Photoinhibition as a function of the ambient redox potential in Tris-washed PS II membrane fragments

The mode of photoinhibition as a function of the ambient redox potential (E_ambient) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, ΔA^total ₈₃₀, as a measure of the capacity to form the `stable' radical pair, P680^+ċ Q^−ċ _A, drastically decreases during a 10 min photoinhibition at E_ambient values below +350 mV; (b) conversely, the normalized extent of the 18 μs relaxation kinetics, ΔA^{18 μ s} ₈₃₀ as a measure of the electron transfer from Y_Z to P680^+ċ becomes highly susceptible to light stress when E_ambient exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K₃[Fe(CN)₆] and K₄[Fe(CN)₆] in the suspension during photoinhibition but rather depends on the E_m-values; (e) qualitatively similar features are observed when the redox buffer system K₃[Fe(CN)₆]/Na₂S₂O₄ is replaced by K₂[IrCl₆]/Na₂S₂O₄; (f) the characteristic E_ambient-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559. This revised version was published online in June 2006 with corrections to the Cover Date.     

Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation

Transient expression of liposome-encapsulated DNA in liver after intravenous injection to rats and mice has raised questions concerning the intracellular fate of this DNA. Electron microscope autoradiography shows that at 10 min after injection the highest concentration of liposomal DNA which is taken up by the liver is associated with lysosomes and vesicles. The proportion of DNA associated with the mitochondria steadily increases for 1 h after injection, up to 48% of the exogenous DNA found in the tissue. Part of this DNA follows the subcellular fractionation profile of the mitochondrial matrix marker, malate dehydrogenase. In contrast, 14% of the liposomal DNA taken up by the liver is found in the nuclei at 3 min after injection, and this percentage decreases over a period of 1 h. These results permit us to establish the distribution of liposome-encapsulated DNA among subcellular organelles in liver at different times after injection.     

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39 μm along the lateral direction and 1.3 μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94 K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90 K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790 nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800 nm spectral region.     

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia: II. Biochemistry and spectroscopy

A novel chlorophyll a containing pigment–protein complex expressed by cells of Chromera velia adapted to growth under red/far-red illumination [1]. Purification of the complex was achieved by means of anion-exchange chromatography and gel-filtration. The antenna is shown to be an aggregate of ~ 20 kDa proteins of the light–harvesting complex (LHC) family, unstable in the isolated form. The complex possesses an absorption maximum at 705 nm at room temperature in addition to the main chlorophyll a maximum at 677 nm producing the major emission band at 714 nm at room temperature. The far-red absorption is shown to be the property of the isolated aggregate in the intact form and lost upon dissociation. The purified complex was further characterized by circular dichroism spectroscopy and fluorescence spectroscopy. This work thus identified the third different class of antenna complex in C. velia after the recently described FCP-like and LHCr-like antennas. Possible candidates for red antennas are identified in other taxonomic groups, such as eustigmatophytes and the relevance of the present results to other known examples of red-shifted antenna from other organisms is discussed. This work appears to be the first successful isolation of a chlorophyll a-based far-red antenna complex absorbing above 700 nm unrelated to LHCI.     

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates a variety of lysophospholipids including bis(monoacylglycero)phosphate

When the lysoglycerophospholipid (GPL) acyltransferase At1g78690 from Arabidopsis thaliana is over-expressed in Escherichiacoli a headgroup acylated GPL, acyl phosphatidylglycerol (PG), accumulates despite that in vitro this enzyme catalyzes the transfer of an acyl chain from acyl-CoA to the sn-2 position of 1-acyl phosphatidylethanolamine (PE) or 1-acyl PG to form the sn-1, sn-2, di acyl PE and PG respectively; it does not acylate PG to form acyl PG. To begin to understand why the overexpression of a lyso GPL acyltransferase leads to the accumulation of a headgroup acylated GPL in E. coli we investigated the headgroup specificity of At1g78690. Using membranes prepared from E. coli overexpressing At1g78690, we assessed the ability of At1g78690 to catalyze the transfer of acyl chains from acyl-coenzyme A to a variety of lyso GPL acyl acceptors including lyso-phosphatidic acid (PA), -phosphatidylcholine (PC), -phosphatidylserine (PC), -phosphatidylinositol (PI) and three stereoisoforms of bis(monoacylglycero)phosphate (BMP). The predicted products were formed when lyso PI and lyso PC were used as the acyl acceptor but not with lyso PC or lyso PA. In addition, At1g78690 robustly acylates two BMP isoforms with sn-2 and/or sn-2′ hydroxyls in the R-stereoconfiguration, but not the BMP isoform with the sn-2 and sn-2′ hydroxyls in the S-stereoconfiguration. This strongly suggests that At1g78690 is stereoselective for hydroxyls with R-stereochemistry. In addition, this robust acylation of BMPs by At1g78690, which yields acyl PG like molecules, may explain the mechanism by which At1g78690 so strikingly alters the lipid composition of E. coli.     

重楼皂甙Ⅱ对狼疮性肾炎患者外周血CD4~＋CD25~＋T调节细胞表达的细胞因子的影响

目的：研究重楼皂甙Ⅱ对狼疮性肾炎患者外周血CD4＋CD25＋Treg分泌的细胞因子IL-10和TGF-β的影响。方法：10例健康人为正常对照组,20例LN病人随机分为四组：LN对照组（n=5）、重楼干预组1（n=5,接种细胞数为1×105/1.5ml/孔）、重楼干预组2（n=5,接种细胞数为2×105/1.5ml/孔）、重楼干预组3（n=5,接种细胞数为5×105/1.5ml/孔）。分离外周血单个核细胞,利用免疫磁珠法分离CD4＋CD25＋Treg。重楼干预组予重楼皂甙Ⅱ干预,正常对照组和LN对照组不予处理,各组培养72h。取各组上清,用ELISA分别检测IL-10和TGF-β的水平。结果：与正常组比较,其余各组TGF-β和IL-10的水平明显降低（P〈0.01）。与LN对照组比较,重楼皂甙Ⅱ干预后各组TGF-β和IL-10的水平升高（P〈0.05）。重楼干预组之间比较,重楼干预组2的TGF-β和IL-10水平较其他两组明显升高（P〈0.01）,而重楼干预组1和3的TGF-β和IL-10水平变化无显著性差异（P〉0.05）。结论：重楼皂甙Ⅱ可上调LN患者TGF-β和IL-10的水平,上调的幅度受接种细胞数量的影响。     

急性肺损伤患者血清高迁移率族蛋白B-1与APACHEII的相关性研究

目的：研究急性肺损伤后血清高迁移率蛋白B-1（HMGB-1）的水平变化,并探讨其与APACHEⅡ评分的相关性。方法：测定10例正常成人与40例急性肺损伤病人伤后第1、4、7天的血清HMGB-1水平,同时评定其APACHEⅡ分值。在此基础上进行统计学分析,了解HMGB-1水平变化与-APACHEⅡ分值的相关性。结果：以APACHEⅡ20为分组界限,急性肺损伤组伤后第4、7天HMGB-1水平与APACHEⅡ明显相关（P〈0.01）。而创伤后第1天,HMGB-1水平与APACHEⅡ评分无显著相关性（P〉0.05）。结论：伤后第4、7天的HMGB-1水平与APACHEⅡ评分显著相关（P〈0.01）,常规检测创伤后血清HMGB-1水平并联合评定APACHEⅡ评分有助于对创伤后脏器功能不全的预测。     

Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions

The close association of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes (PMNs) and other apoptotic cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, ammonium ions inhibit in a concentration-dependent manner the hypochlorous acid-mediated formation of aldehyde and nitrile products from 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS). Concomitantly, the formation of monochloramine (NH₂Cl) raises with increasing NH₄⁺ concentrations. A transchlorination from monochlorinated O-phospho-l-serine to NH₄⁺ with the formation of NH₂Cl occurs only when extraordinary high NH₄⁺ concentrations are applied. Due to the low rate of 0.044 M^− 1 s^− 1 for this process, a transhalogenation reaction from transient chlorinated intermediates of the serine moiety to NH₄⁺ can be ruled out as an important process contributing to the HOCl-mediated formation of NH₂Cl. A significant formation of NH₂Cl by myeloperoxidase interacting with DPPS in the presence of ammonium ions takes only place at acidic pH values around 5, a scenario that may occur in phagosomes of macrophages after the uptake of apoptotic PMNs.     

Common basis for the mechanism of metallo and non-metallo KDO8P synthases

The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (P_i) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggest that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of “minimalist evolution”. Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains.