Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins.

17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.

Rotational spectra of the van der Waals complexes of molecular hydrogen and OCS.

The a- and b-type rotational transitions of the weakly bound complexes formed by molecular hydrogen and OCS, para-H2-OCS, ortho-H2-OCS, HD-OCS, para-D2-OCS, and ortho-D2-OCS, have been measured by Fourier transform microwave spectroscopy. All five species have ground rotational states with total rotational angular momentum J=0, regardless of whether the hydrogen rotational angular momentum is j=0 as in para-H2, ortho-D2, and HD or j=1 as in ortho-H2 and para-D2. This indicates quenching of the hydrogen angular momentum for the ortho-H2 and para-D2 species by the anisotropy of the intermolecular potential. The ground states of these complexes are slightly asymmetric prolate tops, with the hydrogen center of mass located on the side of the OCS, giving a planar T-shaped molecular geometry. The hydrogen spatial distribution is spherical in the three j=0 species, while it is bilobal and oriented nearly parallel to the OCS in the ground state of the two j=1 species. The j=1 species show strong Coriolis coupling with unobserved low-lying excited states. The abundance of para-H2-OCS relative to ortho-H2-OCS increases exponentially with decreasing normal H2 component in H2He gas mixtures, making the observation of para-H2-OCS in the presence of the more strongly bound ortho-H2-OCS dependent on using lower concentrations of H2. The determined rotational constants are A=22 401.889(4) MHz, B=5993.774(2) MHz, and C=4602.038(2) MHz for para-H2-OCS; A=22 942.218(6) MHz, B=5675.156(7) MHz, and C=4542.960(7) MHz for ortho-H2-OCS; A=15 970.010(3) MHz, B=5847.595(1) MHz, and C=4177.699(1) MHz for HD-OCS; A=12 829.2875(9) MHz, B=5671.3573(7) MHz, and C=3846.7041(6) MHz for ortho-D2-OCS; and A=13 046.800(3) MHz, B=5454.612(2) MHz, and C=3834.590(2) MHz for para-D2-OCS.

Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins.

Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.

Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins.

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.

Nuclear hyperfine interaction of rotating hydrogen: a spectroscopic investigation of hydrogen-OCS van der Waals complexes.

The rotational spectra of five weakly bonded hydrogen-OCS complexes (paraH(2), orthoH(2), HD, orthoD(2), and paraD(2)) are measured. Hyperfine structure is resolved and analyzed in all except the complex with paraH(2), where I=0. For the two j=1 species, orthoH(2)-OCS and paraD(2)-OCS, nuclear hyperfine coupling constants are found to be d(a)=21.2(2) and 8.4(2) kHz, respectively, indicative of nearly free uniaxial rotation of the hydrogen around the b-inertial axis. Similar analyses for HD-OCS and orthoD(2)-OCS yield the quadrupole coupling constants eqQ(a)=16(2) and 30(2) kHz, respectively, showing that the internal rotational motions of HD and orthoD(2) in the complex are slightly hindered producing a small nonspherical distribution. For orthoD(2)-OCS, the observed hyperfine structure indicates that the nuclear spin states I=0 and 2 are strongly coupled in the rotation of the complex.

Structure and energy difference of two isomers of He-CH3F.

The intermolecular potential surface of He-CH(3)F is investigated through ab initio calculations and microwave and millimeter-wave spectroscopies. The intermolecular potential is calculated at the fourth-order Møller-Plesset level with a large basis set including bond functions. Three minimums exist, the deepest of which is at the carbon end of the C-F axis and has a depth of 46.903 cm(-1), the second deepest is in a T-shaped position relative to the C-F axis with a depth of 44.790 cm(-1), and the shallowest is at the fluorine end of the C-F axis with a depth of 30.929 cm(-1). The barrier to internal rotation of the CH(3)F subunit about its C-F axis is very low, thus leading to essentially free internal rotation and two separate sets of bound states correlating to ortho-CH(3)F (|K| = 3n) for the ground, or A, internal rotor state upon which this study focuses, and to para-CH(3)F (|K| = 3n +/- 1) for the excited, or E, internal rotor state. Bound-state calculations of the A state performed using two different techniques show the lowest-energy state to have the helium localized in the T-shaped well with an energy of -11.460 cm(-1), while two excited configurations of the A state have the helium localized either in the well at the carbon end ("linear") with an energy of -7.468 cm(-1) or in the well at the fluorine end ("antilinear") with an energy of -4.805 cm(-1). Spectroscopic observations confirm the predicted energy-level structure of the ground and first excited states. Sixteen transitions between 12 distinct energy levels have been observed, including pure rotational transitions of both the T-shaped ground state and the linear excited state, as well as rovibrational transitions between the ground state and the linear excited state. The energy difference between the T-shaped state and the linear state is measured to be 132 374.081(16) MHz. There is significant Coriolis mixing of the ground state J(K(a)K(c)) = 2(20) and the linear J(K) = 2(0) levels which aided in the observation of the T to linear transitions. This mixing and the T to linear energy difference are sensitive probes of the relative well depths of the two lowest minimums and are well predicted by the ab initio potential. Improved agreement between experiment and theory is obtained by morphing the correlation energy of the potential. He-CH(3)F is one of just a few atom-molecule complexes for which the ground-state geometry does not coincide with the global potential minimum.

The rotational spectrum and dynamical structure of LiOH and LiOD: a combined laboratory and ab initio study.

Millimeter wave rotational spectroscopy and ab initio calculations are used to explore the potential energy surface of LiOH and LiOD with particular emphasis on the bending states and bending potential. New measurements extend the observed rotational lines to J=7<--6 for LiOH and J=8<--7 for LiOD for all bending vibrational states up to (03(3)0). Rotation-vibration energy levels, geometric expectation values, and dipole moments are calculated using extensive high-level ab initio three-dimensional potential energy and dipole moment surfaces. Agreement between calculation and experiment is superb, with predicted Bv values typically within 0.3%, D values within 0.2%, ql values within 0.7%, and dipole moments within 0.9% of experiment. Shifts in Bv values with vibration and isotopic substitution are also well predicted. A combined theoretical and experimental structural analysis establishes the linear equilibrium structure with re(Li-O)=1.5776(4) A and re(O-H)=0.949(2) A. Predicted fundamental vibrational frequencies are v1=923.2, v2=318.3, and v3=3829.8 cm(-1) for LiOH and v1=912.9, v2=245.8, and v3=2824.2 cm(-1) for LiOD. The molecule is extremely nonrigid with respect to angular deformation; the calculated deviation from linearity for the vibrationally averaged structure is 19.0 degrees in the (000) state and 41.9 degrees in the (03(3)0) state. The calculation not only predicts, in agreement with previous work [P. R. Bunker, P. Jensen, A. Karpfen, and H. Lischka, J. Mol. Spectrosc. 135, 89 (1989)], a change from a linear to a bent minimum energy configuration at elongated Li-O distances, but also a similar change from linear to bent at elongated O-H distances.

Influence of a polar near-neighbor on incipient proton transfer in a strongly hydrogen bonded complex.

Rotational spectroscopy and ab initio calculations have been used to characterize the complexes H(3)N-HF and H(3)N-HF-HF in the gas phase. H(3)N-HF is a C(3v) symmetric, hydrogen bonded system with an NF distance of 2.640(21) A and an N...H hydrogen bond length of 1.693(42) A. The H(3)N-HF-HF complex, on the other hand, forms a six-membered HN-HF-HF ring, in which both the linear hydrogen bond in the H(3)N-HF moiety and the F-H-F angle of (HF)(2) are perturbed relative to those in the corresponding dimers. The N...F and F...F distances in the trimer are 2.4509(74) A and 2.651(11) A, respectively. The N...H hydrogen bond length in H(3)N-HF-HF is 1.488(12) A, a value which is 0.205(54) A shorter than that in H(3)N-HF. Similarly, the F...F distance, 2.651(11) A, is 0.13(2) A shorter than that in (HF)(2). Counterpoise-corrected geometry optimizations are presented, which are in good agreement with the experimental structures for both the dimer and trimer, and further characterize small, but significant, changes in the NH(3) and HF subunits upon complexation. Analysis of internal rotation in the spectrum of H(3)N-HF-HF gives the potential barrier for internal rotation of the NH(3) unit, V(3), to be 118(2) cm(-1). Ab initio calculations reproduce this number to within 10% if the monomer units and the molecular frame are allowed to fully relax as the internal rotation takes place. The binding energies of H(3)N-HF and H(3)N-HF-HF, calculated at the MP2/aug-cc-pVTZ level and corrected for basis set superposition error are 12.3 and 22.0 kcal/mol, respectively. Additional energy calculations have been performed to explore the lowest frequency vibration of H(3)N-HF-HF, a ring-opening motion that increases the NFF angle. The addition of one HF molecule to H(3)N-HF represents the first step of microsolvation of a hydrogen bonded complex and the results of this study demonstrate that a single, polar near-neighbor has a significant influence on the extent of proton transfer across the hydrogen bond. As measured using the proton-transfer parameter rho(PT), previously defined by Kurnig and Scheiner [Int. J. Quantum Chem., Quantum Biol. Symp. 1987, 14, 47], the degree of proton transfer in H(3)N-HF-HF is greater than that in either (CH(3))(3)N-HF or H(3)N-HCl but less than that in (CH(3))(3)N-HCl.

Kinetics of diesel nanoparticle oxidation.

The technique of high-temperature oxidation tandem differential mobility analysis has been applied to the study of diesel nanoparticle oxidation. The oxidation rates in air of diesel nanoparticles sampled directly from the exhaust stream of a medium-duty diesel engine were measured over the temperature range of 800-1140 degrees C using online aerosol techniques. Three particle sizes (40, 90, and 130 nm mobility diameter) generated under engine load conditions of 10, 50, and 75% were investigated. The results show significant differences in the behavior of the 10% load particles as compared to the 50 and 75% load particles. The 10% load particles show greater size decrease at temperatures below 500 degrees C and significant size decrease at temperatures between 500 and 1000 degrees C in a non-oxidative environment, indicating release of adsorbed volatile material or thermally induced rearrangement of the agglomerate structure. Activation energies determined are 114, 109, and 108 kJ mol(-1) for the 10, 50, and 75% load particles, respectively. These activation energies are lower than for flame soot (Higgins et al. J. Phys. Chem. A 2002, 106, 96), but the preexponential factors are lower by 3 orders of magnitude, and the overall oxidation rates are slower by up to a factor of 4 over the temperature range studied. Possible reasons for the differences are discussed in the text.