用聚乙二醇分离富集microRNA的方法探讨

目的探讨用聚乙二醇（polyethylene glycol,PEG）溶液分离富集miRNA的操作方法和分离富集效果,并与Ambion公司的miRNA分离试剂盒分离效果进行比较。方法用PEG溶液和Ambion公司的miRNA分离试剂盒从肝脏组织总RNA中分离富集miRNA,用变性琼脂糖和变性聚丙烯酰胺凝胶电泳鉴定分离效果,并在富集的miRNA中用RT—PCR扩增miR-122以鉴定是否有效地回收了miRNA。结果PEG和Ambion公司的miRNA分离试剂盒都能有效地富集miRNA,PEG富集的RNA片段比Ambion公司的试剂盒的大。结论PEG溶液能有效地分离富集miRNA,和Ambion公司的miRNA分离试剂盒分离效果相当,并具有操作简便、快捷及成本低廉的优点。     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

改进的稳定小分子量RNA（LMW）指纹图谱技术

通过对稳定小分子量RNA这项技术进行改进,使其与以往的分离技术相比,在时间上缩短到4.5 h,最高电压降低到1400V,胶板的长度降到30 cm;使其可操作性更强,更加适用于大量的样品的分析。     

聚乙二醇沉淀剂有效分离尿外泌体

尿外泌体是病毒大小的胞外囊泡,是非侵入性获得肾及泌尿生殖道细胞生理病理信息的重要靶标。聚乙二醇沉淀 剂可经济高效地分离富集血清等外泌体,但未见用于尿外泌体富集的详细报道。本研究采用聚乙二醇沉淀剂分离鉴定尿外泌体,并对其RNA组分进行检测,以期建立一个经济、高效、简便的尿外泌体分离富集方法。采集10例健康志愿者晨尿20 mL,聚乙二醇沉淀剂分离尿外泌体。透射电镜观察到直径30～100 nm双层膜包绕的囊性小泡,中央有直径5～15 nm高电子密度区。Western印迹检测到外泌体标记蛋白CD63、CD9、TSG101、ADAM10和内标蛋白β-肌动蛋白的表达。纳米粒径仪测定粒子直径介于30～130 nm,并可见25.37 nm和95.07 nm二个粒子峰。qRT-PCR扩增得到β-肌动蛋白和RNU6 RNA产物带。上述结果表明,聚乙二醇沉淀剂可分离富集尿外泌体,该法简单、高效,不需要超速离心机等昂贵设备,且采用该法富集到的外泌体可用于后续蛋白质与核酸分析。该方法可望加速液体活检应用,尤其是肾及泌尿生殖道病变的无创检测。     

Utilization of Hydrocarbons by Microorganisms

Corynebacterium hydrocarboclastus S10BI or S489BI can accumulate a good deal of Lglutamate in a thiamine-deficient medium at the expence of hydrocarbon, but can not form L-glutamate in a thiamine-sufficient medium in spite of rapid cell growth, as already reported. In order to establish the optimal culture condition for L-glutamate formation, the influence of the following factors was first studied: hydrocarbon concentration, pH control, nitrogen sources, temperature, aeration and supplement of metal ions or amino acids. Then L-glutamate production from a variety of hydrocarbons or petroleum fractions was examined. Sodium oleate was found to stimulate growth remarkably instead of thiamine. Ferrous ion was found to be obligatory for L-glutamate formation and was suggested to have taken part in the earliest step of n-alkane oxidation.     

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin,by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

The Utilization of P--N Compounds by Plants: I. Root-mediated Hydrolysis of Phosphodiamidate

The uptake and utilization of sodium phosphodiamidate, a compoundcontaining covalent P—N bonds, was investigated. Growthanalysis showed that this compound could serve as the sole sourceof phosphorus for tomato plants grown in culture solutions,although the growth rate of plants supplied with phosphodiamidatewas slightly less than that of plants utilizing diammonium phosphate.Chromatographic analysis of xylem sap showed that phosphodiamidatewas not transported in the unhydrolysed form in the sap of tomatoplants supplied with this compound. Tomato plants supplied withphosphodiamidate as the sole source of both phosphorus and nitrogenassimilated some nitrogen in a form other than unhydrolysedphosphodiamidate. Comparison with plants supplied with diammoniumphosphate showed that the plants receiving phosphodiamidatehad lower nitrogen contents, suggesting that the rate of hydrolysisof the compound may have been limiting nitrogen assimilationby the plants. Measurements of the hydrolysis of phosphodiamidatein culture solutions in the absence of plants showed that theplants assimilated more nitrogen than that released by normalchemical hydrolysis of the compound occurring in the absenceof plants. The excess nitrogen assimilated, over and above thatproduced by normal chemical hydrolysis, could not be accountedfor solely by the uptake of unhydrolysed phosphodiamidate asthis would require the concomitant uptake of more phosphorusthan was actually present in the plant. Thus it was inferredthat the presence of the plant roots in the culture solutionsomehow caused extra hydrolysis of the phosphodiamidate.     

Antimicrobial Characteristics of Chitosans against Food Spoilage Microorganisms in Liquid Media and Mayonnaise

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

Crystallographic study of the iron-sulfur flavoprotein trimethylamine dehydrogenase from the bacterium W3A1

Large single crystals of trimethylamine dehydrogenase, containing both [4Fe-4S]²⁺ centers and covalently bound FMN, have been prepared by the macro seeding technique. The crystals are monoclinic, space group P2₁ with cell parameters $\text{a = 147.63}\text{A}\text{?}\text{, b = 71.96}\text{A}\text{?}\text{, c = 83.66}\text{A}\text{?}\text{and}\text{β = 97.64 °}$, and diffract to at least 2.0 Å resolution. There is one dimer of approximately 166,000 M_m per asymmetric unit. A 5.0 Å resolution anomalous scattering difference Patterson has been computed which shows the presence and position of two [4Fe-4S]²⁺ centers in the asymmetric unit. A self-rotation function computed at 6.0 Å resolution indicates a non-crystallographic 2-fold axis relating the two subunits. These results show trimethylamine dehydrogenase to be composed of two identical or very similar subunits each containing one [4Fe-4S]²⁺ center.     

Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes

Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry     

Modification of Lipase by Polyethylene Glycol

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.     

Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification

-Chymotrypsin was modified with cyanuric chloride activated monomethoxypolyethylene glycol (MPEG) with molecular weights 1900 and 5000. Using the higher molecular weight MPEG a product that was soluble in benzene at moderate levels of modification was obtained, whereas with MPEG 1900 almost all the enzyme's amino groups had to be modified for dissolving the conjugate. The catalytic activity decreased with increasing degree of substitution. Apparent V_max was considerably higher for the less modified enzyme preparation than for the more modified one, while K_m,app stayed almost constant. The modified enzyme was used for peptide synthesis. The reaction was dependent on the content of dissolved water. Both V_max,app and K_m,app increased with increasing water content. It was possible to achieve a process with complete conversion of substrate to dipeptide.     

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.     

Carbohydrates of the seaweeds,Desmarestia ligulata and D. firma

Crystalline mannitol and some oligosaccharides were separated from ethanolic extracts of Desmarestia ligulata and D. firma. Laminaran, ‘fucans’ and alginic acid were also isolated from both species. The laminaran from D. ligulata comprised both M- and G-chains but no M-chains were found in the laminaran from D. firma. In both species the amount of ‘fucan’ was small, particularly in D. firma. Both ‘fucans’ contained glucuronic acid, galactose, xylose and fucose and that from D. ligulata also contained mannose. After sequential extraction of D. ligulata with water, acid and alkali evidence was obtained for the presence of cellulose, a uronan, and protein in the residual material.     

低分子肝素的抗炎作用及机制

低分子肝素(low molecular weight heparin, LMWH)除作为抗凝血和抗血栓药在临床上广为应用外,近年来其抗炎活性也颇受重视.LMWH抗炎机制涉及炎症细胞、炎症因子和黏附分子等环节.目前对LMWH的抗炎机制研究还处在初级阶段,但是LMWH独特的性质使其有望成为有效且安全的新型抗炎药物.     

The measurement of water relations of plant cell culture. I. Water release in response to centrifuge-induced water potentials

Water loss by cell suspensions during centrifugation is well defined by simple physical principles. The major factors affecting water release during centrifugation are: duration of centrifogation, depth of the cell mass, density of cells, relative centripetal acceleration and centripetal force. Water release during centrifugation was best described by an exponential decay process with a decay constant that increases with acceleration from 0.31 ± 0.01 to 0.66 ± 0.12 min^?1 (mean ± SE) between 4 825 and 19 300 m s^?2, respectively. The cell mass relative water content (RWC) at equilibrium was not a function of rate of water loss and was constant for each acceleration. A centripetal force was generated by the mass of the cells being accelerated away from the axis of rotation. This force generated a pressure that removed some of the cell wall and symplast water, by compression at contact points between the cells and by compression of the cytoplasm. Pressure induced by centripetal forces ranging from ?0.02 to ?0.23 MPa gave a linear relationship (r² > 0.99) between force and RWC. The slope (0.900 MPa) was proportional to the cell wall modulus of elasticity (±). and the intercept was interpreted to give the mass of the cells at full turgor without interstitial water (RWC=1). This interpretation is supported by the findings, of two independent experiments. Centrifuged cells suspended at 100% relative humidity for over 48 h reached the same water content as predicted by the intercept. Interstitial water was labelled with solutions of polyethylene glycol (PEG. M_r 8 000), the diameter of which was too large to enter the pores of plant cell walls. Centripetal accelerations greater than 10 900 m s^?2 removed PEG-labelled water to levels below 0.9% of cell water content. Removal of interstitial water and other loosely bound water provided a convenient method for determination of growth, RWC and ±. The centrifugal methods provide the foundation for new quantitative methods for cell culture water relations analyses.