[The Open House campaign for skin examinations resulted in valuable experiences].
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Evaluation Studies as Topic↗
Biomedical subjects
H Rorsman
Publications and source records attributed to H Rorsman.
The neuromelanin of the human substantia nigra.
The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.
Chromatography, High Pressure Liquid↗
Hydrogen peroxide as a mediator of dopac-induced effects on melanoma cells.
Dopac increases tyrosinase activity and exerts cytotoxic effects in cultures of human melanoma cells. The possible role of hydrogen peroxide in these actions was examined. Catalase (100 micrograms/ml) completely reversed the cytotoxic action of 0.3 mM dopac and reduced its tyrosinase-stimulating effect by approximately one half. The results show that extracellular hydrogen peroxide is a mediator of both the tyrosinase-stimulating and cytotoxic actions of dopac. Analysis of the degradation products of melanin from dopac-treated melanoma cells after hydriodic acid (HI) hydrolysis revealed the presence of aminohydroxy-phenylacetic acid (AHPAc). This substance is obtained by HI hydrolysis of melanin formed by oxidation of cysteinyl-dopac. Thus, the presence of AHPAc indicates that dopac is transported into the melanocytes where it serves as a substrate for tyrosinase.
3,4-Dihydroxyphenylacetic Acid↗
A sensitive tyrosinase method for human skin.
A sensitive competitive radioimmunoassay for quantitative analysis of tyrosinase in biological tissues has been developed. The binding sites of antityrosinase antibodies were competed for by 125I-labelled human tyrosinase and a known (standard) or unknown (sample) amount of tyrosinase in solution. A sensitivity range of about 5-500 pmol/l was obtained when a solubilized melanoma tyrosinase with a molecular weight of 66,000 D was used as a standard. The method was used on 0.5 cm2 skin samples from which tyrosinase was solubilized with Nonidet P-40. The method is useful for measuring the content of tyrosinase in crude samples where the enzyme activity may be affected by inhibitors or by unspecific oxidation.
Animals↗
Glutathione in human melanoma cells. Effects of cysteine, cysteine esters and glutathione isopropyl ester.
Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl-L-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.
Acetylcysteine↗
Isolation of tyrosinase from bovine eyes.
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.
Amino Acid Sequence↗
Enzyme release from cultured human melanoma cells.
The lysosomal enzyme beta-hexosaminidase and the melanocyte specific enzyme tyrosinase were examined in human melanoma cell cultures. The beta-hexosaminidase activity of the medium was approximately 40% of the total cellular activity after 24 h, while after 48 h the activity in the medium was twice that of the cells. The tyrosinase activity in the medium was 5% and 19% of the total cellular activity after the 24 h and 48 h incubation, respectively. The low level of lactate dehydrogenase activity in the medium after 24 as well as 48 h of incubation indicated that the release of beta-hexosaminidase and tyrosinase was not due to membrane injury. The data suggest, that 1) beta-hexosaminidase may be a candidate for tumor markers in malignant melanoma, and 2) the tyrosinase activity found in sera of melanoma patients may be due, at least partly, to enzyme release by living cancer cells.
Cell Line↗
Isolation of soluble tyrosinase from human melanoma cells.
In the human melanoma cell tyrosinase exists in a membraneous and a soluble form. The membraneous enzyme has an N-terminal amino acid sequence identical to that predicted from a human c-DNA clone by Kwon et al.. The soluble form has now been isolated by a technique mainly based on the trypsin resistence of the enzyme and the use of hydrophobic interaction chromatography. The specific dopa oxidase activity of the soluble enzyme was 300 mumol/min x mg protein. On isoelectric focusing the enzyme was found in at least ten bands, pI between 3.8-4.6. The molecular weight was found to be 53,000 D. The N-terminal amino acid sequence was the same as that found in the membrane bound form of the enzyme, i.e. the protein maps at the c-albino locus.
Amino Acid Sequence↗
5-S-cysteinyldopac in human urine.
5-S-Cysteinyldopac, a compound hitherto demonstrated only in brain tissue, has been isolated and quantified in urine. The urines from 12 individuals were found to contain 20 +/- 9.1 micrograms 5-S-cysteinyldopac/24 hours. Incubation of 5-S-cysteinyldopamine with MAO-containing tissue did not give any formation of 5-S-cysteinyldopac, indicating that this compound is formed by nucleophilic addition of cysteine directly to dopac. The findings give further evidence for a small but significant non-specific oxidation of endogenous catechol derivatives in vivo, a fact to be considered when using 5-S-cysteinyldopa as a measure of pigment metabolism.
3,4-Dihydroxyphenylacetic Acid↗
Tyrosinase activity in serum from patients with malignant melanoma.
A preparation procedure is presented for the determination of tyrosinase-catalysed stereo-specific dopa oxidase activity in serum. Purification is obtained by separation on a Phenyl-Sepharose hydrophobic interaction column, followed by Con-A-Sepharose chromatography. Five out of seven sera from patients with widespread melanoma metastases were found to contain detectable quantities of tyrosinase. There was no tyrosinase activity in seven sera from patients with other malignancies, nor in six other control sera from individuals without malignancies. One serum which showed high tyrosinase activity was processed as above and studied by SDS-PAGE. A dopa-reactive band with an apparent MW of 66 kD was present in the gel, i.e. at the same place as that of the soluble tyrosinase of cultured human malignant melanoma cells. The protein was found to have the same pI at isoelectric focusing, and eluted in the same way from the preparation columns used, as did soluble tyrosinase.
Aged↗
Isolation of human tyrosinase from cultured melanoma cells.
Tyrosinase was isolated from cultured melanoma cells using a procedure involving solubilization of the enzyme by means of Triton X-100, followed by different types of chromatography and tryptic digestion to make the enzyme soluble even in the absence of detergent. Starting with a membranous material containing 72 mg protein, 0.21 mg tyrosinase was obtained. The recovery of tyrosinase was 36% of the quantity found in the membranous starting material. In order to acquire a completely purified enzyme preparation suitable for amino acid sequence analysis, SDS-PAGE followed by blotting onto a polyvinylidene difluoride membrane was performed as a final step. The apparent molecular weight was found to be 66,000. Determination of the amino acids of the aminoterminal portion by automated Edman degradation showed the following sequence: His-Phe-Pro-Arg-Ala-X-Val-Ser-Ser-Lys-Asn-Leu-Met-Glu-Lys-Glu-X-X-Pro-Pr o-The enzyme purified has an amino acid sequence identical with that of human tyrosinase deduced from c-DNA by Kwon et al. Striking similarities between our amino acid sequence and that predicted by Yamamoto et al. from mouse tyrosinase c-DNA were also observed.
Amino Acid Sequence↗
Modulation of glutathione level in cultured human melanoma cells.
The effects of buthionine sulphoximine (BSO) treatment on cellular glutathione (GSH) content and on the cytotoxic action of menadione were investigated in cultured IGRI human melanoma cells. Addition of BSO (10(-8)-0.5 X 10(-3) M) to the cultures resulted in a dose- and time-dependent depletion of cellular GSH. BSO (10(-5) and 10(-6) M) did not influence cell multiplication up to 48 h, as determined by trypan blue staining. Menadione (3 X 10(-5) M) treatment decreased the cellular GSH concentration and also reduced cell number after a 24 h exposure. Its cytotoxicity was increased by BSO (10(-5), 10(-6) M), though the potentiating effect was moderate.
Antimetabolites↗
Adenylate cyclase activity in homogenates of human melanoma cells. Effect of alpha-MSH and isoprenaline.
The effects of the alpha-melanocyte-stimulating hormone (alpha-MSH) (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoprenaline (10(-9)-10(-4) M) on adenylate cyclase (AC) activity were investigated in homogenates of the human IGR 1 melanoma cells with or without additional GTP. Basal AC activity was increased by the administration of 10 microM GTP. Alpha-MSH had no effect on cyclic AMP (cAMP) accumulation, while isoprenaline stimulated AC activity in a dose-dependent manner.
Adenylyl Cyclases↗
Stimulation of tyrosinase by dihydroxy phenyl derivatives.
Isoprenaline (0.3 mM) moderately increased the tyrosinase activity of cultured human melanoma cells, while another beta-adrenoceptor agonist, terbutaline (1-3 mM) and a catechol compound, dopac (0.1-0.3 mM) induced a several fold increase in the enzyme activity. Isoprenaline (0.3-1 mM) and dopac (0.3-1 mM) also exerted pronounced toxic effects on the cells. The data suggest: 1) a possible role for beta-adrenoceptors in the regulation of human melanogenesis; 2) two different ways of action for isoprenaline in inducing tyrosinase elevation; 3) the possible usefulness of dopac as a chemotherapeutic agent.
3,4-Dihydroxyphenylacetic Acid↗
Biochemical observations in UV-induced pigmentation.
The biochemical events in the melanocytes resulting from stimulation of pigmentation by exposure to ultraviolet (UV) irradiation are correlated to changes in specific metabolites in the plasma and in the urine. A compound produced in the melanocyte by nucleophilic addition of cysteine to dopaquinone, 5-S-cysteinyldopa, is present in increased quantities in the plasma and in the urine after different types of pigment-producing UV exposure. Similarly, a methylated indolic metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid, appears in increased amounts in the urine after UV exposure of the skin. UVA does not produce inflammation or pigmentation in skin that is anoxic at the time of irradiation. In contrast, inflammation and pigment response to UVB and PUVA are not dependent on oxygenation of the skin at the time of exposure.
Cysteinyldopa↗
Dopaquinone addition products in cultured human melanoma cells.
The concentrations of dopa, cysteinyldopas, 5-S-glutathionyldopa, gamma-glutamyl-5-S-cysteinyldopa and 5-S-cysteinylglycinedopa, were analysed in homogenates of cultured human melanoma cells and in culture media. Cysteinyldopas were found to be the major catechol in the cells, with a molar concentration more than a hundred times that of dopa. 5-S-Glutathionyldopa was found in the same amount as dopa, while the quantity of 5-S-cysteinylglycinedopa was one order of magnitude less. gamma-Glutamyl-5-S-cysteinyldopa was not present in detectable amounts. In the medium the concentrations of dopa, 5-S-cysteinylglycinedopa and of 5-S-glutathionyldopa were about one half of those in the cells, while the concentration of cysteinyldopas was about 2%. The ratio between 2-S-cysteinyldopa and 5-S-cysteinyldopa when incubating dopa and cysteine with tyrosinase was identical with the ratio between the analogically synthetised isomers of glutathionyldopa. Consequently, from the calculation of these ratios in cells and media one cannot deduce whether cysteinyldopas arise from the direct addition of cysteine to dopaquinone, or from degradation of glutathionyldopa. Oxidation of 5-S-glutathionyldopa gives a red chromophore with maximum absorption at 480 nm which develops into a black pigment.
Benzoquinones↗
Oxidation of dopa in the skin of black and albino mice.
The dopa oxidase activity of tyrosinase in the skin from albino and black mice was assayed using a technique based on the formation of two diastereomers of 5-S-cysteinyldopa when incubating tissue extracts with both L-dopa and D-dopa as substrates in the presence of cysteine. The amounts of 5-S-L-cysteinyl-L-dopa and 5-S-L-cysteinyl-D-dopa formed were then determined. In the extract from black mouse skin the L-L-diastereomer was produced in more than ten times the amount of the L-D-diastereomer. This stereospecific dopa-oxidation is indicative of the presence of tyrosinase and corresponds well with earlier determinations of the rates of oxidation for human tyrosinase, using L-dopa and D-dopa as substrates. Stereospecific dopa oxidation was absent in albino skin, and the nonspecific dopa-oxidation was two orders of magnitude less than the dopa oxidation in black skin. The study demonstrates the lack of tyrosinase activity in albino skin, and quantifies the non-specific dopa oxidation. The lack of tyrosinase activity in the eluates from albinotic skin was found not to be due to the presence of a tyrosinase inhibitor.
Albinism↗
Trichochromuria in melanosis of melanoma.
Two patients with metastasizing melanoma and diffuse melanosis have previously been reported to excrete large quantities of trichochromes in the urine. The present study describes 2 further melanoma patients with diffuse melanosis and trichochromuria. The hair of one of the patients which had been red in childhood and turned brown in adult age returned to red with the appearance of melanosis. Normal excretion of a methylated melanocytic metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid, was observed in this patient, possibly indicating exhaustion of the methylating system. The other patients excreted large quantities of 6-hydroxy-5-methoxyindole-2-carboxylic acid. Both patients showed highly increased excretion of 5-S-cysteinyldopa. Both patients with melanosis exhibited fine electrone-dense granules in lysosomes of dermal histiocytes. The findings support the concept that trichochromes or similar pigments in dermal histiocytes are responsible for diffuse melanosis in melanoma patients.
Adult↗