Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report on the culturing of melanocytes from suction blisters from the uninvolved skin of localized (focal and segmental) vitiligo patients and from the foreskins of newborns and adults, over a long period of time using a modified culture medium composed of F-12 medium supplemented with insulin (5 mg/mL), cholera toxin (40 ng/mL), transferrin (5 mg/mL), hydrocortisone (1 mM), epithelial growth factor (20 ng/mL), endothelial cell growth supplement (ECGS) (15 mg/mL), retinol (1 x 10(-7) M), 12-o-tetradecanoyl-phorbol-13 acetates (TPA) (85 nM), and 1% fetal calf serum (FCS). This method may be used in in vitro studies on normal human melanocytes and for study of the difference between melanocytes of normal individuals and of those with vitiligo. The ability to culture melanocytes from localized vitiligo, and the inability to grow those of generalized vitiligo with this system reconfirms the difference in the pathogenesis of various types of vitiligo. Thus, culturing may be used for differentiating localized vitiligo from generalized vitiligo and may be used to guide the mode of treatment when vitiligo has just started to develop and only a few depigmented patches have appeared. The results of studies on seeding density effect and serial Dopa reaction on the melanocytes of normal individuals reveal that the use of tyrosinase activity as the assessment parameter requires the use of melanocytes cultured for less than five months at a seeding density of less than 4 x 10(4) cells/cm2. Enlarged and heavily pigmented cells, senescent cells, were observed after very long-term culture (one year and four months).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Growth of human melanocytes from suction blister of localized vitiligo patient and from foreskins of newborns and adults by modified culture medium. 809 31

Certain mono- and dihydroxybenzene derivatives are selectively cytotoxic for melanocytes in vivo, and can cause depigmentation of skin and hair. We produced selective melanocytotoxicity/hair depigmentation in C57Bl mice by injection of 0.032-1.0% p-t-butylcatechol (tBC) or p-hydroxyanisole (MMEH) in physiological saline. No depigmentation occurred on injection of 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenylacetic acid (DOPAC). Light- and electron-microscopic examination of biopsy specimens taken from depigmented areas indicates selective melanocyte damage as early as 2 h post-injection. Melanocytes from anagen hair are most susceptible to depigmentation. All four compounds are substrates for tyrosinase, but only tBC and MMEH generate their respective isolable 1,2-benzoquinones, tBCQ and MMEHQ. These caused depigmentation in C57Bl mice to a comparable degree to the parent compounds. DOPA- and DOPAC-quinones (DOPAQ and DOPACQ) are not spectroscopically detectable in solution, suggesting extremely low steady-state levels of these compounds. The net observed rate of reaction of the respective 1,2-quinone with 300 microM bovine serum albumin (BSA) in vitro varies widely, with tBCQ >> MMEHQ = DOPACQ >> DOPAQ. The results are consistent with a mechanism involving attack of -SH on melanosomal proteins and/or enzymes by tyrosinase-generated 1,2-quinones. This mechanism evidently differs from that involved in in vitro hydroxybenzene melanocytotoxicity of melanoma cells, in which active oxygen intermediates generated by hydroxybenzene autoxidation play a significant role. The most reliable prognosticator of in vivo depigmentation appears to be the ability of the depigmenter to form a spectroscopically stable 1,2-quinone which is capable of reacting with protein -SH.
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PMID:In vivo depigmentation by hydroxybenzene derivatives. 816 83

B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4-dihydroxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.
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PMID:Tyrosinase isoenzymes in mammalian melanocytes. 1. Biochemical characterization of two melanosomal tyrosinases from B16 mouse melanoma. 822 98

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post-grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4-6 weeks. The number of Dopa-positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre-grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (alpha Ty-SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa-positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre- and post-grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw approximately 14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.
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PMID:Histological, biochemical, and ultrastructural studies on hyperpigmented human skin xenografts. 824 20

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.
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PMID:Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. 838 14

Piebaldism was associated with neurofibromatosis 1 (NF-1) in two patients, an association not previously reported. Dopa staining (tyrosinase) and electron microscopy were performed: no melanocytes or melanosomes were found in hypomelanotic skin of patient 2 and in the white forelock skin of patient 1; in patient 2, normal melanocytes and melanosomes were present in the white forelock epidermis but absent from the cortex, cuticles, and inner root sheath of the white forelock hair. Because these structures receive melanosomes from melanocytes in the hair bulb, it was assumed that there were no melanocytes in the hair matrix. Melanocytes and melanosomes were normal by ultrastructural criteria and in terms of their distribution in a normally pigmented macule within a hypomelanotic patch of patient 2. These and earlier report findings led to three conclusions: subtypes of piebaldism exist, including our patients showing a combination of piebaldism and NF-1; the most commonly reported subtype has no melanocytes in the white forelock and hypomelanotic skin, although microscopic islands of melanocytes may exist within hypomelanotic skin; and the ultrastructure of white forelock skin and hair of patient 2 is consistent with a mouse model of piebaldism, in which the hair follicle has no active melanocytes, but the interfollicular epidermis is normally melanized.
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PMID:Ultrastructural study of two patients with both piebaldism and neurofibromatosis 1. 841 98

The effect of ferrous ions on the monophenolase activity of tyrosinase has been studied. Although a shortening of the lag period which characterizes this hydroxylation reaction was observed, no direct effect on the enzyme was found. The reaction between ferrous ions and molecular oxygen in the presence of chelating agents, such as phosphate or EDTA, produces hydroxyl radicals. These radicals can hydroxylate tyrosine to generate L-3,4-dihydroxyphenylalanine (dopa). Catalase and scavengers of hydroxyl radicals inhibited both the shortening of the lag period and dopa formation. On the basis of these results, it is proposed that the influence of ferrous ions on tyrosinase is due to the formation of dopa in the chemical hydroxylation of tyrosine. Dopa transforms the Emet form of the enzyme (Cu2+Cu2+) into the Edeoxy form (Cu1+Cu1+) and, thus, shortens the lag period.
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PMID:Effect of ferrous ions on the monophenolase activity of tyrosinase. 850 69

In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibly involved in betalain biosynthesis, we have evaluated different assays for the detection and quantification of the enzymatic product Dopa with respect to sensitivity, simplicity, and suitability for automatization. A tyrosinase assay including reversed-phase high-performance liquid chromatography with isocratic elution and fluorescence detection has been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm). This improved assay was sensitive (detection limit: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 pmol-20 nmol Dopa). The method proved to be suitable for high-performance liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues of Portulaca grandiflora.
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PMID:Assay for tyrosine hydroxylation activity of tyrosinase from betalain-forming plants and cell cultures. 866 May 89

Kojic acid inhibits effectively the rate of formation of pigmented product(s) and of oxygen uptake when DL-DOPA, norepinephrine and dopamine are oxidized by mushroom tyrosinase. In addition to the direct effect of kojic acid on the enzyme, kojic acid also affects the UV-VIS spectrum of the final product(s) formed, this being due to the ability of the o-quinones of these substrates to oxidize kojic acid to a yellow product(s). Kojic acid can thus prevent the conversion of the o-quinones of DL-DOPA, norepinephrine and dopamine to their corresponding melanin.
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PMID:Effect of kojic acid on the oxidation of DL-DOPA, norepinephrine, and dopamine by mushroom tyrosinase. 878 97

Polyphenols, catecholamines and their oxidation products have a variety of physiological effects and are key components of insect cuticle and other biological structures. Three dihydroxybenzene isomers were easily differentiated using negative ion electrospray mass spectrometry (ESMS) or tandem mass spectrometry (MS/MS). Ions at m/z 107-109 were diagnostic for more complex polyphenols and catecholamines with a dihydroxybenzene moiety. The product ion spectra of other compounds including 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylalanine (DOPA) and N-acetyldopamine (NADA) had product ions at m/z 121-123 corresponding to methylene homologues of dioxygenated benzene. Oxidation of DOPA using tyrosinase, Ag2O or NaIO4 could be followed using ESMS and several proposed intermediates proceeding from DOPA through a quinone methide intermediate, 5,6-dihydroxyindole and dihydroxyindole quinone were confirmed using negative ion ESMS/MS. Spectra of the NaIO4 oxidation products of 1,2,3-trihydroxybenzene (pyrogallol) were consistent with tetrahydroxybenzene, two non-cyclic tetraoxygenated derivatives and a bicyclic polymerization product, purpurogallin. ESMS can be used to analyse complex polyphenolic conjugates commonly encountered in biological specimens.
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PMID:Negative ion electrospray mass spectrometry of polyphenols, catecholamines and their oxidation products. 899 May 24


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