DNA SELF-ASSEMBLING SYSTEMS: BRANCHED OLIGONUCLEOTIDES AS BUILDING BLOCKS AND MONITORING BY PYRENE EXCIMER BAND FORMATION

The construction of a DNA self-assembling system created by four Y-shaped branched oligodeoxynucleotide building blocks has been studied. The assembly was verified by changes in the fluorescence emission spectra and revealed an additive effect in pyrene excimer band formation during DNA self-assembly.     

循环血游离Shope病毒DNA含量与兔VX2肿瘤18F-FDG PET/CT影像表现的相关性分析

目的:探讨循环血中Shope病毒DNA含量与兔VX2肿瘤18F-FDG PET/CT影像学特征间的关系及其临床意义。方法:采用组织块接种法建立兔VX2肿瘤模型,并行18F-FDG PET-CT观测肿瘤大小及糖代谢相关值,实时定量荧光探针PCR法检测肿瘤组织及血浆中Shope病毒特征性DNA片段含量。结果:移植前外周血中未检测出Shope病毒特异性DNA片段;移植后2周,VX2肿瘤组织和循环血中均可以检测到特征性Shope病毒DNA片段。瘤体内DNA含量明显高于循环血中含量。循环血Shope病毒DNA含量与FDG-PET的最大标准摄取值(SUVmax)明显呈正相关(r=0.943,p=0.005),但与肿瘤体积相关性尚不明确(r=0.657,p=0.156)。结论:循环血Shope病毒DNA有望作为一种潜在的VX2肿瘤标志物,其廉价、无创的特性,有望在肿瘤的早期诊断和预后随访中发挥优势。     

GATE:一种用于辐射层析成像的蒙特卡罗模拟技术

描述了GATE的主要特点以及实现这些特点的GATE所具有的层级结构。通过测试由GATE软件包自带的GATE PET Benchmark的模拟结果,验证了GATE模拟引擎的正确性以及不同初始随机数下多次模拟实验的稳定性与可靠性。     

A colorimetric assay method to measure acetyl-CoA synthetase activity: application to woodchuck model of hepatitis virus-induced hepatocellular carcinoma

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorbance at 580 nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-mul sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-(11)C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-(11)C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. A significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P<0.05). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P<0.05) (total soluble fraction: 876.61+/-34.64 vs. 361.62+/-49.97 mU/g tissue; cytoplasmic fraction: 1122.02+/-112.39 vs. 732.32+/-84.44 mU/g tissue; organelle content: 815.79+/-100.77 vs. 547.91+/-97.05 mU/ g tissue; sedimentable fragment: 251.92+/-51.56 vs. 90.94+/-18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.     

Probing structural changes in the α and β domains of copper- and silver-substituted metallothionein by emission spectroscopy and electrospray ionization mass spectrometry

Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted α and β domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag(I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag(I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted α and β domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag(I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted α and β fragments than when the Ag(I)-MT species were formed from the apo-α and apo-β fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag(I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.     

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau (Biophys. J. 65 (1999) 2272-2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.     

Cu+ distribution in metallothionein fragments

The differential distribution of Cu+ between separate alpha and beta domains of metallothionein (the isolated peptide fragments) and the rate of transfer of Cu+ between the two domains using copper-thiolate specific emission spectroscopy are reported. Kinetic data show the rate of transfer of Cu+ from the Cu6alpha to the Cd3beta domain is 2 x 10(-1) s(-1) while the transfer from Cu6beta to the Cd4alpha domain is much slower at 8 x 10(-3) s(-1), indicating the greater binding affinity of Cu+ for the MT beta domain. We report that the emission intensity of Cu6beta is 0.45 the emission intensity of Cu6alpha-MT. Lambda(max) is shown to be a probe of the environment of the Cu+. A series of copper-containing domain intermediates to the formation of the filled Cu6S9-beta and Cu6S11-alpha-clusters are identified. A mechanism is proposed for the formation of Cu12(betaalpha)-MT that involves metal exchange reactions of Cu+ ions from the alpha to the beta domain with initial formation of a Cu4beta-cluster.     

玉米植株对大田温室气体N2O排放的影响

利用封闭式箱法对玉米田Ｎ２Ｏ排放通量的观测表明,大田种植玉米后,对Ｎ２Ｏ排放产生了很大影响,玉米土壤系统的Ｎ２Ｏ排放通量大于不种玉米的土壤．此外,植物根系能明显促进土壤中Ｎ２Ｏ的排放,特别是在玉米生长后期尤为明显．从播种开始到年底,施尿素导致Ｎ２Ｏ排放为３．３ｋｇ·ｈｍ－２,玉米植株为０．６９ｋｇ·ｈｍ－２,占总排放量的１７．３％．     

长白山北坡不同土壤N2O和CH4排放的初步研究

用箱法技术原位测定了长白山北坡不同土壤（苔原土、生草森林土、棕色针叶林土和暗棕色森林土）６—８月间的Ｎ２Ｏ和ＣＨ４排放．结果表明,这些土壤既是Ｎ２Ｏ的源,又同时是ＣＨ４的汇．Ｎ２Ｏ通量变化于６．１７—１２．３３μｇ·ｍ－２·ｈ－３之间（平均９．３７μｇ·ｍ－２·ｈ－１）,ＣＨ４通量为－８５．６３—－７．５８μｇ·ｍ－２·ｈ－１（平均－４１．４５μｇ·ｍ－３·ｈ－１）,并观察到在Ｎ２Ｏ排放和ＣＨ４吸收之间有着相互消长关系．实验室培养实验表明,最大反硝化作用活性存在于土壤上层（０—６ｃｍ）;不同土壤的反硝化作用活性明显不同．山地暗棕色森林土的ＣＨ４吸收作用也主要发生在土壤的上层（０—１２ｃｍ）．     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography