Single dose radiation is more effective for the UV-induced activation and proliferation of melanocytes than fractionated dose radiation.

PURPOSE: To establish whether the effect of fractionating radiation modifies the effects of ultraviolet (UV) radiation on epidermal melanocytes, we compared the clinical and histological effects of single high dose radiation against repeated intermediate to low dose radiation on epidermal melanocytes. METHODS: Three minimal erythema UV doses (MED) were administered to three sites on the buttocks of healthy volunteers. One site was irradiated with 0.5 MED UV every day for 6 consecutive days, another site was irradiated with 1 MED UV every second day, and a third site received a single dose of radiation with 3 MED UV. The treatment was replicated on the other buttock. For the evaluation of UV-induced erythema and pigmentation, erythema and melanin indices were measured at 2 and 14 days post-irradiation. For purposes of histological evaluation, tissue specimens taken from each irradiated site at 2 and 14 days post-irradiation and were stained with monoclonal antibodies against Mel-5, HMB-45 and tyrosinase. Fontana-Masson silver staining, DOPA staining and split DOPA reactions were also performed. RESULTS: At 14 days post-irradiation, UV radiation induced melanocyte activation, proliferation and melanogenesis in proportion to the radiation dose administered to each fraction. The most prominent responses were observed after single high doses of radiation. CONCLUSION: When the total administered dose is identical, fractionation of radiation dose diminishes the effects of UV radiation on epidermal melanocytes. Furthermore, long, uninterrupted doses of UV radiation were found to more effective in inducing melanogenesis and melanocyte activation.

Modulation of catalase in human skin in vivo by acute and chronic UV radiation.

In this study, we demonstrate that catalase is differently regulated either by acute, or chronic UV radiation during the photoaging process. 2MED of UV radiation decreased the activity and expression of catalase gradually in the epidermis and dermis at between 24 and 48 h after the UV exposure. These levels then returned to near normal by 72 h after exposure. The catalase mRNA was also decreased in the skin 24 h after UV irradiation to 50% of the control level, and then started to recover. In contrast, chronic UV irradiation over a lifetime (approximately 50 years) increased the catalase activity in the epidermis and dermis of the human skin in vivo. Our results suggest that catalase might be one of the important enzymes in the skin aging process, and that it plays an important role in the photoprotection of the skin from UV light.

Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis of human skin in vivo.

Photoaged skin contains elastotic materials in the upper reticular dermis. This phenomenon is commonly known as solar elastosis. Little is known about the mechanisms leading to the accumulation of elastotic materials in photoaged skin, however. In this study, it was demonstrated that ultraviolet irradiation induced tropoelastin mRNA expression in the keratinocytes of human skin in vivo and also in cultured human keratinocytes by in situ hybridization and reverse transcriptase polymerase chain reaction. It was also shown by northern blot analysis (n = 5) that there were increased tropoelastin mRNA levels in the forearm (sun-exposed) skin of elderly persons, compared with upper-inner arm (sun-protected) skin of the same individuals. As demonstrated by in situ hybridization compared to sun-protected skin (upper-inner arm) (n = 5), tropoelastin mRNA expression in photoaged skin was higher in keratinocytes as well as in fibroblasts. Therefore, our results suggest that keratinocytes are another source of tropoelastin production after acute and chronic ultraviolet irradiation in human skin in vivo.

Identification of a third metalloprotease toxin gene in extraintestinal isolates of Bacteroides fragilis.

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.

Manganese-containing superoxide dismutase and its gene from Candida albicans.

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.

D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae.

D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size. In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected. The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.