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Coal slurry pipe, being a new type of energy transportation mode, changes of the particle composition and the effect on other parameters are worth studying. Comparison of ring tube test measured data and batch grinding model simulation data shows that batch grinding model can predict the coal particle gradation to a certain extent. The effect of collision and wear of overcurrent components in the centrifugal pump, friction and collision between particles and piping and piping accessories, and initial particle size distribution during hydraulic conveying were also investigated by laboratory studies. Meanwhile, the effect of particle composition change on slurry viscosity, hydraulic resistance and minimum resistance critical velocity was studied based on the experimental data. The experimental and theoretical analysis indicates that the change of slurry particle composition can lead to the change of viscosity, resistance and minimum resistance critical velocity of the slurry. Moreover, based on the previous studies, the calculation model of minimum resistance critical velocity of coal slurry is proposed.

To improve the problem of turbulence in the forebay of the lateral inlet pumping station, a typical lateral inlet pumping station project in Xuzhou, Jiangsu Province, China was taken as the research object. The forebay of the pumping station is a building connecting the river channel and the pumping station into the water tank. Based on the Reynolds-averaged Navier-Stokes (RANS) law and the k-ε turbulence model, the computational fluid dynamics method (CFD) technology compares and analyzes the numerical simulation with or without rectification measures for the forebay of the lateral intake pumping station when multiple units are operating. The three-dimensional model was created by SolidWorks modeling software and the numerical simulation simulated by CFX-ANSYS. To alter the flow pattern in the forebay of the pumping station, various rectification measures were chosen. Internal rectification flow patterns in the forebay under multiple plans, uniformity of flow velocity distribution in the measuring section, and vortex area reduction rates are investigated and compared. Based on the analysis and comparison of numerical simulation results, when the parabolic wall and some rectification piers are set significantly it improves the flow pattern of the forebay of the lateral inlet pumping station. It also makes the flow pattern of the inlet pool better and increases the uniformity of the flow velocity distribution by 8%. Further, it reduced the vortex area by 70%, effectively improving the operating efficiency of the pump. The research results of this paper provide a technical reference for the improvement of the flow pattern in the forebay of the lateral inlet pumping station.

The forebay of a pumping station is an important building connecting the diversion channel and the intake pool. Based on the physical model test and research method of computational fluid dynamics (CFD) based on the improved fluid volume model, the flow field in a forebay of a multi-unit pumping station is analyzed in combination with the engineering practice of the Exi River flood discharge station in the Anhui Province, China. Aiming at the technical problems of a large-scale swing water area in the forebay internal flow field of a lateral intake pumping station, the technical problems are discussed. Different rectification measures are selected to adjust the flow pattern in the forebay of a pumping station. The internal rectification flow pattern in the forebay under different plans, the uniformity of flow velocity distribution in the measurement section, and the reduction rate of the vortex area are studied and compared, and the optimal plan is given. The results show that the flow pattern of the 7.5 m and 15 m solutions of the lengthened inflow wall is still poor, and the ability to eliminate vortices is not strong or even counterproductive. The combination plan of a rectifier sill and a rectifier pier has a better effect and can eliminate more than 90% of the vortex, but the uniformity of flow speed has not been significantly improved at the inlet of the pumping station, the combination plan of a rectifier sill and a diversion wall opening has the best effect, the reduction rate of the vortex area is more than 85%, and the velocity uniformity of three measuring sections is better than that of the original plan. The uniformity of flow rate near the pumping station is increased by 4% and that far away from the pumping station is increased by 13%. The combination plan of a rectifier sill and diversion wall with openings is recommended.

The outlet conduit is an important construction connecting the outlet of the pump guide vane and the outlet pool, in order to study the hydraulic performance of the straight outlet conduit of the axial-flow pump device, this paper adopts the method of numerical simulation and analyzes the influence of the division pier on the pressure and velocity distribution inside and near the wall of the straight outlet conduit based on three design schemes. Four pressure pulsation measuring points were arranged in the straight outlet conduit, and the low-frequency pulsation characteristic information inside the straight outlet conduit with and without the division pier was extracted by wavelet packet reconstruction. The results show that the addition of a division pier has an effect on the hydraulic loss, near-wall pressure and velocity distribution in the straight outlet conduit. A small high-pressure zone is formed near the wall at the starting position of the division pier, and a large high-speed zone is formed on the left side at the starting position of the division pier. The length of the division pier has no significant effect on the flow distribution of the straight outlet conduit and the pressure and velocity distribution near the wall. Under different working conditions, each monitoring point has the maximum energy in the sub-band (0~31.25 Hz). With the increase of the flow rate, the total pressure energy of the straight outlet conduit decreases gradually. Under each condition, the difference of the energy proportion of the horizontal monitoring points of the straight outlet conduit is small, and the difference of the energy proportion of the two monitoring points at the top and bottom of the outlet channel is relatively large. The energy of the two monitoring points in the straight outlet conduit with a division pier is smaller than that of the two monitoring points in the straight outlet conduit without a division pier. There are differences in the main frequency and the power spectrum corresponding to the main frequency of the monitoring points in the straight outlet conduit, and the reasonable setting of the division pier is conducive to reducing the pressure pulsation of the flow in the straight outlet conduit and is beneficial to the safe and stable operation of the pump device.

Due to the complexity and randomness of emergency demands, it is difficult to describe and analyze the Emergency Medical Service Systems (EMSS) just by certain modeling. Therefore, a stochastic modeling and simulating method for EMSS optimizing is proposed based on the Discrete Event System theory. With a fictive and stochastic medical emergency scenario, the relevant simulation model is constructed with the FLEXSIM software quickly and explicitly. And by simulating and analyzing, it is easily to discover the bottlenecks and to optimize the EMSS system.

The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas -known as glioblastoma multiforme (GBM)- an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins.Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis.Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications.The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment.

Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr ¹³C(γ) and ¹⁵N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. χ-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.

Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-delta and PLC-gamma have been studied, the comparable domains of the beta isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-beta 1 and -beta 2 (PH-beta 1 and PH-beta 2, respectively) for lipid bilayers and G-beta gamma subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-beta gamma subunits had little affect on their membrane affinity. In contrast, binding of PLC-delta 1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-beta gamma subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-beta gamma were in the following order: PH-beta 2/= PH-beta 1PH-delta 1; the affinities of the native enzyme were as follows: PLC-beta 2PLC-delta 1PLC-beta 1. Thus, the PH domain of PLC-beta 1 interacts with G-beta gamma in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-beta2 with G-beta gamma is comparable to that of the full-length protein and may play a key role in G-beta gamma recognition.

Lots of studies and researches had focused on the calculation of water hammer pressure phenomenon of single-phase for a pipeline of uniform characteristics, but the less work had addressed on the calculation of slurry water hammer pressure in complex pipelines with slurry flows carrying solid particles. In this paper, according to the characteristics of solid-liquid flow, continuity equation and momentum equation of pseudo-homogeneous flow was presented, and a pseudo-homogeneous water hammer model was built based on the characteristic line method. The characteristics of solid-liquid flow's viscosity, resistance and wave velocity was considered in the model. Through the numerical simulation and analysis of transient process of a long-distance slurry transportation pipeline system, the protective properties of accumulator was presented and discussed. The result shows that the accumulator works well while pumping accident happens, and the accumulator can be an effective protection measure of long-distance slurry transportation pipelines.

The new monomeric azagallatranes ga(RNCH(2)CH(2))(3)N (R = SiMe(3), 10; SiMe(2)-t-Bu, 11) and dimeric azagallatrane [Ga(MeNCH(2)CH(2))(3)N](2), 14, were prepared by two synthetic routes: (i) the transamination reaction of [Ga (NMe(2))(3)](2), with (MeNHCH(2)CH(2))(3)N, 2, and (ii) the transmetalation reaction of [Al(MeNCH(2)CH(2))(3)N](2), 12, with Ga(acac)(3) (acac = penta-2,5-dienoate). Dimeric azaalumatrane 12, monomeric azaalumatranes Al(RNCH(2) CH2)(3)N (R = SiMe(3), 8; SiMe(2)-t-Bu, 9), and azaboratrane B(MeNCH(2)CH(2))(3)N, 6, react with a variety of alkoxides giving transition metal or main-group element azatranes. The molecular structures of 12 and 14 were established by X-ray diffraction experiments, and both possess an unusual cis configuration of the substituents on the central four-membered (M-N)(2) ring. Crystal data: 12, a = 37.372(10) Angstrom, b = 14.379(2) Angstrom c = 8.774(2) Angstrom, V = 4714.9(1) Angstrom(3), Z = 8, orthorhombic space group Pbca, R = 5.0%; 14, a = 9.716(1) Angstrom, b = 11.325(2) Angstrom, c = 11.343(1) Angstrom, alpha = 110.36(2)degrees, beta = 97.87(1)degrees, gamma = 90.34(1)degrees, V = 1157.2(3) Angstrom(3), Z = 2, triclinic space group P1bar, R = 4.7%.

The new monomeric azaaluminatrane Al(tert-BuMe(2)SiNCH(2)CH(2))(3)N, 6, was prepared by the reaction of [Al(NMe(2))(3)](2), 7, and (tert-BuMe(2)SiNHCH(2)CH(2))(3)N, 11, and was characterized by H-1, C-13, Al-27, and Si-29 NMR, and IR spectroscopies and mass spectrometry. Its analogue Al(Me(3)SiNCH(2)CH(2))3N, 3, was structurally characterized and shown to contain a trigonal monopyramidal aluminum center. The dimeric azaaluminatrane [Al(Me(3)SiNCH(2)CH(2))(2)(HNCH2CH2)N](2), 5, possesses an unusual cis configuration of the central four-membered ring, and the aluminum atoms display a distorted trigonal bipyramidal coordination sphere. Crystal data: 3, a = 13.503(5) Angstrom, b = 10.621(4) Angstrom, c = 17.840(6) Angstrom, beta = 93.29(3)degrees, V = 2554(2) Angstrom(3), Z = 4, space group P2(1)/c, R = 5.2%; 5, a = 12.134(3) Angstrom, b = 17.049(4) Angstrom, c = 18.297(5) Angstrom, beta = 100.02(2)degrees, V = 3726(2) Angstrom 3, Z = 4, space group P2(1)/n, R = 4.6%. Al-27 NMR chemical shifts for a series of Al-N compounds with aluminum coordination numbers ranging from three to six show increased shielding with increasing coordination number.

Optically pure (1R)-(-)-9,9-dimethyltricyclo[6.1.1.0[sup 2,6]]deca- 2,5-diene (HPCp) reacts with Fe-(CO)[sub 5] and Ru[sub 3](CO)[sub 12], in the presence of the hydrogen acceptor norbornene, to form the isomorphic and isostructural complexes cis-[PCpM(CO)[sub 2]][sub 2] (M = Fe (1), Ru (8)), in which the metal is coordinated exclusively to the exo face of PCp. These complexes react with iodine to form PCpM(CO)[sub 2]I (M = Fe(2), Ru(9)). Complex 2 reacts with 1-phenyl-3,4-dimethylphosphole (DMPP), diphenylvinylphosphine (DPVP), and phenyldivinylphosphine (DVPP) to form two diastereomers of PCpFe(CO)(DMPP)I (3a,b), PCpFe(CO)(DPVP)I, (4a,b), and PCpFe(CO)-(DVPP)I (5a,b), respectively. Complex 9 reacts with DMPP to form two diastereomers of PCpRu(CO)(DMPP)I (10a,b). Complexes 4a and 4b react with DPVP and DVPP to undergo sequential ligand substitution and intramolecular [4 + 2] Diels-Alder cycloaddition forming four and five diastereomers of [PCpFe(CO)(DMPP)(DPVP)]I (6a-d) and [PCpFe(CO)(DMPP)-(DVPP)]I (7a-e), respectively. Complexes 10a and 10b similarly react with DPVP and AgBF[sub 4] to form four diastereomers of the [4 + 2] Diels-Alder adducts [PCpRu(CO)(DMPP)(DPVP)] BF[sub 4] (11a-d). Little asymmetric induction is observed in these metal-promoted intramolecular [4 + 2] Diels-Alder cycloadditions. 30 refs., 2 figs., 7 tabs.

Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments through lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFP’s ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr 13Cγ and 15N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. χ-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti conformations as in the AFP crystal structure to partial population in the anti conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.

Lots of researchers have paid attention to time series clustering in recent years. This paper studies the stationarity analysis for autoregressive and moving average models of time series with clustering, firstly presents a set of nonlinear functions, or rather the square function along with logarithmic function to better autocorrelation function, secondly clusters time series into stationary and non-stationary with Clustering, finally an automatic mechanism for prejudging the stationarity of time series is presented. The proposed approach has been tested using two datasets, one natural and one synthetic, and is shown to yield useful and robust result of stationarity analysis.

An aluminophosphate with a layer structure has been synthesized hydrothermally. The ratio P/Al = 2 for the framework; its structure consists of Al-centered tetrahedra and P-centered tetrahedra as basic building units to form a two-dimensional layer structure. The protonated ethylenediamine molecules are located in the interlayer space and sustain the framework. The water molecules are located in the space between two layers. Layers are joined together by van der Waals force to form a three-dimensional structure which crystallizes in the space group Pbnb, with a = 8.052(6), b = 8.760(2), c = 17.037(7) {angstrom}, and V = 1201.88 {angstrom}{sup 3}. The crystal structure has been refined to yield values of R = 0.0418, R{sub w} = 0.0447.

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapine-induced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol Cancer Ther 2006;5(3):586–92]

p53R2 is a newly identified small subunit of ribonucleotide reductase (RR) and plays a key role in supplying precursors for DNA repair in a p53-dependent manner. Currently, we are studying the redox property, structure, and function of p53R2. In cell-free systems, p53R2 did not oxidize a reactive oxygen species (ROS) indicator carboxy-H2DCFDA, but another class I RR small subunit, hRRM2, did. Further studies showed that purified recombinant p53R2 protein has catalase activity, which breaks down H2O2. Overexpression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas overexpression of hRRM2 did not and resulted in a collapse of mitochondrial membrane potential. In a site-directed mutagenesis study, antioxidant activity was abrogated in p53R2 mutants Y331F, Y285F, Y49F, and Y241H, but not Y164F or Y164C. The fluorescence intensity in mutants oxidizing carboxy-H2DCFDA, in order from highest to lowest, was Y331F > Y285F > Y49F > Y241H > wild-type p53R2. This indicates that Y331, Y285, Y49, and Y241 in p53R2 are critical residues involved in scavenging ROS. Of interest, the ability to oxidize carboxy-H2DCFDA indicated by fluorescence intensity was negatively correlated with RR activity from wild-type p53R2, mutants Y331F, Y285F, and Y49F. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its fundamental enzymatic activity. (Cancer Res 2006; 66(4): 1900-5)

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation.The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) or over expression of the SOD2 gene.MCF+FIR cells derived from fractionated IR demonstrated increased transactivation of the pGL3CB1EI1 and NF-kappaB controlled reporter activities, relative to the parental cell line. Transfection of dominant negative mutant IkB that inhibits NF-kappaB nuclear translocation, inhibited pGL3CB1EI1 and NF-kappaB activity, indicating the NF-kappaB dependence of pGL3CB1EI1 mediated transcription. In addition, over expression of the human SOD2 gene (MnSOD) inhibited NF-kappaB and pGL3CB1EI1 activity, indicating that superoxide or some species derived from superoxide may have participated in the up-regulation of reporter activity in response to chronic exposure to fractionated ionizing radiation. These results provide evidence suggesting that a signaling pathway involving NF-kappaB and Cyclin B1 may contribute to adaptive radioresistance induced by chronic exposure to fractionated IR and support the conclusion that MnSOD appears to be a negative regulator of this pathway.

Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-kappaB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-kappaB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominant-negative mutant IkappaB (MCF-7/ErbB2/mIkappaB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-kappaB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-kappaB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-kappaB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin+IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin+IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-kappaB in MCF-7/ErbB2/mIkappaB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-kappaB response to IR, and that a specific prosurvival network downstream of NF-kappaB is triggered by treatments using anti-ErbB2 antibody combined with radiation.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

Nuclear factor-kappaB (NF-kappaB) is one of the key regulatory molecules in oxidative stress-induced cell activation. NF-kappaB is normally sequestered in the cytoplasm of nonstimulated cells and must translocate into the nucleus to regulate effector gene expression. A family of inhibitory proteins, IKBs, binds to NF-kappaB and masks its nuclear localization signal domain and therefore controls the translocation of NF-kappaB. Exposure of cells to extracellular stimuli that perturb redox balance results in rapid phosphorylation, ubiquitination, and proteolytic degradation of IkappaBs. This process frees NF-kappaB from the NF-KB/IKB complexes and enables NF-kappaB to translocate to the nucleus where it regulates gene transcription. Many effector genes including those encoding cytokines and adhesion molecules are in turn regulated by NF-kappaB. NF-kappaB is also an essential component of ionizing radiation (IR)-triggered signal transduction pathways that can lead to cell death or survival. The purpose of this review is to briefly summarize the recent progress in the studies of the role of reactive oxygen species (ROS), cytokines and ionizing radiation in NF-kappaB activation.

In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953

c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1–1 (GSTP1–1) (1). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1–1. The apparent K d for full-length JNK was 188 nm and for a C-terminal fragment (residues 200–424) 217 nm. An N-terminal fragment (residues 1–206) did not bind to GSTP1–1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a concentration-dependent increase in the kinase activity of JNK under normal, unstressed growth conditions indicating a dominant-negative effect. This suggests that the fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1–1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentration-dependent co-immunoprecipitation of GSTP1–1 was achieved. These data provide evidence for direct interactions between the C-terminal of JNK and GSTP1–1 and a rationale for considering GSTP1–1 as a critical ligand-binding protein with a role in regulating kinase pathways.

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.

Phospholipase C (PLC)-β 1 and PLC-β 2 are regulated by the G q family of heterotrimeric G proteins and contain C2 domains. These domains are Ca 2+ -binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-β 1 and PLC-β 2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5′-[γ-thio]triphosphate (GTP[γS])-bound] Gα q subunits. The C2 domain of PLC-β 1 effectively suppressed the activation of the intact isozyme by Gα q (GTP[γS]), indicating that the C2-Gα q interaction may be physiologically relevant. C2 affinity for Gα q (GTP[γS]) was reduced when Gα q was deactivated to the GDP-bound state. Binding to activated Gα i1 subunits or to Gβγ subunits was not detected. Also, Gα q (GTP[γS]) failed to associate with the C2 domain of PLC-δ, an isozyme that is not activated by Gα q . These results indicate that the C2 domains of PLC-β 1 and PLC-β 2 provide a surface to which Gα q subunits can dock, leading to activation of the native protein.

Anthony Joseph Gomez Diaz,1 Daniel Tamae,2 Yun Yen,3 JianJian Li,4 Tieli Wang1 1Department of Chemistry and Biochemistry, California State University at Dominguez Hills, Carson, CA, 2Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, 3Department of Clinical and Molecular Pharmacology, Beckman Research Institute of City of Hope National Medical Center, Duarte, CA, 4Department of Radiation Oncology, University of California Davis, Sacramento, CA, USA Abstract: In our previous study, we identified that a protein target, peroxiredoxin II (PrxII), is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells, and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology's property of targeting to specific cancer-related genes. Keywords: siRNA, PrxII, radiation resistance, Ca2+, MCF+FIR3