Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

光声成像在光动力疗法研究中的应用

组织氧合作用和光敏剂应用在疾病诊治中都有着重要的作用,因此其实时在体无损检测很有意义。光动力疗法涉及光敏剂、光和氧分子三大要素,其疗效受组织氧合作用影响。本文对光声成像(PAI)、光声寿命成像(PALI)和多光谱光声层析成像(MSOT)等光声成像技术在光动力疗法的研究和应用中的使用现状进行了综述。对相关设备系统在检测光敏剂、组织氧分压和微血管损伤等方面的应用原理和技术分别进行了介绍,并总结了这些技术的应用前景。     

Time-resolved angular dependent measurement of triggered light scattering changes in biological suspensions

We describe a photometer for time-resolved measurements of small changes in light scattering suited for suspensions of biological material. The time resolution is 35 μs, the amplitude resolution for bovine rot outer segments is typically ΔI/I = 5 · 10^?4 at a scattering angle of ? = 20°. The use of the apparatus is demonstrated by recording the near infrared scattering of bovine rod outer segments after excitation with flashes of green light.Semiconductor detector arrays are arranged centrosymmetrically around a hemispherical cuvette. The optical characteristics of a hemispherical cuvette and the resulting geometry of cuvette and detection are discussed.Calculations of optimal signal transfer and noise of the detectors led to the following arrangement for each scattering angle: pairs of parallel connected photodiodes are fed into several current-to-voltage converters, whose output voltages are summed up by a summing amplifier.For the test of the device so-called N signals of fresh and liquid N₂-frozen and thawed ROS samples were measured at four scattering angles simultaneously. A strong angular dependence (difference scattering curve) of the relative light scattering change is seen for fresh ROS which is transformed into a flat curve by freezing and thawing. It is concluded that the competence of the fresh sample to extend the light-induced local events — presumably rhodopsin conformational changes — into the gross-structural range is terminated by freezing.     

Activation of cGMP phosphodiesterase by purified green rod pigment from frog retina

Activation of cGMP phosphodiesterase(PDE) of frog rod outer segments (ROS) by purified green rod pigment (GRP) was analyzed. GRP activated PDE in a similar manner to purified rhodopsin. This activation required illumination of the pigment and presence of GTP.     

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

应用ESR检测探讨急性热暴露大鼠肝脏氧化还原状态的动态变化

目的:通过电子顺磁自旋共振技术(ESR)动态观察大鼠在过热条件下肝脏的氧化还原状态.方法:将52只雄性Wistar大鼠随机分成4组:①加温组:麻醉后进行整体加温到直肠温达(43.0±0.5)℃,持续15 min;②对照组:只进行麻醉处理;③,MPG预处理组:用抗氧化剂MPG预处理后,再进行与上述①同样条件的加温处理;④非MPG预处理组:在③中用生理盐水代替MPG.经过以上处理后在不同时间点取肝脏制备组织匀浆,测定ESR波谱.结果:与对照组比较,加温组热暴露处理后记录的ESR波谱振幅-时间直线斜率增大,2 h达最大值.以后逐渐恢复,24 h接近对照组水平.经抗氧化剂预处理上述反应减弱.结论:过热能诱导肝脏产生活性氧,增强其氧化还原反应.     

Light regulation of the insulin receptor in the retina

The peptide hormone insulin binds its cognate cell-surface receptors to activate a coordinated biochemical-signaling network and to induce intracellular events. The retina is an integral part of the central nervous system and is known to contain insulin receptors, although their function is unknown. This article, describes recent studies that link the photobleaching of rhodopsin to tyrosine phosphorylation of the insulin receptor and subsequent activation of phosphoinositide 3-kinase (PI3K). We recently found a light-dependent increase in tyrosine phosphorylation of the insulin receptor-β-subunit (IRβ) and an increase in PI3K enzyme activity in isolated rod outer segments (ROS) and in anti-phosphotyrosine (PY) and anti-IRβ immunoprecipitates of retinal homogenates. The light effect, which was localized to photoreceptor neurons, is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IRβ in outersegment membranes, which leads to the binding of p85 through its N-terminal SH2 domain and the generation of PI-3,4,5-P₃. We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis. The studies linking PI3K activation through tyrosine phosphorylation of IRβ now provide physiological relevance for the presence of these receptors in the retina.     

Photoacoustic measurements of the energy-conversion efficiency of photosynthesis in thalli of the green alga Bryopsis maxima

The efficiency of photosynthetic energy conversion in thalli of the green alga Bryopsis maxima was studied with the photoacoustic technique. Photosynthetic O₂ evolution did not interfere with the photoacoustic measurements in this material, most probably owing to its coenocytic cellular organization. The energy yield (defined as the fraction of absorbed photon energy that is stored in photosynthetic products or intermediates relative to the total absorbed photon energy) was estimated from the photoacoustic signals by applying the background-illumination method to obtain a reference without the photochemical capacity (Lasser-Ross, N., Malkin, S. and Cahen, D. (1980) Biochim. Biophys. Acta 593, 330–341). With the monitoring light modulated at 60 Hz, photon energy is mainly stored by redox changes in electron-transport chains because the energy yield was strongly reduced by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and heat treatment of the thalli, whereas KCN, an inhibitor of CO₂ reduction, had no effect, and because a significant lowering of the energy yield occurred in the presence of methyl viologen but the effect of the Photosystem I acceptor was largely reversed on the addition of an uncoupler, methylamine. The maximum energy yield of 0.4 that was obtained with a saturating background light and with a sufficiently weak monitoring light modulated at 100 Hz is explained in terms of electron transfer from electron-donor pools to acceptor pools of the two photosystems with the quantum yield close to unity. A lowering of the modulation frequency decreased the energy yield, indicating that less energy is stored in more stable intermediates.     

鼠疫耶尔森菌毒力蛋白YopE新功能的初步研究

鼠疫耶尔森菌外部蛋白E（YopE）是鼠疫耶尔森菌的6种分泌蛋白之一,主要通过其144位的”精氨酸手指”结构与细胞膜耦联蛋白RhoGTP酶相互作用发挥功能.本文构建YopE及其144位突变体YopE(R144A)的可诱导表达系统,并优化了诱导条件. 用该系统结合流式细胞技术检测YopE和YopE(R144A)对细胞凋亡、细胞周期和细胞活性氧(ROS)水平的影响.结果显示：YopE(R144A)促进HeLa细胞凋亡|使G0/G1期细胞比例上升,G2/M期细胞比例下降;随着YopE(R144A)表达量增加,p21蛋白的表达量也增加| YopE(R144A)也能抑制细胞ROS的产生.研究结果提示,YopE在细胞内可能存在新的致病靶点.     

Photoacoustic detection of photosynthetic activities in isolated broken chloroplasts

Methodology and demonstration how to utilize the photoacoustic technique in photosynthesis research are presented. Photoacoustic signals were obtained from suspensions of isolated broken chloroplasts. In the presence of strong, continuous (non-modulated) background light the signals were normally larger than without the background light. The effect of the background light was saturable and was absent when non-active (e.g. heat-treated) samples were used, showing that the normal smaller signal in the absence of background light is a genuine reflection of the loss of heat due to the competing photochemistry. The effect of the background light is to close the reaction-centers and hence to inhibit the photochemical process. The percent difference of the photoacoustic signal (± background light) is taken as a measure of the photochemical activity (‘photochemical loss’).

Initial results demonstrate the wavelength dependence of the ‘photochemical loss’. As expected there was a ‘red-drop’ decrease of the ‘photochemical loss’ for λ > 690 nm, when the cofactor methyl viologen was present. Surprisingly, however, there was a ‘red-rise’ increase for λ > 690 nm when no cofactor was present. These findings indicate that under the last conditions there is an unsuspected photoactivity of PS I which was not detected hitherto by the conventional techniques. The dependence on the background light intensity confirms this result. This photoactivity can be explained tentatively as a cyclic electron flow around PS I, present without any added cofactor.

Initial results on the modulation frequency dependence in the presence of electron acceptors are also demonstrated.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with χ_POPC = 0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m^− 1 revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with χ_POPC = 0.4 the jump occurs at ∼ 800 pN. Widths of ∼ 2 nm could be established for POPC and χ_POPC = 0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC′) occurring at pressures > 36.5 mN m^− 1. This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force (∼ 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Hunting oxygen complexes of nitric oxide synthase at low temperature and high pressure

The reaction of nitric oxide synthase (NOS) with oxygen is fast and takes place within several steps, separated by ephemeral intermediates. The use of extreme experimental conditions, such as low temperature and high pressure, associated to rapid kinetic analysis, has proven to be a convenient tool to study this complex reaction. Stopped-flow experiments under high pressure indicated that oxygen binding occurred in more than one step. This was further corroborated by the detection of two short-lived oxy-compounds, differing in their spectral and electronic properties. Oxy-I resembles the ferrous oxygen complex known for cytochrome P450, whereas oxy-II appears to be locked in the superoxide form. Subzero temperature spectroscopy, together with an analytical separation method, revealed that the subsequent one-electron reduction of the oxygen complex is carried out by the NOS cofactor tetrahydrobiopterin (BH4). The low-temperature stabilized oxidation product of BH4 was found to be a protonated BH3 radical. Finally, work in the presence of a BH4 analog indicated that proton transfer to the activated oxygen complex is a second essential function of BH4.