Transcriptome expression profiling of fur color formation in domestic rabbits using Solexa sequencing.

Fur color is an important, genetically determined characteristic of domestic rabbits, and rabbit furs are of great economic value. To investigate the molecular genetics associated with fur color determination in domestic rabbits, we used Solexa-sequencing technology to probe gene expression in dorsal skin tissues sampled from full-sibling Rex rabbits of different colors. The number of expressed genes in each sample was approximately 14,700. Among the top 30 genes and transcription factors with the highest reads per kilobase per million values, the elongation factor-alpha 1 gene was highly expressed in all samples, as were genes of the ribosomal protein and keratin gene families. Compared with the chinchilla (C) Rex rabbit control sample, the numbers of genes in the black (B) and white (W) rabbit samples were 1809 and 460, respectively, and the number of common differentially expressed genes was 257. Clustering analysis of these 257 genes revealed that 32 were up-regulated in sample B and down-regulated in sample W. Of these 32 genes, we identified some that are related to fur formation, including Tyrosinase-related protein 1 (TYRP1) and Tyrosinase (TYR), as well as genes with unknown functions. Quantitative real-time polymerase chain reaction was used to verify the expression patterns of those genes. The findings are expected to provide reference for the further study of fur color formation in rabbits.

Sentinel node in melanoma patients: triple negativity with routine techniques and PCR as positive prognostic factor for survival.

Lymph node mapping and sentinel lymph node biopsy are currently used to stage patients with cutaneous malignant melanoma. Immunohistochemical stains contribute to the detection of micrometastases; however, molecular biology techniques are associated with better diagnostic sensitivity. Sixty sentinel lymph nodes were included in this study. The primary lesions were malignant melanoma stage I or II, with a follow-up of longer than 2 years. Sentinel lymph nodes were studied with hematoxylin-eosin, immunohistochemistry for S-100 and HMB-45, and molecular biology techniques (reverse transcription (RT)-PCR) for the detection of tyrosinase messenger RNA. In 15 of 60 cases (25%), tyrosinase was detected by RT-PCR; three of these cases were also positive by immunohistochemistry. The population was divided into three groups: (i) hematoxylin-eosin-/immunohistochemistry+/molecular biology techniques+ (3 cases); (ii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques+ (12 cases); (iii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques- (45 cases). Correlation of the groups with overall survival showed the following: (i) 2 of 3 patients died (67%); (ii) 5 of 12 died (42%), and (iii) all 45 patients are alive, with no lymphadenectomy and a median follow-up of 84 months. The inclusion of molecular biology techniques appears to be of great value for the detection of sentinel lymph node micrometastases in patients with cutaneous malignant melanoma. In our series, those patients who showed negativity with all the three methods had a null recurrence rate. Therefore, this triple negativity could be a positive prognostic factor for overall survival. Our findings suggest the possibility of molecular oncological staging, which would allow the selection of patients with submicroscopic metastases for a complete treatment.

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

Production of laccase as the sole phenoloxidase by a Brazilian strain of Pleurotus pulmonarius in solid state fermentation.

The production of laccase by a Brazilian strain of Pleurotus pulmonarius was studied in solid state fermentation using wheat bran as substrate. Among oxidative and hydrolytic enzymes tested (laccase, aryl alcohol oxidase, lignin peroxidase, Mn peroxidase, xylanase and cellulase), laccase was the main enzyme produced by P. pulmonarius. The most suitable condition for maximum production of laccase (8,600 U/g substrate) was initial moisture content of 75% and 5 days of cultivation at 30 degrees C. The optimum pH and temperature for laccase activity were found to be 6.5 and 50 degrees C, respectively. P. pulmonarius laccase was stable at 50 degrees C for more than 6 hours, and it retained about 73% and 18% of its activity when heated for 1 h at 55 and 60 degrees C, respectively. The enzyme was greatly stable at alkaline pH, but not at acidic pH. The laccase activity appear to be correlated with the ability of crude extract to decolourize several industrial dyes.

Unequal nuclear Sp1/GC box DNA binding activity distinguishes proliferating from differentiated senescent or apoptotic cells.

Terminal differentiation can result in either viable, non-proliferating or apoptotic cells. In B16 melanoma, millimolar L-tyrosine induces tyrosinase, a key enzyme for terminal pigmentation concurrent with either irreversible growth arrest at low cell density, or apoptosis at high cell density. Since the promoter for melanocyte-specific tyrosinase expression contains sites for the Sp1 transcription factor, we have investigated the relationship of Sp1-mediated GC-box DNA binding activity to growth control in undifferentiated and in terminally differentiated viable or apoptotic cells. Nuclear extracts from viable, differentiated cells showed increased retardation of GC box DNA sequence compared with that seen in proliferating cells or those reversibly arrested in early G(1) or late G(1) / S. In contrast, nuclear proteins from dying, differentiated cells showed loss of nuclear GC box DNA binding activity without decrease in binding to TTTGCGCG sequences recognized by the E2F transcription factor, which is known to interact with Sp1. However, cyto-plasmic fractions from apoptotic cells revealed phos-phatase-activated retardation of GC box DNA, which was not evident in similarly treated fractions from undifferentiated cells or sparse differentiated cells. Terminal differentiation also correlated with increase in a slow-migrating phosphorylated Sp1 isoform. Our data suggests that lack of nuclear Sp1/GC box DNA binding activity, may promote apoptosis by diminishing expression of survival-associated genes regulated by GC box DNA promoter sequences in dense terminally differentiated melanoma cells.

Specific tyrosinases associated with melanoma replicative senescence and melanogenesis.

Replicative senescence occurs in normal cells, in contrast to their malignant counterparts which are generally immortal in vitro. We now show that induction of melanogenesis in subconfluent B16 melanoma cells deprived of growth factors can lead to irreversible growth arrest but continued cell viability, concurrent with the expression of specific glycosylated high molecular weight tyrosinases. These tyrosinase activities identify withdrawal from the cell cycle since they were not detected in reversibly arrested quiescent melanocytes, serum-deprived melanoma, or apoptotic melanoma. Our data suggest that different tyrosinases can distinguish cycling and noncycling cells of melanocytic origin and also imply that replicative senescence can be restored in some tumor cells when induced to terminal differentiation in the absence of growth-promoting agents.