Serum levels of S-100 protein and 5-S-cysteinyldopa as markers of melanoma progression.
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Serum S-100 protein is widely used as a marker of melanoma and since 5-S-cysteinyldopa (5-S-CD) is a precursor of melanin its serum and urinary levels can reflect melanoma progression. In this study we examined the concentration changes of serum S-100 protein and 5-S-CD in 252 melanoma patients of different clinical stages. Serum samples were taken from 252 melanoma patients at 860 times, from June 1996 to July 1998. The serum S-100 protein was measured by the immunoluminometric assay, levels of 5-S-CD was determined by HPLC. The value of S-100 protein in patients with primary melanoma (0.11m mg/l) and in patients without symptoms (0.15 m mg/l) ranged around the normal level (0.01 0.12 m mg/l). There was a significant difference between the values of patients with or without symptoms. There was a similarly significant difference between the S-100 values of clinical Stage I (0.11 mg/l) and Stage III (2.91 mg/l) as well as between those of clinical Stage II (0.47 mg/l) and Stage III (2.91 mg/l), respectively. Analyzing the values of patients with symptoms we observed significant difference between the S-100 protein values of patients with primary tumor and those with solitary or multiple distant metastases. In case of 5-S-CD significant difference was found between clinical Stage I and III as well as clinical Stage II and III. Furthermore, there was a significant difference between the mean marker values of patients with primary tumor, lymph node, lung metastasis and clinical stage III. (+info)
New regulatory mechanisms in the biosynthesis of pheomelanins: rearrangement vs. redox exchange reaction routes of a transient 2H-1, 4-benzothiazine-o-quinonimine intermediate.
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Pheomelanins, the typical epidermal pigments of red haired, Celtic-type Caucasians, arise from oxidative cyclization of cysteinyldopas, mainly the 5-S-isomer CD, via 1,4-benzothiazines. However, the mechanism and the relative yields of formation of these intermediates have remained poorly defined. We have now examined the course of the oxidation of CD at physiological pHs, under different reaction conditions. Surprisingly, a consumption of CD far exceeding the stoichiometry of the oxidant was observed at low oxidant-to-substrate ratios, low temperatures and high substrate concentrations. The yields of the 3,4-dihydro-1,4-benzothiazine-3-carboxylic acid DHBCA vs. the non-carboxylated analogue DHB in the oxidation mixture, after NaBH4 reduction, were also found to depend markedly on the reaction conditions. Based on these and other results, a reaction scheme is proposed involving a transient o-quinonimine generated by oxidative cyclization of CD to which three different paths are offered, namely redox exchange with CD to give DHBCA (path A) or intramolecular rearrangement with (path B) or without (path C) decarboxylation, leading to the benzothiazine BTZ and the 3-carboxy analogue BTZCA, respectively. The relative operation of path A vs. path C was assessed by deuterium labeling experiments. These findings point to new mechanisms of regulation of the initial steps of pheomelanogenesis, bearing significant implications on the structure of the final pigment. (+info)
Quantitative analysis of tyrosinase transcripts in blood.
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BACKGROUND: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods. METHODS: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: Ultraspec(TM)-II RNA isolation system, FastRNA(TM) GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2, 4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5'DNP-GGGGAGCCTTGGGGTTCTGG-3') and internal standard (5'DNP-CGGAGCCCCGAAACCACATC-3') and quantified by ELISA. RESULTS: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean +/- SD) 1716+/-1341, 2670+/-3174, and 24 320+/-5332 transcripts/mL of blood. Corresponding values were 527+/-497, 2497+/-1033, 14 930+/-1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120-168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found. CONCLUSIONS: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases. (+info)
Production of cysteinyl-dopamine during intravenous dopamine therapy.
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BACKGROUND: Oxidized dopamine rapidly forms thiol-conjugates with --SH groups on cysteine, glutathione, and proteins. We used cysteinyl-dopamine production as an index of thioester production during intravenous dopamine treatment of critically ill patients. METHODS: Cysteinyl-dopamine and catecholamines were measured by high-performance liquid chromatography with electrochemical detection. The production of cysteinyl-dopamine by purified human neutrophils was measured using dopamine (1 micromol/L) and cysteine (1 mmol/L) concentrations similar to those found during dopamine treatment. To examine the impact of endotoxic shock on cysteinyl-dopamine production, anesthetized rats were given dopamine (12 to 15 microg/kg/min intravenously) with or without endotoxin (50 mg/kg intravenously). RESULTS: In vitro, neutrophils converted 26% of dopamine to cysteinyl-dopamine (30 min at 37 degrees C). Activating neutrophils with zymogen increased dopamine consumption from 26 to 68%, but only 36% appeared as cysteinyl-dopamine. The remainder may have been oxidized to other cysteinyl derivatives. Endotoxin increased cysteinyl-dopamine in rat plasma from 2.5 nmol/L (range <0.2 to 11) to 9.7 nmol/L (range <0.3 to 31, P = 0.1). After four hours, with or without endotoxin, cysteinyl-dopamine was <0.3 nmol/L in cerebrospinal fluid. In the plasma of eight patients receiving dopamine (6 to 20 microg/kg/min for 1 to 3 days), dopamine was 0.5 to 9.9 micromol/L, and cysteinyl-dopamine was 48 to 1660 nmol/L. Cysteinyl-dopamine was 4.3 to 22.6% of dopamine and correlated with leukocyte count (r(2) = 0.388, P = 0.099). CONCLUSIONS: A significant fraction of exogenously administered dopamine reacts with -SH groups of cysteine and probably also with -SH groups on peptides and proteins. During brief dopamine treatment of endotoxic shock in rats, neither dopamine nor cysteinyl-dopamine crossed the blood-brain barrier. (+info)
Metal ions as potential regulatory factors in the biosynthesis of red hair pigments: a new benzothiazole intermediate in the iron or copper assisted oxidation of 5-S-cysteinyldopa.
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In the presence of iron or copper ions, the course of the oxidation in air of 5-S-cysteinyldopa (1), the main biosynthetic precursor of pheomelanins and trichochromes, was markedly changed affording two main products. One of these was identified as the oxobenzothiazine 8, previously obtained under nonphysiologically relevant conditions, while the other was characterized as the novel hydroxybenzothiazole 9. Besides 8 and 9, carboxylated and noncarboxylated benzothiazine products were obtained by persulfate oxidation of 1 in the presence of iron or copper ions. The ratio of formation yields of carboxylated/noncarboxylated benzothiazines, determined after reduction of the mixture, was lower than that of the control reaction run in the absence of metal ions, and much lower than that of the oxidation carried out in the presence of zinc ions, in agreement with a recent report. Notably, 8 and 9 were formed in variable yields under different oxidation conditions including tyrosinase/O(2), peroxidase/hydrogen peroxide, and the hydrogen peroxide/or (9E,11Z,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid/Fe(III) systems. Mechanistic routes to 8 and 9 were proposed based on the results of experiments involving in situ generation of labile benzothiazine intermediates. Overall, these results allow to formulate an improved biosynthetic scheme in which metal ions act as critical regulatory factors determining pheomelanin vs. trichochromes formation. (+info)
Comparison of prognostic significance of serum 5-S-Cysteinyldopa, LDH and S-100B protein in Stage III-IV malignant melanoma.
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5-S-cysteinyldopa is a precursor of pheomelanin. S-100B protein is a low molecular weight, acidic, calcium binding, cytoplasmatic protein. LDH was defined as the most important serum parameter in disseminated melanoma. The aim of the present study was to compare the prognostic values of serum 5-S-Cysteinyldopa, S-100B and LDH concentrations in Stage III-IV melanoma patients. Serum samples were taken from 179 Stage III-IV melanoma patients at diagnosis. Serum 5-S-CD concentrations were determined by HPLC, S-100B protein by immunoluminometric assay while LDH by UV kinetic method. The mean/median concentrations of LDH, S-100B protein and 5-S-CD in Stage III patients ranged around the normal level. In Stage IV, the markers ranked as S100B = 5-S-CD > LDH for sensitivity, S-100B > LDH > 5-S-CD for specificity and LDH = S100B = 5-S-CD for positive predictive value, respectively. Furthermore, mean marker concentrations of patients with progressive disease differed significantly from nonprogresssive cases (when staging categories have been disregarded). Survival analysis indicated, that the initially elevated LDH and S-100B level in Stage IV disease predicts comparably short survival. Results of our study suggest that these serum marker values correlate well with Stages and disease progression. In Stage IV melanoma, the markers had appropriate sensitivity, high specificity as well as important positive predictive value. Among the studied serum markers S-100B protein and LDH proved to be similarly reliable in respect to the clinical outcome. (+info)
Neurocutaneous melanosis associated with malignant leptomeningeal melanoma in an adult: clinical significance of 5-S-cysteinyldopa in the cerebrospinal fluid---case report.
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A 35-year-old male presented with a variant of neurocutaneous melanosis with leptomeningeal malignant melanoma. He had three pigmented nevi from birth. He suffered diplopia followed by headache. T1-weighted magnetic resonance (MR) imaging revealed hydrocephalus and a small linear hyperintense lesion in the right frontal cortex. Several parts of the cortical sulci and the brain surface were slightly enhanced by gadolinium. Ventriculoperitoneal shunting was performed and extensive pigmented leptomeninges were recognized. Open biopsy established the diagnosis of leptomeningeal malignant melanoma. Combined chemoimmunotherapy was repeated every other month with monitoring of the 5-S-cysteinyldopa (5-S-CD) level in the cerebrospinal fluid (CSF). The 5-S-CD level decreased after each treatment, but the basal level steadily increased prior to the next treatment. Two years after the onset, he showed paraplegia caused by an extramedullary mass at the T-6 level. MR imaging showed that melanoma had involved the entire subarachnoid space including the whole spine. He underwent emergent removal of the spinal tumor and showed transient marked improvement. Further intensive chemotherapy was given. However, he died 31 months after the onset of massive proliferation of intracranial leptomeningeal melanoma. Measurement of CSF 5-S-CD levels is valuable for evaluating the therapeutic efficacy and for monitoring the progression of melanoma. (+info)
Dopamine-derived dopaminochrome promotes H(2)O(2) release at mitochondrial complex I: stimulation by rotenone, control by Ca(2+), and relevance to Parkinson disease.
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Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1-0.2 mum)- and respiration-dependent generation of H(2)O(2) at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5-30 nm). The rotenone effect required that contaminating Ca(2+) (8-10 mum) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O(2), generating O(2)(.) and then H(2)O(2). Mitochondrial removal of H(2)O(2) monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca(2+) levels. Thus, the H(2)O(2) formed accumulated in the medium if contaminating Ca(2+) was present; in the absence of Ca(2+), H(2)O(2) was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular O(2)(.) release via activation of NADPH oxidase in the microglia. In turn, O(2)(.) oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described. (+info)