Gas Phase Conformations of Selenocysteine and Related Ions: A Comprehensive Theoretical Study

Extensive ab initio molecular calculations have been first performed to thoroughly characterize the gas-phase potential energy surfaces (PES) of the 21th amino acid selenocysteine and related ions (neutral, protonated and deprotonated). A wide range of trial structures generated by considering the combinations of all internal single-bond rotamers was surveyed at the BHandHLYP/6-31G­(d) level, and then refined at the BHandHLYP/6-311++G­(d,p) level. A total of 76, 23, 38, and 3 unique stable conformers respectively for neutral, protonated, deprotonated, and doubly deprotonated selenocysteine is identified, and neutral zwitterionic forms are found to be as local minima on the gas-phase PES. The properties of the low energy conformers, such as relative energies, dipole moments, rotational constants, and intramolecular hydrogen bonds, were determined and analyzed. The thermochemical properties of proton affinity (PA), gas-phase basicity (GB), proton dissociation energy (PDE), gas-phase acidity (GA), and the vertical ionization energies (VIEs) were computed by the theoretical approaches of BHandHLYP, B3LYP, MP2, and CCSD­(T). Moreover, the conformational equilibrium effect (CEE) on thermochemical properties was analyzed. The statistical simulation predicts that the CEE generally yields a physical correction on about a 1 <i>k</i>_B<i>T</i> scale in GA/GB calculations for multi-conformer systems

Low Energy Conformations and Gas-Phase Acidity and Basicity of Pyrrolysine

The gas-phase conformational potential energy surfaces (PES) of the last, 22nd amino acid pyrrolysine and related derivatives (neutral, deprotonated, and protonated) were extensively searched for the first time. By considering all possible combinations of the single-bond rotational degrees of freedom with a semiempirical and ab initio combined computational approach, a large set of unique low-energy conformers was identified for each pyrrolysine species, and essential properties such as vibrational frequencies, dipole moments, rotational constants, and intramolecular hydrogen bonding configurations were presented and characterized. The conformational electronic energies and thermochemical properties of proton affinity/dissociation energy (PA/PDE) and gas-phase acidity/basicity (GA/GB) were determined by the density functional BHandHLYP, B3LYP, and M062X, and Møller–Plesset MP2 methods. The MP2 and DFT methods are found to predict disparate PES for neutral and protonated conformations and sufficiently different thermochemical data. The measurements of dipole moments and characteristic IR modes at low temperature as well as GA/GB are demonstrated to be feasible approaches to verify the theoretical predictions

Growth curves of tumor in nude mice.

<p>The average volume of tumors derived from CTPE-induced BEAS-2B cells at passage 30 and THP-1 cells increased compared to the CTPE-induced BEAS-2B cells at passage 30 alone at different observation time points (n = 6, *<i>P</i><0.05).</p

The gene levels of NF-κB in the BEAS-2B cells at passages 10, 20 and 30.

<p>(<b>A</b>) NF-κB mRNA of BEAS-2B cells in four groups on agarose gel. M:marker;1–4 lanes: 10^th B(a)P, 10^th CTPE, 10^th DMSO, 10^th Co-culture+CTPE; 5–8 lanes: 20^th B(a)P, 20^th CTPE, 20^th DMSO, 20^th Co-culture+CTPE; 9–12 lanes: 30^th B(a)P, 30^th CTPE, 30^th DMSO, 30^th Co-culture+CTPE. (<b>B</b>) Quantitative comparison of NF-κB mRNA. The level of NF-κB mRNA in Co-culture/CTPE group started to increase at passage 10, which was higher than that of CTPE or B(a)P at passage 20, and kept the highest level at passage 30.(n = 6, *: vs DMSO, <i>P</i><0.05; #: vs CTPE, <i>P</i><0.05; Δ:vs Co-culture+CTPE, <i>P</i><0.05). The data was from two independent experiments.</p

Larger tumor was formed in nude mice transplanted with mixed CTPE-induced BEAS-2B cells at passage 30 and THP-1 cells.

<p>(<b>A</b>) Representative examples of tumor formation in nude mice on the 30^th day after injection (n = 6/group). (<b>a</b>) untrested BEAS-2B cells, (<b>b</b>) BEAS-2B cells induced with DMSO, (<b>c, d</b>) CTPE-induced BEAS-2B cells at passage 20 and 30, (<b>e</b>) CTPE-induced BEAS-2B cells at passage 30 mixed with THP-1 cells. Red arrow showed tumors. (<b>B</b>) Representatives of tumor removed from nude mice after 30 days transplantion. (<b>a–b</b>) CTPE-induced BEAS-2B cells at passage 30, (<b>c-d</b>) CTPE-induced BEAS-2B cells at passage 30 mixed with THP-1 cells.</p

Cell growth rate assays of BEAS-2B cells at passage 10, 20 and 30 in four groups.

<p>BEAS-2B cells were treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the BEAS-2B cells were cultured in the presence or absence of THP-1 cells and passaged. The percentage of S-phase BEAS-2B cells at passage 10, 20 and 30 was determined using flow cytometry. (<b>A</b>) Representatives of DNA content of BEAS-2B cells at passage 20 in four groups. (<b>B</b>) The percentage of BEAS-2B cell in S phase among different passages and groups. The percentage of the S phase cells in Co-culture/CTPE group started to increase at passage 10; was higher than that of CTPE or B(a)P at passage 20, and kept the highest level at passage 30.(n = 6, *: vs DMSO, <i>P</i><0.05; #: vs CTPE, <i>P</i><0.05; Δ:vs Co-culture+CTPE, <i>P</i><0.05). Phenotypes were triplicated.</p

The number of BEAS-2B cells with aneuploidy in Co-culture/CTPE group increased at passage 10, 20 and 30.

<p>Aneulpoidy was observed based on abnormal hromosome numbers, including hypodiploid (<2n) and hyperdiploid (>2n∼<4n). (<b>A</b>) Representative of normal chromosome (2n) and aneuploidy (>2n). Red arrow showed double centromere. (<b>B</b>) The number of cells with aneuploidy in total 100 BEAS-2B cells in Co-culture/CTPE group at passage 10 was more than that of DMSO group, but was not increased compared with CTPE group. At passage 20 and 30, the rates of cells with aneuploidy in 100 BEAS-2B cells in Co-culture/CTPE group were peaked among these four groups. (n = 6, *: vs DMSO, <i>P</i><0.05; #: vs CTPE, <i>P</i><0.05; ♦:vs B(a)P, <i>P</i><0.05.). Phenotypes were triplicated.</p

Representatives of colony in soft agar.

<p>(<b>A</b>) A colony in soft agar, scale bar = 10 µm; (<b>B</b>) A colony in soft agar, scale bar = 50 µm; (<b>C</b>) The representative of colonies of BEAS-2B cells in Co-culture/CTPE group at passage 20 in dish; (<b>D</b>) The representative of colonies of BEAS-2B cells treated with DMSO at passage 20 in dish.</p

The number of colony and percentage of colonies formation of BEAS-2B cells in soft agar.

<p>The number of colony (<b>A–C</b>) and clongenicity percentage (<b>D–F</b>) were significantly increased in Co-culture/CTPE group compared to those of other three groups at passage 20, or passage 30. (n = 6, *: vs DMSO, <i>P</i><0.05; #: vs CTPE, <i>P</i><0.05; Δ:vs Co-culture+CTPE, <i>P</i><0.05).Values were the mean±SD of three independent experiments.</p

CD68 protein expression levels in lung tumor tissue, adjacent tumor tissue and surrounding non-tumorous lung tissue from lung cancer patients.

<p>Scale bar = 20 µm. (<b>A</b>) Adenocarcinoma; (<b>B</b>) Squamous cell carcinoma; (<b>C</b>) adjacent adenocarcinoma tissue; (<b>D</b>) adjacent squamous cell carcinoma tissue; (<b>E</b>) non-tumor. CD68 were mainly detected in cytoplasm of macrophages with brown staining. Positive cell number of CD68 in adjacent lung cancer tissue (<b>C–D</b>) was more than that in lung cancer tissues (<b>A–B</b>) and non-tumorous lung tissues (<b>E</b>) (n = 67, <i>P</i><0.05).</p