We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Thirty patients were treated with a flashlamp-pumped pulsed dye laser, with 0.45 ms pulse width and 585 nm wavelength. Punch biopsies were taken prior to treatment, and the biopsies were examined morphometrically. Three different test sites were exposed to laser light of fluence 5.25, 6.50 and 7.75 J/cm2. The degree of blanching was examined 6-8 weeks after treatment, and each site was retreated four times. Six patients (20%) achieved poor blanching, eight patients (27%) obtained moderate lightening and 16 patients (53%) showed good response. The vessels of the good responders were located significantly more superficially than the vessels of the moderate and poor responders (P < 0.05). The poor responders had significantly smaller vessels than the moderate and good responders (P < 0.01). The moderate responders had deeper, but larger vessels, than the poor responders. Hence, an increasing vessel diameter reduces the negative outcome of increasing vessel depth. The vessel diameter was correlated to the colour (P < 0.01), e.g. the mean vessel diameter was increasing from 16.5 microns in pink lesions to 51.2 microns in purple lesions. The vessel depth was partly reflected in the lesional colour, as the pink and purple lesions had significantly deeper vessels than the red ones (P = 0.02). These results indicate that pink lesions predict poor blanching due to deeply located small vessels, while red lesions predict a good therapeutic result because of more superficially located vessels.

Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase high-performance liquid chromatography to determine the content of the oxidation product 5-formyluracil (5-foU) in DNA. With this method, it is shown that 5-foU residues are formed with high frequency from thymine by quinone-sensitized UV-A photooxidation. Since 5-foU is also induced by ionizing radiation, it appears to be formed under conditions where thymidine radical cations are generated and react with molecular oxygen. It was previously shown that 5-foU is formed directly from [methyl-3H]thymine residues in radioactively labeled DNA by two consecutive transmutations of 3H to 3He. The theoretical basis for the kinetics of such conversion is presented in this paper, and the calculated yields are confirmed experimentally by measuring the content of 5-foU in [methyl-3H]thymine-labeled DNA aged for different time periods. Such DNA contains virtually only 5-(hydroxymethyl)uracil and 5-foU, apart from normal bases, and is therefore very useful for the investigation of repair enzyme activities involved in the repair of 5-foU-containing DNA. Using this substrate, a DNA glycosylase activity was identified in human cell extracts for the removal of 5-foU.(ABSTRACT TRUNCATED AT 250 WORDS)

In involved psoriatic tissue, which is characterized by chronic inflammation in both epidermis and dermis, elevated levels of arachidonic acid and eicosanoids have been measured. This implies that a phospholipase A2 (PLA2) may be involved in the pathogenesis of psoriasis. The PLA2's are a group of enzymes that release unsaturated fatty acids from the sn2-position of membrane phospholipids. Once released, the fatty acids are converted by various enzymes into biologically very important signaling molecules. Release of arachidonate initiates the arachidonate cascade, leading to the synthesis of eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and lipoxines. Eicosanoids are important in a variety of physiological processes and play a central role in inflammatory mediators, such as lyso-PAF (a precursor for PAF) and other lysophospholipids, may also be formed through the action of a PLA2. We report for the first time the detection of transcripts of nonpancreatic phospholipase A2 (npPLA2, type II) and cytosolic (c) PLA2 in human skin, and overexpression of npPLA2 in involved skin from patients with psoriasis (plaque psoriasis and pustular psoriasis). Limited amounts of npPLA2 enzyme are detected immunologically in the uppermost layers of epidermis from healthy persons. Both involved and uninvolved psoriatic epidermis contain higher levels of npPLA2 than normal skin. Positive cells in dermis showed significantly higher levels of npPLA2 than epidermal cells. In dermis from healthy persons, only weak staining of a few cells could be detected. The two PLA2 enzymes detected in psoriatic skin (cytosolic and nonpancreatic) may both be involved in eicosanoid overproduction in psoriatic tissue, and the npPLA2 may also be involved in potentiating cell activation, especially T cells.

Thirty patients were treated with a flashlamp-pumped pulsed dye laser, with 0.45 ms pulse width and 585 nm wavelength. Punch biopsies were taken prior to treatment, and the biopsies were examined morphometrically. Three different test sites were exposed to laser light of fluence 5.25, 6.50 and 7.75 J/cm2. The degree of blanching was examined 6-8 weeks after treatment, and each site was retreated four times. Six patients (20%) achieved poor blanching, eight patients (27%) obtained moderate lightening and 16 patients (53%) showed good response. The vessels of the good responders were located significantly more superficially than the vessels of the moderate and poor responders (P < 0.05). The poor responders had significantly smaller vessels than the moderate and good responders (P < 0.01). The moderate responders had deeper, but larger vessels, than the poor responders. Hence, an increasing vessel diameter reduces the negative outcome of increasing vessel depth. The vessel diameter was correlated to the colour (P < 0.01), e.g. the mean vessel diameter was increasing from 16.5 microns in pink lesions to 51.2 microns in purple lesions. The vessel depth was partly reflected in the lesional colour, as the pink and purple lesions had significantly deeper vessels than the red ones (P = 0.02). These results indicate that pink lesions predict poor blanching due to deeply located small vessels, while red lesions predict a good therapeutic result because of more superficially located vessels.