Tyrosinase cDNA Research Articles

Biochemical and phylogenetic analysis of Eugenia and Syzygium species from Mauritius

The Myrtaceae represents a plant family with several species having important medicinal virtues. In view of these properties four Syzygium species namely S. latifolium, S. commersonii, S. coriaceum, S. petrinense and two Eugenia species namely E. pollicina and E. pyxidata were studied. Phytochemical analysis showed that the methanol extracts had higher content of total phenolics and total flavonoids than the hexane extracts. The in vitro antioxidant activity evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric reducing antioxidant potential (FRAP) assays was significant. The antibacterial activity of the extracts against Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes and Escherichia coli was also promising. Species studied displayed significant tyrosinase inhibitory activity with S. petrinense having the highest activity. Interestingly, when the expression analysis of the tyrosinase cDNA in mouse melanocyte cells was studied it was observed that the methanolic Syzygium extracts inhibited melanogenesis by upregulating or downregulating the tyrosinase gene. Phylogenetic analysis of the ribosomal DNA sequences showed the genetic uniqueness of each plant species reflecting in their unique biological activity. Bioprospecting of endemic Mauritian Eugenia and Syzygium could be envisaged against acne vulgaris, skin hyperpigmentation and possible exploitation as phytotherapeutics.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

Tyrosinase expression during black truffle development: From free living mycelium to ripe fruit body

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.

A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: Technical and operative instructions

Background To develop a new absolute quantitative real-time PCR method for blood mRNA tyrosinase assay and to compare this new method with standard RT-PCR nested. Methods Ten blood of melanoma patients (stages I–III), 5 tissue samples, 2 surgical fresh metastatic skin and 3 lymph nodes paraffin-embedded slices were analysed, and 10 negative controls were used. Ten millilitres of blood was analysed for each individual. Three different protocols for RNA extraction and two reverse transcription methods were used. Specific human tyrosinase cDNA fragment was cloned into pcDNA3+ vector and then titrated for the standard curve construction (from 10 6 to 10 1 copies/μl). Recovery assays for RNA and cells were also performed. Results Our method was able to detect less than 5 cells/10 8 WBC and about 100 fg of tyrosinase RNA. Very low CVs (<1.5%) were obtained on all samples run in triplicate. Sensitivity and specificity were of 100%. The amount of starting volume of blood was crucial for the determination of copy number since large volumes are necessary for patient's monitoring. Conclusions Our absolute qRT-PCR assay could be proposed as a new standardized molecular method for the management of melanoma patients, particularly for the follow up of the highest AJCC stages.

Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice

To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the efficiency of gene transfer. A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5' non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. Of the 11 animals that developed from the injected eggs, two mice (P1 and #8) exhibited pigmented hair (P1) and eyes (P1 and #8), as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5' flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.

Bone Morphogenetic Protein-4, a Novel Modulator of Melanogenesis

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin.

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism.

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.

Coat color-tagged green mouse with EGFP expressed from the RNA polymerase II promoter.

Laborious molecular genotyping and variegated gene expression are two widely encountered issues for transgenic mouse studies. To facilitate genotyping in the FVB/N albino background and to reduce variegated expression, we successfully generated double-tagged transgenic mice for direct visual genotyping with the coat color phenotype derived from tyrosinase cDNA driven by the tyrosinase promoter and with simultaneous high enhanced green fluorescent protein (EGFP) expression driven by the promoter of RNA polymerase II large subunit gene. Incorporation of insulator into a transgene construct achieved high efficiency of transgene expression in more than 90% of the founders. EGFP was detected as early as the one-cell fertilized egg and lasted for the whole embryo development, as well as in all of the adult tissues examined. The coat color-tagged green mice offer opportunities in applications such as tissue transplantation, lineage tracing, chimera biology, RNA interference, and other transgenic studies.

Functional Conservation of the Promoter Regions of Vertebrate Tyrosinase Genes

Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5' flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5' flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5' flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed.

Transfection of nonmelanocytic cells with tyrosinase gene constructs for survival studies.

To better understand the role of melanin in the response of cells to radiation, the vector pcTYR containing the tyrosinase cDNA and a control vector pcTYW with no tyrosinase cDNA were transfected and expressed in nonpigmented CHOK1-A(L) 1282B5 cells. A pigmented clone was selected from the pcTYR transfectants and an antibiotic-resistant clone was selected from the controls. Melanin was assessed qualitatively by electron paramagnetic resonance (EPR) and quantitatively by a 14C-based assay. The EPR signal detectable in pcTYR-containing cells was at least twice that of pcTYW and parental CHOK1-A(L) cells and the tyrosinase activity was found to be at least six times greater. Melanin was classified to be eumelanin. Survivals of the transfectants were compared to those of the parent cells after irradiation by UVC from a germicidal lamp, UVB from TL01 lamps, UVA from Alisun lamps, UVB/UVA from FS20 lamps, and by gamma-rays from a 137Cs source. Compared to the pcTYW-containing cells, the pigmented cells were more sensitive to killing by UVC, and resistant to killing by UVA and gamma-rays. There were no significant differences in survival after the other irradiations. These results suggest that the pigment synthesized by the activity of tyrosinase alone, unmodified by the activities of TRP1 and TRP2, is protective against the types of reactive oxygen species produced by UVA and gamma-rays but not protective against lethal damage from photons in the UVB range and sensitizes to UVC photons.

Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells.

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.

Selective uptake of para-boronophenylalanine increases in amelanotic melanoma cells transfected by the tyrosinase gene

To investigate the mechanism of uptake of para-boronophenylalanine (p-BPA), a capture agent for boron neutron capture therapy (BNCT) of melanoma and brain tumour, into melanoma cells, we studied the relationship between melanin synthesis and the concentration of boron using tyrosinase-deficient mouse amelanotic melanoma cells (A1059) and melanotic melanoma cells (TA1059). A1059 was established from mouse B16F10 cells, and TA1059 was constructed by transfecting human tyrosinase cDNA into A1059. The melanin content of TA1059 was 1.5-fold higher than that of B16F10, and was undetectable in A1059. The order of p-BPA uptake was TA1059 > B16F10 > A1059 at the time points examined, and the boron content of TA1059 was approximately 1.5-fold higher than that of B16F10. Our experimental findings indicated that melanin synthesis is a very important factor for characterizing the increase in accumulation of p-BPA in melanoma cells. A significant difference in boron uptake into TA1059 was observed between p-BPA and meta-BPA (m-BPA), but there were no apparent differences in the case of A1059. The difference in accumulation of p-BPA and m-BPA could be due to differences in the properties of p-BPA as a tyrosine analogue needed for melanin synthesis.

CD83+ human dendritic cells transfected with tumor peptide cDNA by electroporation induce specific T-cell responses: A potential tool for gene immunotherapy.

Dendritic cells (DC) are the most potent immunostimulatory cells, with the capacity to induce primary T-cell responses. Functional autologous DC can be generated from fetal calf serum-free peripheral blood mononuclear cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor and are stimulated with a defined cytokine cocktail for terminal maturation. We were able to establish a nonviral transfection protocol for these DC by electroporation. Using enhanced green fluorescent protein as a reporter gene, we achieved transfection efficiencies of up to 10%. FACScan analyses revealed a stable phenotype, and the expression of major histocompatibility complex class II and CD83 was not affected by the transfection conditions used. Like their untransfected counterparts, DC that were functionally transfected with green fluorescent protein were potent inducers of allogeneic T cells. To assess whether cDNAs transfected into DC are functionally expressed, human tyrosinase cDNA was transfected into DC. Tyrosinase-transfected DC, but not controls, resulted in antigen-specific tumor necrosis factor-alpha release of the tyrosinase-specific cytolytic T-cell clone IVSB. Taken together, the data show that genuine (CD83+) mature DC can be transfected using a nonviral method, and that the DC retain their functionality. These DC are ideal candidates for immunotherapy (e.g., cancer therapy).

The Cytotoxicity and Apoptosis Induced by 4-Tertiary Butylphenol in Human Melanocytes are Independent of Tyrosinase Activity

It has been known for several decades that cutaneous depigmentation, i.e., contact/occupational vitiligo, can be caused by some phenolic derivatives that have a similar structure to tyrosine. Among these phenolic depigmenting agents, 4-tertiary butylphenol is the most potent. The cutaneous depigmentation induced by phenolic derivatives results from the loss of functional melanocytes. Tyrosinase is a melanocyte specific copper-containing enzyme that catalyzes the conversion of the amino acid tyrosine, through a complex series of intermediates, to melanin. In this study we tested the hypothesis that the cytotoxicity induced by 4-tertiary butylphenol is mediated by tyrosinase and occurs via an apoptotic process. Melanocyte cultures derived from African-American and Caucasian donors exhibiting a 3-fold difference in tyrosinase activity and 14-fold difference in melanin content demonstrate comparable concentration-dependent sensitivity to 4-tertiary butylphenol. In addition, cultures of dermal fibroblasts and epidermal keratinocytes exhibited similar and reduced sensitivity, respectively, to 4-tertiary butylphenol compared with autologous melanocytes. Two melanoma cell lines, one melanotic and one amelanotic lacking the expression of both tyrosinase protein and activity, when transfected with the tyrosinase cDNA, exhibited no alteration in its sensitivity to 4-tertiary butylphenol. These data suggest that 4-tertiary butylphenol cytotoxicity is not mediated via tyrosinase. Melanocytes treated with 4-tertiary butylphenol, however, did exhibit plasma membrane blebbing, DNA fragmentation, and phosphatidylserine relocalization indicating that 4-tertiary butylphenol induced melanocyte destruction occurs by an apoptotic process.

Autosomal albino chicken mutation (ca/ca) deletes hexanucleotide (-ΔGACTGG817) at a copper-binding site of the tyrosinase gene

We compared tyrosinase cDNA sequences from a line of autosomal albino and Black Silky chickens isolated from cultured melanocytes by reverse transcription-polymerase chain reaction (RT-PCR). Both sources produce a single DNA fragment of predicted normal tyrosinase size. Direct sequencing of the PCR product showed three mutated sites in the tyrosinase gene of the albino chicken. Two silent point mutations and a deletion of six nucleotides (-ΔGACTGG) at 817 bp in the tyrosinase cDNA sequence were observed when compared with the White Leghorn and Black Silky cDNA sequences. The deduced albino chicken tyrosinase protein lacks two amino acids, aspartic acid and tryptophan. The position of these amino acids is consistent with one of the potential copper-binding sites that should be indispensable for function of the enzyme. We speculate that the six-base deletion is responsible for the inactive tyrosinase in this line of albino chickens.

Sulfur Containing Tyrosine Analogs Can Cause Selective Melanocytotoxicity Involving Tyrosinase-Mediated Apoptosis

Sulfur-containing tyrosine analogs such as 4-S-cysteaminylphenol (4-S-CAP) and its N-acetyl derivative, N-acetyl-4-S-CAP, are tyrosinase substrates and can cause selective cytotoxicity or cell death of melanocytes and melanoma cells. It is not clear, however, if the cytotoxicity derives from a cytostatic or cytocidal effect. The latter can also be either apoptotic or necrotic. This paper summarizes our attempt to clarify the nature of melanocytotoxicity and cell death by using a new derivative of 4-S-CAP, N-propionyl-4-S-CAP (NPr-CAP). The i.p. administration of NPr-CAP caused marked depigmentation of black hair follicles in C57 mice. At 12 h postadministration of NPr-CAP, follicular melanocytes showed histochemical and morphologic features indicative of apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and electron microscopy. The agarose gel electrophoresis of DNA from drug-treated melan a2 cells, an immortal melanocyte line of C57 black mice, showed the nucleosomal DNA ladder pattern. NPr-CAP caused irreversible cytotoxicity in melan a2 and the effect was inhibited by a tyrosinase inhibitor, phenylthiocarbamide. The tyrosinase-mediated cytotoxicity of NPr-CAP was further confirmed by the decreased viability of COS 7 monkey-kidney cells, which expressed a level of high tyrosinase activity through transfection of human tyrosinase cDNA. NPr-CAP, however, also transiently inhibited the proliferation of melan c cells, a control tyrosinase-negative albino melanocyte line, and vector-transfected COS 7 cells. Thus, the major process of NPr-CAP-mediated melanocytotoxicity involves cytocidal apoptosis associated with active tyrosinase. In addition, there is transient, nontyrosinase-mediated cytostatic cytotoxicity.

Identification of Epitopes on Tyrosinase which are Recognized by Autoantibodies from Patients with Vitiligo

The identification of tyrosinase autoantibodies in some patients with vitiligo has previously been reported. In this study we have determined the B cell epitopes on tyrosinase which are recognized by these autoantibodies. Deletion derivatives of tyrosinase cDNA were constructed and then translated in vitro with the concomitant incorporation of [35S]methionine into the protein products. The 35S-labeled tyrosinase derivatives were subsequently used in radioimmunoassays to investigate the reactivity of sera from five vitiligo patients. The epitope regions identified were: three in a central region of tyrosinase (amino acids 240-255, 289-294, and 295-300) and two others towards the C-terminal end of the protein (amino acids 435-447 and 461-479). Computer analysis of the potential B cell epitopes on tyrosinase revealed that the epitope regions recognized by the vitiligo sera were located in areas predicted to be highly antigenic. In addition, the centrally located antigenic regions (amino acids 289-294 and 295-300) had amino acid sequence homology to both tyrosinase-related protein-1 and -2. Thus, the epitopes on tyrosinase recognized by vitiligo patient sera are heterogeneous and include a region with homology to two related proteins which may explain the cross-reactivity previously noted between these antigens.

Promotion of tyrosinase folding in COS 7 cells by calnexin.

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.

Tyrosinase transfection produces melanin synthesis and growth retardation in glioma cells.

Tyrosinase is the key enzyme in melanin biosynthesis in pigmented cells. We transfected 9L rat glioma cells with human tyrosinase cDNA that had been cloned in a high expression vector. Stable transfectants were selected by their resistance to the antibiotic G418. More than a dozen G418-resistant clones were isolated and were screened for tyrosinase expression using dopa-oxidase activity. The clone with the highest tyrosinase activity was selected and expanded for further studies. Northern blot analyses of total RNA from cells showed that the transfected cells had relatively more tyrosinase transcript than SK-MEL-23 human melanotic melanoma cells. The melanin content of the transfected cells was dependent on the concentration of L-tyrosine in the culture medium. In addition, the growth of transfected cells was inhibited when grown in a medium containing high concentrations of L-tyrosine. These results suggest that tyrosinase activity is cytotoxic in a substrate-dependent manner. This may have far reaching therapeutic use for glioma tumours.