茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。     

FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

Suppressing ROS-TFE3-dependent autophagy enhances ivermectin-induced apoptosis in human melanoma cells

Melanoma is an aggressive skin malignancy with a high mortality rate; however, successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to its observed antitumor effects. However, the molecular mechanisms of ivermectin remain poorly understood. In the current study, we tested the involvement of autophagy in the ivermectin mechanism of action in human melanoma cells. We exposed SK-MEL-28 cells to different concentrations of ivermectin (2.5, 5, and 10 μM) for 24 hours. Here, ivermectin-induced apoptosis, as evidenced by the upregulation of cleaved poly (ADP-ribose) polymerase, BAX expression, and caspase-3 activity and downregulation of BCL-2 expression. In line with the apoptosis response, ivermectin triggered autophagy. Pharmacological or genetic inhibition of autophagy further sensitized SK-MEL-28 cells to ivermectin-induced apoptosis. Mechanistically, ivermectin-induced TFE3^(Ser321) dephosphorylation, activated TFE3 nuclear translocation and increased TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes, and subsequently, initiated autophagy in SK-MEL-28 cells. Moreover, N-acetyl-cysteine, an reactive oxygen species (ROS) scavenger, abrogated the effects of ivermectin on TFE3-dependent autophagy. Taken together, we demonstrated that ivermectin increases TFE3-dependent autophagy through ROS signaling pathways in human melanoma cells and that inhibiting autophagy enhances ivermectin-induced apoptosis in human melanoma cells.     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.     

Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor

A mixed culture of moderately thermophilic microorganisms was enriched from acid mine drainage samples collected from several chalcopyrite mines in China. Such mixed culture can be used to effectively extract copper from chalcopyrite. Furthermore, after being adapted to gradually increased concentration of chalcopyrite concentrate, the tolerance of the mixed culture to chalcopyrite concentrate was brought up to 80 g/L. The effects of several leaching parameters on copper recovery in stirred tank reactor also had been investigated. The results of the investigation show that it was possible to achieve a copper extraction rate of 75% in 44 days at a pulp density of 8%. The leaching rate of chalcopyrite concentrate tended to increase with dissolved total iron concentration. At low pH ranges, more microscopic counts of microorganisms were found in the solution. Furthermore, the analysis of leached residues indicates that the passivation of chalcopyrite concentrate was mainly due to a mass of jarosite and PbSO(4) on the mineral surface, other than the elemental sulphur layer. The bacterial community composition was analyzed by using Amplified Ribosomal DNA Restriction Analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus with abundance of 67% and 33% in the bio-pulp, respectively.     

中国表珠甲螨属二新记录种记述（甲螨亚目：珠甲螨科）

记述表珠甲螨属Epidamaeus 2中国新记录种:变表珠甲螨Epidamaeus variabilis Fujikawa & Fujita,1985及蒙古表珠甲螨Epidamaeus mongolicus Bayartogtokh, 2000,并分别对两种做了重新描述。     

CTAB-PEG DNA Extraction from Fungi with High Contents of Polysaccharides

Polysaccharides influence concentration and purity of extracted DNA. Here we present rapid and efficient protocol for DNA extraction from samples rich in polysaccharides. The technique has been developed using cultures of Schizophyllum commune and involves a modification of known Cetyltrimethyl Ammonium Bromide (CTAB) protocol. To remove polysaccharides, Polyethylene Glycol (PEG) 8000 was added during DNA precipitation. Genomic DNA obtained with the CTAB-PEG method had high integrity, with average fragment size >30 kb, the concentration higher than 100 ng/μL, and the yield more than 30 μg/g. Presented technique is suitable for DNA extraction from fungi, bacteria, archaea or even mollusks with high polysaccharide content.