Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Functional assessment of tyrosinase variants identified in individuals with albinism is essential for unequivocal determination of genotype-to-phenotype correlation.

BACKGROUND: Oculocutaneous albinism type 1 (OCA1), caused by pathogenic variations in the tyrosinase gene (TYR), is the most frequent and severe form of hypopigmentary disorder worldwide. While OCA1A manifests as a complete loss of melanin pigment, patients with OCA1B show residual pigmentation of the skin, hair and eyes. Limited experimental evidence suggests retention of TYR in the endoplasmic reticulum (ER) causes OCA1 pathogenesis. However, a comprehensive functional analysis of TYR missense variations and correlation with genotype is lacking. OBJECTIVES: Functional characterization of nonsynonymous tyrosinase variants in patients with OCA1 reported in the Albinism Database, dbSNP and the published literature, and an attempt to correlate them with reported and predicted phenotypes. METHODS: Thirty-four reported missense variants of TYR were subcloned by site-directed mutagenesis, and the dual-enzyme activities of the variant proteins were compared with the wild-type. The degree of ER retention was also checked for each of the variants through endoglycosidase H (Endo H) digestion followed by immunoprecipitation and densitometric analysis. RESULTS: Functional studies revealed one reported OCA1A variation with nearly 100% enzyme activity, 10 OCA1B variants lacking any enzyme activity, eight nonsynonymous single-nucleotide polymorphisms (SNPs) with ~30-70% of enzyme activity, and three SNPs that completely lacked activity altogether. The Endo H assay corroborated these results. CONCLUSIONS: Loss of enzyme activity of TYR variants was completely in agreement with ER retention across all variants examined. The results of the assay clearly established that determination of the biological activity of identified variants in patients with OCA is essential to correlate the identified suspect genotype with the obvious phenotype of the disease.

Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity.

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktail.

Assessment of tyrosinase variants and skin cancer risk in a large cohort of French subjects.

BACKGROUND: Tyrosinase (TYR) is a key pigmentation gene that is highly polymorphic and responsible for the most common form of autosomal recessive albinism, OCA1. OBJECTIVE: To assess the role of frequent and rare TYR variants in predisposition to skin cancer (SK) in the French population. METHODS: We genotyped a frequent TYR variant (p.R402Q) in 1273 patients {1047 cutaneous melanoma (CM) and 226 basal cell carcinoma (BCC)} and 925 controls, and the full coding region of TYR was sequenced in 287 patients suspected of genetic predisposition to SK (familial and/or multiple SK and/or onset before 40 years) and 187 controls. RESULTS: The homozygous p.R402Q variant was significantly associated with SK risk (P value=0.008; OR=1.57), and was mostly associated with multiple CM risk (P value=0.021; OR=2.50) and familial CM risk (P value=0.022; OR=2.16). In addition, 19 rare TYR variants, mainly albinism mutations, were identified in 15 patients and 8 controls. Among these, 3 clearly deleterious mutations (1 non-sense and 2 affecting mRNA splicing) were identified in 3 patients, one of which was homozygous. CONCLUSION: Our data confirmed the association of TYR p.R402Q with SK risk in the French population, and support that rare deleterious TYR variants may also play a role in multi-factorial genetic predisposition to SK. These results should be confirmed by replications studies.

Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART-1 and tyrosinase messenger RNA.

PURPOSE: Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment. EXPERIMENTAL DESIGN: Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR. RESULTS: Using histopathology, SNs and patients were allotted to three diagnostic groups: (a) metastasis positive, (b) BNI positive, and (c) melanocyte-free. Median MART-1 and tyrosinase mRNA levels in SNs were significantly different in patients with metastasis compared with patients with BNIs (P < 0.05) and patients without melanocytic lesions (P < 0.001). However, a "gray-zone" was observed where distinction, based on mRNA levels, could not be made between the three groups. For both genes, the highest mRNA level recorded in each RT-PCR-positive patient was positively correlated with Breslow's tumor thickness. For SNs with metastases, tumor burden was significantly correlated to the mRNA level. Using the presence of a MART-1 RT-PCR signal to detect patients with metastases, a sensitivity of 100% and a negative predictive value of 100% were achieved when extensive immunohistology was used as reference. CONCLUSIONS: Quantitative RT-PCR MART-1 and tyrosinase mRNA analysis cannot be used alone for SN diagnosis because of its poor specificity for melanoma metastasis. However, in approximately one third of cases without RT-PCR evidence of MART-1 expression, extensive histopathologic SN investigation is not necessary, thus substantially reducing the cost of SN analysis. The level of melanocyte-associated mRNA is associated with both tumor thickness and tumor burden as measured histopathologically, suggesting that this may be of prognostic value.

Quantitative detection of circulating tumor cells in cutaneous and ocular melanoma and quality assessment by real-time reverse transcriptase-polymerase chain reaction.

PURPOSE: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells. EXPERIMENTAL DESIGN: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples. RESULTS: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/ microl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples). CONCLUSIONS: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma.

Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival.

Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction.

The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed cDNA in the different samples after RNA isolation and reverse transcription, a beta-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.

A polymerase chain reaction-based semiquantitative assessment of malignant melanoma cells in peripheral blood.

Malignant melanoma cells can be detected with high sensitivity in peripheral blood of patients using reverse transcription-PCR. The detection of tyrosinase mRNA that is actively expressed only in melanocytes and melanoma cells indicates the presence of melanoma cells in peripheral blood. As shown previously, tyrosinase transcripts can be found in a variety of patients with metastatic malignant melanoma. For semiquantitative analysis of these cells in peripheral blood and evaluation of possible influence of immunotherapy on the amount of circulating cells, we describe an assay combining reverse transcription-PCR and Southern blotting. In this system, the amount of circulating tumor cells was determined by interpolating the amplified tyrosinase signal strength of patient samples to an equivalent tyrosinase signal of diluted SK-mel 28 cells. We found that the amount of circulating tumor cells correlates with the tumor burden. Furthermore, in patients with regression of melanoma metastases after immunotherapy, a decrease of the amount of tumor cells in the peripheral blood was observed. Quantitative estimates of residual disease may be an accurate and sensitive predictor for the clinical course.