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Notes: Abstract: Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.

Notes: Brain levels of the 5-S-cysteinyl adducts of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine were determined in several mammalian species. The low levels of the compounds and the risk of artifacts during sample preparation necessitated rather profound modifications of the assaying method. The refined method has made it possible to present more accurate data than those previously reported from this laboratory. The occurrence of low levels of the 5-S-cysteinyl adducts in dopamine-rich brain areas, but not in cerebellum, is indirect evidence of in vivo autoxidation of DOPA, DOPAC, and dopamine. The products generated during catechol autoxidation, including quinones and reduced forms of oxygen, are known to be potentially cytotoxic.

Notes: Abstract: Extracellular levels of endogenous serotonin (5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in the caudate-putamen of anesthetized and awake rats using intracerebral microdialysis coupled to HPLC with fluorimetric detection. A dialysis probe (of the loop type) was perfused with Ringer solution at 2 μl/min, and samples collected every 30 or 60 min. Basal indole levels were followed for up to 4 days in both intact and 5,7-dihydroxytryptamine (5,7-DHT) lesioned animals. Immediately after the probe implantation, the striatal 5-HT levels were about 10 times higher than the steady-state levels that were reached after 7-8 h of perfusion. The steady-state baseline levels, which amounted to 22.5 fmol/30 min sampling time, remained stable for 4 days. In 5,7-DHT-denervated animals, the steady-state levels of 5-HT, measured during the second day after probe implantation, were below the limit of detection (〈10 fmol/60 min). However, during the first 6h post-implantation, the 5-HT output was as high as in intact animals, which suggests that the high 5-HT levels recovered in association with probe implantation were blood-derived. As a consequence, all other experiments were started after a delay of at least 12 h after implantation of the dialysis probe. In awake, freely moving animals, the steady-state 5-HT levels were about 60% higher than in halothane-anesthetized animals, whereas 5-HIAA was unaffected by anesthesia. KCI (60 and 100 mM) added to the perfusion fluid produced a sharp increase in 5-HT output that was eight-fold at the 60 mM concentration and 21-fold at the 100 mM concentration. In contrast, 5-HIAA output dropped by 43 and 54%, respectively. In 5,7-DHT-lesioned animals, the KCl-evoked (100 mM) release represented less than 5% of the peak values obtained for the intact striata. Omission of Ca2+ from the perfusion fluid resulted in a 70% reduction in baseline 5-HT output, whereas the 5-HIAA levels remained unchanged. High concentrations of tetrodotoxin (TTX) added to the perfusion medium (5-50 μM) resulted in quite variable results. At a lower concentration (1 μM), however, TTX produceda 50% reduction in baseline 5-HT release, whereas the 5-HIAA output remained unchanged. The 5-HT reuptake blocker, indalpine, increased the extracellular levels of 5-HT sixfold when added to the perfusion medium (1 μM), and threefold when given intraperitoneally (5 mg/kg). By contrast, the 5-HIAA level remained unaffected during indalpine infusion. Application of TTX (1 μM) under simultaneous 5-HT uptake blockade induced a decrease in 5-HT output by 62–71%. p-Chloroamphetamine (2.5 mg/kg, i.p.) induced a 12-fold increase in 5-HT release and reduced the 5-HIAA output by about 50%. The p-chloroamphetamine-induced increase in 5-HT release was 10 times lower in the 5,7-DHT-denervated striatum. Pargyline (75 mg/kg, i.p.) increased the extracellular levels of 5-HT 11-fold within 6 h, and reduced the 5-HIAA levels by 80%. The 5-HT receptor agonist, 5-methoxy-N,N-dimethyltryptamine (1 mg/kg, i.p.), produced an immediate reduction of about 50% in 5-HT release and a small (11 %) decrease in 5-HIAA output. It is concluded (1) that intracerebral microdialysis coupled to HPLC with fluorimetric detection provides a useful method for the study of extracellular 5-HT and 5-HIAA levels; (2) that steady-state levels of 5-HT and 5-HIAA recovered in the dialysis perfusate are neuronally derived, but these steady-state levels are reached only after a minimum of 7–8 h after probe implantation; (3) that changes in striatal extracellular levels of 5-HT are closely related to changes in serotonergic synaptic activity; and (4) that extracellular levels of 5-HIAA are a poor indicator of synaptic activity, and instead primarily reflect intraneuronal metabolism.

Notes: Abstract: UVB light was used to induce an experimental inflammation in normal human skin in order to investigate its correlation with the activity of the newly described enzyme d-dopachrome tautomerase (DDT) in the fluid of experimental blisters. Macrophage migration inhibitory factor (MIF) activity was determined as a closely related marker of inflammation. DDT and MIF activities were demonstrated in blister fluids in all 10 healthy subjects. All but one of these subjects showed increased activity of DDT and MIF after three minimal erythemal doses (MED) of UVB. The mean activity of DDT increased approximately twofold and the mean activity of MIF also increased twofold after UVB in our experimental model. We found a strong correlation between DDT and MIF activities. The presence of DDT in epidermis and its increase at UV irradiation was confirmed by immunohistochemical studies. In this study, DDT is for the first time demonstrated in the skin. It is also the first time DDT can be related to inflammation, and its covariation with MIF strengthens this observation.

Notes: Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.

Notes: Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. l-Tyrosine and d-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of l-tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of d-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the l-isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with l- and d-tyrosine, human tyrosinase, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether d-tyrosine or l-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.

Notes: The mouse b locus controls black/brown coat coloration. Its product, the b-protein or TRP-1, has significant homology to tyrosinase, and this has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b-protein. We were unable to con-firm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.

Notes: Pigmented tissues from bovine eye were used as a source for isolation of tyrosinase from normal melanocytes. Tyrosinase is highly hydrophobic and the isolation procedure is mainly based on the use of hydrophobic interaction chromatography. The bovine enzyme is, in contrast to the human melanoma tyrosinase, mainly soluble. The predominant part of the ocular enzyme from cow has a molecular weight and isoelectric behavior similar to that of the soluble tyrosinase in the human melanoma cells. The N-terminal amino acid sequence of isolated bovine tyrosinase was determined by automated Edman degradation. The N-terminal amino acid sequence from normal bovine tyrosinase was identical to the sequence of an N-terminal region of mouse melanoma tyrosinase predicted from a c-DNA clone by Kwon et al. (1988). The amino acid sequence of bovine tyrosinase shows homology to that of human tyrosinase (Wittbjer et al., 1989), but three amino acids of the 16 residues determined by us differed. Histidine was the N-terminal amino acid.

Notes: The effects of N-acetyl-l-cysteine (l-NAC), N,N-diacetyl-l-cystine (oxidized form of l-NAC) and N-acetyl-d-cysteine on the intracellular glutathione (GSH) level and their toxicity were investigated in the human melanoma cell culture IGR1. l-NAC applied in 3 mM concentration for 24 hr decreased; when applied for 48 hr it did not alter the intracellular GSH level. Treatment with 1 mM l-NAC for 24 hr had no effect on cellular glutathione, whereas the same concentration applied for 48 hr resulted in an increase in the level of GSH. Both concentrations also induced cell injury as determined by protein assay and trypan blue staining. N,N-diacetyl-l-cystine (0.5 and 1.5 mM, 24 hr) induced a decrease in cellular glutathione content without any apparent cell toxicity. d-NAC (1 and 3 mM, 24 hr) did not influence the GSH level of the melanoma cells; however, it had toxic effects resulting in cell loss.