DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Abstract A phr-gene from the filamentous fungus Neurosporu crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,1O-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurosporu photolyase is shifted from ca 380 nm to 391 nm (t = 34 800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiue photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurosporu photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.     

PHOTOCHEMISTRY, PHOTOPHYSICS, AND MECHANISM OF PYRIMIDINE DIMER REPAIR BY DNA PHOTOLYASE

Abstract— DNA photolyases photorepair pyrimidine dimers (PyroPyr) in DNA as well as RNA and thus reverse the harmful effects of UV-A (320–400 nm) and UV-B (280–320 nm) radiations. Photolyases from various organisms have been found to contain two noncovalently bound cofactors; one is a fully reduced flavin adenine dinucleotide (FADH^-) and the other, commonly known as second chromophore, is either methenyltetrahydrofolate (MTHF) or 8-hydroxydeazaflavin (8-HDF). The second chromophore in photolyase is a light-harvesting molecule that absorbs mostly in the near-UV and visible wavelengths (300–500 nm) with its high extinction coefficient. The second chromophore then transfers its excitation energy to the FADH^-. Subsequently, the photoexcited FADH^- transfers an electron to the Pyr<>Pyr generating a dimer radical anion (Pyr<>Pyr^-) and a neutral flavin radical (FADH^-). The Pyr<>Pyr^- is very unstable and undergoes spontaneous splitting followed by a back electron transfer to the FADH^-. In addition to the main catalytic cofactor FADH^-, a Trp (Trp277 in Escherichia coli ) in apophotolyase, independent of other chromophores, also functions as a sensitizer to repair Pyr <> Pyr by direct electron transfer.     

PHOTOREPAIR OF NONADJACENT PYRIMIDINE DIMERS BY DNA PHOTOLYASE

Abstract— Photolyases reverse the harmful effects of UV light on cells by converting pyrimidine dimers (Pyr[]Pyr) into two pyrimidine monomers by utilizing near-UV and visible light. Previous work has shown that photolyase repairs T[c,s]T and T[t,s]T in DNA as well as U[]U in RNA, all of which are formed by joining the two adjacent pyrimidines in a light-dependent reaction. In this report, we show that Pyr[]Pyr formed in nonadjacent pyrimidines are also substrates for DNA photolyase.     

KINETICS OF BACTERIAL BIOLUMINESCENCE AND THE FLUORESCENT TRANSIENT

Abstract— The addition of FMNH₂ to Vibrio harveyi luciferase at 2°C in the presence of tetradecanal results in the formation of a highly fluorescent transient species with a spectral distribution indistinguishable from that of the bioluminescence. The bioluminescence reaches maximum intensity in 1.5 s and decays in a complex manner with exponential components of 10^-1s^-1, 7 × 10^-3s^-1, and 7 × 10 ⁴s^-1. The fluorescent transient rises exponentially at 7 × 10^-2s^-3 and decays at 3 × 10^-4s^-1. The slowest bioluminescence component, comprising the bulk of the bioluminescence, decays at twice the rate of the fluorescent transient under all variations of reaction conditions: concentration of reactants, temperature 2–20°C, and aldehyde chain length—decanal, dodecanal and tetradecanal. The activation energy for both the slowest bioluminescence decay and the transient fluorescence decay is 80 kJ-mol^-1. An energy transfer scheme is proposed to explain the results where two distinct chemically energized species utilize the fluorescent transient as emitter for the slower bioluminescences, and for the faster process a fluorophore present in the protein preparation. Kinetic observations suggest that typical preparations of V. harveyi luciferase comprise 15% active protein.     

大叶桉酚甲的结构和合成

从大叶桉的叶中分得大叶桉酚甲,为一种新的酰基间苯三酚衍生物的二缩聚体,通过紫外光谱、红外光谱、核磁共振谱和质谱的解析以及碱液分解产物的鉴定,推定其结构为1,并经全合成确证.大叶桉酚甲对鼠疟原虫有抑制作用.     

大孔吸附树脂结合酶解法分离纯化虎杖中白藜芦醇的研究

研究了大孔吸附树脂结合酶解法提取和纯化虎杖中白藜芦醇的方法,采用HPLC法测定虎杖中白藜芦醇的含量,考查了β-糖苷酶对虎杖药材酶解前后白藜芦醇含量的变化,并经静态吸附考察了4种树脂,最后确定以H1020作为提取分离白藜芦醇的树脂.此树脂吸附量较高,脱附容易,有利于得到质量较好的白藜芦醇产品,经该树脂吸附解吸,饱和吸附量可达51.4mg/g,解吸率达92.5%.大孔树脂分离纯化白藜芦醇的含量可达71.5%,而上柱前粗提物中白藜芦醇含量为8.71%,说明采用本法分离纯化虎杖中白藜芦醇是可行的.     

FLUORESCENT TAUTOMERS AND THE APPARENT PHOTOPHYSICS OF ADENINE AND GUANINE

Abstract— Fluorescence, absorption and fluorescence excitation spectra, and quantum yields of 0.02 mM solutions of adenine, 7-methyladenine (7-MA), guanine and 7-melhylguanine (7-MG) are presented for excitation with240–300 nm light. The solvent is neutral ethylene glycol-water (70:30 v/v) in the temperature range140–165 K. Phosphorescence spectra of adenine and 7-MA at 140 K are also presented. The excitation spectrum of adenine shows vibrational structure, whereas the absorption does not. However, the fluorescence of adenine shows the vibrational structure, as do the absorption, fluorescence and excitation spectra of 7-MA. The results confirm (and reinforce) the notion that luminescence from adenine under these conditions is from the N₇–H tautomer, instead of the more abundant N₉–H form. In a similar fashion, the data from guanine and 7-MG strongly suggest that the luminescence from guanine is also mostly from the N₇–H tautomer.     

A NEW FLUORESCENT CHOLESTEROL DERIVATIVE: STRUCTURE AND BLOOD PLASMA ANALYSIS

A fluorescent cholesterol derivative produced by reaction with gaseous HCl and zinc chloride in ethyl acetate is shown to be a chlorine substituted B-ring diene. The species forms relatively rapidly via an i-steroid rearrangement, requiring a temperature around 70 degrees C. A weak, possibly dimeric pi-allyl zinc complex exists in solution, leading to a red shift in the fluorescence emission. The application of the derivative to the determination of cholesterol in bovine plasma provides good sensitivity and precision and requires notably small sample volumes.     

RESTRICTION ENZYME CLEAVAGE OF UV-IRRADIATED DNA: A COMPARISON OF FAR-UV AND MID-UV WAVELENGTHS

UV-irradiated DNA is less susceptible to restriction by Type II endonucleases than unirradiated DNA presumably due to photolesions formed in the recognition sites. Previous reported studies have used 254 nm radiation or 313 nm plus acetophenone, both treatments which introduce pyrimidine dimers in preference to other photolesions. To assess the effect of a longer wavelength, at which the ratio of pyrimidine dimer formation to the formation of other photolesions is reduced, two different DNAs were irradiated with UV of either 254 or 313 nm and restricted with suitable restriction endonucleases. Restriction patterns were analysed for novel fragments resulting from UV-induced alteration of enzyme recognition sites. EcoRI restriction of 254 nm irradiated lambda DNA produced six novel bands, only three of which were observed following restriction of 313 nm irradiated lambda. These three represented the largest fragments resulting from single site blocks. Novel fragments involving adjacent site blocks observed at 254 nm were not found with 313 nm radiation. Comparison of 254 nm irradiated pSV₂gpt to that irradiated at 313 nm, both restricted with Dral, revealed a more complex pattern. Although all sites were singly blocked by radiation of both wavelengths, multiple site blocks produced by 313 nm radiation did not occur in the order predicted by the 254 nm radiation dose response. These data suggest that certain sites in pSV₂gpt may be more refractory to multiple site blocks than others when irradiated at 313 nm.     

聚苯类共轭聚合物的合成及荧光特性研究

聚苯类共轭聚合物的合成及荧光特性研究李建科阳明书漆宗能王佛松（中国科学院化学研究所工程塑料国家重点实验室北京１０００８０）关键词聚对苯，共轭聚合物，吸收光谱，荧光光谱，发光共轭聚合物在非线性光学、光电化学池、二次电池等方面有着广泛的应用前景，近...     

EVALUATING THE STABILITY AND REACTIVITY OF A LIGHT-SENSITIVE PROBE BY ENZYME ANALYSIS

Abstract— Enzymes can provide well defined functional targets for determining the availability and reaction characteristics of light sensitive probes. Trypsin and chymotrypsin are inhibited by flash photolysis with a photoprobe, 4-fluoro-3-nitrophenylazide (FNPA). Inhibition is competitive in the dark, and non-competitive (irreversible) following photolysis of the FNPA enzyme solution. Photoinactivation is dependent upon the concentration of FNPA, the flash rate, and the time of photolysis. The enzymes can be protected from photolytic inactivation with FNPA if photolysis is carried out in the presence of 2,4-dinitrophenol or 4-fluoro-3-nitroaniline which compete with FNPA for binding sites. The photo-probe is effective over a wide pH range (i.e. pH 2–11), and provides a sensitive tool for probing conformational and charge adjustments which increase or decrease the affinity of the binding site. Chymotrypsinogen was also sensitive to photolysis, indicating that FNPA binding sites are present in the zymogen structure.     

ISOLATION AND CHARACTERIZATION OF A STRUCTURAL SUBUNIT FROM THE CORE LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides 2.4.1 AND puc705-BA

A method for isolating a structural subunit, B825, from the B875 core light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1 (wild-type) and a B800-B850(-) mutant, puc705-BA, is presented. This method, based on one developed to prepare a similar subunit, B820, from the core LHC of Rhodospirillum rubrum [Miller et al., Biochemistry 26, 5055-5062 (1987)], requires the dissociation of treated chromatophores with the detergent, octyl-glucoside. A subsequent gel filtration step separates B800-850 (if present), reaction centers, and free bacteriochlorophyll from the subunit complex. B825 was quantitatively reassociated into an 873 nm absorbing form which resembled the in vivo complex as judged by its absorption properties. The polypeptides in B825 and B800-850 were isolated by HPLC and identified by N-terminal amino acid sequencing. Two polypeptides, alpha and beta, were found in each complex in a 1:1 ratio. The spectral and biochemical properties of the subunits isolated from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus are compared.     

六氟丙烯的等离子体聚合和聚合物结构的研究

应用外部电容耦合式聚合装置,研究了六氟丙烯(HFP)的聚合规律,找到了较好的聚合条件。实验中发现在聚合过程中,随功率增加脱氢作用增大,这可利用氢等离子气体加以控制。X-射线衍射法和元素分析结果表明,在辉光区中形成的PHFP具有高度支化和交联的结构。从性能研究中看出,等离子体PHFP膜具有较好的光学性能,疏水性和较高的热稳定性。     

土槿甲酸的分子结构及晶体结构的研究

土槿甲酸是抗真菌土槿皮酸的成分之一.晶体属四方晶系,空间群P422晶胞参数a=b=8.956Å,c=51.834Å,α=β=γ=90°,z=∑a_n=4564.土槿甲酸的粗结构模型和化学结构式通过直接法测定.     

A RAPID ASSAY FOR DNA PHOTOLYASE USING A MEMBRANE-BINDING TECHNIQUE*

Abstract— A rapid assay for DNA photolyase was developed that makes use of the membrane binding technique of A. D. Riggs, H. Suzuki and S. Bourgeois, J. Mol. Biol. 48 , 67–83 (1970). The complex formed between UV-irradiated bacteriophage T7 DNA-³H and the enzyme is trapped on a nitrocellulose filter after a five-minute equilibration and is counted. Using this technique, one can calculate the molar concentration of enzyme in an enzyme preparation and the saturation constant (K) for the complex formation. The latter value is 2.6 cyclobutadipyrimidines per genome (T7) for yeast DNA photolyase; thus approximately 5.2 dipyrimidines/genome are necessary for the full retention of the DNA on the membrane. The speed and reproducibility of the new method makes it a convenient one for assaying column effluents and as many as one hundred samples can be handled routinely in an afternoon.     

A CHARACTERIZATION OF THE FLUORESCENT PROPERTIES OF CIRCULATING HUMAN EOSINOPHILS

Abstract— This paper presents a characterization of the fluorescence properties of human eosinophils isolated from peripheral blood of normal donors over a wide range of excitation and emission wavelengths. Circulating eosinophils possess three fluorescence excitation emission maxima: one at 280 nm excitation, 330 nm emission, attributable to tryptophan fluorescence, and currently unassigned peaks at 360 nm excitation, 440 nm emission and 380 nm excitation, 415 nm emission. Fluorescence microscopy studies show that the Huorescence of eosinophils may be site dependent; specifically, when observed at 365 nm excitation, circulating eosinophil Huorescence appears blue-violet, while the fluorescence of tissue-dwelling eosinophils appears amber-gold. These results should be considered in developing an optical biopsy technique to identify eosinophils in human tissue.