Cross-linking of collagen by singlet oxygen generated with UV-A.

Singlet oxygen (1O2), a highly reactive and toxic intermediate, may play a role in photo-induced aging. We examined singlet oxygen generation from hematoporphyrin (HP) with UV-A, by monitoring the emission at 1,268 nm corresponding to 1O2 --> 3O2. Singlet oxygen was formed HP-dose-dependently in this system. We then investigated the reaction of singlet oxygen generated by UV-A irradiation with collagen, which is related to skin elasticity and softness. Collagen from skin was rapidly and dose-dependently cross-linked by singlet oxygen. The reaction was inhibited by NaN3, a selective quencher of singlet oxygen. In contrast, SOD (superoxide dismutase) and mannitol had no effect. These results suggested that cross-linking of collagen was caused by UV-A-generated singlet oxygen, not by any other reactive oxygen species. Compared with another multisubunit protein, alcohol dehydrogenase, collagen was cross-linked much more efficiently. Further, the finding that semicarbazide inhibited cross-linking of collagen showed that cross-links were formed between photooxidized histidyl residues and amino groups. Singlet oxygen generated by UV-A irradiation may contribute to cross-linking of collagen in the process of skin photoaging.

Singlet oxygen (1 delta g) generation from coproporphyrin in Propionibacterium acnes on irradiation.

Although singlet oxygen has been postulated to be a highly reactive and toxic intermediate, there has been no evidence of considerable generation of singlet oxygen in vivo level except for special cases. In this work, we firstly measured the near-infrared emission spectra corresponding to the O2(1 delta g) --> O2(3 epsilon g-) transition of singlet oxygen of cutaneous Propionibacterium acnes (P. acnes) porphyrin under laser excitation. A comparison of the singlet oxygen production of coproporphyrin, which is produced predominantly from P. acnes, with that of other photosensitizers revealed coproporphyrin to be a highly efficient singlet oxygen generator under ultraviolet light A irradiation on the skin. These results suggest that singlet oxygen can be generated on the skin surface from P. acnes porphyrin under ultraviolet irradiation and induce serious damage to the skin.

Measurement of 1268 nm emission for comparison of singlet oxygen (1 delta g) production efficiency of various dyes.

Singlet oxygen generation from laser-excited photosensitive dyes was measured directly using a sensitive near-infrared emission spectrometer to monitor the O2(1 delta g)-->O2(3 sigma -g) transition at 1268 nm. The emission intensity was proportional to both the laser power and the concentration of the dyes. The singlet oxygen producing ability of the dyes was compared with that of eosin YS as a standard in methanol. The relative efficiencies of singlet oxygen generation were determined for rose bengal, erythrosine B, phloxine B and eosin YS as 2.39, 1.73, 1.38, 1.00, respectively, while uranine showed no emission in this spectral region. Using rose bengal, erythrosine B, phloxine B and eosin YS, the efficiency of singlet oxygen generation correlated with the photobleaching reaction rate of azo-dyes by these dyes, suggesting singlet oxygen to be a species responsible for causing the photobleaching of azo-dyes. The halogen substituent effect on the efficiency of singlet oxygen generation from laser-excited photosensitive dyes was also examined systematically.

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium.

A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na2SeO3 (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lentil lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linked N-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium. alpha 1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9 +/- 1.5 pmol.h-1.mg-1 protein) than cultured with FBS-containing media (18.2 +/- 1.2 pmol.h-1.mg-1 protein). These results have indicated that the selective increase of alpha 1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.