BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.
Dihydroxyphenylalanine ; Humans* ; Levodopa ; Melanins ; Melanocytes* ; Melanoma ; Monophenol Monooxygenase ; Tics ; Tyrosine*

BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.
Dihydroxyphenylalanine ; Humans* ; Levodopa ; Melanins ; Melanocytes* ; Melanoma ; Monophenol Monooxygenase ; Tics ; Tyrosine*

Tatbion is a tripeptide, reduced form of Glutathione(GSH or p-glutamyl-cysteiny1 -glycine). Glutathione(SH compound)is believed to inhibit melanin formation by combining witb the copper in tyrnsinase whicb is essential in the conversion of tyrosine to DOPA (3, 4-dihydroxyphenylalanine) and DOPA to DOPA-quinone or by forming cornplex with the intermediate in the tyrosine-to-melaa.in reaction. The effect of Tathion in the treatment of melasma has not been reported in Korea. We have observed the effect of Tatbion in 150 patients with melasma. After the average duration of Gwks of treatment(50-100mg tree tirnes/daily), we were abIe to grade the results as follow. Excellent(Pigmentation almost disappeared): 17. 4 % Good (Pigmentation markedly improved): 56. 7% Fair (Pigmentation slightly improved: 7. 3% None (No effect) : 18.6% The result showed relatively good effects of Tathion in the treatment of 122pts (81.4%) with melasma in total. The brief review of literature on the treatment of melasma was undertaken.
Copper ; Dihydroxyphenylalanine ; Glutathione* ; Humans ; Korea ; Melanins ; Melanosis* ; Tyrosine

The goal of this study was to determine whether gypenosides (GPS) exert protective effects against dopaminergic neuronal cell death in a 6-hydroxydopamine (OHDA)-lesioned rat model of Parkinson's disease (PD) with or without long-term 3,4-dihydroxyphenylalanine (L-DOPA) treatment. Rats were injected with 6-OHDA in the substantia nigra to induce PD-like symptoms; 14 days after injection, groups of 6-OHDA-lesioned animals were treated for 21 days with GPS (25 or 50 mg/kg) and/or L-DOPA (20 mg/kg). Dopaminergic neuronal cell death was assessed by counting tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra and measuring levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. Dopaminergic neuronal cell death induced by 6-OHDA lesions was ameliorated by GPS treatment (50 mg/kg). L-DOPA treatment exacerbated 6-OHDA-induced dopaminergic neuronal cell death; however, these effects were partially reversed by GPS treatment (25 and 50 mg/kg). These results suggest that GPS treatment is protective against dopaminergic neuronal cell death in a 6-OHDA-lesioned rat model of PD with long-term L-DOPA treatment. Therefore, GPS may be useful as a phytotherapeutic agent for the treatment of PD.
3,4-Dihydroxyphenylacetic Acid ; Animals ; Cell Death* ; Dihydroxyphenylalanine ; Dopamine ; Dopaminergic Neurons* ; Homovanillic Acid ; Levodopa* ; Models, Animal* ; Norepinephrine ; Oxidopamine ; Parkinson Disease* ; Rats* ; Substantia Nigra ; Tyrosine 3-Monooxygenase

Melanin is a pigment produced from tyrosine in melanocytes. Although melanin has a protective role against UVB radiation-induced damage, it is also associated with the development of melanoma and darker skin tone. Tyrosinase is a key enzyme in melanin synthesis, which regulates the rate-limiting step during conversion of tyrosine into DOPA and dopaquinone. To develop effective RNA interference therapeutics, we designed a melanin siRNA pool by applying multiple prediction programs to reduce human tyrosinase levels. First, 272 siRNAs passed the target accessibility evaluation using the RNAxs program. Then we selected 34 siRNA sequences with ΔG ≥−34.6 kcal/mol, i-Score value ≥65, and siRNA scales score ≤30. siRNAs were designed as 19-bp RNA duplexes with an asymmetric 3′ overhang at the 3′ end of the antisense strand. We tested if these siRNAs effectively reduced tyrosinase gene expression using qRT-PCR and found that 17 siRNA sequences were more effective than commercially available siRNA. Three siRNAs further tested showed an effective visual color change in MNT-1 human cells without cytotoxic effects, indicating these sequences are anti-melanogenic. Our study revealed that human tyrosinase siRNAs could be efficiently designed using multiple prediction algorithms.
Dihydroxyphenylalanine ; Gene Expression ; Humans ; Melanins ; Melanocytes ; Melanoma ; Monophenol Monooxygenase* ; RNA ; RNA Interference ; RNA, Small Interfering* ; Skin Pigmentation ; Tyrosine ; Weights and Measures

Segawa disease, hereditary progressive dystonia with marked diurnal fluctuations or defined dopa-responsive dystonia has age-dependent clinical courses, which are characterized with marked progression in the first one and half decades, its subsiding in the third decade and almost stationary courses after the fourth decade. Also, it has characteristic diurnally fluctuating symptoms, aggravated towards the evening and alleviated after sleep. This autosomally dominantly inherited dystonia is caused by abnormalities of the gene of GTP cyclohydrolase I. The heterozygotic gene's abnormality induces partial decrement of tetrahydrobiopterin and affects synthesis of tyrosine hydroxylase(TH) rather selectively. The reduction of TH induces decrement of dopamine and disfacilitates the D1 receptor-striatal direct pathway. The pathognomonic finding in biochemical examination is the decrease of neopterin in the cerebrospinal fluid(CSF). Levodopa, by replacing dopamine contents at the terminal, alleviates motor symptoms completely and the effects sustain without any side effects. We experienced a girl diagnosed as Segawa disease with typical clinical courses and a decrease of neopterin in the CSF.
Dopamine ; Dystonia* ; Female ; Genetic Diseases, Inborn ; GTP Cyclohydrolase ; Humans ; Levodopa ; Neopterin ; Tyrosine

This study investigated the effects of ombuoside, a flavonol glycoside, on dopamine biosynthesis in PC12 cells. Ombuoside at concentrations of 1, 5, and 10 µM increased intracellular dopamine levels at 1 – 24 h. Ombuoside (1, 5, and 10 µM) also significantly increased the phosphorylation of tyrosine hydroxylase (TH) (Ser40) and cyclic AMP-response element binding protein (CREB) (Ser133) at 0.5 – 6 h. In addition, ombuoside (1, 5, and 10 µM) combined with L-DOPA (20, 100, and 200 µM) further increased intracellular dopamine levels for 24 h compared to L-DOPA alone. These results suggest that ombuoside regulates dopamine biosynthesis by modulating TH and CREB activation in PC12 cells.
Animals ; Carrier Proteins ; Dopamine ; Levodopa ; PC12 Cells ; Phosphorylation ; Tyrosine 3-Monooxygenase

BACKGROUND: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. METHODS: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. RESULTS: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T cell lysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. CONCLUSION: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.
Cell Proliferation ; Cytoplasm ; Killer Cells, Natural ; Phosphotyrosine ; Protein Tyrosine Phosphatases ; T-Lymphocytes ; Transfection

OBJECTIVE: This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). MATERIALS AND METHODS: Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA, embryoid bodies (EB, for 4 days) derived from TH#2/MB03 cells were exposed to RA (10-6 M)/AA (5x10-2 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14 or 21 days. Exp. II) When b-FGF was used, neuronal precursor cells were expanded at the presence of b-FGF (10 ng/ml) for 6 days followed by a final differentiation in N2 medium for 7, 14 or 21 days. Neuron differentiation was examined by indirect immunocytochemistry using neuron markers (NF160 & NF200). RESULTS: After 7 days in N2 medium, approximately 80% and 20% of the RA or b-FGF induced Th#2/MB03 cells were immunoreactive to anti-NF160 and anti-NF200 antibodies, respectively. As differentiation continued, NF200 in RA treated cells significantly increased to 73.0% on 14 days compared to that in b-FGF treated cells (53.0%, p<0.05), while the proportion of cells expressing NF160 was similarly decreased between two groups. However, throughout the differentiation, expression of TH was maintained (~90%). HPLC analyses indicated the increased levels of L-DOPA in RA treated genetically modified hES cells with longer differentiation time. CONCLUSION: These results suggested that a genetically modified hES cells (TH#2/MB03) could be efficiently differentiated in vitro into mature neurons by RA induction method.
Antibodies ; Blotting, Western ; Cell Differentiation* ; Cell Line ; Chromatography, High Pressure Liquid ; DNA, Complementary ; Embryoid Bodies ; Embryonic Stem Cells* ; Humans* ; Immunohistochemistry ; Levodopa ; Neurons ; Transfection ; Tretinoin ; Tyrosine 3-Monooxygenase* ; Tyrosine*

BACKGROUND: Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson's disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. METHODS: To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recom-binant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. RESULTS: The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 nM) but more sensitive to the inhibition at high concentration of dopamine (10 mM). CONCLUSIONS: The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibi-tion by the end product.
Amino Acids ; Blotting, Western ; Cycloheximide ; DNA, Complementary ; Dopamine ; Fruit ; GTP Cyclohydrolase ; Humans ; Levodopa ; Parkinson Disease ; Polymerase Chain Reaction ; Retroviridae ; Staphylococcal Protein A ; Tyrosine 3-Monooxygenase* ; Tyrosine* ; Zidovudine