两种不同红藻的R—藻红蛋白激发强度相关的皮秒（10^…

本文研究了分别从红藻多管藻和条斑紫菜中提取的两种不同光谱类型的Ｒ－藻红蛋白Ｒ－ｐｈｙｃｏｅｒｙｔｈｒｉｎ激发强度相关的皮秒（１０＾－１２秒）荧光衰减动力学过程。结果发现：随激光强增大,单重态－单重态激子湮灭发生（其衰减过程约为６０－８０皮秒）,并引起荧光量子产率下降。这两种Ｒ－藻红蛋白在相同光强激发下,表现出不同的单重态－单重态激子湮灭过程,主要因它们处于激发态的发色团数目不同所致。     

红藻条斑紫菜R-藻红蛋白晶体的时间分辨偏振荧光研究

本文在皮秒时域内研究红藻条斑紫菜Ｒ－藻红蛋白单晶在不同晶轴取向下的偏振荧光动力学过程。荧光衰减表现为二指数过程,即与色素之间的能量传递和激发平衡有关的快过程（τ１≤６０ｐｓ）和色素荧光跃迁过程（τ２～３００ｐｓ）;由于色素间的能量传递,荧光明显退偏振;由于晶体中色素的光学跃迁矩取向倾向于沿主晶轴方向分布,在不同的晶轴的取向下各向异性荧光衰减过程明显不同。     

红藻条斑紫菜R—藻红蛋白晶体的时间分辩偏振荧光研究

本文在皮秒时域内研究红藻条斑紫菜Ｒ－藻红蛋白单晶在不同晶轴取向下的偏振荧光动力学过程。荧光衰减减表现为二指数过程,即与色素之间的能量传递和激平衡有关的快过程和色素荧光跃迁过程;由于色素间的能量传递,荧光明显退偏振;由于晶体中色素的光学跃迁距取向倾向于沿主晶轴方向分布,在不同的晶轴的取向下各向异性荧光衰减过程明显不同。     

R－藻红蛋白──金（AUC4）和汞（PCMS）重原子衍生物的制备和分析

在获得适合Ｘ射线衍射分析用的Ｒ－藻红蛋白单晶体的基础上,用重原子浸泡法,将晶体浸入含重金属金和汞的０．０５ｍｏｌ／Ｌ磷酸钠－硫酸铵的母液内,经对晶体衍射点强度的分析．找出有明显变化的重原子衍生物。最终得到了与Ｒ－藻红蛋白晶体同晶型的含金和汞的两种重原子衍生物。用面探测仪分别收集了这两种重原子衍生物的衍射数据。通过差值Ｐａｔｔｅｒｓｏｎ图分析,分别确定了重金属金和汞的位置,经对重原子位置参数精化后,给出的品质因子结果表明,含金和汞的重原子衍生物均可被用于多对同晶置换法求解Ｒ－藻红蛋白晶体母体相角的计算。     

R－藻红蛋白──金（AUC4）和汞（PCMS）重原子衍生物的制备和分析

在获得适合Ｘ射线衍射分析用的Ｒ－藻红蛋白单晶体的基础上,用重原子浸泡法,将晶体浸入含重金属金和汞的０．０５ｍｏｌ／Ｌ磷酸钠－硫酸铵的母液内,经对晶体衍射点强度的分析。找出有明显变化的重原子衍生物。最终得到了与Ｒ－藻红蛋白晶体同晶型的含金和汞的两种重原子衍生物。用面探测仪分别惧了这两种重原子衍生物的衍射数据。通过差值Ｐａｔｔｅｒｓｏｎ图分析,分别确定了重金属金和汞的位置,经对重原子位置参数精化化,给     

~（60）Co－γ射线诱变钝顶螺旋藻的研究

用不同剂量的６０Ｃｏ－γ射线诱变钝顶螺旋藻Ｓｐｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ出发株（Ｓｐ）ＩＳ－９００１０,筛选获得两株抗高光抑制突变株（Ｓｐ）ＡＩｐ－９００１０和（Ｓｐ）ＡＩｐ－９００１１,然后比较出发株和突变株的一般形态和生理生化特性。出发株和突变株的一般形态有较大的差异,与出发株相比,两个突变株藻丝体显著变短,螺旋数目大大减小。出发株是对高光敏感的品系,而突变株表现明显的抗高光抑制,１３０００ｌｘ光强下出发株和两个突变株的代时分别为２９．４、２０．８和２２．２ｈ。（Ｓｐ）ＡＩｐ－９００１１的光合作用和呼吸作用与出发株相似,而（Ｓｐ）ＡＩｐ－９００１０明显地表现出高光合、低呼吸作用。突变株和出发株都属于中温品系,最适温度为２８℃,具有较广的温度适应范围（２３～３５℃）以及相同的耐盐性,但突变株的生长速率比出发株快、代时短。此外三者的蛋白质含量、氨基酸组成差别不大：（Ｓｐ）ＡＩｐ－９００１１的可溶性多糖比出发株减少４０％。而在（Ｓｐ）ＡＩｐ－９００１０中其含量提高６０％。     

神经节苷脂（Gangliosides）对人红细胞膜Ca~（2＋）－Mg~（2＋）ATPase的影响

神经节苷脂（Ｇａｎｇｌｉｏｓｉｄｅｓ）是红细胞膜Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的一种激活剂,这种激活作用也是依赖于Ｃａ~（２＋）存在。在２００μｍｏｌ／ＬＣａ~（２＋）存在的反应体系中,１００μｇ／ｍＬＧａｎｇｌｉｏｓｉｄｅｓ对Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的激活作用最大,为基本酶活性的１５０％以上。实验还发现ＣａＭ拮抗剂三氟拉嗪（ＴＦＰ）、粉防已碱（Ｔｅｔ）等也同样抑制Ｇａｎｇｌｉｏｓｉｄｅｓ的这种激活作用。其抑制的ＩＣ_（５０）值为２５μｍｏｌ／Ｌ和３０μｍｏ１／Ｌ;而此浓度下抑制剂存在的反应体系中,对Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的基本活性影响不大。     

人神经生长因子低亲和力受体（p~（75）NGFR）cDNA的克隆及其在神经细胞中表达活性的初步研究

利用酸性异硫氰酸胍－酚－氯仿一步法从人胎儿基底前脑中提取总ＲＮＡ,用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出人神经生长因子低亲和力受体ｐ~（７５）ＮＧＦＲ基因ｃＤＮＡ,在限制性内切酶ＳｍａⅠ存在下的连接体系中,将扩增出的ｃＤＮＡ片段克隆入ｐＵＣ１２的ＳｍａⅠ位或,经限制性内切酶ＥｃｏＲⅠ和ＰｓｔⅠ酶切鉴定是否插入以及ＨｉｎｄⅢ酶切鉴定方向。将重组质粒中的ｐ~（７５）ＮＧＦＲ的ｃＤＮＡ再次亚克隆至ｐＵＣ１２载体中后,以其双链ＤＮＡ为模板,用末端终止法测出其全部核苷酸顺序,证实其核苷酸编码的ｐ~（７５）ＮＧＦＲ除两个碱基突变外,其余与文献完全一致。完整的ｐ~（７５）ＮＧＦＲ的ｃＤＮＡ分两步克隆到逆转录病毒表达载体ｐＸＴ－１,经ＰＡ３１７包装细胞株体外包装后、收集病毒上清转染条件不死性大鼠小脑神经细胞系Ｒ２．初步结果表明转染了ｐ~（７５）ＮＧＦＲ的Ｒ２细胞株去除ＮＧＦ培养时出现程序化死亡的典型特征梯型ＤＮＡ带。     

Epstein－Barr病毒潜伏膜蛋白（LMP）基因在哺乳动物传代细胞中的表达

ＥＢ病毒潜伏膜蛋白（ＬＭＰ）是由病毒编码的主要的与病毒致宿主细胞潜伏感染有关的蛋白之一。我们用基因重组技术,把含有ＬＭＰ基因（ＢＮＬＦ１）３个外显子（ｅｘｏｎ）开放阅读框架（ＯＲＦ）的长１．８０ｋｂｐ的ＤＮＡ片段,和能分解ＨｙｇｒｏｍｙｃｉｎＢ的含有ＳＶ４０早期启动子和ＨｇｒｙｏｍｙｃｉｎＢ磷酸转移酶全基因（长１０２５ｂｐ）的ＤＮＡ片段（长１．６０ｂｐ）,同时重组于亚克隆载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ（ｐＢＳ）中,并使该重组质粒ｐＢＳ－ＬＭＰ－Ｈｙｇ（长５７６７ｂｐ）在乳地鼠肾传代细胞（ＢＨＫ）中获得表达。ＢＨＫ细胞在经此重组质粒转染后,ＬＭＰ阳性细胞是２％,在ＨｙｇｒｏｍｙｃｉｎＢ的持续压力下,ＬＭＰ表达细胞率可达２０％。３个月后,ＬＭＰ表达细胞逐渐减少。５个月后,不能测到ＬＭＰ表达细胞。经免疫荧光和蛋白印迹（Ｗｅｓｔｅｍｂｌｏｔ）实验证实,人鼻咽癌、风湿性关节炎和正常人血清中不含有抗ＬＭＰ抗体。     

白介素10的临床应用研究

１９８９年,Ｆｉｏｒｅｎｔｉｏ等发现Ｔｈ２细胞分泌一种能抑制Ｔｈ１细胞功能的未知因子,称为细胞因子合成抑制因子（ｃｙｔｏｋｉｎｅｓｙｎｔｈｅ－ｓｉｓｉｎｈｉｂｉｔｏｒｙｆａｃｔｏｒ,ＣＳＩＦ）。Ｍｏｏｒｅ等发现ＣＳＩＦ与ＥＢ病毒（ＥＢＶ）中ＢＣＲＦ－...     

Bimolecular quenching of excitons and fluorescence in the photosynthetic unit.

The recent results of Campillo et al. and Mauzerall on the quenching of the fluorescence of chlorophyll a in Chlorella pyrenoidosa as a function of the intensity of the laser excitation pulses are rationalized by applying a model invoking singlet-singlet exciton annihilation.     

Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach.

A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.     

Theory of exciton annihilation in complexes of a finite number of molecular sites

A theory of the kinematics of singlet exciton annihilation in complexes of a finite number of molecular sites is developed. The theory is based on a specific scheme suggested earlier by Gülen, Wittmershaus, and Knox [Biophys J. 49:469-477 (1986)]. It is adequate to address the excitation kinetics and dynamics in such systems, especially under high excitation intensities. A Pauli master equation is formulated and is solved to give explicit expressions for observables such as quantum yield and fluorescence intensity. The excitation intensity dependence of the observables is taken into account by introducing Poisson statistics. Details relevant to its application to the annihilation of excitons in photosynthetic systems and its connection to earlier theories are presented.     

Theory of Picosecond-Laser-Induced Fluorescence from Highly Excited Complexes with Small Numbers of Chromophores

The problem of singlet excitation kinetics and dynamics, especially at high excitation intensities, among a small number of chromophores of a given system has been addressed. A specific scheme for the kinetics is suggested and applied to CPII, a small chlorophyll (Chl)a/b antenna complex the fluorescence lifetime of which has been reported to be independent of excitation intensity over a wide intensity range of picosecond pulses. We have modeled the kinetics from the point of view that Chla molecules in CPII are Förster coupled so that a second excitation received by the group of Chla's either creates a state with two localized excitons or raises the first one to a doubly excited state. The data on CPII can be understood on the basis of a kinetic model that does not exclude exciton annihilation during the excitation pulse. The implied annihilation rate is consistent with our theoretical estimates of that rate obtained by applying excitation transfer theory to pairs of molecules both initially excited.     

Picosecond fluorescence of cryptomonad biliproteins. Effects of excitation intensity and the fluorescence decay times of phycocyanin 612, phycocyanin 645, and phycoerythrin 545.

The fluorescence of purified biliproteins (phycocyanin 645, phycocyanin 612, and phycoerythrin 545) from three cryptomonads, Chroomonas species, Hemiselmis virescens, and Rhodomonas lens, and C-phycocyanin from Anacystis nidulans has been time resolved in the picosecond region with a streak camera system having less than or equal to 2-ps jitter. The fluorescence lifetimes of phycocyanins from Chroomonas species and Hemiselmis virescens are 1.5 +/- 0.2 ns and 2.3 +/- 0.2 ns, respectively, regardless of the fluence of the 30 ps, 532-nm excitation pulse. (Fluence [or photons/cm2] = f intensity [photons/cm2s]dt.). In contrast, that of C-phycocyanin is 2.3 +/- 0.2 ns when the excitation fluence is 8.2 X 10(11) photons/cm2 and decreases to a decay approximated by an exponential decay time of 0.65 +/- 0.1 ns at 7.2 X 10(16) photons/cm2. The cryptomonad phycoerythrin fluorescence decay lifetime is also dependent on intensity, having a decay time of 1.5 +/- 0.1 ns at low fluences and becoming clearly biphasic at higher fluences (greater than 10(15) photons/cm2). We interpret the shortening of decay times for C-phycocyanin and phycoerythrin 545 in terms of exciton annihilation, and have discussed the applicability of exciton annihilation theories to the high fluence effects.     

Energy transfer and fluorescence mechanisms in photosynthetic membranes

Energy transfer in photosynthetic membranes involves the migration of excitons from light‐harvesting antenna chlorophyll‐protein complexes to the reaction center complexes. Recent efforts have focused on determining the time of arrival of excitons (trapping times) at the reaction centers following excitation with a single picosecond laser pulse. Three different approaches have been utilized: (1) determination of appearance of separated charges within the reaction centers by differential absorbtion spectroscopy, (2) determination of appearance of separated charges by fast photoemf measurements, and (3) kinetics of decay of fluorescence. The first two methods provide more direct information on exciton trapping by reaction centers than fluorescence methods, but are experimentally difficult to realize. Therefore, much activity has centered around the accurate measurement and analysis of fluorescence‐decay profiles by single‐photon counting methods. In green plants, about three different components with lifetimes of about 100 psec, 200 to 500 psec, and >1 nsec, have been reported. The first two components are believed to be related to trapping rates by reaction centers, while the third component is attributed to a charge recombination (Klimov) mechanism. Results from photoemf and exciton‐exciton annihilation experiments are consistent with the interpretation that the first decay component reflects exciton‐trapping rates. A critical analysis and discussion of these fast energy‐transfer phenomena in photosynthetic membranes of green plants are offered in this review.     

Lifetime of fluorescence from light-harvesting chlorophyll a/b proteins. Excitation intensity dependence.

The fluorescence from a purified, aggregate form of the light-harvesting chlorophyll a/b protein has a lifetime of 1.2 +/- 0.5 ns at low excitation intensity, but the lifetime decreases significantly when the intensity of the 20-ps, 530-nm excitation pulse is increased above about 10(16) photons/cm2. A solubilized, monomeric form of the protein, on the other hand, has a fluorescence lifetime of 3.1 +/- 0.3 ns independent of excitation intensity from 10(14)-10(18) photons/cm2/pulse. We interpret the lifetime shortening in the aggregates and the lack of shortening in monomers in terms of exciton annihilation, facilitated in the aggregate by the larger population of interacting chlorophylls.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10¹³ photons · cm^?2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10¹³–10¹⁶ photons · cm^?2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Excitation energy transfer and charge separation in photosystem II membranes revisited

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation.