A MICROSPECTROFLUOROMETRIC STUDY OF PORPHYRIN-PHOTOSENSITIZED SINGLE LIVING CELLS—II. METABOLIC ALTERATIONS

The transient change in the NAD(P)H NAD(P) equilibrium following microinjection of mitochondrial (malate) or extra-mitochondrial (6-phosphogluconate) substrates into single living cells is strongly modified a few seconds after the onset of 365 nm irradiation in the presence of hematoporphyrin. There is no major difference in the time frame for the alterations of the Krebs cycle and pentose pathway. In view of the complex interrelationships between mitochondrial and extramitochondrial pathways, there is a reasonable chance that effects on both pathways are not unrelated- However there is no definite evidence for a direct correlation between the photoeffects on these two pathways.     

BINDING OF HEMATOPORPHYRIN DERIVATIVE TO BRAIN TUMOR CELLS—A FLUORESCENCE SPECTROSCOPIC STUDY

The binding of hematoporphyrin derivative (HpD) to brain tumor cells and their photosensitivity was studied as a function of HpD concentration, time of incubation and growth phase of cells. Upon binding to cells, HpD showed three fluorescence bands at 616, 636 and 678 nm. In plateau phase cells a fluorescence band at 636 nm was predominant, which was further enhanced by increasing HpD concentration and/or increasing incubation time. In exponential phase cells the maximum fluorescence was exhibited at 616 nm. After 1 h incubation of exponential phase cells with increasing HpD concentration an overall intensity enhancement occurred with no change in the distribution of bands, whereas longer incubation time caused an increase in relative intensity of the 636 nm band similar to that observed in plateau phase cells. After 1 h incubation with HpD plateau phase cells were more photosensitive than exponential phase cells, although cell bound HpD was much less in the former case. Incubation of cells for 24 h drastically enhanced the photosensitivity irrespective of the growth phase. Our results suggest a relationship between the fluorescence emission band of HpD at 636 nm and photosensitivity of cells.     

PHOTODYNAMIC ACTION OF HEMATOPORPHYRIN ON YEAST CELLS—A KINETIC APPROACH

Abstract— By a technique which combines rapid mixing of cells and hematoporphyrin (HP) with a short duration of illumination, the photodynamic inactivation of yeast cells was investigated, particularly, in seeking for the information of the location of HP at the time of action. The fluence-survival curves obtained under the conditions where the reaction mixture was kept in the dark for Is, 60s and even 35 min before illumination were indistinguishable from each other, indicating no interaction between cells and sensitizers took place in about 30 min in such a way that the photodynamic efficiency could be modified. It is unlikely that HP acted intracellularly, since the protective effect of N^?₃ was observed at concentrations as low as 0.5 mM. Furthermore, the rate constant k_p related to the protective effect of NJ, was estimated to be 1 × 10⁸M^?1 s^?1 under the assumption that ¹O₂ was the active intermediate and had a lifetime of 2 μs under the present conditions. This value of k_p is rather close to that of k_q, the quenching rate constant of N^?₃ for ¹O₂, of which the accepted value is 2 × 10⁸M^?1s^?1 in the homogeneous aqueous system. This information, together with the absence of uptake of HP by cells and a well response of survival upon illumination to the D₂O fraction of the reaction mixture, provide strong bases for the argument that direct interaction of HP with yeast cells is of minor importance in the photodynamic processes, and the photodynamic action is largely mediated by an intermediate (¹0₂) generated in bulk medium.     

电极防吸附膜研究

在电化学分析及电极过程动力学研究中,表面活性物质在电极上的吸附,常使测量结果受到严重影响^[1,2]。在天然水的反向极谱分析中,由于天然水,特别是海水,含有大量表面活性物质及悬浮体,并且分析溶液又进行搅拌,因此对电极的灵敏度及重演性影响特别严重^[3-5]。用汞膜电极测定一个新采集的海水样品后,即需重新涂汞^[3,4]。     

FLUORESCENCE OF Euglena gracilis PHOTORECEPTOR PIGMENT: AN in vivo MICROSPECTROFLUOROMETRIC STUDY

Abstract— The spectroscopic characterization of the photoreceptor pigment is one of the main questions in the study of the photosensory transduction chains in photomotile microorganisms. One of the possible techniques that can be used is in vivo microspectrofluorometry. By means of a tunable dye-laser microspectrofiuorometer developed by us, we have investigated some of the spectroscopic properties of the photoreceptor pigment of the green flagellate Euglena gracilis. The in vivo fluorescence excitation spectrum has been determined and the fluorescence quantum yield has been measured. The results show that flavins are indeed present in the paraflagellar body of E. gracilis and that their fluorescence quantum yield is much lower than that of a free flavin. An estimate of the order of magnitude of the rate constants for primary molecular reactions is tentatively given.     

杯芳烃膜电极研究

用吸附法将 C-十一烷基间苯二酚杯 [4]芳烃固定在玻碳电极上制成杯芳烃膜电极 ,对DL-去甲肾上腺素、尿酸、DL-酪氨酸、邻苯二酚进行了测定 ,该电极对 DL-去甲肾上腺素和邻苯二酚具有良好的响应 ,其线性范围分别为 4.0× 1 0 -5～ 8.0× 1 0 -4 和 2 .0× 1 0 -4 ～ 2 .0× 1 0 -3mol·L-1,检测下限分别为 1 .2 5× 1 0 -5和 1 .0× 1 0 -5mol· L-1,对尿酸、DL -酪氨酸没有响应 ,试验发现杯芳烃膜电极不但具有选择性 ,而且有较快的响应速度、良好的重现性和稳定性。     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.     

乳化液膜清除分离胆红素的研究

以液体石蜡为膜相,司班８０为表面活性剂,乙酸乙酯为转移载体,氢氧化钠水溶液为内相制备了一种新型的乳化液膜,用于清除分离对人体有害的胆红素,其分离过程具有不可逆的特点,分离的速度快,清除率高,对游离胆红素的清除率为９９．８％,对未结合胆红纱的清除率为８３．０％,分离的速度和清除率优于其他分离方法。     

THE USE OF EXOGENOUS FLUORESCENT PROBES FOR TEMPERATURE MEASUREMENTS IN SINGLE LIVING CELLS

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazo1-4-y1 (NBD) and 6-dodeca-noy1-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2°C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1°C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.     

聚乙烯－葡萄糖氧化酶膜的制备和性能研究

聚乙烯－葡萄糖氧化酶膜的制备和性能研究朱如瑾，殷弘浩，刘永盛，黄家湛（成都高分子材料国家重点实验室成都科技大学高分子研究所成都６１００６５）关键词聚乙烯，等离子体，固定化酶，葡萄糖氧化酶固定化酶是６０年代发展起来的生物工程技术Ｉ‘］．酶的固定化方法主...     

动态单滴法研究乳状液液膜的稳定性

乳状液液膜作为化学分离的一种手段,自七十年代发现以来已有了许多进展,但是如何将其工业化还有许多课题有待于进一步研究.目前,除了乳状液液膜的水静态渗透性质以外,对乳状液液膜的稳定性和溶胀性质的研究主要采用搅拌法.虽然得到较好的规律性,但是搅拌法具有乳状液滴粒径分布广的弱点,对于不同的搅拌方式及条件,乳状液液膜的有效面积不同,对液膜作用的机械强度不同,因此结果会有很大差别.我们自行设计的动态单滴法实验装置,可定量地研究乳状液液膜的溶胀、稳定性及水渗透性质.     

聚乙烯－葡萄糖氧化酶膜的制备和性能研究

聚乙烯－葡萄糖氧化酶膜的制备和性能研究朱如瑾，殷弘浩，刘永盛，黄家湛（成都高分子材料国家重点实验室成都科技大学高分子研究所成都６１００６５）关键词聚乙烯，等离子体，固定化酶，葡萄糖氧化酶固定化酶是６０年代发展起来的生物工程技术Ｉ‘］．酶的固定化方法主...     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

PHOTOSENSITIZATION BY PORPHYRINS DELIVERED TO L CELL FIBROBLASTS BY HUMAN SERUM LOW DENSITY LIPOPROTEINS. A MICROSPECTROFLUOROMETRIC STUDY

Human serum LDL were used as vehicles to deliver protoporphyrin and hematoporphyrin dimer to L cell mouse fibroblasts. Topographic analysis by microspectrofluorometry on single living cells shows that after digestion of LDL, protoporphyrin is localized in cytoplasmic areas. Protoporphyrin and hematoporphyrin dimer are readily bleached by 420 ± 60 nm radiations at the high fluence rate used. Complex bleaching kinetics are observed. Spectral studies using the same technique demonstrate that an intense fluorescent emission (λ_max= 450 nm) is produced immediately after the onset of irradiation with 365 ± 2 nm or 420 ± 60 nm radiations using LDL loaded with protoporphyrin or Photofrin II. These fluorescent products have been previously identified as lipofuscin-like pigments formed by reaction of lipid photoperoxides with amino groups. The permeation of lysosomal membranes is also induced after delivery of the porphyrins by LDL. This permeation can be strongly inhibited not only by the lysosomal inhibitors chloroquine and monensin but also by a-tocopherol. On the other hand, neither α-tocopherol nor chloroquine or monensin inhibit the lipofuscin-like pigment formation.     

CHARACTERIZATION OF THE 32,000 DALTON MEMBRANE PROTEIN—I. EARLY SYNTHESIS DURING PHOTOINDUCED PLASTID DEVELOPMENT OF SPIRODELA

The kinetics of ³⁵S methionine incorporation into soluble and membrane proteins during the transition from steady state dark growth to greening was studied in Spirodela. A sharp increase in the rate of incorporation occurred at 3 h, which was several h before major increases in chlorophyll were apparent. Part of this enhanced incorporation was due to enhanced synthesis of a 32 ,000 dalton membrane protein. This synthesis was paralleled by a temporal increase in in uitro template capacity for this protein and an increase in 0.5 × 10⁶ dalton plastid messenger RNA.     

DCMU-INDUCED STIMULATION OF PHOTOSYSTEM I ELECTRON TRANSPORT. INVOLVEMENT OF MEMBRANE STRUCTURAL CHANGES

DCMU-induced stimulation of the rate of photosystem I (PS I) electron transport in DCIPH₂→ MV photoreaction occurs through the action of DCMU on the rate-limiting step which contains the site of electron donation of DCIPH₂ (Ramanujam et al. , 1981). The magnitude of stimulation of the rate by 50 μ M DCMU decreased with increasing concentration of chlorophyll (Chl), implying that DCMU is stoichiometrically related to Chl with respect to the stimulation of the PS I rate.
DCMU-induced stimulation was sensitive to the ionic condition of the thylakoids, the effect being reduced at low cation concentration. Cation-induced scattering changes in thylakoid suspension were partially reversed by DCMU, and the percent Chl in the 10 K fraction of the thylakoid decreased upon addition of DCMU, indicating that grana structure is disrupted by DCMU. Hydroquinone-mediated reduction of cytochrome f in thylakoids in the dark was accelerated in the presence of DCMU. The DCMU effect was not observed in isolated PS I particles.
It is concluded that DCMU binds to the thylakoid membranes and brings about structural changes leading to unstacking of the thylakoids accompanied by an altered interaction of the electron transfer chain components with the added electron donor. This binding of DCMU must have an affinity lower than the well-known binding of DCMU to photosystem II (PS II), because the concentration required is markedly higher.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

A MICROSECOND KINETIC STUDY OF THE PHOTOGENERATED MEMBRANE POTENTIAL OF BACTERIORHODOPSIN WITH A FAST RESPONDING DYE

Abstract— Bacteriorhodopsin is a light activated proton pump which generates proton and electric gradients across the cytoplasmic membrane of Halobacterium halobium. In this study, a dye whose fluorescence intensity responds rapidly to membrane potential was used to follow the evolution of the potential on liposomes reconstituted with bacteriorhodopsin, in the microseconds time domain. By comparing the formation kinetics of the potential to those of the long-lived intermediate species in the bacteriorhodopsin photocycle, M₄₁₂, both in H₂O and ₂H₂O suspensions, we can draw the following conclusion: the electric potential onset time is 20 μs after initiation of the illumination. The triggering of the potential is not the formation of the M₄₁₂ intermediate, which was hitherto considered to be the first species in the bacteriorhodopsin cycle which has an unprotonated Schiff base linkage at the retinal chromophore. Rather, the potential forms at the transition of the L₅₅₀ intermediate to the species X which precedes M₄₁₂ or even at the preceding conversion of K₅₉₀ to L₅₅₀.     

单电子转移活性自由基聚合制备支化聚丙烯酸甲酯的研究

以丙烯酸2-(2-溴异丁酰氧基)乙酯(BIEA)为引发剂单体(inimer),丙烯酸甲酯(MA)为单体,Cu0/CuBr2和N,N,N',N″,N″-五甲基二亚乙基三胺(PMDETA)为催化体系,二甲亚砜(DMSO)为溶剂,在常温(25℃)下通过单电子转移活性自由基聚合(SET-LRP)合成支化聚丙烯酸甲酯.聚合反应过程中,采用气相色谱(GC)、核磁共振(1H-NMR)和三检测体积排除色谱(TD-SEC)等测试手段跟踪分析和表征支化聚合物的结构.研究结果表明,采用SET-LRP方法,铜粉作为催化剂,常温下聚合反应就能快速进行,130 min之内MA的转化率已达99%以上,制备出高分子量支化聚合物.随着反应的不断进行,聚合物支化程度不断提高,相比较同分子量下的线型聚合物其黏度不断下降,Mark-Houwink特征常数α最小可达0.290.此外,低分子量聚合物组分随着反应不断减少,在高单体转化率下,聚合体系中以高支化度的聚丙烯酸甲酯为主.