Regulation of gene expression in prostate cancer cells with an artificially constructed promoter responsive to radiation.

A promoter library was developed that is composed of DNA fragments constructed by randomly elongating the cis-acting elements of transcription factors presumably activated in prostate cancer by radiation, and linking to the TATA-box sequence. One promoter with the strongest reactivity to X-ray in the LNCap cells of the library was chosen and improved by the introduction of random mutations. The resultant promoter was designated clone 880-8, showing the highest dose-dependent activity enhancement with X-ray irradiation (X-irradiation). A recombinant retrovirus expressing the luciferase gene under the control of clone 880-8 was infected into LNCap cells that showed 9.12±0.36-fold enhancement of luciferase activity 12 h after X-irradiation at 10 Gy. When the infected cells were inoculated onto nude mice, enhancement of luciferase expression was 4.27±1.36-fold 12 h after X-irradiation at 10 Gy. When LNCap was infected with another recombinant carrying the fcy::fur gene downstream from clone 880-8, fcy::fur expression was enhanced by X-irradiation. It was also shown to increase the dose-dependent cell killing ratio with 5-FC as compared with a counterpart without X-irradiation. These results suggest that the method used in this study is effective to construct a promoter responsive to stimulation. Such promoters can be used for stimulation-controlled gene therapies.

Substituted 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase.

A series of 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives have been synthesized and evaluated for their ability to selectively inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The 1-phenyl moiety participates in a hydrophobic interaction where an optimum size is required. At this position, 3,4-dimethylphenyl is the best moiety for inhibiting chymase and showed high selectivity compared with chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrogen-bond acceptors such as nitrile and methoxycarbonyl enhances its activity. Molecular-modeling studies on the interaction of 3-[(4-chlorophenyl)sulfonyl]-1-(4-chlorophenyl)-imidazolidine-2,4-dione (29) with the active site of human heart chymase suggested that the 1-phenyl moiety interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, and the 4-carbonyl of the imidazolidine ring and sulfonyl group interact with the oxyanion hole and the His-45 side chain of chymase, respectively. The complex model is consistent with the structure-activity relationships.

Highly purified photosynthetic reaction center (PscA/cytochrome c551)2 complex of the green sulfur bacterium Chlorobium limicola.

The photosynthetic reaction center (RC) complex that forms a homodimer of core and cytochrome c subunits was isolated from Chlorobium limicola f. thiosulfatophilum, strain Larsen. The complex showed only two subunit bands at 68 (PscA core) and 21 kDa (cytochrome c551) on SDS-PAGE analysis, indicating the complete deletion of the light-harvesting bacteriochlorophyll a (BChl a) protein as well as the iron-sulfur protein. It contained 27 +/- 3 molecules of BChl a, 7 +/- 1 Chl-670, 3 +/- 1 carotenoids, and 1.6 +/- 0.1 c-type hemes per the primary electron donor P840. The complex showed a light-induced charge separation and recombination between P840 and the acceptor Chl-670 at 77 K as follows: P840*Chl-670-->P840+Chl-670(-)-->P840TChl-670-->P84 0 Chl-670. Pigment compositions and their function in the (PscA/cytochrome c551)2 complex were studied by absorption, circular dichroism, and fluorescence spectroscopy.

Substituted 3-phenylsulfonylquinazoline-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase.

A series of 3-phenylsulfonylquinazoline-2,4-dione derivatives have been synthesized and evaluated for their ability to inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The phenyl moiety of quinazoline participates in a hydrophobic interaction where an optimum size is required. In this moiety, 7-chloroquinazoline is the best moiety for inhibiting chymase, chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrophobic electron-withdrawing groups at the 4-position potentiated the activity. Anthranil moiety also enhanced the activity. Pyridylmethyl and N-pyridylacetamide at the 1-position gave an IC50 in the order of 10(-8)M. Molecular modeling studies on the interaction of 7-chloro-3-(4-chlorophenylsulfonyl) quinazoline-2,4(1H, 3H)-dione (4) with the active site of human heart chymase suggested that the phenyl moiety of quinazoline interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, the moiety at the 1-position locates in the S2-S3 subsites and the 4-carbonyl and 3-sulfonyl group interact with the oxyanion hole and the His57 side-chain of chymase, respectively.