Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.     

斑点嗜蓝孢孔菌化学成分、生物活性及水提物荧光猝灭研究

对斑点嗜蓝孢孔菌乙醇提取物进行分级萃取,利用柱层析技术得到6个单体化合物。经核磁数据比对为β-谷甾醇(1)、麦角甾醇(2)、麦角甾-7,22-二烯-3β-醇(3)、5,8-过氧麦角甾-6,22-二烯-3β-醇(4)、3-乙酰齐墩果酸(5)、白桦脂醇(6)。采用Alamar Blue法检测各有机相及单体化合物对NCI-H460人非小细胞肺癌细胞增殖的影响,用DPPH自由基清除法检测其抗氧化活性。利用荧光光谱及紫外-可见光光谱研究方法在模拟人体生理条件下,研究了斑点嗜蓝孢孔菌水提物与人纤维蛋白原的相互作用,实     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y_D^ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S₂Q_A⁻ and S₂Q_B⁻ charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y_D⁺Q_A⁻ recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S₁-to-S₂-transition and S₂-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q_A to Q_B electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

Photoprotection and xanthophyll‐cycle activity in three marine diatoms1

Light is one of the most important factors affecting marine phytoplankton growth, and its variability in time and space strongly influences algal performance and success. The hypothesis tested in this work is that the activity of the xanthophyll cycle and the development of nonphotochemical quenching could be considered a functional trait of algal diversity. If this hypothesis is true, a relationship must exist between fast‐activated pigment variations linked to photoprotective behavior and the ecology of the species. This assumption was tested on three diatoms: Skeletonema marinoi Sarno et Zingone, Thalassiosira rotula Meunier, and Chaetoceros socialis Lauder. These three diatoms occupy different ecological niches. Strains of these diatoms were subjected to five changes in irradiance. Xanthophyll‐cycle activity, quantum yield of fluorescence, and electron transport rate were the main parameters determined. There were marked interspecific differences in xanthophyll‐cycle activity, and these differences were dependent on the light history of the cells. Chaetoceros socialis responded efficiently to changing irradiance, which might relate to its dominance during the spring bloom in some coastal areas. In contrast, T. rotula responded with a slower photoprotection activation, which seems to reflect its more offshore ecological distribution. The photoresponse of S. marinoi (a late‐winter coastal species blooming in the Adriatic Sea) was light‐history dependent, becoming photoinhibited under high light when acclimated to low light, but capable of reaching a high photoprotection level when acclimated to moderate light. Our hypothesis on the photoprotection capacity as a functional trait in microalgae seems to be validated given the results of this study.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Function of plastoquinone in heat stress reactions of plants

The effect of high temperature treatment (40 °C, 3 h, illumination at 100 μmol m^− 2 s^− 1) on the photosynthetic electron flow in barley seedlings of different age was investigated. Thermoinduced inhibition of the liner electron flow due to partial impairment of the water oxidizing complex (WOC) and the increase in the extent of Q_A⁻ reoxidation by Tyr_z^ox in thylakoids isolated from 4-day-old leaves was shown by measurements of oxygen evolution using benzoquinone or potassium ferricyanide as electron acceptors, as well as by following Q_A⁻ reoxidation kinetics in the absence and presence of exogenous electron acceptors, DCBQ and DMBQ. Using HPLC analysis, an increase in the oxidation of the photoactive plastoquinone pool in young leaves under heating was shown. In older, 11-day-old leaves, heat treatment limited both photosynthetic electron flow and oxygen evolution. The same effects of heat shock on oxygen evolution caused an inhibition of electron flow on the donor side of PSII only. However, a rise in the proportion of PSII with Q_A⁻ reoxidized through recombination with the S₂/S₃ state of the WOC was observed. The addition of exogenous electron acceptors (DCBQ and DMBQ) and a donor (DPC) showed that the thermoinduced decrease in the electron transport rate was caused by an impediment of electron flow from Q_A⁻ to acceptor pool. The decrease in size of the photoactive PQ-pool and a change in the proportions of oxidized and reduced PQ in older leaves under heat treatment were shown. It was suggested that a thermoinduced change of the redox state of the PQ-pool and a redistribution of plastoquinone molecules between photoactive and non-photoactive pools are the mechanisms which reflect and regulate the response of the photosynthetic apparatus under heat stress conditions.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.