ideal rotation tillage systems to balance GWP decreases and grain yield improvements in the North China Plain region.     

Cellular entry and nuclear targeting by a highly anionic molecular umbrella

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.     

人粒细胞集落刺激因子受体膜外区在昆虫细胞中的表达

以胎盘组织提取的mRNA为模板,RTPCR扩增人粒细胞集落刺激因子(G-CSF)受体膜外区CRH的cDNA片段,克隆于供体质粒pF_ASTB_AC1,与杆状病毒表达载体Bacmid同源重组后,转染昆虫细胞SF9,获得重组杆状病毒并证明了CRH的高效表达。表达产物经G-CSF亲和层析进一步纯化,纯度可达90%以上。受体竞争性结合实验结果表明,该表达产物能特异性结合G-CSF,具有较高的亲和力(Kd=3.8nmol/L)。     

氧化修饰HDL对培养人主动脉平滑肌细胞胆固醇流出的影响

大量研究显示,高密度脂蛋白(highdensitylipoprotein,HDL)具有抗动脉粥样硬化(atherosclerosis,AS)作用.这是由于HDL能够促进外周组织如血管壁内皮细胞、平滑肌细胞(smoothmusclecell,SMC)及巨噬细胞储集的胆固醇流出,并将其转移到肝脏通过胆汁分泌而排出体外[1],这一过程...