Surface plasmon resonance-based immunoassay for 17β-estradiol and its application to the measurement of estrogen receptor-binding activity

A rapid and simple surface plasmon resonance (SPR)-based immunoassay for detection of 17-estradiol was developed. The assay was designed as an inhibitive format, in which 17-estradiol–BSA conjugates are immobilized on an SPR sensor chip and the binding of antibody to the chip is measured. The binding was inhibited by 17-estradiol in the concentration range 0.468 to 21.4 nmol L^–1 with an IC₅₀ value of 2.29±0.10 nmol L^–1. Although not as sensitive as traditional radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA), this method requires no separation and washing after addition of the antibody, steps which are relatively time-consuming. Estrogen receptor (ER)-binding was then investigated using this SPR immunoassay for the determination of the amount of unbound 17-estradiol after competition with test compounds for the ER-binding. Inhibition of the binding of 17-estradiol to ER by diethylstilbestrol (DES) was successfully measured by injecting the reaction mixture into the SPR sensor after addition of the antibody. This binding assay requires no separation of unbound 17-estradiol from the mixture and no radioisotope- or fluorescence-labeling of 17-estradiol. These results show the potential usefulness of the SPR sensor both detecting 17-estradiol and evaluating the ER-binding activity of xenoestrogens such as DES in a single assay system.     

Redox-induced reversible [2 + 2] cycloaddition of an etheno-fused diporphyrin

3,5-Ethenoporphyrin is a π-extended porphyrin containing a fused ethene unit between the meso- and β-positions, exhibiting unique contribution of macrocyclic antiaromaticity. We have recently reported that its analogue, etheno-fused diporphyrin, underwent thermal [2 + 2] cycloaddition to furnish X-shaped cyclobutane-linked tetraporphyrins. Here we demonstrate that the cyclobutane-ring formation is dynamically redox-active. Namely, the tetraporphyrin underwent two-step four-electron oxidation to afford two etheno-fused diporphyrin dications. The reduction of the resulting dication regenerated the cyclobutane-linked tetraporphyrin. The dication was sufficiently stable to allow its isolation under ambient conditions. The structure of the dication has been confirmed by ¹H NMR spectroscopy and X-ray diffraction analysis. Importantly, the simultaneous double C–C bond cleavage in the cyclopropane ring in the tetraporphyrin is exceptional among dynamic redox (dyrex) systems to achieve large structural changes, thus offering new insights for the design of novel redox-active functional organic materials for electrochromic dyes, organic batteries, and organic memories.

A four-electron oxidation of an X-shaped tetraporphyrin affords stable etheno-fused diporphyrin dications through double C–C bond cleavage. The reduction of the dication recovers the tetraporphyrin via a thermal [2 + 2] cycloaddition.     

Molecular dynamics simulations of bovine cathepsin B and its complex with CA074

To promote our better understanding of the dynamic stability of the bovine cathepsin B structure, which is characterized by an extra disulfide bond at Cys148-Cys252 from the other species, and of the binding stability of CA074 (a cathepsin B-specific inhibitor), molecular dynamics (MD) simulations were performed for the enzyme and its CA074 complex, assuming a system in aqueous solution at 300 K. The MD simulation covering 400 ps indicated that the existence of a Cys148-Cys252 disulfide bond increases the conformational flexibility of the occluding loop, although the conformational stability of the overall structure is little affected. The structural characteristics of the complex elucidated by X-ray analysis were suggested to be also intrinsic and stable in the dynamic state; the hydrogen bonding/electrostatic interactions between the main and side chains of CA074 and the Sn and Sn' subsites of the enzyme were maintained throughout the MD simulation. Furthermore, the simulation made clear that the binding of CA074 significantly restricted the conformational flexibility of the substrate binding region, especially the occluding loop, of cathepsin B. Statistical analyses during the simulation suggest that the selectivity of CA074 for cathepsin B stems from the tight P1'-S1' and P2'-S2' interactions, assisted in particular by double hydrogen bonds between the carboxyl two oxygens of the CA074 C-terminus and the imidazole NH groups of His110 and His111 residues.     

Mono- and di-t-butyl substituted 2,2′-bis(1,3-dithia[3]ferrocenophane-2-ylidene) derivatives: TTF-like ferrocene analogs

Mono- and di-t-butyl substituted 2,2′-bis(1,3-dithia[3]ferrocenophane-2-ylidene) derivatives were prepared. These derivatives showed the E_1/2(1) in lower potential region than that of differrocenyl-ethene and -ethyne. Also, the ΔE_1/2 value declined according to the increase of a t-butyl group.     

Development of hardware accelerator for molecular dynamics simulations: a computation board that calculates nonbonded interactions in cooperation with fast multipole method

Evaluation of long-range Coulombic interactions still represents a bottleneck in the molecular dynamics (MD) simulations of biological macromolecules. Despite the advent of sophisticated fast algorithms, such as the fast multipole method (FMM), accurate simulations still demand a great amount of computation time due to the accuracy/speed trade-off inherently involved in these algorithms. Unless higher order multipole expansions, which are extremely expensive to evaluate, are employed, a large amount of the execution time is still spent in directly calculating particle-particle interactions within the nearby region of each particle. To reduce this execution time for pair interactions, we developed a computation unit (board), called MD-Engine II, that calculates nonbonded pairwise interactions using a specially designed hardware. Four custom arithmetic-processors and a processor for memory manipulation ("particle processor") are mounted on the computation board. The arithmetic processors are responsible for calculation of the pair interactions. The particle processor plays a central role in realizing efficient cooperation with the FMM. The results of a series of 50-ps MD simulations of a protein-water system (50,764 atoms) indicated that a more stringent setting of accuracy in FMM computation, compared with those previously reported, was required for accurate simulations over long time periods. Such a level of accuracy was efficiently achieved using the cooperative calculations of the FMM and MD-Engine II. On an Alpha 21264 PC, the FMM computation at a moderate but tolerable level of accuracy was accelerated by a factor of 16.0 using three boards. At a high level of accuracy, the cooperative calculation achieved a 22.7-fold acceleration over the corresponding conventional FMM calculation. In the cooperative calculations of the FMM and MD-Engine II, it was possible to achieve more accurate computation at a comparable execution time by incorporating larger nearby regions.     

A novel carbon skeletal trichothecane, tenuipesine A, isolated from an entomopathogenic fungus, Paecilomyces tenuipes

Tenuipesine A (1), a novel trichothecane with an unprecedented carbon-migrated skeleton that embodies of a cyclopropane ring, was isolated from cultivated fruiting bodies of Paecilomyces tenuipes (Isaria japonica), a popular entomopathogenic fungi employed in folk medicine and health foods in China, Korea, and Japan. The structure was determined on the basis of two-dimensional NMR data. Its stereochemistry was elucidated by spectroscopic data and the chemical transformation of the coexisting trichothecene, 4beta-acetoxy-12,13-epoxytrichothec-9-ene-3alpha,15-diol (2). [structure: see text]     

Influence of melatonin on reproductive behavior in male rats.

The influence of melatonin on reproductive behavior was examined by administering it to pinealectomized male rats for 30 consecutive days. After the 30-d administration of melatonin or vehicle, each male rat was introduced into a female rat's cage on the day of proestrus and allowed to mate overnight. All rats administered the vehicle and the pinealectomized rats copulated; five of the twelve melatonin (8.0 mg/kg)-treated rats did not copulate. These findings suggest that melatonin inhibits the reproductive behavior of male rats.     

Radioactive nuclides in the marine environment--distribution and behaviour of 95Zr, 95Nb originated from fallout

To investigate behaviour of 95Zr, 95Nb in the marine environment, various samples have been collected and measured by means of Ge(Li) gamma-ray spectrometry and/or radiochemical analysis during a period from 1974 to 1982 at coastal area of Tokai-mura, Ibaraki prefecture. Concentration of the nuclides in seaweeds increased remarkably after atmospheric nuclear detonation by P.R. of China, and the activity ratio between the nuclides changed by time was not fit well by the transient decay equation. Concentration variation in sea water was smaller than that in sea weeds, and the minimum change in sea sediment. Increase of concentration in these environmental samples was observed in chronological order of sea water, sea weeds then sediment after detonations, suggesting that the uptake of the nuclides by these sea weeds from sea water is faster than that via root. Observed concentration factors on the nuclides by sea weeds were calculated from the observed concentrations in sea water and sea weeds. Maximum values on 95Zr and 95Nb were 2110, 2150, respectively for Ecklonia cava and Eisenia bicyclis.     

Design and synthesis of a caged Zn2+ probe, 8-benzenesulfonyloxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant 1,4,7,10-tetraazacyclododecane, and its hydrolytic uncaging upon complexation with Zn2+

8-Benzenesulfonyloxy-5- N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (BS-caged-L(4), BS = benzenesulfonyl) was designed and synthesized as a "caged" derivative of a previously described Zn(2+) fluorophore, 8-hydroxy-5- N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (L(4)) (cyclen = 1,4,7,10-tetraazacyclododecane). In the absence of metal ions and in the dark, BS-caged-L(4) (10 microM) showed negligible fluorescence emission at pH 7.4 (10 mM HEPES with I = 0.1 (NaNO3)) and 25 degrees C (excitation at 328 nm). Addition of Zn(2+) induced an increase in the UV/vis absorption of BS-caged-L(4) (10 microM) at 258 nm and a significant increase in fluorescence emission at 512 nm. These responses are results from the formation of Zn(H-1L(4)) by the hydrolysis of the sulfonyl ester at the 8-position of the quinoline unit promoted by the Zn(2+)-bound HO(-). Improvement of cell membrane permeation in comparison with L(4) is also described.