Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels.
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PURPOSE: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker. EXPERIMENTAL DESIGN: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value. RESULTS: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II). CONCLUSIONS: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage. (+info)
Coating proteins: structure and cross-linking in fp-1 from the green shell mussel Perna canaliculus.
(10/26)
The protein family known as fp-1 provides mussel byssus with a protective outer coating and has drawn much attention for its water resistant bioadhesive properties in vitro. A new fp-l isolated from the green shell mussel Perna canaliculus (pcfp-1) reveals a composition dominated by only four amino acids: 3,4-dihydroxyphenyl-L-alanine (dopa), lysine, proline, and valine at approximately 20 mol % each. SDS-PAGE and MALDI-TOF mass spectrometry detected size variants at 48 and 52 kDa in preparations of purified Pcfp-1. The N-terminal sequence enabled construction of oligonucleotide primers for PCR and RACE-derived cDNAs from which the complete sequence of four variants was deduced. pcfp-1 deviates from all known homologues in other mussels in several notable respects: its mass is half, most of its sequence is represented by 75 tandem repeats of a tetrapeptide, i.e., PY*VK, in which Y* is dopa, prolines are not hydroxylated, and thiolate cysteines are clustered in homologous sequences at both the amino and carboxy termini. Amino acids in the repeat sequence show a striking resemblance to proline-rich cell wall proteins with tandemly repeated PPVYK pentapeptides [Hong, J. C., Nagao, R. T., and Key, J. L. (1987) J. Biol. Chem. 262, 8367-8376]. Cysteine plays a key role in cross-linking pcfp-1 by forming adducts with dopaquinone. Significant 5-S-cysteinyldopa and smaller amounts of 2-S-cysteinyldopa were detected in hydrolysates of the byssal threads of P. canaliculus. The cross-links could also be formed by oxidation of pcfp-1 in vitro using mushroom tyrosinase. Cysteinyldopa cross-links were present in trace amounts only in the byssus of other mussel species. (+info)
UVA-induced modification of catalase charge properties in the epidermis is correlated with the skin phototype.
(11/26)
The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products. (+info)
Pigmentation effects of solar-simulated radiation as compared with UVA and UVB radiation.
(12/26)
Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro.
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Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0.2mM-L-dopa (L-dihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis. After this treatment, the amount of pheomelanin was 3.7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyl-dopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0.2mM-L-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome. (+info)
Identification of 5-S-cysteinyldopa by high performance liquid chromatography in biopsies from patients with dysplastic melanocytic nevi.
(15/26)
The appearance of 5-[(L)-S-cysteinyl]dopa, a major product in pheomelanogenesis was examined in affected and nonaffected skins from 20 patients with clinical signs of dysplastic melanocytic nevi. Analysis by high performance liquid chromatography and electrochemical detection showed that 20 of the 35 lesions had a pathological formation of 5-[(L)-S-cysteinyl]dopa (0.04-28.86 ng/micrograms acid soluble protein). 5-[(L)-S-cysteinyl]dopa was not detected in any of the normal uninvolved skin samples analyzed. (+info)
A new amino acid, 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine, from the tapetum lucidum of the gar (Lepisosteidae) and its enzymic synthesis.
(16/26)
The tapetum lucidum of the alligator gar Lepisosteus was shown by t.l.c. to contain a new phenolic amino acid, which is apparently a major constituent of the reflecting material. It was isolated in a yield of 0.5 mg/eye and its physical and chemical characteristics, especially reductive hydrolysis with hydriodic acid giving dopa (3,4-dihydroxyphenylalanine) and cysteine, suggested that it might to SS-dicysteinyldopa. Tyrosinase oxidation of L-dopa in the presence of an excess of L-cysteine yielded, in addition to known 5- and 2-S-cysteinyldopa, the same amino acid as that isolated from the eye of the gar, thus confirming the gross structure. The position of the two cysteine residues was established by the fact that tyrosinase oxidation of catechol and cyteine gave 3-S-cysteinylcatechol and 3,6-SS-dicysteinylcatechol. The natural amino acid is therefore formulated as 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine (2,5-SS-dicysteinyldopa), which may be formed by two consecutive additions of cysteine, first to dopaquinone and then to 5-S-cysteinyldopaquinone. The enzymic synthesis of 2,5-SS-dicysteinyldopa in vitro suggests that it may also be involved in the biosynthesis of phaeomelanin. (+info)