辐射诱导的HeLa细胞旁效应信号通路研究

随着肿瘤认识的不断深入，肿瘤外科学、肿瘤放射治疗学、肿瘤化学治疗学构成了现代肿瘤治疗学的三大支柱，而放射治疗学的研究对于肿瘤的治疗有重要的临床意义。本文通过对HeLa进行辐射来观察其产生的旁效应信号通路，从而对临床工作的起到一定的帮助。辐照过的细胞通过释放信号分子引起周围未辐照细胞产生一系列的生物学反应的现象，称之为辐射诱导的旁效应。活性氧（Reactive oxygen species, ROS）和一氧化氮（Nitric oxide, NO）是辐射诱导的旁效应信号通路中的两个重要信号分子。本文研究了这两种重要的信号分子在辐射诱导的HeLa细胞旁效应信号通路中的关系。通过微核实验，我们发现X射线以及12C6+ 辐照过的HeLa细胞及其旁观者细胞的微核形成明显增加，而1%的二甲亚砜（Dimethyl sulfoxide，DMSO，ROS清除剂）预处理X射线辐照的细胞则显著抑制了受辐照细胞及其旁观者细胞的微核形成。1 Gy的X射线辐照能够抑制细胞的增殖速率而0.5%和1%的DMSO预处理则能减少X射线的增殖抑制作用，并且DMSO预处理的效果与浓度有关：1%的DMSO比0.5%的DMSO处理更大程度的恢复了受辐照细胞的增殖速率。另一方面，接受条件培养基（Conditioned medium）的旁观者细胞的增殖速率增加，而DMSO预处理产生条件培养基的受辐照细胞则使旁观者细胞的增殖速率降低，且DMSO预处理的效果同样与其浓度相关：浓度越高，条件培养基的刺激生长作用越小。Western blotting和DAF-FM DA荧光探针检测分别显示了辐照过后细胞的诱导型一氧化氮合酶（Inducible nitric oxide synthase, iNOS）和NO水平均升高，而DMSO预处理则降低其水平。因此，我们推测在X射线辐照的HeLa细胞旁效应信号通路当中ROS是NO的上游信号。另外，我们采用培养基转移后立即加DMSO或BMS－345541（IKK/NF-κB抑制剂）的方法研究了旁观者细胞当中的旁效应信号通路。我们发现DMSO和BMS－345541均显著抑制了旁观者细胞的NO水平。因此，在旁观者细胞当中ROS与NF-κB均为NO的上游信号

Production of active oxygen species from Taxus cuspidata induced by far-UV radiation

In order to understand the role of active oxygen species in mediating plant injuries induced by far-UV radiation, seedlings of Taxus cuspidata Sieb. et Zucc. were irradiated by far-UV rays in laboratory for 4 weeks. The production of organic free-radicals in detached needles, and the production of O-2(radical anion) and O-1(2) in isolated chloroplasts were detected weekly by electron spin resonance (ESR) to evaluate their relative importance. The results show that the cumulative effect of far-UV irradiation, is best indicated by the production of organic free radicals in the needles, O-2(radical anion) production in chloroplasts is the next. The enhancement of O-1(2) production in chloroplasts by the cumulative far-UV irradiation seems to be not so important as O-2(radical anion) in mediating injuries induced by, far-UV radiation because of its high background value.

A STUDY OF SURFACE OXYGEN SPECIES FROM LA-ME-O (ME=MG, CA, SR, BA) CATALYSTS FOR METHANE OXIDATIVE COUPLING

CO2-TPD was used to study the surface basicity of La-Me-O mixed oxides and O-2-TPD, CH4-TPD were employed to study the surface active oxygen species. Comparing the CO2-TPD with O-2-TPD, we can see that the basicity of catalyst is in parallel with the catalystic activity. The stronger basicity is more profitable for the catalyst to adsorb oxygen to form active oxygen species and to activate CH4 by breaking a C-H bond, By comparing the catalytic activity, the results showed that La-Ba-O(La/Ba=7/3) catalyst had the strongest basicity, and it gave the highest CH4 conversion and C-2 selectivity, The results from the pulse reaction showed that the lattice oxygen participated in the OCM reaction without gas oxygen, and it was the selective oxygen species.

A STUDY OF CATALYTIC PROPERTIES OF PEROVSKITE-TYPE OXIDES LAMNYCO1-YO3 .1. THE RELATIONSHIP BETWEEN THE TYPE OF OXYGEN SPECIES AND THE CATALYTIC PROPERTY IN AMMONIA OXIDATION

The type of oxygen species in perovskite-type oxides LaMnyCo1-yO3 (y = 0.0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0) has been studied by means of XRD, XPS and TPD. The catalytic activity in ammonia oxidation was also investigated. It was found that there were three desorption peaks in TPD curve corresponding to three types of oxygen species (alpha, beta, beta'). The desorption temperatures were 293 K less-than-or-equal-to T(alpha) less-than-or-equal-to 773 K, 773 K less-than-or-equal-to T(beta) less-than-or-equal-to K and T(beta') greater-than-or-equal-to 1073 K respectively. The relationship among the composition, structure and the catalytic property of.the catalyst was correlated and could be explainned with a model based on solid defect reaction and the interaction between Co and Mn ions. The adsorption strength and quantity of a oxygen are proportional to the catalytic activity. The, result indicates that the synergetic effect between B-site ions seems to the benefit of the ammonis oxidation reaction.

Examination of Antioxidative System's Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant Reaumuria soongorica (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H2O2), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H2O2 in the early phase of drought and enzymes of ascorbate-glutathione cycle scavenge H2O2 in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H2O2 under drought stress.

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Laminaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as Alterornonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that: (1) The blades of L. japonica exhibited symptoms of lesion, bleaching and deterioration when infected by the bacterium, and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species (ROS) generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde (MDA) and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. (2) The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase (SOD) and catalase (CAT) increased greatly when infected by the bacterium, but glutathione peroxidase (Gpx) and ascorbate peroxidase (APX) did not exhibit active responses to the bacterium throughout the experiment. (3) The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

Formation of subsurface oxygen species and its high activity toward CO oxidation over silver catalysts

Silver is well known to show peculiar catalytic activities in several oxidation reactions. In the present paper, we investigate the catalytic activity of silver catalysts toward CO-gelective oxidation in H-2. XRD, TEM, TPD, and in situ FTIR techniques were used to characterize the catalysts. The pretreatment of the catalysts was found to have great influence on their performance. The pretreatment in 02 improves the activity of the silver catalyst, whereas He pretreatment at 700 degreesC or direct hydrogen pretreatment shows an inverse effect. Silver catalysts undergo massive structural change during oxygen pretreatment at high temperatures (> 500 degreesC), and there is solid evidence for the formation of subsurface oxygen species. The existence of this silver-subsurface oxygen structure facilitates the formation of active sites on silver catalysts for CO oxidation, which are related to the size, morphology, and exposed crystal planes of the silver particles. Its formation requires a certain temperature, and a higher pretreatment temperature with oxygen is required for the silver catalyst with a smaller particle size. It is observed, for the first time, that adsorbed CO on the surface of silver particles can directly react with subsurface oxygen species at low temperatures (e.g., RT), and the surface oxygen can migrate into and refill these subsurface sites after the consumption of subsurface oxygen by the reaction with CO. This finding provides a new reaction pathway for CO oxidation on silver catalyst. (C) 2004 Published by Elsevier Inc.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: New insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily .

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range.The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg+2 binding first (Kd =140 ± 40 M), are kcat = 105 ± 2 s-1 and P-pyr Km = 5 ± 1 M. PEP (slow substrate kcat = 2 × 10-4 s-1), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 ± 0.1 mM, 17 ± 1 M, and 210 ± 10 M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (/)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

Generation of Atomic Oxygen in the Effluent of an Atmospheric Pressure Plasma Jet

The planar 13.56MHz RF-excited low temperature atmospheric pressure plasma jet (APPJ) investigated in this study is operated with helium feed gas and a small molecular oxygen admixture. The effluent leaving the discharge through the jet’s nozzle contains very few charged particles and a high reactive oxygen species��� density. As its main reactive radical, essential for numerous applications, the ground state atomic oxygen density in the APPJ’s effluent is measured spatially resolved with two-photon absorption laser induced fluorescence spectroscopy. The atomic oxygen density at the nozzle reaches a value of ~1016 cm-3. Even at several centimetres distance still 1% of this initial atomic oxygen density can be detected. Optical emission spectroscopy (OES) reveals the presence of short living excited oxygen atoms up to 10 cm distance from the jet’s nozzle. The measured high ground state atomic oxygen density and the unaccounted for presence of excited atomic oxygen require further investigations on a possible energy transfer from the APPJ’s discharge region into the effluent: energetic vacuum ultraviolet radiation, measured by OES down to 110 nm, reaches far into the effluent where it is presumed to be responsible for the generation of atomic oxygen.

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca2+ channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage. (c) 2006 by Radiation Research Society.