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A short review on the history of pigment cell research at Charles University (Ch.U.) in Prague is presented. The famous Czech physiologist and professor J.E. Purkyne started the pigment cell research at Ch.U. already in 1837. He discovered melanin granules in the cells of substantia nigra of the brain. Later, in 1858, a Czech professor of medicine at Ch.U., B. Eiselt, as the first, described melanogenuria in 3 patients with generalized melanoma. Also some German professors at Ch.U. contributed to the research of melanins and melanogenuria in the past, especially H. Waelsch (1932). After the World War II, a Czech professor of medical chemistry at Ch.U., A.F. Richter with his young assistant J. Duchon continued in the chemical exploration of melanins (1954) and J.D. with Z. Pechan, B. Matous and S. Pavel devoted their attention to melanogenuria in melanoma patients (1962-1980). In 1967 they identified 2 new metabolites in melanoma urine: 5-hydroxy-6-methoxy and 6-hydroxy-5-methoxy-indole-2-carboxylic acids. J. Duchon with J. Borovanský and P. Hach also studied morphology and chemical composition of different melanosomes (1972-1979) and brought the first evidence that melanosomes consist of several proteins (1972). In 1980's 4 groups devoted to the pigment cell research and originated from Ch.U. were formed. The groups of J.B., of B.M. and of J. Vachtenheim in Prague and the group of S.P. who moved to the Netherlands (Leiden). As for the clinical aspects of the pigment cell research, the s.c. Hermanský-Pudlák syndrome published in 1959 and the histopathological classification of malignant melanomas estimated by J. Trapl (1957), should be mentioned. Therefore it is not surprising that, as a result of the tradition of pigment cell research at Ch.U., the 3rd European Workshop on Melanin Pigmentation was held in Prague already in 1981 and that, in 1998 again, Ch.U. was entrusted with the arrangement of the 8th Meeting of the European Society for Pigment Cell Research at the occasion of the Ch.U. 650th anniversary.

A short review on the pigment cell research at Charles University in Prague is presented.

Melanogenesis is a multistep biochemical process resulting in the formation of melanin in pigment cells in the skin and the eye. Three melanogenic factors, tyrosinase, TRP1, and TRP2 participate in the pathway. Here, the regulation of gene expression of these melanocyte-specific markers is shortly reviewed.

A short review on the development of the study of melanogenuria in malignant melanoma is presented.

The authors point out that melanogenuria, i.e. the excretion of specific phenolic and, particulary, indole metabolites in melanoma, is undoubtedly due to elevated tyrosinase activity in this disease. Other enzymes, in particular glucuronyltransferase, catechol-O-methyltransferase and possibly hydroxyindole-O-methyltransferase, also evidently participate in the further biochemical conversion of the resultant metabolites, however.

The Thormählen positive melanogen previously determined as "A" has been isolated from melanotic urine by means of column chromatography on Amberlite IR 45, DEAE cellulose and Dowex 50. According to its behavior during a study with 1H--NMR spectra it has been found that the isolatd compound is 6-methoxy-5-indolyglucosiduronate, the metabolite of 5,6-dihydroxyindole. The possible formation and biotransformation of this compound is discussed.

A simple spectrophotometric method for a rapid determination of tyrosinase (EC 1.14.18.1) is described. The basis of the assay is the incubation of the enzyme with L-dopa in the presence of an optimal concentration of Zn2+ ions and the measurement of the formation of melanochrome, as indicated by the rise in absorbance at 540 nm. Final absorbance change reflects probably two activities of tyrosinase: the oxidation of dopa to dopaquinone and the conversion of 5,6-dihydroxyindole to melanochrome. Using a purified preparation from hamster melanoma, the assay was found to be more sensitive than the commonly used dopachrome assay. Comparison with some other currently available methods for assaying tyrosinase is presented and potential applications of the assay are discussed.

Urinary melanogens are indolic and phenolic compounds which are excreted in an elevated amount in the urine of melanoma patients. Urinary melanogens can be divided into two principal groups according to their behavior during Thormählen test--Thormählen positive melanogens (TPM) and Thormählen negative melanogens (TNM). When comparing TPM and TNM (represented by homovanillic and vanillactic acids) urinary excretion in stage III of melanoma patients we have found two significantly different relations for melanogens excretion, i. e. predominating TPM urinary excretion in melanotic forms of melanomas and predominating homovanillic acid and vanillactic acid urinary excretion in amelanotic forms of melanomas. We assumed that it could be an indirect evidence of the different catalytic activity of either cresolase or catecholase activities of tyrosinase.

Polyclonal antibodies to hamster melanoma tyrosinase were raised in rabbits, and series of immunoinhibition experiments with a purified enzyme and specific immunoglobulins were carried out. Tyrosinase activity was determined by a set of radiochemical and spectrophotometric methods utilizing tyrosine, dopa, dopamine, or dihydroxyindole (DHI) as substrates. The quantitative data obtained indicated that the complexing of tyrosinase with its specific antibody inhibited melanogenesis in a specific manner: dopachrome formation from dopa and dopamine conversion to melanin were not affected and all other enzyme activities comprising the DHI oxidation step were inhibited to various degrees. Additionally, tyrosine hydroxylation was also slightly inhibited. The data obtained implied that melanogenesis was restricted at the point of DHI oxidation. From observations on the immunoinhibition of a DHI oxidation at varying dopa-cofactor concentrations, we propose that dopa-cofactor may be bound at separate site than DHI and thus may act as a positive allosteric effector for DHI oxidation by tyrosinase. Study of tyrosinase immunoinhibition by the antibodies against the enzyme thus seems to provide a valuable system for investigating the tyrosinase-mediated melanogenesis.

Melanogens which are considered as probable precursors or their metabolites in the metabolic pathway toward melanin are excreted in an increasing amount during the melanotic melanoma growth in hamsters. Moreover, an increased excretion of zinc was observed. The evaluation of dependence between zinc and Thormählen positive melanogens excretions during the melanoma growth suggests an exponential type of dependence. Likewise as in the case of human melanoma the hyperzincuria proved to be a laboratory sign of tumor disease progression in Syrian hamsters as well. The cause of an increased zinc excretion during the melanoma growth seems to consist in an increased lysis of pigment cells. The presumed destruction of tumor cells was not accompanied by a higher excretion of magnesium, though. Concentration of creatinine proved to be a useful mean for expressing of standard excretion of various substances also in hamsters.

Strategy of protein determination in melanin containing samples was analyzed, and the prerequisites under which the Lowry procedure could be used were established. (a) Complete solubilization of proteins from melanosomes was not reached until the treatment with 3% sodium dodecylsulfate at 100 degrees C was prolonged to 5 h. (b) It is necessary to correct the data obtained with Folin reaction to eliminate melanin interference. (c) The correction is based upon the determination of the extent of solubilized melanin and subtraction of the corresponding Folin-Ciocalteu reaction values. (d) The extent of correction due to the melanin interference was found to be about 40% of the absorbance value. It was concluded that correction of the data obtained with Lowry assay is possible, but the procedure is complicated and time-consuming. However, a more simple modification has not been developed.