Amount Of Tyrosinase Research Articles

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration.

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese-dependent superoxide dismutase (MnSOD), a mitochondrial-resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract-induced regulation of melanogenesis.

Effect of Delta-Tocotrienol on Melanin Content and Enzymes for Melanin Synthesis in Mouse Melanoma Cells

In the present study, we investigated the dose-dependent effect of delta-tocotrienol long term (48, 72 h) on the melanin content of cells treated with delta-tocotrienol, and whether cells treated with delta-tocotrienol for long a time show cytotoxicity. We also examined whether other enzymes responsible for melanin biosynthesis, tyrosinase-related protein-1 (TRP-1) and -2 (TRP-2), are involved in the decrease in melanin levels. Protein levels in cells treated with 25 or 50 microM delta-tocotrienol for 48 h or 72 h were similar to those in control cells. Melanin content decreased by 44 (25 microM delta-tocotrienol) to 50% (50 microM) at 48 h, and by 14 to 21% at 72 h, compared to control levels. Tyrosinase activity, amounts of tyrosinase and TRP-1 decreased dependent on dose : by 50 (25 microM delta-tocotrienol) to 75% (50 microM), 20 to 45% and 42 to 82% at 48 h, and by 25 to 50%, 75 to 80% and 78 to 77% at 72 h, respectively. Although the amount of TRP-2 increased by 20% on treatment with 25 microM delta-tocotrienol for 48 h, it decreased by 52% on treatment with 50 microM delta-tocotrienol for 48 h. The amount of TRP-2 dose-dependently decreased by 55% and 75% on 72 h by treatment with 25 and 50 microM delta-tocotrienol, respectively. From these findings, delta-tocotrienol at up to 50 microM dose-dependently caused a reduction in melanin content by the decrease of TRP-1 and TRP-2 as well as tyrosinase, and no cytotoxicity.

Delta-Tocotrienol Causes Decrease of Melanin Content in Mouse Melanoma Cells

We examined the effect of δ-tocotrienol on melanin content in mouse melanoma B16 cells. Melanin content was significantly reduced in cells treated with 50 and 100 μM δ-tocotrienol, but not 10 μM δ-tocotrienol. The activity and amount of tyrosinase also significantly decreased in cells treated with 10, 50, and 100 μM δ-tocotrienol. Furthermore, the mRNA level of tyrosinase as measured using realtime PCR was significantly decreased compared to controls in cells treated with 100 μM δ-tocotrienol, but not 10 or 50 μM δ-tocotrienol. These results indicated that at first δ-tocotrienol caused tyrosinase degradation, and then caused a further decrease in the tyrosinase protein level via both tyrosinase degradation and a decrease in the mRNA level of tyrosinase. We conclude that the decrease of melanin content in the cells by δ-tocotrienol was the result of the decrease of the protein level of tyrosinase (tyrosinase degradation is more important than the decrease of mRNA).

Down-regulation of melanin synthesis by a biphenyl derivative and its mechanism.

Down-regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2'-dihydroxy-5,5'-dipropyl-biphenyl (DDB), down-regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse-chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down-regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation.

Analysis of the UV-induced melanogenesis and growth arrest of human melanocytes.

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm2 UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G2-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 microM alpha-melanocyte stimulating hormone (alpha-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of alpha-MSH on the UV-induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.

Effects of melanocyte-stimulating hormone on tyrosinase expression and melanin synthesis in hair follicular melanocytes of the mouse.

Tyrosinase mRNA, synthesis and activity were measured in the skin during the first 2 weeks of life of C3H-HeAvy mice. Tyrosinase mRNA levels were found to peak on days 3-4 and were followed by increases in tyrosinase synthesis and activity which peaked on days 6-7 and 7-8 respectively. These changes in tyrosinase expression were presumably associated with the growth of the first coat of hair that in neonatal C3H-HeAvy mice is yellow in colour as a result of the increased proportion of phaeomelanin. By the time hair growth had ceased there was no expression of tyrosinase at both mRNA and protein levels. Daily administration of alpha-melanocyte stimulating hormone (alpha-MSH) enhanced the expression of tyrosinase mRNA transcripts, tyrosinase synthesis and activity. The increase in tyrosinase activity paralleled the change in the amount of tyrosinase, suggesting that the primary action of alpha-MSH is to stimulate new synthesis of the enzyme. This induction of tyrosinase was associated with the growth of hair that was darker in colour than that of the controls and contained an increased proportion of eumelanin. This increase in eumelanin reflected a decrease in phaeomelanin content. It was concluded that, through its actions on the enzyme tyrosinase, alpha-MSH is able to switch the synthesis of phaeomelanin to that of eumelanin in hair follicular melanocytes.

Melanocyte-stimulating properties of arachidonic acid metabolites: possible role in postinflammatory pigmentation.

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D2, leukotriene (LT) B4, LTC4, LTD4, LTE4, thromboxane (TX) B2 and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC4 was particularly strong compared to that of PGE2, about which we have previously reported. On the other hand, PGE1, PGF2 alpha and 6-ketoPGF1 alpha did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC4, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.

Dopa reaction test in hair bulbs of fetuses and its application to the prenatal diagnosis of albinism

No information is available on the amount of tyrosinase normally present in fetuses. A dopa reaction test in hair bulbs from the scalp of normal fetuses obtained after abortion showed that tyrosinase is present in fetuses as early as 17 weeks. Only faint activity was detected in skin specimens other than from the scalp. This assay can serve as a quick and reliable method for the prenatal diagnosis of tyrosinase-negative albinism.

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.

Optimization of Conditions for Preparing Synthetic Pheomelanin

Synthetic melanins are often used as the standards for natural melanins. Conditions for preparing synthetic pheomelanin from L-dopa and L-cysteine were studied under different temperatures (25 and 37 degrees C), different amounts of cysteine (1.5 and 2.0 eq), and different amounts of tyrosinase (10 and 20 mg). Effects of addition of catalase and of replacement of oxygen with air were also examined. The best preparation of pheomelanin was obtained in a good yield, when 1 mmol of L-dopa was oxidized at 25 degrees C under oxygen current in 100 ml of a pH 6.8 buffer by 20 mg of mushroom tyrosinase inn the presence of 1.5 mmol of L-cysteine. The pheomelanin thus obtained had an elemental composition and an absorption spectra that were very similar to those from a pheomelanin prepared by tyrosinase oxidation of 5-S-cysteinyldopa. Hydrolysis of synthetic pheomelanins and pheomelanic tissues with hydriodic acid gave aminohydroxyphenylalanines (AHP) as a major product and hydroxybenzothiazolylalanines as a minor product. It is proposed that AHP can be used as a specific indicator of purity of pheomelanins.

Quantitative analysis of mouse tyrosinase by enzyme-linked immunosorbent assay.

A sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505 +/- 106 (S.D.) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.

Stimulation of melanogenesis by cholecalciferol in cultured human melanocytes: a possible mechanism underlying pigmentation after ultraviolet irradiation.

An increase in the amount of tyrosinase was demonstrated in the cultured human melanocyte after 6-day culturing with cholecalciferol (vitamin D3) by increased intensity of the immunofluorescent staining using monoclonal antibody against tyrosinase. Furthermore, the melanocytes became more dendritic as noted in those in the skin after the irradiation of ultraviolet. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha, 25-dihydroxy-vitamin D3 (activated vitamin D3) did not induce any such effect on the cultured human melanocytes. Since cholecalciferol is known to be photo-chemically converted by the ultraviolet irradiation from pro-vitamin D3 produced in the skin, the so-far-unknown mechanism of human skin pigmentation after the ultraviolet irradiation may be partly explained by this stimulating effect of vitamin D3 on the melanocytes.

EFFECT OF ENVIRONMENTAL CONDITIONS UPON TYROSINASE ACTIVITY IN GLOMERELLA CINGULATA

PREVIOUS EXPERIMENTS with Glomerella cingulata have established that tyrosinase activity first appears in plate culture about 120 hr. after inoculation and that its formation is probably adaptive in nature (Sussman and Markert, 1953). On the other hand, cytochrome oxidase activity is present at all times in the organism and in roughly equivalent quantities. In an earlier investigation (Markert, 1950) it was noted that the amount of tyrosinase in the mycelium of Glomerella varies with the conditions of nutrition, aeration, and temperature under which the mycelium is grown. More recently, other workers have called attention to the importance of temperature (Horowitz and Shen, 1952) in the formation of tyrosinase in Neurospora, and in the formation of other fungous enzymes (Fries, 1953). The following experiments were designed, therefore, to explore the effects of these factors and others upon tyrosinase formation in Glomerella. METHODS.-The organism used in these experiments was the standard type (A1B13) of Glomerella cingulata which is a conidiating double mutant from the wild type. Surface cultures were grown from mass conidial inoculum, usually on 30 ml. of agar medium in Petri dishes, and incubated at 200C. until harvested. Where divergence from these techniques occurred, mention will be made before the results are presented. The complete Neurosporac medium (Ryan, 1950) was used, except where otherwise noted. Mycelia were harvested and handled as described by Sussman and Markert (1953). Enzyme extracts were made by grinding the weighed mycelial mats in 1/15 M phosphate buffer at pH 7.2 with a glass homogenizer. The brei was centrifuged at 100 X g for 15 min. after which the supernatant fluid was collected and used. Tyrosinase activity was determined colorimetrically by the method of Markert (1950) as modified (Sussman and Markert, 1953) to employ a Beckman spectrophotometer. In this investigation the model DU was used in place of the model B. The reaction mixture consisted of 1 ml. of 0.02 M DL-dihydroxyphenylalanine (dopa), 0.1 ml. of the enzyme preparation, and 1.5 ml of 1/15 M phosphate buffer at pH 6.5. In certain instances, less enzyme was used because of the preparation's high activity; when this was done, enough buffer was added to bring the total volume to 3.0 ml. The substrate was added at zero time and readings were made at 480 mln at 10-sec. intervals. The change in