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The expression of genes encoding antioxidant and/or phase 2 detoxifying enzymes can be enhanced in response to various environmental stresses. The main transcription factor involved in this response is nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activity is negatively regulated by the protein Kelch-like-Ech-associated-protein 1 (Keap1). While the roles of Nrf2 and phase 2 genes in chemoprevention of carcinogenesis have been well described; only few studies have dealt with their role in skin cancer. Normal human keratinocytes (NHK) and melanocytes (NHM) were treated by chemical inducers of the Nrf2 pathway or by small interfering RNAs (siRNA) used to knock down Keap1 mRNA. The above treatments resulted in significant stimulation of NQO-1 (NADPH-Quinone-Oxidoreductase 1) gene expression. GCL (gamma-Glutamyl-cysteinyl-ligase) gene was also induced but interestingly increased mRNA encoding the catalytic, heavy subunit GCLC was mainly stimulated in NHK, whereas the mRNA encoding the modifier, light subunit GCLM was mostly induced in NHM. HO-1 (Heme Oxygenase 1) gene induction was relatively strong in NHM, but generally absent in NHK, except when the cells were subjected to cytotoxic doses of the above chemicals. Exposure to solar UV (UVB + UVA, 300-400 nm) or to UVA alone (320-400 nm) confirmed this trend, but interestingly, at doses where cell growth reduction was comparable, UVA was generally more efficient than solar UV in inducing phase 2 genes. When siRNAs directed against Nrf2 were used, a strong down-regulation of NQO-1 expression was observed in both, NHM and NHK, whereas reduction of HO-1 expression was mainly detected in NHM. To our knowledge, this is the first study comparing phase 2 gene modulation in NHK and NHM. The results hereby presented should contribute to a better understanding of the molecular mechanisms involved in skin adaptation to environmental stress.

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Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.

An extremely thermophilic archaeon, strain AL662T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at a latitude of 21"N. This strain is a strictly anaerobic coccus, and its cells range from 0.8 to 2 pm in diameter. The optimum temperature, pH, and Sea Salt concentration for growth are 85"C, 6, and 20 to 40 g/liter, respectively. Strain AL662T grows preferentially on proteolysis products, on a mixture of 20 amino acids, and on maltose in the presence of elemental sulfur. The membrane lipids consist of di-and tetraether glycerol lipids. The DNA G+C content is 58 mol%. Sequencing of the 16s rRNA gene showed that strain AL662T belongs to the genus Thermococcus. On the basis of hybridization results, we propose that this strain should be placed in a new species, Thermococcus hydrothermalis.Since the discovery of hyperthermophilic archaea, strains belonging to the order Thermococcales have been isolated from both terrestrial and marine hot environments and described. Species belonging to the genus Thermococcus have been isolated from various deep-sea and shallow marine hydrothermal areas and described. All of the strains have quite similar physiological characteristics and can be divided into the following two groups on the basis of their G + C contents: (i) a group of strains with high G + C contents (50 to 58 mol%), including strains belonging to two species from shallow marine environments, Therrnococcus celer (34) and Thermococcus stetteri (20), and three species from deep-sea environments, Thermococcus profundus (14), "Thermococcus peptonophilus" (S), and Thermococcusfumicolans (7); and (ii) a group of strains with low G+C contents (38 to 47 mol%), including strains belonging to two species from deep-sea environments, "Thermococcus chitonophagus" (1 1) and "Thermococcus alcaliphilus" (13), and an organism from a shallow marine environment, "Thermococcus litoralis" (22). All of these species are able to use proteinaceous carbon substrates in the absence or presence of sulfur. Some of these strains are able to grow in the absence of elemental sulfur, but sulfur greatly enhances growth. In this paper, we describe a new hyperthermophilic heterotrophic archaeal species that was isolated from a deep-sea hydrothermal vent on the East Pacific Rise at a latitude of 21"N. This organism was selected on the basis of the results of a preliminary screening for alcohol dehydrogenase (ADH) activity in 71 hyperthermophilic heterotrophic strains that were isolated at 80 and 95°C from various deep-sea hydrothermal vent areas (15). MATERIALS AND METHODSReference strains. Thermococcus celer DSM 2476', "Thermococcus litoralis" DSM 5474T, Thermucoccus stetteri DSM 5262.", Sulfolobus acidocaldarius DSM 639", and Thermotugu maritima DSM 310gT were obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig-Stockeim, Germany. Other strains were directly obtained from researchers. DT5432T was provided by Tetsuo Kobayashi (RIKEN, Wako, Japan). Thermococcus fumicolans CIP104680~" was isolated in our...

An extremely thermophilic archaeon, strain ST557T (T = type strain), was isolated from a deep-sea hydrothermal vent in the North Fiji Basin. This strain is a strictly anaerobic coccus whose cells are about 0.8 to 2 pm in diameter. The optimum temperature, pH and sea salt concentration for growth are 85"C, 8.5, and 20 to 40 @liter, respectively. Strain ST557T grows preferentially in the presence of elemental sulfur on proteinaceous substrates and on a mixture of 20 amino acids. It grows slowly on pyruvate and maltose. Growth is inhibited by rifampin. The DNA G+C content is 54 to 55 mol%. Sequencing of the 16s rRNA gene revealed that strain ST557T belongs to the genus Thermococcus. We propose that this organism should be placed in a new species, Thermococcus fumicolans.Since the discovery of deep-sea hydrothermal vents in 1977, a number of hyperthermophiles have been isolated from various sites in the deep sea, including sites on the East Pacific Rise, in the southwest Pacific Ocean and on the Mid-Atlantic Ridge (for a review see reference 39). The deep-sea hyperthermophiles that have been described previously belong to the archaeal domain. These organisms are methanogens belonging to the genera Methanococcus (23, 24) and Methanopyrus (28); sulfate reducers, such as Archaeoglobus profundus (7); and sulfur metabolizers, such as members of the genera Staphylothermus (14), Desulfirococcus (21), Pyrococcus (12, 22), Orodictium (37). and Therrnococcus (15, 20, 27) and isolated ES1 and ES4 (35,36). Most of the genera mentioned above are not restricted to deep-sea vents, and members of these genera also occur in shallow-water or terrestrial hot spring systems.During the French-Japanese "Starmer II" cruise in July 1989 in the North Fiji Basin, new hydrothermal sites were explored by the manned submersible "Nautile" (10). At these sites, temperatures up to 288°C were measured. During this cruise, many samples (including samples of chimney walls, rock debris, hydrothermal fluid, and animals) were recovered and used to inoculate enrichment cultures for hyperthermophilic sulfur metabolizers that were incubated at 80 and 95°C (1,12). In this paper, we describe a new hyperthermophilic heterotrophic archaeal species that was isolated from chimney wall fragments. MATERZALS AND METHODSReference strains. Thermococcus celer DSM 2476= (T = type strain), "Thermococcus litoralis" DSM 5474T, Thermococcus stetteri DSM 5262=, qVrococcus furiosus DSM 3638T, Pyrococcus woesei DSM 3773T, Sulfolobus acidocaldarius DSM 639T, and Thermotoga maritima DSM 31091 were obtained from the Deutsche Sammlung von Mikroorganismen,