Milligram Production and Biological Activity Characterization of the Human Chemokine Receptor CCR3

Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycline-inducible stable mammalian cell line. A cell clone with high hCCR3 expression was selected from 46 stably transfected cell clones and from this cell line pure hCCR3 on a milligram scale was obtained after two-step purification. Circular dichroism spectrum with a characteristic shape and magnitude for α-helix indicated proper folding of hCCR3 after purification. The biological activity of purified hCCR3 was verified by its high binding affinity with its endogenous ligands CCL11 and CCL24, with K _D in the range of 10⁻⁸ M to 10⁻⁶ M.     

A site-specific genomic integration strategy for sustained expression of glucagon-like peptide-1 in mouse muscle for controlling energy homeostasis

The incretin hormone glucagon-like peptide-1 (GLP-1) exerts important functions in controlling glucose and energy homeostasis. Endogenous GLP-1 has a very short half-life due to DPP-IV-mediated degradation and renal clearance, which limits the therapeutic use of native GLP-1. We have shown previously that immunoglobulin fragment-fused GLP-1 (GLP-1/Fc) is a structurally stable GLP-1 analog. Here, we report a non-viral GLP-1/Fc gene therapy strategy utilizing a REP78-in-trans and REB-in-cis element system to achieve a site-specific genomic integration. For this purpose, the GLP-1/Fc expression cassette, which is fused with the RBE element, was co-injected with the Rep78 plasmid into the muscles of transgenic mice carrying the AAVS1 locus of human chromosome 19. The Rep protein-mediated site-specific integration was demonstrated by nested PCR, dot-blot, and Southern blotting. We found that this approach reduced weight gain and improved lipid profiles in the AAVS1-mice on high-fat diet challenge. Our observations reveal a new GLP-1 therapeutic strategy with an apparent absence of side effects, which may find applications in diabetes treatment and obesity prevention     

大鼠降结肠上皮细胞γ-氨基丁酸及谷氨酸脱羧酶的表达与意义

目的检测γ-氨基丁酸(gamma-aminobutyric acid,GABA)和谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)在大鼠降结肠上皮的表达及分布特征,并探讨GABA与上皮细胞分化增殖的关系。方法用免疫荧光及激光共聚焦显微扫描技术,检测GABA、GAD65及GAD67在大鼠降结肠上皮中的表达,并以麦芽凝聚素组织化学染色与免疫荧光结合的双重染色显示GABA和GAD65表达细胞的分布特征。同时,用RT-PCR方法检测GAD mRNA的表达。此外,用3H-胸腺嘧啶放射自显影及增殖细胞核抗原(PCNA)免疫组化方法显示降结肠上皮的增殖带。结果RT-PCR显示降结肠粘膜中GAD65及GAD67mRNA均为阳性。GABA及GAD65免疫反应阳性细胞主要分布在降结肠的腔面和隐窝的上1/3上皮细胞的胞浆,而GAD67阳性细胞仅分布腔面,此外,GABA及GAD65阳性染色也见于黏膜固有层。双重染色显示杯状细胞中GABA及GAD65均为阴性3。H-胸腺嘧啶及PCNA标记阳性细胞主要在隐窝的中下段。结论GABA及GAD65分布在大鼠降结肠上皮的成熟带及功能带,GABA系统可能参与上皮细胞的分化与增殖的调节。     

Functionalization of sawdust with monosodium glutamate for enhancing its malachite green removal capacity

In this paper, waste sawdust was functionalized by monosodium glutamate for improving its cationic sorption capacity. The functionalized sawdust (FS) and crude sawdust (CS) were compared for their malachite green (MG) sorption behaviors with a batch system. The effects of various experimental parameters (e.g. initial pH, sorbent dose, dye concentration, contact time, and temperature etc.) were investigated and the sorption kinetic and thermodynamic characteristics were understood. The MG removal ratios on FS and on CS increased with increasing initial pH and came up to the maximum value beyond pH 6 for FS and pH 8 for CS, respectively. The ratio of sorbed MG kept above 95% for 250 mg/l of MG solution when 2.0 g/l or more of FS was used. The MG removal percentage decreased more on CS than on FS with increasing initial MG concentration. The isothermal data of MG sorbed on FS and on CS followed the Langmuir model. By functionalizing, the sorption capacity (Q(m)) of sawdust for MG was increased from 85.47 to 196.08 mg/g and the sorption equilibrium time of MG was shortened from 23 to 4.5 h. The MG sorption processes on FS and on CS followed the pseudo-second-order rate kinetics. The sorptions of MG on FS and on CS were spontaneous and exothermic processes and lower temperatures were favorable for the sorption processes.     

Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum-ethyl acetate-methanol-water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3mg of cryptotanshinone, 16.2mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3mg of miltirone were obtained from 400mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.     

Polycyclic Aromatic Hydrocarbons in Surface Soils of Kunming,China: Concentrations,Distribution, Sources,and Potential Risk

The concentrations, distribution, possible sources, and potential risk of polycyclic aromatic hydrocarbons (PAHs) in surface soils were studied in Kunming, which is situated in a lake basin of the Yunnan-Guizhou plateau. 15 PAHs were analyzed in 40 surface soil samples (0–5 cm layer) collected from six types of land uses in the Kunming urban area. Meanwhile, the potential sources of PAHs in surface soils of Kunming were investigated by PAH composition ratios, isomer ratios, and principal component analysis (PCA). The total concentrations of 15 PAHs ranged from 101.64 to 6,208.25 ng/g (dry weight basis). The concentrations in different land uses increased in an order as: green space (541.43 ng/g) < education area (756.49 ng/g) < business area (810.17 ng/g) < residential area (1,034.36 ng/g) < industrial area (1,166.79 ng/g) < roadside greens (2,146.76 ng/g). The results of sources identification suggested that PAHs in surface soils of the Kunming urban area were greatly affected by combustion activities which came mainly from coal combustion and vehicular traffic. In addition, the toxic equivalency factors (TEFs) were used to estimate benzo[a]pyrene-equivalent concentration in surface soils of Kunming, and the risk level of PAHs in Kunming's urban surface soils was low as a whole.     

应用PCR-DGGE技术分析BKS-2型糖尿病模型小鼠盲肠内容物微生物菌群结构

通过对BKS-DB 2型糖尿病小鼠盲肠内容物微生物群落结构的分析,探讨基因突变模型鼠肠道微生物群落与糖尿病发生的相关性。提取BKS-DB 2型糖尿病小鼠与同窝对照小鼠盲肠内容物细菌总DNA,PCR扩增16S rRNA基因V3区,DGGE方法检测扩增产物,并挑选特异条带测序,对图谱作UPGAMA聚类分析,运用SPSS软件分析多样性。结果显示,UPGAMA聚类分析表明,对照组样本之间相似度0.66-0.91,糖尿病组样本之间相似度0.61-0.88,两组之间最高相似度为0.51。特异性条带测序后经比对与Parabacteroides菌属亲缘关系最近。SPSS统计分析糖尿病组和健康对照组小鼠的DGGE条带数、多样性指数(H’)、丰富度(R)和优势度(D)的差异性显著,具有统计学意义(P<0.05)。糖尿病组小鼠与对照组小鼠的盲肠内容物微生物群落多样性存在差异,糖尿病对肠道微生物的多样性有影响。     

Kinetics and thermodynamics of basic dye sorption on phosphoric acid esterifying soybean hull with solid phase preparation technique

In this paper, the solid phase preparation method of a cationic sorbent, which bears hydroxyl groups of phosphoric acid derived from esterified soybean hull (ESH), was reported. The sorption kinetics and thermodynamics of two basic dyes, acridine orange (AO) and malachite green (MG), from aqueous solution onto ESH were investigated with a batch system. The isothermal data of dye sorptions followed the Langmuir model better than the Freundlich model. The maximum sorption capacity (Q(m)) of ESH for AO and MG was 238.1 mg/g and 178.57 mg/g, respectively. The dye sorption processes could be described by the pseudo-second-order kinetic model. The thermodynamic study indicated that the dye sorptions were spontaneous and exothermic. Lower temperatures were favorable for the sorption processes.     

Uptake of cationic dyes from aqueous solution by biosorption onto granular kohlrabi peel

A new, low cost, locally available biomaterial was tested for its ability to remove cationic dyes from aqueous solution. Granules prepared from kohlrabi peel had been utilized as a sorbent for uptake of three cationic dyes, methylene blue (MB), neutral red (NR) and acridine orange (AO). The effects of various experimental parameters (e.g., dye concentration, particle size, initial pH, contact time and other factors) were investigated and optimal experimental conditions were ascertained. Above the value of initial pH 4, three dyes studied could be removed effectively. The isothermal data fitted the Langmuir model in the case of NR sorption and the Freundlich model for all three dyes sorption. The biosorption processes followed the pseudo-first-order rate kinetics. The results in this study indicated that kohlrabi peel was an attractive candidate for removing cationic dyes from the dye wastewater.     

Predictive Mapping of Topsoil Organic Carbon in an Alpine Environment Aided by Landsat TM

The objective of this study was to examine the reflectance of Landsat TM imagery for mapping soil organic Carbon (SOC) content in an Alpine environment. The studied area (ca. 3*10⁴ km²) is the upper reaches of the Heihe River at the northeast edge of the Tibetan plateau, China. A set (105) of topsoil samples were analyzed for SOC. Boosted regression tree (BRT) models using Landsat TM imagery were built to predict SOC content, alone or with topography and climate covariates (temperature and precipitation). The best model, combining all covariates, was only marginally better than using only imagery. Imagery alone was sufficient to build a reasonable model; this was a bit better than only using topography and climate covariates. The Lin’s concordance correlation coefficient values of the imagery only model and the full model are very close, larger than the topography and climate variables based model. In the full model, SOC was mainly explained by Landsat TM imagery (65% relative importance), followed by climate variables (20%) and topography (15% of relative importance). The good results from imagery are likely due to (1) the strong dependence of SOC on native vegetation intensity in this Alpine environment; (2) the strong correlation in this environment between imagery and environmental covariables, especially elevation (corresponding to temperature), precipitation, and slope aspect. We conclude that multispectral satellite data from Landsat TM images may be used to predict topsoil SOC with reasonable accuracy in Alpine regions, and perhaps other regions covered with natural vegetation, and that adding topography and climate covariables to the satellite data can improve the predictive accuracy.