Early Picosecond Events in the Photocycle of Bacteriorhodopsin

The primary processes of the photochemical cycle of light-adapted bacteriorhodopsin (BR) were studied by various experimental techniques with a time resolution of 5 × 10^-13 s. The following results were obtained. (a) After optical excitation the first excited singlet state S₁ of bacteriorhodopsin is observed via its fluorescence and absorption properties. The population of the excited singlet state decays with a lifetime τ₁ of ~0.7 ps (430 ± 50 fs) (52). (b) With the same time constant the first ground-state intermediate J builds up. Its absorption spectrum is red-shifted relative to the spectrum of BR by ~30 nm. (c) The second photoproduct K, which appears with a time constant of τ₂ = 5 ps shows a red-shift of 20 nm, relative to the peak of BR. Its absorption remains constant for the observation time of 300 ps. (d) Upon suspending bacteriorhodopsin in D₂O and deuterating the retinal Schiff base at its nitrogen (lysine 216), the same photoproducts J and K are observed. The relaxation time constants τ₁ and τ₂ remain unchanged upon deuteration within the experimental accuracy of 20%.     

Non-Isomerizable Artificial Pigments: Implications for the Primary Light-Induced Events in Bacteriorhodopsin

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the critical C₁₃=C₁₄ bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C₁₃=C₁₄ torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C₁₃=C₁₄ isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.     

细菌视紫红质的光电响应特性和机制

在ITO导电玻璃上制备定向细菌视紫红质 (BR)电泳沉积膜或LB膜组成光电池系统 ,在短脉冲激光照射下 ,测定其脉冲响应光电压 ;在间断光照射下 ,测定其对光强变化产生的微分响应信号。对脉冲光电响应和微分响应的机理及其关系进行理论分析和解释 ,认为脉冲响应是BR分子内部生色团快速光极化引起的电荷分离和希夫碱及其周围氨基酸去质子化和再质子化过程引起的质子定向运输产生的位移电流 ,是一个快反应过程 ,是微分响应的早期反应和基础。微分响应则是由于菌紫质的光驱动质子泵产生的连续质子流在光开和光关瞬间引起光电池系统充放电以及测量电路的耦合特性引起的 ,是一个慢变化过程     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

Internal Water Molecules as Mobile Polar Groups for Light-Induced Proton Translocation in Bacteriorhodopsin and Rhodopsin as Studied by Difference FTIR Spectroscopy

FTIR spectroscopy is advantageous for detecting changes in polar chemical bonds that participate in bacteriorhodopsin function. Changes in H-bonding of Asp85, Asp96, the Schiff base, and internal water molecules around these residues upon the formation of the L, M, and N photo-intermediates of bacteriorhodopsin were investigated by difference FTIR spectroscopy. The locations and the interactions of these water molecules with the amino acid residues were further revealed by use of mutant pigments. The internal water molecules in the cytoplasmic domain probably work as mobile polar groups in an otherwise apolar environment and act to stabilize the L intermediate, and carrying a proton between the Schiff base and the proton acceptor or donor. Similar internal water molecules were shown to be present in bovine rhodopsin.     

Transient Features in Nanosecond Pulsed Electric Fields Differentially Modulate Mitochondria and Viability

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0–80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.     

光敏蛋白菌紫质固相支撑人工膜系统的界面电荷

以固相支撑的菌紫质人工膜系统,在分子电子器件研究中占有重要地位。本文对其两种类型,Ｎ固相型和Ｃ固相型的界面电荷进行了研究,并提出了相关的模型。     

Gene Expression and Signal Transduction in Water-Stress Response

We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8-12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.     

Hormonal Relations in the Phototropic Response IV. Light-Induced Changes of Endogenous Auxins in the Coleoptile

Beside indoleacetic acid (IAA), 3 auxins were found by chromatographic resolution of acidic fractions of Avena and Zea coleoptile tips. One of these auxins, designated P, occurred at levels of activity approaching those of IAA. The other 2 auxins, termed F and M, occurred at lower levels of activity. When the auxins of the excised coleoptile tips were isolated immediately after equilateral or unilateral irradiation with blue light at first positive energies, the ratio of IAA to the other auxins increases. This rise is the result of a decrease in P and F, and probably an increase in IAA. Light did not affect materially the total auxin content. It is suggested that P and F might be associated with the basipetal transport inequalities of IAA in phototropism.

P has been partially characterized. Its R_F on chromatograms developed in ammoniacal isopropanol is about 0.65. It is converted to IAA in vitro by heat. The ultraviolet absorption spectrum of chromatographically resolved P also suggests an indolyl complex. P is not readily transported basipetally, and the slope of its relative concentration-response curve (Avena section test) is lower than that of IAA. P does not appear to be any of the chemically characterized native auxins.

     

Cutaneous Papilloma and Squamous Cell Carcinoma Therapy Utilizing Nanosecond Pulsed Electric Fields (nsPEF)

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7–30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.     

Basic Fibroblast Growth Factor Expression is Implicated in Mesenchymal Stem Cells Response to Light-Induced Retinal Injury

Neurotrophic factors are involved in neuroprotection and its expression in mesenchymal stem cells (MSCs) may change during light-induced retinal injury. In this study, neurotrophic factor expression in MSCs was investigated after stimulation by supernatants of homogenized retina (SHR) from normal and light-injured rats. Conditioned media from control MSCs (CM-MSCs), MSCs stimulated by normal SHR (CM-NSHR), and MSCs stimulated by light-injured SHR (CM-ISHR) were examined regarding their ability to prevent degeneration of retinal explants. Basic fibroblast growth factor (bFGF) in MSCs was knockdown by lentivirus-mediated mRNA interference. Transfected MSCs were stimulated by SHR, and retinal preservation was reevaluated in the resultant conditioned media. We detected significant up-regulation of bFGF in CM-ISHR, accompanied by superior retinal neurotrophic effects in CM-ISHR over CM-NSHR and CM-MSCs. Down-regulation of bFGF in MSCs effectively inhibited this protective effect. Adding neutralizing antibody against bFGF to CM-ISHR also induced a similar effect. It is thus concluded that retinal injury may enhance neurotrophic factor expression in MSCs and promote the repair process. bFGF may play a critical role in MSCs’ response to retinal injury.     

Combination of Microsecond and Nanosecond Pulsed Electric Field Treatments for Inactivation of Escherichia coli in Water Samples

Inactivation of microorganisms with pulsed electric fields is one of the nonthermal methods most commonly used in biotechnological applications such as liquid food pasteurization and water treatment. In this study, the effects of microsecond and nanosecond pulses on inactivation of Escherichia coli in distilled water were investigated. Bacterial colonies were counted on agar plates, and the count was expressed as colony-forming units per milliliter of bacterial suspension. Inactivation of bacterial cells was shown as the reduction of colony-forming units per milliliter of treated samples compared to untreated control. According to our results, when using microsecond pulses the level of inactivation increases with application of more intense electric field strengths and with number of pulses delivered. Almost 2-log reductions in bacterial counts were achieved at a field strength of 30 kV/cm with eight pulses and a 4.5-log reduction was observed at the same field strength using 48 pulses. Extending the duration of microsecond pulses from 100 to 250 μs showed no improvement in inactivation. Nanosecond pulses alone did not have any detectable effect on inactivation of E. coli regardless of the treatment time, but a significant 3-log reduction was achieved in combination with microsecond pulses.     

Separation of Light-Induced [C]ent-Kaurene Metabolism and Light-Induced Germination in Grand Rapids Lettuce Seeds

The effect of light on the metabolism of [¹⁴C]kaurene in light-requiring lettuce seeds (Lactuca sativa L. cv Grand Rapids) was investigated. Seeds were soaked in a solution of [¹⁴C]ent-kaurene in methylene chloride with 0.01% Tween-20, dried, and incubated in 20% polyethylene glycol (PEG) to prevent seedling development. Labeled metabolites were extracted and analyzed by high performance liquid chromatography and gas chromatography-radio counting. [¹⁴C]ent-Kaurenol and [¹⁴C]ent-kaurenal were identified in seeds incubated in constant white light, while no ethyl acetate-soluble metabolites were found in seeds incubated in the dark. In time course experiments using acid scarified seeds, metabolism began after 18 hours of incubation and greatly increased after 24 hours of incubation in 20% PEG. By 48 hours, several unidentified, more polar metabolites were found. Germination was induced in seeds imbibed in 20% PEG by 4 hours of red or 4 hours of white light following 20 hours in the dark, and was fully reversed by 2 hours of far red light. However, in metabolism experiments, [¹⁴C]ent-kaurene oxidation was observed only with constant white light. These results indicate that although ent-kaurene oxidation is a light sensitive step in the biosynthesis of gibberellins in Grand Rapids lettuce seeds, ent-kaurene metabolism is not required for light-induced germination.     

Effects of Inhibitors on the Light-Induced Changes in Electric Potential in Pulvinar Motor Cells of Phaseolus vulgaris L.

To analyze the mechanism of the light-induced changes in electricpotential in motor cells of the pulvinus of Phaseolus vulgarisL., inhibitors were applied to the pulvinus by the xylem perfusionmethod. The membrane potential was –60 to –80 mV,which indicated that the polarization was less than that ofcells of a pulvinus in air. A pulse (30 s) of blue light (BL)induced transient depolarization of the membrane in the motorcells. Red light (RL) caused hyperpolarization of the membrane.The magnitude of BL pulse-induced transient depolarization wasgreater under the hyperpolarized state caused by the RL. The membrane was depolarized to –30 to –40 mV onperfusion with the respiratory inhibitor NaN₃ (1 mM) and a pulseof BL or irradiation with RL did not cause any change in thepotential in the depolarized state. Hyperpolarization of themembrane by RL was inhibited by perfusion with DCMU (50 µM),an inhibitor of electron transport in photosynthesis. However,the magnitude of the depolarization induced by the pulse ofBL was not affected. Perfusion with a proton ionophore CCCP(100µM) depolarized the membrane and no change in thepotential was induced by a pulse of BL or by RL in the depolarizedstate. The extent of the BL pulse-induced depolarization of the membranewas proportional to the magnitude of the membrane potentialat the time of which the pulse of BL was applied. It is suggestedthat the active component of the membrane potential was inhibitedby the pulse of BL. The experimental results further supportthe hypothesis that BL inhibits the activity of the proton ATPaseand, thus, causes loss of the electrogenic component of themembrane potential of the pulvinar motor cells. (Received June 22, 1992; Accepted August 24, 1992)     

机械伤害诱导的植物防御机制和信号转导

茉莉酸是植物伤反应的特异激素, 在植物伤反应中具有核心作用, 其下游调控机制已经比较清晰。在番茄(Lycopersicon esculentum)伤反应中, 系统素和茉莉酸协同启动相关基因的表达, 行使系统性防御功能。拟南芥(Arabidopsis thaliana)信号肽是新发现的一类信号物质, 可以激活植物的初始免疫反应, 但其在伤反应中的作用机制有待进一步研究。脱落酸位于茉莉酸上游, 单独或者协同茉莉酸参与植物的防御反应。另外, 植物中还存在以核糖核酸酶为代表的且不依赖于茉莉酸的伤反应信号转导途径。该文对植物伤反应的防御机制和信号转导做了详细概述。     

Oxygen- and Light-Induced Proteolysis in Isolated Oat Chloroplasts

The influence of light and O₂ on the degradation of proteinsin isolated oat chloroplasts was studied by SDS-polyacrylamidegel electrophoresis. Proteolysis increased as irradiance andthe concentration of O₂ increased. Dark treatments preventedprotein degradation. Neutral and alkaline aminopeptidase activitiesand neutral endopeptidase activity decreased with the time andwith the increase in the concentration of O₂. However, acidendopeptidase activity increased as the level of O₂ increased,accounting, at least in part, for the proteolysis observed underhigh irradiance and high concentrations of O₂. Acid endopeptidaseactivity was strongly associated with thylakoid membranes. Treatmentwith H₂O₂ increased thylakoid-bound acid endopeptidase activitybut had no effect on the solubilized activity. It is suggestedthat active species of oxygen generated in illuminated chloroplastsmay enhance proteolysis by inducing alterations in membraneswhich, in turn, increase the endopeptidic activity. (Received October 18, 1989; Accepted February 19, 1990)     

罂粟科植物自交不亲和反应中信号转导的研究进展

自交不亲和性是植物特异性地识别并拒绝自花或亲缘关系很近的花粉的一种遗传机制,该特异性受S基因座控制.在前人的研究基础上总结了罂粟科植物自交不亲和反应过程中信号转导的研究进展,将参与其信号级联反应的信号分子分为与S-位点连锁(包括花柱S-蛋白和花粉S-受体)和不连锁的信号分子(Ca2+、p26、p68、MAPK、细胞骨架以及PCD),并综述了各个信号分子之间的相互作用.