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 have determined a molecular defect to be the likely basis for inactivity of the tyrosinase (EC 1.14.18.1) from a patient with tyrosinase-negative oculocutaneous albinism. A single base (thymine) was inserted in exon 5 of the tyrosinase gene following codon 471 in the putative transmembrane coding region. This insertion caused a shift in the reading frame of 19 amino acids at the 3' end and introduced a premature termination signal that would be expected to truncate the protein by 21 amino acids at the carboxyl terminus. The albino tyrosinase was not recognized by antibodies directed to the carboxyl terminus of tyrosinase. Furthermore, as shown by gel electrophoresis of the immunoprecipitated protein, the tyrosinase was approximately 3 kDa smaller than normal. Similar immunoprecipitation data were obtained when cloned normal and mutant tyrosinases were expressed in COS-1 cells.
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PMID:A single base insertion in the putative transmembrane domain of the tyrosinase gene as a cause for tyrosinase-negative oculocutaneous albinism. 171 Dec 23

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.
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PMID:Cloning and expression of cDNA encoding mouse tyrosinase. 300 90

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.
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PMID:Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1. 777 68

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV-B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV-B (2.5-5.0 mJ/cm2) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SK-MEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Coordinated mRNA and protein expression of human LAMP-1 in induction of melanogenesis after UV-B exposure and co-transfection of human tyrosinase and TRP-1 cDNAs. 788 4

We constructed two genes specific to melanogenesis, human tyrosinase (HT) and tyrosinase-related protein-1 (TRP-1) genes, into two separate expression vectors so that the cloned genes were under the control of a human cytomegalovirus promoter and enhancer. Monkey kidney COS-7 cells and human amelanotic and melanotic melanoma cells were then cotransfected by both HT and TRP-1 or transfected individually with each gene. The transfectants were examined for mRNA expression by reverse transcription-mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectants and cotransfectants of the two genes. Both light and electron microscopic observations indicated that degeneration and premature death of melanocytes occurred in HT transfectants, but not in TRP-1 transfectants or in HT and TRP-1 cotransfectants. Cotransfected cells from five cell lines revealed numerous granular reaction products with an anti-TRP-1 antibody and lysosomal granules with electron-dense material. Our melanin assay confirmed the new production of melanin pigments in these cells, indicating that the lysosomal granules would contain melanin pigments. The gene expression studies of lysosomal protein (beta-galactosidase, CD63, Lamp-1, and Lamp-2) revealed a dramatically elevated gene expression of Lamp-1, which is associated with the membrane receptor of lysosomal granules, in HT- and TRP-1-cotransfected cells. Conversely, the treatment of melanoma cells with antisense oligodeoxynucleotides against Lamp-1 resulted in a decreased expression of TRP-1 protein by immunoprecipitation, supporting the observations of the HT and TRP-1 cotransfection study regarding the up-regulation of Lamp-1 expression. We conclude that HT, TRP-1, and Lamp-1 gene products may function together, being expressed as a multiprotein complex within the melanosomal compartment. Specifically, HT and TRP-1 may function together via Lamp-1 by stabilizing the enzyme-protein complex within the melanosome and prevent the premature death of melanocytes due to tyrosinase-mediated cytotoxicity.
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PMID:Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1. 802 May 95

Anti-melanoma cytolytic T-lymphocyte (CTL) clones were derived from peripheral blood lymphocytes of HLA-A2 melanoma patient LB265 after stimulation with the autologous tumor cell line LB265-MEL, which showed high expression of melanocyte-lineage specific genes. Of 55 CTL clones, 46 recognized HLA-A2-restricted antigens. These 46 CTL clones were studied for their ability to specifically release tumor necrosis factor in the presence of COS cells cotransfected with the HLA-A2 gene and the cDNA of either tyrosinase, Melan-A/MART1, Pmel17/gpl00, gp75/TRP1, or MSH receptor. Six CTL clones recognized the Melan-A/MART1 antigen, whereas the remaining 40 CTL clones recognized a Pmel17/gp100 antigen. These 40 anti-PmelI7/gpl00 CTL clones were all able to lyse T2 cells pulsed with the antigenic peptide YLEPGPVTA, as previously reported. The T-cell receptor beta chain hypervariable region was sequenced and found to be identical in the 15 CTL clones analyzed. Taken together, these data show a high frequency of Pmell7/gp100-specific T cells in autologous antitumor CTL clones derived from peripheral blood of a melanoma patient.
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PMID:The majority of autologous cytolytic T-lymphocyte clones derived from peripheral blood lymphocytes of a melanoma patient recognize an antigenic peptide derived from gene Pmel17/gp100. 875 41

A method to concentrate drugs, DNA, or other materials with target cells in two-phase polymer systems for high-efficiency electroloading is described. The two-phase polymer system is utilized for cell and loading material selection, as well as for cell aggregation before electrofusion. The phase mixing of several water-soluble polymers is characterized, and the polyethylene glycol-Dextran (PEG m.w. 8,000 + Dextran m.w. 71,000) mixture is selected to illustrate the advantage of the two-phase systems. Fluorescently labeled Dextran or DNA is loaded into Chinese hamster ovary (CHO) and JTL cells, using electroporation in either the two-phase polymer system or the conventional single-phase suspension. The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range. Transfections of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-galactosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer system and the conventional single-phase electroporation method, are compared. The former method is far superior to the latter in terms of efficiency. The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former method is more favorable in terms of equipment requirement and safety. Electrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improvement from the conventional electrofusion method with dielectrophoresis. The two-phase polymer method is, therefore, a valuable technique for gene delivery to a limited cell source, as in ex vivo gene therapy.
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PMID:High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems. 884 49

In the present study we describe the detection of TRP-2 antibodies in vitiligo patients using in vitro 35S-labelled human TRP-2 in a radioimmunoassay. Of 53 vitiligo sera examined in the assay, three (5 9%) were found to be positive for TRP-2 antibodies. In contrast, 20 control sera, sera from 10 patients with Hashimoto's thyroiditis and sera from 10 patients with Graves' disease were all negative. All three patients positive for TRP-2 antibodies (mean age 54 years, age range 50-63 years) had had vitiligo of the symmetrical type for more than 1 year and all of them also had an associated autoimmune disorder: Graves' disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzyme tyrosinase were present in their serum. To examine any immunological cross-reactivity between TRP-2 and tyrosinase, the three vitiligo sera positive for TRP-2 antibodies were preabsorbed with COS-7 cell extract containing either expressed TRP-2 or tyrosinase, and subsequently used in the radioimmunoassay. These absorption studies indicated that preincubation with both proteins inhibited the immunoreactivity of the positive sera in the immunoassay using in vitro translated 35S-TRP-2. This antibody cross-reactivity suggests the humoral response to the two melanogenic enzymes in these patients may not be entirely independent.
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PMID:Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2). 932 28

In the present study we describe the in vitro transcription-translation of human melanocyte-specific protein Pmel17 cDNA and subsequent use of the resulting 35S-labelled Pmel17 in an RIA to analyse vitiligo sera for the presence of Pmel17 antibodies. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for Pmel17 antibodies. In contrast, sera from 20 healthy controls, 10 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease (GD) were all negative for Pmel17 antibodies. All three patients positive for Pmel17 antibodies (aged 50-63 years) had had vitiligo of the symmetrical type for > 1 year and all of them also had an associated autoimmune disorder: GD in one and autoimmune hypothyroidism in two. In addition, all three patients had antibodies to the melanogenic enzymes tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) in their serum. Absorption studies indicated that preincubation with COS-7 cell extract containing expressed Pmel17 absorbed out the immunoreactivity of the three sera positive in the RIA, confirming the anti-Pmel17 reactivity of the sera from these patients. In contrast, COS-7 cell extracts containing either expressed tyrosinase, TRP-1 or TRP-2 did not remove the anti-Pmel17 reactivity of the three sera in the RIA. This lack of cross-reactivity suggests that the humoral response to Pmel17 in these patients is specific and independent of the antibody reactivity to tyrosinase, TRP-1 and TRP-2.
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PMID:Autoantibodies to human melanocyte-specific protein pmel17 in the sera of vitiligo patients: a sensitive and quantitative radioimmunoassay (RIA). 984 40

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
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PMID:Promotion of tyrosinase folding in COS 7 cells by calnexin. 988 Aug 1


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