Direct evidence of inter-molecular vibrations by THz spectroscopy

Abstract The time-dependent THz spectra of some chemical materials after being dipped in solvents or after application of spots of solvent were observed. Some specific peaks disappeared, increased in intensity, or newly appeared. These peaks showed different features after spots of different solvents were applied. The evidence suggests that these peaks can be attributed to the vibration of inter-molecules, and shows that the solvents significantly affect inter-molecular vibrations. This method will help in assigning the THz band, and will be useful in researching molecular interactions

A novel aromatic carboxylic acid inactivates luciferase by acylation of an enzymatically active regulatory lysine residue.

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53, reported here for the first time, substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modifies Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53. The insights from this study about the novel inhibitory modification mechanism might help not only to avoid misinterpretation of the results of Luc-based reporter screening assays but also to explain the pharmacological and toxicological effects of carboxylic acid-containing drugs

Active gas sensing with sub-terahertz waves reflected from a wall

We present active gas sensing with a sub-terahertz (THz) spectrometer. The transmitter and receiver of the system are composed of a photonic-base source and a Schottky barrier detector, respectively, and their arrangement is monostatic. From the intensity of the absorption peak of the received sub-THz signal, the density of the sample gas (N1

Effect of a kinase inhibitor on firefly luciferase (Luc) modification by F-53.

<p>A: The structure of PD98059. B: Effect of PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor for F-53 inhibition. PD98059 was added to HeLa cells transiently transfected with pCMVluc+ and pCMVβ to normalize differences in transfection efficiency at 3 h prior to the addition of F-53 (0.1 µM). Luc activity was determined after the addition of PicaGene LT 2.0 and normalized relative to β-gal activity. The results (Y-axes) are shown as the percentage of Luc activity relative to the DMSO control. C: Western blotting after native PAGE. PD98059 was added to HeLa cells transiently transfected with pCMVluc+ and pCMVβ at 1 h prior to F-53 or DMSO treatment for 3.5 h. HeLa cells lysates were subjected to native PAGE and western blotting. Luc: Luciferase. Each value represents the mean±SEM of three transfection experiments.</p

Chemical synthesis of F-53 (1) and its CoA conjugate (2), and the structure of PTC124.

<p>Conditions: a) pyridine, EtOH, reflux, 24 h, 62.3%; b) H₂, 10% Pd-C, benzene, RT, 93.6%; c) HCHOaq., NaBH₃CN, AcOH, THF, RT, 7.5 h, 88.4%; d) NaOH aq., EtOH, RT, 10 h, 77.6%; e) 3LiCoASH, PyBOP, K₂CO₃, THF, RT, 50%</p

Proposed mechanism for the reaction of F-53 with firefly luciferase (Luc).

<p>The mechanism might involve F-53 activation to its CoA-derivative by Luc, which functions as an acyl-CoA synthetase in living cells. F-53-CoA is proposed to be transferred to lysine-529 of Luc by an unknown cellular acetyltransferase. Lysine-529 might be the most important lysine for the regulation of enzyme activity by acetylation and deacetylation. We also suggest that after F-53-CoA is activated by native acyl-CoA synthetase in mammalian liver microsomes, F-53-CoA may be transferred to the lysine residue, which induces the acetylation of the same enzyme.</p

Effect of F-53 on recombinant luciferase (Luc) activity <i>in vitro</i>.

<p>The inhibitory effects of F-53 on recombinant Luc enzymatic activity in lysis buffer (A) and HeLa cell lysate (B) are shown. The fold increase (Y axes) in Luc activity after treatment with either F-53 or PTC124 was normalized relative to the level obtained after DMSO treatment. The values shown above the bars represent the mean, and the error bars indicate SEM of three independent experiments. C: Western blotting after native PAGE by using the Luc protein sample used in (A). We added protein samples for native PAGE (12.5 ng protein), obtained from HeLa cells, to each Luc protein sample used in (A) because the band representing recombinant Luc protein alone was not clearly visible on western blotting of native PAGE gels, and then used western blotting to analyze these proteins after native PAGE. Cont represents samples with 12.5 ng of HeLa protein only; no Luc band was detected in this control.</p

Effect of F-53 on reporter enzymes.

<p>The effects of F-53 on firefly luciferase (A), Renilla luciferase (B), β-galactosidase (β-gal) (C), and chloramphenicol acetyl transferase (CAT) (D) are shown. A–C: All individual reporter plasmids, that is, pCMVluc+ (A), pRL-CMV (B), and pCMVβ (C), were transfected into HeLa cells. The relevant enzymatic activity in cell lysates was measured after treatment with DMSO or F-53 (1 and 10 µM), in the presence of the relevant enzymatic substrate. D: pSG5-hRARα1 DR5G-TK-CAT, and pCMVβ were transfected into COS-1 cells. After treatment with DMSO, F-53, and LE540 (RAR pan-antagonist) with or without Am80 (RARα,β-selective agonist), CAT activity in cell lysates was measured by a CAT ELISA, and the results were normalized relative to β-gal activity. The asterisks indicate statistically significant differences (** p<0.01) between F-53 and DMSO treatments, which were determined using post-hoc analysis with Fisher's LSD test. D: DMSO, CAT: chloramphenicol acetyl transferase, RLU: relative light unit, RFU: relative fluorescence unit. Each value represents the mean±SEM of three transfection experiments.</p

Time course of changes in firefly luciferase (Luc) activities.

<p>A: Protocol for treating cells and measuring Luc activity. HeLa cells were transiently transfected with the pCMVluc+ and the pCMVβ to normalize differences in transfection efficiency. The cells were then treated with F-53 (B: 1 µM, D: 0.1–10 µM,) or PTC124 (C: 0.1 µM) for the incubation periods indicated in the figure (B–C). B and C: Luc activity was determined by measurement-1 in (A). D: Luc activity after the addition of F-53 in culture medium was determined by measurement-1 (black bar) and measurement-2 (white bar) in (A). Luc activity after the addition of F-53 at the same time as lysis was determined by measurement-3 (black bar) and measurement-4 (white bar) in (A). Luc activity after the addition of F-53 after lysis was determined by measurement-5 in (A). Treatment with F-53 after cell lysis involved treatment with a solution of F-53 in lysis buffer solution. Luc activity was normalized relative to β-gal activity. The results (Y-axes) are expressed as the fold increase in Luc activity after F-53 or PTC124 treatment, relative to the increase after treatment with DMSO. Each value represents the mean±SEM of three transfection experiments.</p

<i>In vitro</i> production of F-53-CoA.

<p>The typical HPLC/UV chromatograms obtained from standard mixture (A), the assay samples without enzyme (negative control, B), by using recombinant luciferase (0.133 µM, C), and by using mouse liver microsomes (27.6 µg, D) with ATP, MgCl₂, and CoA-SH are shown. Enzymatic <i>in vitro</i> assays were performed at 37°C for 15 min with F-53 (20 µM) as the substrate. The formation of F-53-CoA (product) was measured by HPLC/UV at 260 nm by using 10%–90% CH₃CN in water with 10 mM AcONH₄ (pH 7.0) as the mobile phase. Mass analysis was performed by HPLC/MS (positive-mode ESI) using the same mobile phase.</p